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Background We investigated serous effusions occurring during the course of an already known hematologic neoplasia or as a first manifestation of it. We correlated cytology results with flow cytometry results, when available. In the absence of flow cytometry, our correlation was based on clinical follow up information obtained retrospectively. We evaluated our results in relation to the data of the literature and we considered some new suggestions for the improvement of cytology service. Methods Serous effusions in hematologic patients were retrieved from the files of the Department of Cytology, Laiko Hospital, for a period of 2 years. All patients had enrolled either a previous hematologic history, or a suspicious clinical and imaging status. Seventy-three serous effusions were included. Cytology reports consisting of morphology and immunocytochemistry assessment were correlated to flow cytometry results and, occasionally, to clinical follow-up. Results In the group of patients with previous history, sensitivity was 82.76%, positive predictive value was 100%, specificity 100%, and negative predictive value was 58.33%. In the group of patients without previous history, sensitivity and positive predictive value were both 91%, whereas specificity and negative predictive value could not be estimated. Conclusion We provide evidence that the diagnostic accuracy of cytology with the adjunct of immunocytochemistry is high compared to flow cytometry for detecting hematologic malignancies. In order to improve clinical performance, it is suggested that a cytology triage of serous effusions in all patients with hematologic malignancy must be implemented.

We aimed to determine whether Alemtuzumab-induced immune reconstitution affects immunoglobulin and complement levels in the serum of Relapsing-Remitting Multiple Sclerosis (RRMS) patients. IgG4-levels were increased 24-months after treatment initiation compared to baseline levels in twenty-nine patients. Alemtuzumab-treated patients with the highest IgG4-levels were more prone to thyroid-related autoimmune manifestations and specific autoimmune adverse events such as Crohn's disease, Graves' disease, and hemolytic anemia. Compared to baseline, total IgG-levels showed a trend towards reduced levels following two-courses of Alemtuzumab, but no significant change of C3 and/or C4-levels was observed. In conclusion, monitoring of IgG4-levels can serve as a marker for secondary autoimmunity risk in multiple sclerosis patients treated with Alemtuzumab.

Aims To examine for differences in circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs) in patients with and without diabetic peripheral neuropathy (DPN). Methods A total of 105 participants were included: 50 patients with type 2 diabetes (T2DM) and DPN, 30 patients with T2DM without DPN and 25 healthy individuals. CPCs and 6 different EPCs phenotypes were assessed with flow cytometry. We also measured plasma levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), vascular cell adhesion protein-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM) and tumor necrosis factor a (TNFa). Results No difference was observed in the number of CPCs among the 3 groups. Patients with DPN had higher numbers of all 6 EPCs phenotypes when compared with patients without DPN and higher number of 5 EPCs phenotypes when compared with healthy individuals. Plasma VEFG, VCAM-1, ICAM-1 and TNFa levels did not differ among the 3 groups. Patients with DPN had lower SDF-1 levels in comparison with healthy individuals. Conclusion Circulating EPCs are increased while SDF-1 levels are decreased in the presence of DPN. Our findings suggest that DPN may be associated with impaired trafficking of EPCs and impaired EPCs homing to the injured endothelium.

ObjectiveTo investigate the pathophysiologic mechanism of encephalopathy and prolonged comatose or stuporous state in severally ill patients with coronavirus disease 2019 (COVID-19).MethodsEight COVID-19 patients with signs of encephalopathy were tested for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the serum and CSF using a Food and Drug Administration-approved and independently validated ELISA. Blood-brain barrier (BBB) integrity and immunoglobulin G (IgG) intrathecal synthesis were further tested using albumin and IgG indices. The CSF was also tested for autoimmune encephalitis antibodies and 14-3-3, a marker of ongoing neurodegeneration.ResultsAll patients had anti–SARS-CoV-2 antibodies in their CSF, and 4 of 8 patients had high titers, comparable to high serum values. One patient had anti–SARS-CoV-2 IgG intrathecal synthesis, and 3 others had disruption of the blood-brain barrier. The CSF in 4 patients was positive for 14-3-3-protein suggesting ongoing neurodegeneration. In all patients, the CSF was negative for autoimmune encephalitis antibodies and SARS-CoV-2 by PCR. None of the patients, apart from persistent encephalopathic signs, had any focal neurologic signs or history or specific neurologic disease.ConclusionsHigh-titer anti-SARS-CoV-2 antibodies were detected in the CSF of comatose or encephalopathic patients demonstrating intrathecal IgG synthesis or BBB disruption. A disrupted BBB may facilitate the entry of cytokines and inflammatory mediators into the CNS enhancing neuroinflammation and neurodegeneration. The observations highlight the need for prospective CSF studies to determine the pathogenic role of anti–SARS-CoV-2 antibodies and identify early therapeutic interventions.

Dissemination of lymphomas in serous effusions is quite common. Cytology aims to contribute in the clinical management of haematologic patients, providing an accurate and rapid diagnosis. Ancillary techniques such as immunocytochemistry and flow cytometry are essential to classify the lymphoma entity. Comprehensive awareness of the clinical picture and previous histologic documentation are essential for a lymphomatous effusion diagnosis. We report an unusual case of monomorphic epitheliotropic intestinal T-cell lymphoma, formerly known as enteropathy associated T-cell lymphoma (EATL) type II, spreading in the pleural cavity. Cell morphology and immunohistochemistry of the pleural fluid were consistent with the histology of a jejunal tumor previously excised. Flow cytometry data were consistent, though not pathognomonic for the disease. Serous effusions with evidence of lymphoma involvement should be thoroughly examined with cytology and adjuvant techniques to provide diagnosis for proper therapeutic strategies.

Background. Serum free light chains (sFLC), the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients.Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients’ series.Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet stage A, B and C, respectively. Median patients’ followup was 32 months (range 4–228).Results. Increased involved (restricted) sFLC (iFLC) was found in 42% of patients, while the summated FLC-kappa plus FLC-lambda was above 60 mg/dL in 14%. Increased sFLC values as well as those of summated FLC above 60 were related to shorter time to treatment (P=0.0005andP=0.000003, resp.) and overall survival (P=0.05andP=0.003, resp.). They also correlated withβ2-microglobulin (P=0.009andP=0.03, resp.), serum albumin (P=0.009for summated sFLC), hemoglobin (P<0.001), abnormal LDH (P=0.037andP=0.001, resp.), Binet stage (P<0.05) and with the presence of beta symptoms (P=0.004for summated sFLC).Conclusion. We confirmed the prognostic significance of sFLC in CLL regarding both time to treatment and survival and showed their relationship with other parameters.

Changes in the production of monoclonal intact immunoglobulin (M-Ig) or of serum free light chains (sFLC) during symptomatic MM patients’ relapse eventually reflect molecular myeloma cells changes and / or reveal clonal cell competition. We aimed to study changes in Ig production (CIg) in symptomatic MM patients and to evaluate related frequency and corresponding specific disease characteristics. Patients and Methods 232 symptomatic MM patients with available follow-up sFLC measurements, were retrospectively studied. Light chain escape (LCE) was defined as sFLC increase with stable or falling M-Ig concentrations, M-Ig escape (MCE) as decreasing sFLC with increasing M-Ig, de-differentiation (DD) as clinical relapse with normal or decreased M-Ig and sFLC and Clonal Domination (CD) as normalization of formerly increased IgG, IgA or FLC in relapsing patients presenting increase of another component. Survival was calculated from diagnosis or from CIg date to last follow-up or death, survival curves were plotted by Kaplan-Meyer Method and assessed by the log-rank test. Results There were 94 women and 128 men, median aged 66 years; 29%, 42%, 29% and 22%, 30%, 48% were in Durie-Salmon and ISS stages I, II, III and 1, 2, 3 respectively. MM type was IgG MM in 59%, IgA in 21%, light chain only in 17%, IgD in 2% and non-secretory in1%. Median survival of the whole cohort was 46,4 months. CIg was observed in 39 patients (17%), consisting in LCE in 15 patients (6%), MCE in 7 (3%), DD in 5 (2%) and CD in 10 (4%); two additional patients (1 IgD and 1 LC) transiently produced another monoclonal component that was IgG in both cases, while in stringent complete remission. In CD patients, the dominated clone was IgG in 9 out of 10 patients, while the dominating one was LC in 8 and IgA in 2. LCE and MCE were more frequent in IgG patients. The median number of treatment lines received prior CIg was 5 for LCE, 4 for MCE, 2 for DD and 1 for CD. LCE and MCE patients had all received novel agents and/or ASCT. The median time from CIg to last follow-up or death was 2,6 months (2,2-3) for LCE, 3,3 months (2,2-4,4) for MCE, 6,3 months (1,1-11,6) for DD and 31,1 months (23,6-38,6) for CD. Patients presenting LCE, MCE and DD had a considerably shorter survival after CIg compared to patients presenting CD (p=0,0002). However because CIg was usually a late event in the course of the disease the overall survival of CIg patients was 60,6 months. In conclusion, LCE, MCE and DD are late events in the course of MM, mainly observed in patients whose previous treatments included with novel drugs. They reflect a very aggressive disease behavior with shortened survival thereafter, probably due to the emergence of a new resistant clone. CD was mainly observed in patients secreting low IgG levels and FLCs, and possibly reflect IgG clone remission in biclonal patients, given that thereafter, the disease behaves as a usual multiple myeloma, secreting however the other clone. Disclosures: No relevant conflicts of interest to declare.

Abstract 2927 Background and Aims: Multiple Myeloma (MM) is characterized by bone marrow (BM) plasma cell infiltration and the presence of serum/urine monoclonal immunoglobulin (Ig). The depth of response has been associated with longer PFS in MM causing subsequent prolonged survival. Recently novel M-based biomarker immunoassays have been developed (Freelite™, Hevylite™) and their significance in MM diagnosis and prognosis has been demonstrated.1,2 Furthermore serum Free Light Chains (sFLC) are used for better assessment of treatment response, thus patients are considered to achieve stringed Complete Response (sCR) by having CR criteria plus normal serum Free Light Chains Ratio (sFLCR) and absent clonal cells on BM.3 The significance of Hevylite™ on response has not been assessed so far. Patients in nCR or better do not automatically restore their ratio of intact monoclonal Ig/intact polyclonal Ig of the same class (Hevylite™ or HLCR). We therefore investigated the importance of sFLCR and HLCR normalisation at plateau on PFS, in a series of patients with intact Ig MM. Patients and Methods: 50 intact immunoglobulin MM patients were studied from diagnosis to last follow up. Immunofixation was IgG (26 -kappa and 12 –lamdba) and IgA (6 –kappa and 6 -lambda). All patients were symptomatic at diagnosis. Sera samples (n=312) were analyzed for sFLC-kappa and sFLC-lambda with Freelite™ and sFLCR were calculated, and for IgGkappa, IgGlambda IgAkappa, IgAlambda with Hevylite™ and ratios IgGkappa/IgGlambda, IgGlambda/IgGkappa, IgAkappa,/IgAlambda and IgAlambda/IgAkappa (HLCRs) were calculated. sFLCRs and HLCRs values above the 95%-ile of normal individuals were considered abnormal. Statistical analysis was performed using SPSS ver 15.0. File data were reviewed. Results: At diagnosis sFLCR was abnormal in 86% of patients while HLCR was abnormal in all. All treatment lines were initiated according to standard criteria and median lines of therapy were 2 (range 1–11). Median follow up was 33 months (7–145). During patients' cumulative follow-up, 145 lines of therapy were studied and the subsequent responses were estimated. Thirty eight percent of responses were sCR, CR and nCR, 20% PR, 18% MR and 24% refractory and progressive disease. HLCR normalized in 44% of patients with sCR, CR and nCR. The depth of response correlated to PFS and patients in sCR, CR and nCR had longer PFS than the others (p Conclusion: Serum HLCR normalization at plateau reflects prolonged responses in intact Ig MM. Disclosures: Harding: Binding Site: Employment. Bradwell:The Binding Site: shareholder Other.

Abstract 4585 Background and Aims: Reduced serum IgM levels were proposed as an unfavorable prognostic factor in multiple myeloma1. We therefore investigated the possible impact of decreased serum IgM concentrations on the outcome of Chronic Lymphocytic Leukemia (CLL) patients, a disease that is frequently characterized by severe hypogammaglobulinemia. Serum IgM levels were measured by 2 methods. Patients and methods: 188 patients with CLL were studied. Of them 54%, 27%,10%, 7% και 2% where in RAI stage 0, 1, 2, 3, 4 respectively while 65%, 22% and 13% in Binet stages A, B and C. IgM was determined at diagnosis by both classical nephelometry and the new Hevylite™ methods; the first measures total IgM levels while the second one measures the immunoglobulin fractions bound to either kappa or lambda light chains therefore enabling us to determine the ratio of IgM-kappa/lambda (HLCR). Hevylite™ measurements were retrospectively performed in 69/188 patients with available frozen sera aliquots from drawn at the time of diagnosis. Of them the Ig subclasses IgMkappa and IgMlambda, IgGkappa and IgGlambda, IgAkappa and IgAlambda (HLC) were measured by Hevylite™ antibodies nephelometrically. Light chain restriction (kappa or lambda) was determined by flow cytometry or bone marrow/lymph node biopsy immunohistochemistry. The ratios (HLCRs) were calculate with the monoclonal immunoglobulin as numerator, i.e IgMkappa/IgMlambda, IgGkappa/IgGlambda and IgAkappa,/IgAlambda in kappa-restricted patients and IgMlambda/IgMkappa, IgGlambda/IgGkappa, IgAlambda/IgAkappa in lambda-ones. Serum IgM concentration by both methods were related to time to first treatment (TFT) and overall survival (OS). Statistical analysis was performed conventionally; survival curves were drawn by Kaplan-Meyer method and compared by the log-rank test. Results: Total IgM ranged from 12 to 246 mg/dl (median 47.6). The sum of IgMkappa plus IgMlambda from 12 to 369 (median 60). IgMkappa from 7.9 to 349 mg/dL (median 41) in kappa-restricted patients and IgMlambda from 7.6 ωζ 98 mg/dL (median 22) in lambda restricted ones. Low total IgM level ( Finally IgM-HLCR was abnormal in 35% of patients. Of them, 25% had also abnormal IgG-HLCR and/or IgA-HLCRs leading to the hypothesis of multiple clones coexistence within the neoplastic B-cell lymphocytic population. Conclusions: Low IgM levels determined by novel immunoassays (Hevylite™) correlate to shorter TFT and OS in patients with CLL. Their use may reveal new disease characteristic and are suggestive of a multi-oligoclonal disorder. Disclosures: No relevant conflicts of interest to declare.

Abstract 4568 Background and Aim: Symptomatic CLL patients need treatment immediately. For these patients, molecular-genetic factors (mutated-unmutated, ZAP 70, ATM, p53) are important prognostic factors of response and survival. Nevertheless, 2/3 of newly diagnosed patients are asymptomatic and require only of follow up that can last for months or years. For these patients overall survival (OS) depends on the time to first treatment (TFT). The most frequent paraprotein produced in CLL is serum free light chain in 50% of the patients. It has recently been shown that serum free light chains (sFLC) and their sum above 60 (κ+λ above 60) are useful prognostic factors for TFT. We therefore studied the eventual prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients' series. Patients and Methods: 143 CLL patients were studied of which 18 needed immediate treatment while 37 more needed treatment during their follow up. 64% and 72%, 28% and 18%, 7.5% and 10%, were in stage 0 and A, 1 and β, 2 and C according to Rai and Binet respectively. Median patients' follow up was 32 months (range 4–228). Light chain restriction was established by flow cytometry or bone marrow biopsy immunohistochemistry. Serum free light chain values were retrospectively determined by nephelometry (Freelite™, the Binding Site Birmingham, UK) in frozen sera drawn at diagnosis. Elevated sFLC values were defined using as cut-off values the 95th percentile range of healthy individuals. Statistical analysis was performed using SPSS v15.0. Hazard ratios and prognostic significance of abnormal sFLC, HLC and ratios were determined by univariate Cox regression analysis. Kaplan Meier method was used for pictorial representation of survival and time to treatment. Results: Increased sFLC were found in 45% of the patients while the summated FLC-kappa plus FLC-lambda was higher than 60 mg/dl in 14%. Increased sFLC values as well as values of FLC κ+λ>60 were related to shorter TFT (p=0,0005 and p=0,000003 respectively). In addition, high levels of sFLC and FLC κ+λ >60 correlated with β2-microglobulin (r=0.2, p=0.009 και r=0.2, p=0.03 respectively), serum albumin (r=0.2, p=0.009 only for FLC κ+λ > 60), negatively with hemoglobin (r=-0.3, p=0.000003 και r=-0.2, p=0.0002 respectively), increased LDH (p=0.037 και 0.001 respectively), Rai stage (p=0.03 και 0.003 respectively) and Binet stage (p=0.02 only for FLC κ+λ > 60) and with the presence of beta-symptoms (p=0.004 only for FLC κ+λ > 60). Finally, increased sFLC and FLC κ+λ>60 values correlated with shorter OS (p=0.05 and p=0.003 respectively). Conclusion: The results of our study confirmed the significance of sFLC in CLL with regard to TFT and their relationship with adverse prognostic clinical and laboratory parameters but also demonstrated for the first time their impact on OS. Disclosures: No relevant conflicts of interest to declare.

Background HevyLite™ is a new, recently developed method that facilitates separate quantification of the kappa- and lambda-bounded amounts of a given immunoglobulin (Ig). Using this method, we measured intact immunoglobulin (heavy/light chain; HLC) IgG-kappa, IgG-lambda, IgA-kappa, IgA-lambda individually, as well as their deriving ratios (HLCR) in a series of IgG or IgA multiple myeloma (MM) patients, to investigate and assess the contribution of these tests to disease evaluation. Patients and methods HevyLite™ assays were used in sera from 130 healthy individuals (HI) and 103 MM patients, at time of diagnosis. In patients, the level of paraprotein was IgG in 78 (52 IgG-kappa, 26 IgG-lambda) and IgΑ in 25 (13 IgΑ-kappa, 12 IgΑ-lambda). Durie-Salmon and International Staging System stages were evenly distributed. Symptomatic patients (n = 77) received treatment while asymptomatic ones (n = 26) were followed. Patients' median follow-up was at 32.6 months. HLCR was calculated with the involved Ig (either G or A) as numerator. Results In HI, median IgG-kappa was 6.85, IgG-lambda 3.81, IgA-kappa 1.19 and IgA-lambda 0.98 g/L. The corresponding median involving HLC values in MM patients were 25.8, 23.45, 28.9 and 36.4 g/L. HLC-IgG related to anemia, high serum free light chain ratio and extensive bone marrow infiltration, while high HLCR correlated with the same plus increased β2-microglobulin. In addition, increased HLCR and the presence of immunoparesis correlated with time to treatment. Patients with high HLCR had a significantly shorter survival (p = 0.022); HLCR retained its prognostic value in multivariate analysis. Conclusions HLC and HLCR quantify the precise amount of the involved immunoglobulin more accurately than other methods; moreover, they carry prognostic information regarding survival in MM patients.

We have recently shown that serum free light chain ratio (sFLCR) provides independent prognostic information in patients with newly diagnosed MM (Kyrtsonis et al, Br J Haematol, 137: 240–243, 2007). The aim of the present study was to extend our previous observations in a multicenter setting and to investigate the potential additive effect of sFLCR to the ISS system, in determining the prognosis of patients with MM. We analyzed 214 newly diagnosed MM patients (125 kappa-, 89 lambda-). Serum free light chain levels were measured in sera drawn at diagnosis, using a latex-enhanced immunoassay (The Binding Site, Birmingham, UK). Then, the sFLCR was calculated, accordingly as kappa/lambda or lambda/kappa, depending on the monoclonal light chain type of the patient. Based on our previous study “high” sFLCR was defined as ratios ≥3.57 and ≥45.09 for kappa- and lambda- MM respectively. The median age of the patients was 68 years (33–92), 51% were males, 28%, 30%, and 42% had Durie-Salmon stages I, II, and III, 14% creatinine >2 mg/dl, and 13% had Bence-Jones MM. ISS stage was 1, 2, or 3 in 33%, 33%, and 34% of the patients, 48% had CRP ≥4 mg/l, 18% elevated LDH, 31% hemoglobin DSS According to the Combined sFLCR and ISS System Patient Subgroup Pts (#,%) 3-yr DSS (%) 5-yr DSS (%) p “Low” sFLCR and ISS