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A new method for specific reamplification of DDRT-PCR products is presented. After transient ligation of the primary DDRT-PCR fragments into a T-vector, the cDNAs of interest were reamplified by hemi-nested PCR and thermally asymmetric cycles. In contrast to the originally described protocol, this method of reamplification is specific, sensitive, reproducibly gives a high yield of DNA and allows direct sequencing of the reamplified product without purification or cloning. We used the differential display technique to investigate the effect of a blood meal on the expression of Anopheles gambiae midgutspecific genes and thereby identify genes potentially involved in the sporogonic cycle of the human malaria parasite Plasmodium falciparum. The differential display technique (DDRT-PCR) (1‐3) is a method that can be used for analysis of changes in gene expression by detecting differential production of mRNA and which requires a small amount of RNA. This method has been used successfully to identify a variety of differentially expressed genes (2,4‐6). However, it has some drawbacks such as the large number of false positives generated, and different modifications of the original protocol have been proposed to limit the production of false positives or to facilitate the identification of putative positives (2,7‐10). Another disadvantage is the difficulty in selectively re-amplifying the target cDNA, which is a necessary step in verifying the expression pattern of the DDRT-PCR band ( 8,11). The original procedure for reamplification resulted, in our hands, in poor yields even after two rounds of re-amplification which in most cases generated non-specific PCR products. Furthermore, such direct reamplification often led to the production of fragments which contained the same arbitrary upstream primer at both ends ( 8,9 and unpublished results). These products have no T-tail and may correspond either to mRNA or to contaminating DNA. To circumvent these problems, we developed a procedure for reamplification at high annealing temperature using a transient ligation step (11) and a modified oligo-dT anchored primer (DDT3). To increase the specificity and the yield of the reaction, three hemi-nested reactions are performed with thermal asymmetric cycles (12). Total RNA was isolated from mosquito midguts dissected prior to

Background Immunoglobulin D (IgD) multiple myeloma represent uncommon immunoglobulin isotypes, accounting for 2% of cases with limited information is available regarding the prognosis of these isotypes. They have been considered to have a more aggressive course than the more common Immunoglobulin of IgG and IgA isotypes in the era of conventional chemotherapy. Several studies have reported the outcome of IgD MM after autologous hematopoietic stem cell transplantation (ASCT) in recent years, but none in the context of novel agents currently the backbone of almost all if not all lines of therapy upfront. We sought to study the prognostic impact of novel agents used upfront on IgD MM treated in several IFM (Intergroupe Francophone du Myélome) centres. Method This study has included a group of 21 patients with IgD MM that were treated from 1994 to 2012 across 5 IFM centres. IgD MM was diagnosed as outline in international consensus criteria. Response and survival criteria were used as depicted in the IMWG consensus criteria published. Results Median age was 59 years (min max 38-80), sex ratio was 1.7, 75% had lambda light chain, 65% had severe bone disease and 20% one to 3 bone lesions, 55% had severe renal insufficiency, 30% had hypercalcemia, 55% profound anemia. Finally, 39% had adverse FISH either del17p or t(4;14) on bone marrow plasma cells and 50% were ISS 3 with beta2M greater than 5mg/L. 80% had receive novel agents containing regimens upfront, including 70% in the context of ASCT and 75% a bortezomib-based regimen. Overall, 65% reached at least VGPR with a median time to first response at 4 months (IQ 1-5). IgD MM had a greater chance to reach VGPR on bortezomib-based regimen, 80% versus 50% for patients on thalidomide-based regimen; furthermore, ASCT also improved the depth of response rate with 85% versus 20% that reached VGPR, respectively (p=0.022). The presence of renal insufficiency at initiation did not affect the incidence and depth of response rate as expected. With a median follow-up of 65 months, 40% have relapsed and 35% have died. The median TTP and OS was 3 years (CI95% 3;7) and 6 years (CI95% 5;9), respectively. Although not significant due to the limited number of patients the median OS was much shorter in the group treated with conventional agents as to novel agents, 5 years (2;8) versus not reached with a 6-year event free of 53%. Similarly, the patients that have received a thalidomide-based regimen upfront had a shorter OS as to bortezomib-based regimen, 1.9 years (1;nr) versus 5.8 years (2;9) although not statistically significant. Interestingly, ASCT improved TTP but not OS, with 1.6 years (1;2.5) versus not reached (6-year event free is 63%) as median TTP in patients non ASCT and ASCT, respectively (p=0.045). Conclusion Despite the retrospective analysis and the small number of patients, our study showed that the use of ASCT and bortezomib-based regimen improve the prognosis of IgD MM. This data analysis confirms that IgD MM should be treated according to current guidelines upfront that recommend use of a bortezomib-based regimen in the context of ASCT in fit patients. Treatment options including the role of consolidation and maintenance may further improve the outcomes of these patients. Disclosures: No relevant conflicts of interest to declare.

Invasive aspergillosis (IA) remains a major concern among patients with hematological malignancies who receive cytotoxic chemotherapy or allogeneic stem cell transplantation (allo-SCT). With a view toward better understanding the differences in terms of clinical significance and outcomes of IA between allo-SCT recipients and patients treated for leukemia, we report a single-center study of 739 unselected consecutive patients treated in our unit between August 2000 and February 2004. IA episodes were classified according to the 2008 EORTC/MSG criteria. Patients were hospitalized either for allo-SCT (n=135), or for the treatment of acute leukemia (n=378), chronic lymphocytic leukemia (n=116), or myelodysplasic syndrome (n=105). Probable or proven IA were observed in 29 patients, among whom 7 were undergoing allo-SCT (5.2%), 20 were treated for acute leukemia (5.3%), 1 for chronic lymphocytic leukemia (0.8%), and 1 for myelodysplastic syndrome (0.95%). Most of the clinical signs were mild and nonspecific, especially in allo-SCT recipients. Chronologically, thoracic computerized tomography (CT) showed the first signs of IA in 38% of cases, and was positive the same day as at least one biological test (bronchoalveolar lavage [BAL] and serum galactomannan antigen) in 41% of cases. BAL was contributive to the diagnosis in 66% of the 29 cases. Comparison of patients undergoing allo-SCT to the others revealed significant differences. In allo-SCT recipients, the onset of IA occurred later than in the other patients, after the neutropenic period. The median time between the last hematologic treatment and the diagnosis of IA were 231 days (range, 68-341) in allo-SCT recipients and 17 days (6-57) in the other patients (p First line therapy was liposomal amphotericin B (n=22), caspofungin (n=8), voriconazole (n=6), or itraconazole (n=1), either alone (n=21) or in combination (n=8). Complete remission of IA on CT was observed in 12 cases, stable lesions in 8 cases and progression in 9 cases. Mortality directly related to IA was 24% in our study and occurred early after the onset of infection. The 100-day, 1-year, 2-year and 10-year overall survival (OS) were 42.9%, 28.6%, 0%, 0% for allo-SCT recipient, respectively, compared to 68.1%, 40.9%, 18.2%, 13.6% for the other patients, respectively (P-values ≥ 0.05). These poor outcomes were mainly attributable to the non-relapse mortality (NRM), especially in allo-SCT recipients, who had a 2-year NRM of 100% compared to 54.6% in the other patients, p=0.034 (2-year cumulative incidence of relapse were 0% and 70.2%, respectively). In conclusion, our data seem to distinguish 2 types of IA. One has an early onset in neutropenic leukemia patients. The second, observed in allo-SCT recipients, occurs later, after GVHD development and is associated with a very high NRM incidence, suggesting that those patients remain fragile. Thus, systematic broad-spectrum anti-fungal prophylaxis is highly recommended in patients undergoing allo-SCT who develop GVHD. Disclosures: Berthon: CELGENE: Research Funding.

Abstract 2964 Background. Lenalidomide is an oral IMiD®, immunomodulatory compound, approved for use in combination with dexamethasone (Len/Dex) in patients with RRMM who have received one prior therapy. Len/Dex is indicated until evidence of disease progression at the best-tolerated dose of both Len and Dex (Dimopoulos et al. Leukemia 2011). However, the tolerability profile of long term exposure to Len/Dex is not well described, and evidence that long term exposure to Len/Dex would improve on the response rate and survival has yet to be determined. We sought to determine the efficacy and safety profile of long term exposure to Len/Dex in RRMM pts in a multicentre study. Method. We retrospectively reviewed the medical records of 50 RRMM pts treated with Len/Dex and remaining on Len for ≥2 years with a special focus on pts receiving Len for ≥3 years. All pts included had complete follow up records. Results. The median (range min-max) age was 58 years (39–79) with 30% (n=15) > 65 years (elderly MM), the sex ratio M/F was 1.2, 49% (n=24) ISS 2 and 3, 12% (n=6) severe renal insufficiency (CrCl < 30mL/min), and 8% (n=4) adverse FISH [del17p and/or t(4;14)]. Overall, 25 pts (50%) had Len/Dex at first relapse, 19 pts (38%) at second relapse and 6 (9%) pts in subsequent relapses. Len/Dex was given at first relapse in 10 (66%) elderly patients. The median time from diagnosis to starting Len/Dex was 4.5 years (1–16) for overall cohort and 3 years (1–8) for elderly patients (p=0.05). 28 pts (56%) received Len/Dex for ≥3 years. The median duration on Len/Dex was 3 (2–7) years for the overall cohort, and was 4 (3;7) years for patients exposed to Len ≥3 years. Treatment duration was similar across age categories and across number of previous relapses. With a median follow up of 4 years, 19 patients had stopped Len/Dex. The response rate (ORR, ≥PR) was 96% (n=48), including 37 (74%) patients with ≥VGPR, similar across age categories. Interestingly, the ORR and ≥VGPR were similar irrespective of whether patients have stopped Len/Dex in our study. The ORR was also similar across number of previous relapses, but the ≥VGPR rate was lower in patients at third relapse and beyond, (50%; p=ns). The ORR and ≥VGPR rate was 93% and 77% in patients exposed to Len ≥3 years, similar to the whole cohort. The median time to first response and best response were 2 (1–5) months and 4.5 (2–9) months, respectively. Overall, 9 (18%) patients stopped treatment due to toxicity, 9 (18%) progression of MM, and 1 (0.5%) patient decision. With a median follow up of 4 years, the median (95%CI) TTP was not reached, the estimated 4-yr TTP was 51.5%. There was no imbalance in the incidence of toxicity based on age, number of previous relapses, and patients exposed to Len ≥3 years did not discontinue more often due to toxicity, 14% versus 19% for those receiving Len < 3 years. The hematological safety profile was similar across age categories, number of previous relapses, and patients exposed to Len ≥ 3 years; overall, 8 (16%) patients experienced grade 3–4 neutropenia, 6% thrombopenia, and 6% anemia. Ten (20%) patients experienced a thromboembolic event (VTE), all of them of venous type. Two patients had previous history of VTE, but none of them experienced VTE on Len/Dex, likely related to adequate VTE prophylaxis. The median time to first occurrence was 5 (1;28) months, although 4/10 occurred in patients with ≥3 years on Len. All VTE occurred while on VTE prophylaxis except for 1 patient, 5 on aspirin, 2 on prophylactic doses of LMWH, and 2 on VKA (target INR 2–3). The incidence rate of second primary malignancy (SPM) was 3 (6%) (larynx, lung, and MDS). The SPMs occurred at a median time of 4 years from start of Len, while Len was already stopped in 2/3 patients, the latter stopped len at time SPM was diagnosed. Interestingly, none of the patients with more than 3 years exposure on Len had SPM. Conclusions. The current study provides estimates of responses, TTP and safety in a series of MM pts with long-term exposure to Len-based regimen at relapse. 62% of patients remained on Len beyond 3 years reflecting the efficacy and good safety profile of Len in relapsed MM, irrespective of age and number of prior therapies. Furthermore, no excess of long term side effects, including SPM, was observed with a prolonged long follow-up in this study. Disclosures: Robinson: Celgene: Employment. Miljkovic:Celgene: Employment. Morel:Celgene: Employment. Boccacio:Celgene: Employment. Facon:millenium: Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees. Hulin:celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees. Leleu:Onyx: Honoraria, Speakers Bureau; Sanofi: Honoraria; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; LeoPharma: Honoraria, Speakers Bureau.

Abstract 3970 Background: Measurement of serum M-spike is used to assess response to therapy and treatment-free survival in IgA myeloma. However, its resolution on SPEP and the presence of IgA-dymers can make accurate measurement difficult. The more the M-spike decreases on SPEP, the more imprecise the M-spike measurement is using SPEP. IgA M-spike often migrates in the betaglobines that renders the M-spike measurement often complicated to analyze. Furthermore, quantification of the clonal IgA chain by nephelometry (IgAneph), which inherently includes monoclonal and polyclonal immunoglobulins, does not accurately reflect the tumour burden. Currently, there is a need in myeloma with IgA isotype (approximately 30–40% of patients) to identify new markers that better reflect the disease burden and the response to treatment, and correlate to patients' outcome. Hevylite® measures IgAkappa and IgAlambda and might provide precise quantitative measurement of the IgA M-spike. We sought to determine whether Hevylite® can be used as a reliable marker for diagnosis and response to therapy in IgA myeloma as compared to the M-spike measurement on SPEP and nephelometry. Methods: We conducted a retrospective analysis on 113, smoldering or symptomatic, IgA myeloma patients at diagnosis referred to our department from 1997 to 2011. All serum samples were collected prior to treatment or at relapse (for sequential data) and were kept frozen since collection. Hevylite® measurements were made at The Binding Site Ltd, Birmingham, UK. A normal range was produced from normal (blood donor) sera (n=138), and were for IgA kappa 0.48–2.82g/L, IgA lambda 0.36–1.98g/L and IgA K/L ratio 0.80–2.04. For ease of comparison we have studied the IgA hevylite ratios expressed as IgA K/L ratio. Results: The median age at diagnosis was 65 years (range: 33–93) and the M/F sex ratio was 0,91. Forty-eight percent of patients had an ISS greater than 2 (n=86). On nephelometry the median IgA level was 22g/L (range min-max, 0–100). Fifty patients had M-spikes migrating among the betablobulines, 27 among the gammaglobulines, 10 migrated in both and 23 unspecified. Fourteen patients had oligosecretory disease (M-component < 10g/L). Forty-two patients had an IgA kappa clonal chain, 70 others an IgA lambda and for one patient the data was not available. Among IgA kappa patients the mean HCLratio was 616.6 (median 117.7 [0.021–4323.7]) whereas it was 0.61 (0.21 [0.004–0.455]) among the IgA lambda patients. Across the entire population, 58 patients were identified by SPEP and HCL ratios. Among the 55 patients whose M-component was not quantified on SPEP, HCL ratios were abnormal in 53/55 (96%) cases. In the subgroup of patients whose M-protein migrates in the beta-region 29 out of 50 are identified by HCL ratios and SPEP. Another 19 patients (38%) had abnormal HCL ratios while unquantified on SPEP. The same was seen in patients whose M-protein migrates among the gammaglobulins with 16/27 and 11/27 (40%), respectively. In the last subgroup of patients with beta and gamma migrating M-proteins, all (10/10) were identified by HLC and SPEP. More interestingly, among the oligosecretory MM patients identified by an M-component < 10g/L, all (14 patients) had an abnormal HCL ratio. In our series of IgA myeloma, 51% of patients were accurately quantified on SPEP. When using HCLratios, an extra 47% of patients became measurable, with an abnormal HLC ratios allowing IgA myeloma in up to 98% of cases. When considering IgA measurement using nephelometry, 102 patients had both high IgA levels (90%) and abnormal HCL ratios. Interestingly, 7 patients had normal IgA levels with IgAneph and abnormal HCL ratio offering a useful diagnostic tool for 96% of patients as compared to IgAneph. In our series, IgA myeloma was neither associated with a poor outcome (median [range] OS: 119 months [0,5–604]) nor a poor response to therapy (median [range] TTP: 16 months (1–92). HCL involved chain ROC analysis identified a 37g/L cut-off as prognostic in IgA myeloma(p=0.039). Conclusion: Hevylite® is a new and reliable marker for diagnostic and monitoring of IgA myeloma. It enables to quantify accurately up to 98% of IgA-MM patients. These preliminary data need confirmation in further prospective trials in order to monitor further the impact of this marker in IgA myeloma patients before it becomes the gold standard to monitor the IgA M-protein in years to come. Disclosures: Boyle: Chugai: Consultancy, Honoraria. Combat:The Binding Site: Employment. Pietrantuono:The Binding Site: Employment. Facon:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees. Harding:The Binging Site: Employment. Leleu:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Sanofi: Honoraria; Onyx: Honoraria, Speakers Bureau; LeoPharma: Honoraria, Speakers Bureau.

We report in vitro culture of zoonotic Babesia sp. EU1 from blood samples of roe deer in France. This study provides evidence of transovarial and transstadial transmission of the parasite within Ixodes ricinus, which suggests that this tick could be a vector and reservoir of EU1.

Background Light chain amyloidosis (AL) is characterized by a median overall survival (OS) from diagnosis of approximately 3 years, but with clinically overt cardiac involvement this is reduced to 1 year. Bortezomib (B) has shown great promise in the treatment of AL, especially of cardiac involvement. However, efficacy might be hampered by severe safety issues with use of B, primarily of neuropathy type. We sought to study the prognostic impact of the safety profile of B in AL with attention to cardiac involvement. Method This study has included 27 patients with AL, of these 18 had cardiac and 9 kidney but no cardiac involvement. AL was diagnosed as outline in international consensus criteria, as to the hematologic and organ responses. B was given IV twice weekly at the starting dose of 1.3 mg/m², in combination to weekly cyclophosphamide in 13 patients (52%). Results The median age was 63, sex ratio was 18/9, all cardiac AL had Mayo Cardiac Stage III but 3, and none in patients with no cardiac AL. 70% were at diagnostic. Seven patients died during the first month, all of them but one had cardiac AL. We then looked at the safety profile of B in the studied population, and found that 25% and 75% experienced some degree of hematotoxicity in cardiac and in no cardiac AL (p=NS). The non hematology toxicity rate was 62% and 38%, respectively (p=NS). 26% of patients needed dose reduction of B and 33% dose interruption of B in the study before cycle 4, all related to non hematological toxicity of neuropathy, fatigue and GI AEs; and was similar in cardiac as compared to no cardiac AL. The overall hematologic response rate (ORR) was 56% and at least VGPR (with >90% decrease in difference between involved/uninvolved light chain) 41%. The responses were 56% and 39% in cardiac AL, similar to patients with no cardiac involvement (56% and 44%), respectively. The median duration of response was 13 months overall, 10 and 20 months in cardiac and in no cardiac AL, respectively (p=NS). Non hematological toxicity did not impact the response rate or the duration of response. With a median follow-up of 41 months from start of B, 70% relapsed and 59% died in the study as a whole, and 67% and 67% in the cardiac group, respectively. The median time to progression was 20 (95CI 4;35.5) months as a whole, and 13 (9;17) months and 20 (0;43) months in cardiac and in no cardiac AL (p=NS). The median OS was not reached in the cohort as a whole and in patients with no cardiac involvement, but was 5 months in cardiac AL (p=NS); the estimated 4-year OS was 55%, 62% and 50%, respectively. Interestingly, all cardiac AL that survived beyond 6 months remain alive at F-up date. Several variables negatively impacted survival in univariate analysis in the group with cardiac AL, including decreased LVEF (p=0.025), NYHA greater than 2 (p=0.007),Mayo Cardiac Stage III (p=0.028), no hematological (p=0.002) and no organ responses (p=0.05), occurrence of non hematological toxicity (p=0.002) with the consequence of dose reduction of B (p=0.09) and dose interruption of B (p=0.04). In multivariate analysis, independent variables that impacted survival were hematological response (OR = 5.1, 95%CI = 1.5-18; p = 0.011) and non hematological toxicity (OR = 3.6, 95%CI = 0.8-14; p = 0.05). Conclusion Bortezomib is a very rapid and effective therapy for AL particularly of cardiac involvement. However, patients may develop severe side effects with Bortezomib that preclude efficacy of Bortezomib given IV, and consequently impact negatively survival. This data favours use of sub cutaneous Bortezomib in AL although not validated in this indication yet. Disclosures: Leleu: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy, Honoraria; Leopharma: Consultancy, Honoraria; Millennium : Honoraria; Amgen: Honoraria; Novartis: Honoraria.

Background Solitary plasmacytoma (SP) is characterized by a localized proliferation of monoclonal plasma cells resulting in a mass in either bone (SBP: Solitary Bone Plasmacytoma) or soft tissue (EMP: Extra-Medullary Plasmacytoma), without evidence of Multiple Myeloma (MM). The prognosis of SP is marked by a high risk of transformation to MM. SBP (as compared to EMP), older age, tumor size > 5 cm, and persistence of monoclonal immunoglobulin after treatment were prognostic for progression to MM. More recently, studies demonstrated that presence of focal lesions on magnetic resonance imaging (MRI) favored progression to MM in patients (pts) with plasmacytoma. The exact role of FGD-PET CT (PET) in MM remains debatable, although it clearly provides additional valuable information to assess plasmacytoma in the context of MM, compared with MRI. The prognostic role of PET in identifying progression to MM of SP has not been formally demonstrated. We aimed to determine the impact of PET pre and post therapy on the risk of transformation of SP to overt MM. Method We retrospectively reviewed the medical records of 43 pts diagnosed with one SP clinically, confirmed with histology, either EMP (10 pts) or SBP (33 pts). All pts had one SP clinically, and were treated locally with surgery and/or radiotherapy. PET and MRI were performed at diagnosis prior to (initial) and at the end of therapy. SP was diagnosed as outlined in international consensus criteria. All pts had complete follow up records pre and post therapy. Results The median age was 57.5 years with 33% pts older than 65, the sex ratio was 1.8, IgG kappa was the most frequent isotype, the maximum M-spike value was 30g/L, 48% pts had abnormal involved serum free light chain (isFLC) value and 64% had abnormal sFLC ratio (K/L). 33% had 2 hypermetabolic lesions on initial PET, and 20% had 2 focal lesions on initial MRI. Out of the SBP, 56% were localized on spine or pelvis. With a median follow-up of 50 months (mo), the median overall survival (OS) was not reached for the whole cohort, with a 6-year OS at 79.4%. The median time to MM progression (TTMM) was 71 mo (95%CI: 59;101). The TTMM was not significantly different in SBP versus (vs) EMP although the 5-year TTMM was 58% and 83%, respectively. The TTMM for the 2 hypermetabolic lesions on initial PET group was 23 mo (9;37) vs not reached otherwise (p=0.003). Conversely, MRI at diagnosis did not have any impact on TTMM in our study, although the median TTMM for the 2 focal lesions on initial MRI group was 30 mo (9;51) vs not reached otherwise. Age had no impact on TTMM, but abnormal initial K/L ratio (p=0.022) and abnormal initial isFLC (p=0.002) did impact TTMM, 36 mo (14;58) and 21 mo (0;42) vs not reached otherwise, respectively. A normalized PET at completion of treatment did not reach significance, as to normalized MRI, but the absence of normalized isFLC value also impacted TTMM, 21 mo (10;32) vs not reached otherwise (p=0.016). Using multivariate analysis, independent variables that impacted TTMM were abnormal initial isFLC (OR=10, 95%CI=1-87; p=0.008) and initial PET (OR=5, 95%CI=0-9; p=0.032). Interestingly, initial PET did not influenced OS, median 71 mo for the 2 hypermetabolic lesions on initial PET group vs not reached otherwise, respectively (p=ns). This data suggested that pts with SP that transformed into MM did not have a worse prognostic at time of MM. The median OS of pts with SP from start of MM was not reached, the 4-year OS was 66%. With a special focus on SBP, we identified the exact same prognostic factors for TTMM as to the whole cohort in univariate and multivariate analysis. The localization of SBP in the spine is usually considered of poor prognosis, but we did not find any confirmation of this observation in our study. Conclusion FDG-PET CT and involved sFLC value at diagnosis of SP are important predictors of the risk of progression to MM. This data analysis may lead to a different management of SP for patients with one or the 2 abnormal indicators, irrespective to the solitary clinical aspect of SP. One may consider to embrace treatment of MM for SP when FDG-PET CT and involved sFLC value are abnormal at diagnosis, while surgery and/or radiotherapy would remain the appropriate therapeutic procedure for SP otherwise. Disclosures: Facon: JANSSEN: Honoraria, Speakers Bureau; CELGENE: Honoraria, Speakers Bureau. Leleu:JANSSEN: Honoraria; CELGENE: Honoraria.

Background Quantification of monoclonal immunoglobulins in serum is the foundation of IMWG multiple myeloma (MM) response criteria for patients with intact immunoglobulin disease. However, electrophoretic methods for quantifying M-Ig in serum are subject to well documented limitations including inaccuracy at high concentrations (due to dye saturation) and poor sensitivity at low concentrations. Moreover, the methods require skilled interpretation and can be time consuming; however their clinical utility is well established. Recently heavy/light chain (HLC) assays, which quantify kappa and lambda isotypes of intact immunoglobulins have become available. Here we compare responses assigned by the immunoassays to IMWG criteria and evaluate the clinical impact of discordance. Methods Sequential sera, from enrolment to progression, were available for 107 MM patients (59 IgGκ, 29 IgGλ, 12 IgAκ, 7 IgAλ) enrolled onto either the IFM 2009-02 end-stage relapsed or refractory MM or the IFM 2010-02 in del17p and t(4;14) relapsed or refractory MM trials. The inclusion criterion was a measurable intact immunoglobulin MM according to IMWG criteria (M spike ≥ 10 g/L), using serum and/or urine protein electrophoresis. IgA HLC (IgAκ and IgAλ) and IgG HLC (IgGκ and IgGλ) analysis was compared to historic SPEP, IFE, UPEP, uIFE and serum free light chain (sFLC) results (measured using polyclonal antisera based assays). Responses were assigned at approximately 90 days (median 90, range 61-107 days) and at maximum response (if different) according to IMWG criteria using changes in monoclonal protein concentrations measured either by SPEP or dHLC (clonal - non clonal). Complete response was assigned either by the absence of monoclonal protein on IFE or by a normal HLC ratio. Results At the time of enrolment, 87/88 IgG and 17/17 IgA patients had abnormal HLC ratios which were concordant with IFE results, and quantification of the monoclonal protein by dHLC was similar (median (range): 29.2 (2.5-77.5) g/L) and SPEP (30.7 (1.8-66.9) g/L). After 3 months of treatment, responses assigned by HLC/FLC assays showed near-perfect agreement with responses assigned using SPEP and IFE (Weighted Kappa >0.81, p At maximum response there was near-perfect agreement between the responses assigned using HLC/FLC or SPEP, IFE, UPEP, uIFE (Weighted Kappa >0.81, p Conclusion Changes in dHLC can be used in place of SPEP quantification to assign IMWG responses in patients with intact immunoglobulin MM. Minor discordances had an impact on the model fit favouring the HLC assigned response over response assigned using SPE/IFE. Changes in dHLC identified response and relapse earlier than clinically assigned in a number of patients. Considering the small number of patients, we suggest that larger prospective trials be conducted to validate these findings. Disclosures Leblond: GSK: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau. Snell:The Binding Site Group Ltd: Research Funding. Leleu:Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; BMS: Honoraria; Amgen: Honoraria.

Introduction: CD38 is highly and uniformly expressed on myeloma cells (1). Daratumumab is a human anti-CD38 IgG1κ monoclonal antibody that has previously shown a favourable safety profile as a single agent in patients with relapsed and refractory (RR) multiple myeloma (MM) (2). This study further assesses the efficacy of Daratumumab in combination with Dexamethasone in heavily pre-treated myeloma patients that are refractory to Lenalidomide, Pomalidomide, and Bortezomib. Methods: This study is an ongoing, open-label phase II study of Daratumumab in combination with Dexamethasone (NCT02626481). Sixty-four, heavily pretreated Patients were recruited in thirty centres in France and Belgium from November 2015, to receive Daratumumab and Dexamethasone. Daratumumab 16 mg/kg was administered weekly during the first two 28-day cycles, every other week during Cycles three through six, and monthly in Cycle seven and beyond until disease progression or unacceptable toxicity. Patients were all refractory to Lenalidomide (Len), Pomalidomide (Pom) (defined by a progression within 60 days from last drug dosing) and Bortezomib (Bz) (defined by a progression within 6 months from last drug dosing). The primary objective was overall response rate as per the International Myeloma Working Group criteria. A planned safety and efficacy interim analysis was performed after the first 19 patients were enrolled. The last patient was enrolled on the 1stof August 2016. Results: Sixty-four patients were recruited onto the study. The median age (range) at screening was 61 (30-80). The median number (range) of prior lines of therapy was 6 (2-9). Sixty-seven percent of patient had previously received an autologous stem-cell transplant. At the time of screening, 20% of patients (n=13) had a t(4;14) and 12.5% (n=8) a del(17p). Planned interim analysis after the first 19 patients were enrolled did not find any unexpected toxicity. Safety and efficacy results (data cut May 15, 2016) of Daratumumab 16 mg/kg are presented here. No patient discontinued treatment due to Treatment Emergent Adverse Event such as infusion related reactions. Ten (15%) patients discontinued treatment due to disease progression after a median of one-cycle. The most common non-haematological TEAEs included infusion related (IRR, n=5, 8%), and fatigue (n=6, 9.3%). All patients with IRRs recovered and continued to receive treatment. Only six (9.5%) patients experienced hyperthermia. Thrombocytopenia and neutropenia were the most frequently reported grade 3 or 4 TEAE (11 and 5% respectively). Planned interim efficacy assessment showed a response rate (defined as a Partial Response (PR) or greater) in 3/19 patients at the end of the first cycle and 4/19 at the end of the second cycle, and a clinically relevant response (Stable Disease (SD) or greater) at the end of the second cycle for 11 of 19 patients, thus meeting the planed futility criteria and enabling the trial to go forward. As per the 15thof May, among the 40 evaluable patients (that had received at least 2 treatment cycles or progressed within the first) the overall response rate (3) was 25%, with eight (20%) partial responses (PR) and two (5%) very good partial responses (VGPRs) after a median of two cycles (range 1-5). An additional seven patients (17.5%) obtained a Minimal Response (MR) according to the EBMT criteria (4). This is consistent with prior results. Updated results will be presented at the time of ASH. Conclusions: Daratumumab in combination with Dexamethasone is a safe treatment option with a favourable benefit/risk profile for the treatment of triple relapsed or refractory (Len, Pom and Bz) myeloma patients. 1. Stevenson GT. CD38 as a Therapeutic Target. Mol Med. 2006;12(11-12):345-6. 2. Lokhorst HM, Plesner T, Laubach JP, Nahi H, Gimsing P, Hansson M, et al. Targeting CD38 with Daratumumab Monotherapy in Multiple Myeloma. N Engl J Med. 2015 Sep 24;373(13):1207-19. 3. Kyle R, Rajkumar S. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leuk Off J Leuk Soc Am Leuk Res Fund UK. 2009 Jan;23(1):3-9. 4. Bladé J,et al. Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation. Myeloma Subcommittee of the EBMT. Br J Haematol. 1998 Sep;102(5):1115-23. Disclosures Boyle: Novartis: Honoraria; Pfizer: Honoraria; Takeda: Honoraria; Janssen: Honoraria. Leleu:Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria; TEVA: Membership on an entity's Board of Directors or advisory committees. Hulin:Amgen: Honoraria; Janssen: Honoraria; Bristol: Honoraria; celgene: Honoraria; takeda: Honoraria. Moreau:Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Fohrer:amgen: Consultancy; celgne: Consultancy. Decaux:SIEMENS: Honoraria, Other: supply of free light chain assays , Research Funding; The Binding Site: Other: supply of free light chain assays , Research Funding. Avet-Loiseau:celgene: Consultancy; amgen: Consultancy; janssen: Consultancy; sanofi: Consultancy. Attal:celgene: Consultancy, Research Funding; amgen: Consultancy, Research Funding; janssen: Consultancy, Research Funding; sanofi: Consultancy. Facon:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: travel and expense, Speakers Bureau.

Ixodes ricinus tick species, the most abundant and widespread tick in Europe, is an important vector of pathogens affecting both animal and human health. To replace the use of acaricides that generate environmental contamination and resistances, new environmentally sustainable approaches providing broad protection against ticks and tick-borne pathogens (TBP) are urgently needed. Such development requires improved understanding of the biology of ticks and more particularly of their interactions with vertebrate hosts and TBP. Tick saliva is an essential biofluid for ticks, as its proteolytic, anticoagulant, immunomodulatory, analgesic and anti-inflammatory activities allow ticks to acquire their blood meal under optimal conditions. Moreover, injection of saliva during blood feeding represents the principal route by which TBP are transmitted to the host. To understand the molecular mechanisms involved in TBP transmission, as well as to identify putative vaccine candidates against I. ricinus, salivary glands from bacteria infected and uninfected ticks were previously compared by high throughput transcriptomics. The most up-regulated transcript following infection was IrSPI, which belongs to the Kunitz/BPTI inhibitor family. Functional analyses via RNAi knockdown experiments revealed that IrSPI enhances both blood feeding and bacterial burden in the salivary glands. This present PhD work concerns then the structural, biochemical and functional characterization of IrSPI as a molecule involved in tick-host-pathogen interactions. Our aim was first to define the structure of IrSPI gene but, unfortunately, while our results have led to progress on this issue, we have not been able to get the full sequence. Then, the dynamic of IrSPI expression was evaluated during both tick feeding and colonization of ticks by pathogens, showing that its expression is induced by blood feeding and TBP but not by Escherichia coli that is not transmitted by I. ricinus. In addition, our results shown the expression of IrSPI in several tick organs, suggesting its implication in several functions in tick physiology. Among them, the discovery of the injection of IrSPI, through the saliva, to the vertebrate host allowed us to consider a role in host responses to tick bite. Evaluation of IrSPI effect on host showed no impact on coagulation through extrinsic pathway, as determined by analysis of thrombin generation time and by fibrinolysis, or in angiogenesis. However, it inhibited the proliferation of mitogen-stimulated CD4+ lymphocytes and increased unstimulated-B cell proliferation. In addition, IrSPI also modulated cytokine production from macrophages and splenocytes, repressing significantly most of proinflammatory cytokines and chemokines. Thus, we demonstrated that IrSPI plays a role in modulating the host immune response during blood feeding. Finally, preliminary results in the identification of the protein’s interactants open many research perspectives for understanding how IrSPI acts in tick physiology and counteracts host responses to tick injury and pathogen transmission., Ixodes ricinus est l’espèce de tique la plus abondante et ayant la plus vaste répartition géographique en Europe. Elle est le vecteur de nombreux agents pathogènes d’importance en santé publique et vétérinaire. Le remplacement des acaricides générant pollution environnementale et apparition croissante de résistances requiert le développement urgent de nouvelles stratégies de lutte efficaces contre les tiques et les agents pathogènes qu’elles transmettent. La découverte de telles stratégies passe nécessairement par une meilleure connaissance des interactions entre les tiques, leurs hôtes et les agents pathogènes transmis. La salive de tique, à l’interface de ces interactions, est un fluide essentiel pour ces arthropodes et possède notamment des propriétés protéolytiques, anticoagulantes, immunomodulatrices, analgésique, et anti-inflammatoires qui permettent à la tique de réaliser ses repas sanguins extrêmement longs. Afin de comprendre les mécanismes moléculaires impliqués dans la transmission des agents pathogènes et pour identifier de possibles candidats vaccinaux contre I. ricinus, une étude transcriptomique comparative entre des glandes salivaires infectées et non infectées par la bactérie Bartonella henselae a été antérieurement réalisée. Le transcrit le plus surexprimé suite à cette infection était IrSPI, un inhibiteur de sérine protéase de la famille des Kunitz. Les analyses fonctionnelles par ARN interférence ont montré l’implication de ce gène dans le gorgement et de l’infection des glandes salivaires par B. henselae. Ainsi, les travaux de thèse présentés ici ont concerné l’analyse structurelle, biochimique et fonctionnelle de IrSPI en tant que molécule impliquée dans les interactions tick-hôte-pathogène. Le premier objectif était de définir la structure et la séquence du gène IrSPI mais, malheureusement, bien que nos résultats aient permit des avancés sur cette question, nous n'avons pu obtenir la totalité de sa séquence. Dans un second temps, la dynamique d’expression d’IrSPI a été évaluée au cours du gorgement et de l’infection des tiques par différents agents pathogènes, montrant que son expression est induite par le repas sanguin, par des agents transmis par la tique mais pas par Escherichia coli, bactérie non transmise. De plus, nos résultats ont montré l’expression de IrSPI dans plusieurs organes de la tique, suggérant son implication dans diverses fonctions au sein de ce vecteur. Parmi elles, la mise en évidence d'une injection, par la salive, de la protéine à l'hôte vertébré nous a permis d'envisager un rôle sur les réponses de l'hôte à la piqûre de tique. Nos résultats n’ont montré aucune implication dans la voie extrinsèque de la coagulation ni dans la fibrinolyse, ni dans l’angiogenèse. En revanche, ils ont démontré que IrSPI inhibe la prolifération des lymphocytes TCD4+ sous stimulation monogénique quand chez des lymphocytes B non stimulés IRSPI, il induit une hausse de la prolifération. De plus IrSPI a montré une action négative significative sur la production de la majorité des cytokines et chimiokines pro-inflammatoires produites par les macrophages et les splénocytes. Ainsi, IrSPI, correspond à un des composants salivaires d’I. ricinus lui permettant de moduler la réponse immune de l’hôte pour lui permettre de prélever son repas sanguin tout en favorisant la transmission des agents pathogènes. Enfin, des résultats préliminaires dans l'identification des interactants de IrSPI à la fois chez la tique et l’hôte vertébré ouvre de nombreuses voies de recherche quant à la compréhension de ses fonctions.

http://web.oie.int/boutique/index.php?page=ficprod&id_prec=1407&id_produit=1516&lang=fr&fichrech=1; International audience; Vector-borne diseases are principally zoonotic diseases transmitted to humans by animals. Pathogens such as bacteria, parasites and viruses are primarily maintained within an enzootic cycle between populations of non-human primates or other mammals and largely non-anthropophilic vectors. This 'wild' cycle sometimes spills over in the form of occasional infections of humans and domestic animals. Lifestyle changes, incursions by humans into natural habitats and changes in agropastoral practices create opportunities that make the borders between wildlife and humans more permeable. Some vector-borne diseases have dispensed with the need for amplification in wild or domestic animals and they can now be directly transmitted to humans. This applies to some viruses (dengue and chikungunya) that have caused major epidemics. Bacteria of the genus Bartonella have reduced their transmission cycle to the minimum, with humans acting as reservoir, amplifier and disseminator. The design of control strategies for vector-borne diseases should be guided by research into emergence mechanisms in order to understand how a wild cycle can produce a pathogen that goes on to cause devastating urban epidemics.