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Summary Aim of the study To determine the usefulness of the combination of serum free light chain (FLC) assays and serum protein electrophoresis (SPE) in identifying serum monoclonal protein in a population of hospitalized patients. Patients and methods One hundred and thirty-five consecutive sera sent to our laboratory were tested by total protein and Ig quantification, FLC assays, capillary zone electrophoresis and immunofixation. Results of SPE and FLC assays were compared with the standard immunofixation method for identifying monoclonal protein. Results In our hospital population, 31% of patients’ sera have a monoclonal protein revealed by immunofixation. Sensitivity and specificity in detecting monoclonal protein were respectively 88% and 82% for SPE, 69% and 55% for FLC assays, 93% and 52% for combination of SPE and FLC assays. Conclusion In our hospital population, various clinical and therapeutic situations require the search and/or the biological follow-up for monoclonal protein. Combination of SPE, immunofixation and, according to clinical criteria, serum FLC assays are recommended.

Resume But de l’etude Le but de ce travail a ete de comparer les performances de lecture d’un logiciel (Neurosoft-Sebia) a celle du biologiste pour la detection d’une anomalie qualitative a l’electrophorese des proteines seriques, anomalie pouvant justifier la recherche d’un constituant monoclonal. Materiel et methodes A partir de 440 serums, les interpretations des electrophoreses du biologiste et du logiciel ont ete comparees entre elles, et confrontees aux resultats de l’immunofixation, utilisee comme test de reference pour la recherche d’un constituant monoclonal. Resultats La concordance des resultats entre la lecture du biologiste et du logiciel est de 81,6 %. Pour la mise en evidence d’une anomalie qualitative, en rapport avec la presence d’un constituant monoclonal, la lecture du logiciel apparait plus sensible que celle du biologiste (96 versus 91 %) mais moins specifique (52 versus 76 %). Les valeurs predictives negatives sont respectivement de 96 et 94 % et les valeurs predictives positives de 50 et 66 %. Discussion L’emploi du logiciel permet une standardisation des lectures et des procedures de validation. Par sa grande sensibilite et sa bonne valeur predictive negative, la validation d’un resultat negatif est fiable et rapide. Pour le depistage d’un constituant monoclonal on observe une meilleure performance diagnostique avec l’utilisation du logiciel.

Background. POEMS syndrome is a rare form of B cell dyscrasia combining a proliferation usually of plasma cells, a polyneuropathy, osteocondensing bone lesions and multiple other clinical signs. The pathogenesis is not well understood but VEGF plays a major role. In patients with one or two sclerotic plasmacytoma and no bone marrow involvement, first line therapy should include radiation. For patients with diffuse sclerotic lesions, bone marrow involvement or absence of any bone lesion and for those who have not demonstrated stabilization of their disease 3 to 6 months after completing radiation systemic therapy is indicated, the most effective being high dose chemotherapy with autologous stem cell transplant (ASCT). Radiation of a single lesion is effective in about every other case and is accompanied by a fairly slow improvement of the neurological symptoms, often after initial worsening. ASCT seems to be accompanied by a number of important complications, in particular engraftment syndrome. Outside these 2 treatments there is no consensus therapy. Lenalidomide (LEN), a drug without serious neurological toxicity, has the advantage of being both anti-angiogenic and cytotoxic to malignant plasma cells (Richardson PG, Blood 2002;100(9):3063-7). We have recently reported a series of 20 French patients with POEMS syndrome treated by LEN with a good efficacy. We now report the first 27 patients of a prospective phase II trial using LEN + dexamethasone (LEN-DEX), 2 cycles preceding radiation or high dose treatment trying to obtain a rapid clinical response and to avoid engraftment syndrome or 9 cycles followed by 1 year LENalone in patients who cannot receive radiation or ASCT. Methods. Newly diagnosed or relapsing patients with POEMS syndrome who needed to be treated were eligible. Patients who can be treated by local radiation or intensive treatment with stem cell support receive two 28 day cycles of LEN 25 mg PO Days 1-21 and DEX 40 mg PO Days 1,8,15,22 before radiation or intensive treatment (Group 1), the other patients receive 9 cycles of the same LEN-DEX (Group 2) and then 12 cycles of continuous low dose LEN (10 mg). LEN dose was tapered to 10 mg for patients with a creatinine clearance between 30 and 50 ml/min and DEX to 20 mg for patients above 75 years of age and for those who were frail patients. Main eligibility criteria included a diagnosis of POEMS syndrome according to criteria by Dispenzieri et al (Am J Hematol 2012;87(8):804-14), an age of 18 or more, a creatinine clearance above 30 ml/min, no prior treatment with or contraindication to LEN and no uncontrolled thrombosis. Serum and plasma VEGF, serum electrophoresis, immunofixation and free light chain measurements were centrally monitored. Neurologic evaluations were performed using the Overall Neuropathy Limitations Scale (ONLS), the Neurological Impairment Scale (NIS) and the 10 meter walk test (10MWT). The primary endpoint was evaluation of the effectiveness of LEN-DEX combination using biological responses (decrease of monoclonal protein and serum VEGF level) and secondary endpoints were clinical and particularly neurological responses. Results. Twenty-seven patients have been included in 12 centres, median age was 61 (range 32-75), the median follow-up was 6.6 months (range 2-24). Eighteen patients were in group 1, with radiotherapy in 10 patients and ASCT in 8 patients; 9 patients were in group 2. Nineteen patients were in first line and 8 already treated. Only 2 patients experienced grade 3-4 adverse events due to LEN (cytopenia) and 2 patients had allergic rashes, no thrombotic event occurred. No engraftment syndrome was noted in the 5 patients already treated with ASCT. To date, no patient have died. Evolution of VEGF median values in serum and plasma, M-spike and dFLC levels and evolution of neurological measurements are reported in table 1. Neurological improvement was very rapid in some patients, using ONLS and 10MWT 11/18 evaluable patients had a neurological improvement after 2 cycles with an improvement of 1 or more of the ONLS score and/or change of 0.1 m/s or more in the 10MWT. Only one patient who progressed after nine cycles received another therapy. Conclusion. This is the first prospective trial of LEN-DEX in POEMS syndrome. This combination seems well tolerated in this disease with a good efficacy on VEGF measurements and rapid neurological improvement in the majority of patients. Updated data will be presented at the meeting. Download : Download high-res image (32KB) Download : Download full-size image Figure 1 . Disclosures Jaccard:Celgene: Drug supply to Trial Other. Tournilhac:mundipharma: Honoraria, Other, Research Funding; GSK: Honoraria, Other, Research Funding; Roche: Honoraria, Other, Research Funding. Moreau:celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Resume Objectif. L’identification de cristaux de cryoglobuline IgG1-κ dans le liquide articulaire d’une femme souffrant d’une arthropathie destructrice nous a fait rechercher si le caractere cristallise de cette cryoglobuline lui conferait des proprietes pro-inflammatoires, notamment en stimulant la production du facteur de necrose tumorale-α (TNF-α) par les monocytes humains. Methodes. Les monocytes normaux isoles par adhesion au plastique ont ete cultives en micropuits. Ils ont ete mis en presence, apres sterilisation, de 150 μg/mL ou de 2 mg/mL de la cryoglobuline cristallisee ou d’une cryoglobuline non cristallisee de type I, de 2 mg/mL de microcristaux d’urate de sodium (UMS), de 10 μg/mL de LPS ou de milieu seul. La concentration de TNF-α dans les surnageants de cultures a ete mesuree par methode Elisa apres 6, 8 et 24 heures d’incubation. Resultats. Il n’a pas ete observe de stimulation de la secretion du TNF-α par la cryoglobuline cristallisee. En revanche, en presence des deux concentrations de la cryoglobuline non cristallisee, le taux de TNF-α a augmente au cours du temps de facon dose dependante. Cette production etait significativement inferieure (p

Objectives. We recovered an IgG1-κ cryocrystalglobulin in synovial fluid and membrane specimens from a patient with destructive arthropathy. In the present study, we investigated its proinflammatory properties by measuring its effects on TNF-α production by normal human monocytes. Materials and methods. Normal human monocytes isolated by plastic adhesion were cultured in microtiter plates. Adherent monocytes were cultured for 6, 8, and 24 hours with sterile cryocrystalglobulin (150 μg/mL and 2 mg/mL), type I noncrystallised cryoglobulin (same concentrations), monosodium urate (MSU) crystals (2 mg/mL), LPS (10 μg/mL), or medium alone. Supernatant TNF-α concentrations were assayed using an ELISA. Results. Cryocrystalglobulin had no effect on TNF-α production by normal human monocytes. Noncrystallised cryoglobulin increased TNF-α levels in supernatants in a time-dependent and concentration-dependent fashion. This increase was significantly less marked than the increases achieved with MSU crystals or LPS. Conclusion. IgG1κ cryocrystalglobulin has no effect on TNF-α production by normal human monocytes. Fc region changes within the cryocrystalglobulin molecule may explain this finding.

Sensory chronic inflammatory demyelinating polyneuropathy (CIDP) can be difficult to diagnose.We report 22 patients with chronic sensory polyneuropathy with ≥1 clinical sign atypical for chronic idiopathic axonal polyneuropathy (CIAP) but no electrodiagnostic criteria for CIDP.Clinical signs atypical for CIAP were: sensory ataxia (59%), generalized areflexia (36%), cranial nerve involvement (32%), rapid upper limb involvement (40%), and age at onset ≤55 years (50%). Additional features were: normal sensory nerve action potentials (36%), abnormal radial/normal sural pattern (23%), abnormal somatosensory evoked potentials (SSEPs) (100%), elevated cerebrospinal fluid (CSF) protein (73%), and demyelinating features in 5/7 nerve biopsies. Over 90% of patients responded to immunotherapy. We conclude that all patients had sensory CIDP.Sensory CIDP patients can be misdiagnosed as having CIAP. If atypical clinical/electrophysiologic features are present, we recommend performing SSEPs and CSF examination. Nerve biopsy should be restricted to disabled patients if other examinations are inconclusive.

We present the case of a woman who had a chronic infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). She developed severe polyarthralgias associated with type II mixed cryoglobulinemia during therapy with an HIV-1 protease inhibitor. This therapy resulted in a dramatic increase in her CD4+ T cell count, from 70/mm3 to 567/mm3, which was composed of a high proportion (88%) of naive CD45RA+ CD62L+ cells, together with a recovery of her CD4+ T cell reactivity to antigenic stimulation. This may suggest that rapid recovery of immune competence in the CD4+ T helper subset might participate in the development of immunopathologic events such as this patient's cryoglobulinemia.

Abstract 3872 Poster Board III-808 Introduction POEMS syndrome is a rare disease characterized by peripheral neuropathy, organomegaly, endocrinopathy, monoclonal plasma cells, skin changes, papilledema, volume overload, sclerotic bone lesions, thrombocytosis, and high serum VEGF level. Efficient treatments consist in irradiation for patients with localized solitary plasmocytoma and high-dose chemotherapy with autologous stem cell transplantation for appropriate candidates without a focal lesion. Conventional myeloma chemotherapy can only control the disease in a limited number of patients. Results of monoclonal anti-VEGF antibodies, which seem to be attractive due to the role of VEGF in this disease, are controversy with efficacy in 3 patients but treatment related deaths in 2 other patients. Thalidomide effectiveness has been reported in Japanese patients but enthusiasm for its use is tempered by the high incidence of thalidomide-induced peripheral neuropathy. Lenalidomide, which efficacy has been described in one observation (Dispenzieri, Blood 2007 110: 1075-1076), has the advantage of being anti-angiogenic, cytotoxic to malignant plasma cells and with a much lower risk of peripheral neuropathy. We reported here a multicentric French experience with this drug in POEMS syndrome. Patients and Methods There were 3 women and 6 men treated with Lenalidomide in 7 French centres. Median age was 60 (41-76). All patients had sensitive polyneuropathy with motor deficiency in 5 patients. A monoclonal component was present in all cases (IgA lambda in 7 patients, IgG lambda and lambda light chain only in 1 patient each). Other manifestations of POEMS syndrome included sclerotic bone lesions in 6 patients, endocrinopathies in 7 patients, skin changes in 8 patients, oedema in 7 patients, organomegaly in 5 patients, papilledema in 5 patients, thrombocytosis in 3 patients. VEGF serum level was elevated in 4 among 5 patients with a dosage. Previous treatments were high-dose chemotherapy with autologous stem cell transplantation in 3 patients, Melphalan-Prednisone in 3 patients because of advanced age, and prolonged steroid treatment in 2 patients. One patient received Lenalidomide as primary treatment before high-dose therapy. Lenalidomide was given during 21 days each month and sequentially associated with dexamethasone, 5 patients received 25 mg/day and 4 patients received 10 or 15 mg, for a median of 5 cycles (1 to 11). Results Serious side effects were noted in 3 patients with 2 hematologic toxicities (grade III and IV) and a cutaneous allergy. Six patients could be evaluated for hematologic response and all responded, complete response in 3 patients and partial in 3 (>25%). Clinical responses occurred early, before 3 months of treatment, in 6 cases among 8 (1 patient is not yet evaluable), with a marked improvement in performance and in neurological syndrome. Other manifestations of POEMS syndrome improved, especially oedema in 5 cases among 6. VEGF level (normal value < 500 pg/ml) could be serially measured in 4 patients with a normalization in 1 patient and a significant decrease in 3 patients, median 7100 pg/ml (2100-10100) before treatment to 887 pg/ml (304-3270). In 1 of these 3 patients VEGF level increased to initial value while he was still taking Lenalidomide. A second patient experimented a relapse 5 months after ending Lenalidomide, he is still in good response after Lenalidomide reintroduction. With a median follow-up of 12 months (1-26) all patients are alive. Conclusion Lenalidomide seems to be a very promising therapy in POEMS syndrome. It should be tested in larger studies in patients with a systemic disease, who are not able to receive high dose therapy, in relapsing patients and before high dose treatment to avoid transplant related morbidity, particularly engraftment syndrome. Disclosures: Jaccard: Celgene: Membership on an entity's Board of Directors or advisory committees. Facon:Celgene: Membership on an entity's Board of Directors or advisory committees and Speakers Bureau. Moreau:Celgene: Membership on an entity's Board of Directors or advisory committees. Fermand:Celgene: Speakers Bureau.

Abstract 4389 The immunophenotypic characterization is an essential tool in the diagnosis of hematological malignancies but the immunophenotypic profile of Waldenstrom's macroglobulinemia (WM) B cells remains not clearly defined. In order to improve the diagnosis of WM by an immunophenotypic peripheral blood test, we studied within a multicentric protocol the expression of costimulatory molecules CD80 (B7.1) and CD86 (B7.2) on blood B-cells of WM patients diagnosed by monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow (BM) infiltration. Results were compared to those obtained in healthy controls and other chronic B-cell lymphoproliferative disorders (LPD). Immunophenotypic analysis was performed by flow cytometry using six-colour staining. The CD80 and CD86 staining was concomitant with usual analysis of standard antigens (kappa/lambda immunoglobulin light chains, CD20, CD19, CD79b, CD5, CD10, CD23, FMC7, CD38, CD25, CD43, CD11c, CD1c, CD27, IgM, IgD). We first analyzed 65 cases of WM and compared results to 13 healthy control subjects. Among the WM patients, 45 (69%) showed a monotypic kappa or lambda peripheral blood B-cell population with a Matutes score '3. In these patients, CD80 was always positive (>21% of B-cells) and CD86 negative ( Disclosures: No relevant conflicts of interest to declare.

The immunophenotypic characterization is an essential tool in the diagnosis of hematological malignancies but the immunophenotypic features in Waldenstrom’s macroglobulinemia (WM) remain not clearly defined. We studied 96 cases of WM diagnosed by monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow infiltration, and we compared results to 33 cases of other chronic B-cell lymphoproliferative disorders (LPD), including marginal zone (MZL)(n=23), mantle cell (MCL)(n=8) and follicular (FL)(n=2) lymphomas. Patients with a Matutes score >3 (chronic lymphocytic leukemia) and with pathognomonic immunophenotype (hairy cell leukemia) were excluded. Immunophenotypic analysis was performed by flow cytometry using six-colour staining (FACS Canto II, Becton Dickinson). In WM and LPD groups, a monoclonal B-cell population was identified in blood (31 and 28 patients, respectively), blood and bone marrow (28 and 4 patients) or bone marrow samples (23 and 1 patients). Overall, 61% of WM patients showed a monoclonal B-cell population in blood. Neoplastic cells of WM and LPD patients with blood and/or bone marrow involvement expressed a monoclonal immunoglobulin light chain kappa (in 70% and 73% of cases respectively) or lambda (30% and 27%). The intensity of expression of the light chain was heterogeneous in both groups (high, normal or low expression in 43%, 27% or 30% of WM, and in 52%, 33% or 15% of LPD, respectively). All pan-B antigens (CD20, CD19, CD79b) were positive for at least 97% of patients. Results obtained with other antigens in WM compared to LPD were: CD10 = 10% vs 7% of patients, CD23 = 33% vs 56%, CD5 = 14% vs 26%, FMC7 = 76% vs 89%, CD38 = 56% vs 41%, CD25 = 86% vs 84%, CD43 = 12% vs 16%, and CD11c = 10% vs 36%. The intensity of expression of these antigens was heterogeneous in both groups. Among the antigens only tested in the WM group, CD1c and CD27 were positive for 70% of patients, IgM and IgD for 95% of patients, and CD103 as well as CD117 were negative in all cases. No difference was found between blood and bone marrow for all previous antigens. Plasma cells (CD38/CD138 positive cells) were found at low levels (less than 2.5% of B-cells) for 46% of WM in blood and/or bone marrow samples. Among the 10 WM patients tested for ZAP-70 expression, 9 were negative and 1 showed a low intensity expression. These results confirm that the immunophenotypic analysis usually performed with standard antigens does not allow defining a typical profile of WM. In order to tentatively identify the WM among the B-cell malignancies, we studied the expression of molecules known to be involved in B-cell development or in costimulatory pathways of antigenic activation, namely CD69, CD83, CD80 and CD86. We first analyzed blood samples of 24 WM patients showing a peripheral monoclonal B-cell population. CD80 was positive (> 20% of B-cells) in all cases and CD83, CD69 and CD86 were always negative. Among these WM patients, 13 were also studied for the bone marrow phenotype. No difference was found between blood and bone marrow phenotype in 11/13 WM cases. We then studied 11 LPD with blood tumoral involvement (MZL(n=7), MCL(n=2) and FL(n=2)). In these LPD, CD69 and CD83 were always negative and, in most cases (9/11 patients), CD80 and CD86 were also negative. Interestingly, CD80 was found positive in 2 patients with MZL, but the CD80 positivity was always associated to the CD86 positivity. Altogether, these data suggest that the inclusion of CD80 and CD86 in the panel of cytometric analysis allow to discriminate WM from other B-LPD with peripheral blood involvement.