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Introduction: Immunotherapy with Chimeric Antigen Receptor T cells (CAR-Τ) is a promising innovative treatment for refractory B cell malignancies offering a considerable chance for long-term survival in patients (pts) with an otherwise dismal prognosis. Since January 2020, two anti-CD19 CAR T cell products have been introduced into clinical practice in Greece: a) tisagenlecleucel (Kymriah) for adults with relapsed/refractory diffuse large B-cell Lymphoma (r/r DLBCL) including transformed follicular lymphoma (TFL), as well as for children or young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL), and b) axicabtagene ciloleucel (Yescarta) for adults with r/r DLBCL including TFL and primary mediastinal B-cell lymphoma (PMBCL). The aim of this study was to present the real-world experience of the initial application of CAR T cell therapy in adult pts in Greece, with special focus on early toxicity and disease outcomes. Methods: Data from all consecutive pts were collected from the two transplant centers which were initially accredited for CAR T cell therapies in adult pts: Evangelismos Hospital, Athens and George Papanicolaou Hospital, Thessaloniki. Between November 2019 and July 2021, 51 pts were referred for CAR T cell treatment. In 41 pts lymphocyte collection was performed and product manufacturing was successfully completed in 35; in 2 pts insufficient cell expansion was noted and in 4 manufacturing was terminated due to disease progression and patient death. Results: From January 2020 until July 2021, CAR T cells were infused in 27 pts; 3 pts could not receive the product due to clinical deterioration/death and 5 pts are presently being scheduled for infusion. Of the 27 treated pts, 16 received tisagenlecleucel and 11 axicabtagene ciloleucel. The median age of infused pts was 49 (18-69) years. Diagnosis was DLBCL (n=16), PMBCL (n=3), TFL (n=2), B-ALL (n=6), and the median number of previous lines of treatment was 4 (2-5). Five pts with lymphoma had undergone autologous, and 4 pts with B-ALL allogeneic stem cell transplantation. In total 18/27 pts received bridging therapy, including radiotherapy (n=5), chemoimmunotherapy (n=9), steroids (n=3), and inotuzumab ozogamicin (n=1). The median time from leukapheresis to product delivery and infusion was 35 (15-81) and 59 (35-152) days, respectively. All pts received lymphodepleting therapy before CAR T cell infusion with combination of cyclophosphamide and fludarabine. For patient monitoring, prophylactic therapy and management of toxicity, the EBMT (Yakoub-Agha I, et al. Haematologica 2020) and MD Anderson (Neelapu S, et al. Nat Rev Clin Oncol 2018) guidelines were adopted. Twenty-six pts developed neutropenia (grade II: 2, grade IV: 24) and 20 thrombocytopenia (grade I: 7, grade II: 3, grade III: 1, grade IV: 9), with a median duration of 11 (4-132) and 20 (3-150) days, respectively. Cytokine release syndrome (CRS) and neurotoxicity (ICANS) were noted in 21 (grade I: 8, grade II: 7, grade III: 6) and 5 (grade I: 3, grade III: 2) pts, respectively. Tocilizumab was administered for CRS according to guidelines, and steroids were additionally required for CRS and/or ICANS in 12 pts. In 2 pts, persistent ICANS necessitated further treatment with anakinra (n=2), siltuximab (n=1), and cyclophosphamide (n=1). Hypogammaglobulinemia was encountered in 14/27 pts. With a median follow-up of 7.3 (1-17) months, overall response rate was 48% with 12 (45%) pts being currently in complete remission (CR). No treatment related mortality was observed. Disease-free (DFS) and overall survival (OS) were 52% and 85.3% at 1-year, respectively. DFS and OS were significantly associated with baseline LDH levels (p=0.017 and 0.050, respectively) and grade II/III CRS (p=0.041 and 0.015, respectively, Figure). Conclusions: Despite the limited experience in the real-world setting, CAR T cell therapy can be administered safely and may successfully rescue patients with DLBCL or B-ALL who lack alternative treatment options. Close monitoring of patients and prompt recognition and management of side effects are mandatory for achieving the benefits of therapy. Figure 1 Figure 1. Disclosures Baltadakis: WinMedica: Other: Travel Grants; Gilead: Other: Travel Grants; Genesis Pharma: Other: Travel Grants; Abbvie: Honoraria; Novartis: Honoraria; Gilead: Honoraria; Pfizer: Honoraria, Other: Travel Grants; Astellas: Honoraria; Alexion: Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Baxalta Hellas: Other: Travel Grants. Gavriilaki: Pfizer Corporation: Research Funding; Gilead Corporation: Honoraria; Alexion, Omeros, Sanofi Corporation: Consultancy. Tzannou: Allovir: Current equity holder in publicly-traded company; Gileas: Honoraria. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials .

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Introduction: The preferred method for haploidentical stem cell transplantation (haploSCT) is currently the use of post-transplantation cyclophosphamide (PTCY) since it obviates the need for depletion of T lymphocytes, which is associated with profound immunosuppression. Despite preservation of non-alloreactive donor T cells, reconstitution of pathogen-specific immunity may be delayed even after T cell replete haploSCT. The incidence and clinical sequelae of viral reactivation may thus compromise the outcomes of the procedure. Patients and Methods: The study included 47 patients, who underwent haploSCT with PTCY from 12/2013 until 05/2019 and achieved stable donor engraftment. Median age at transplant was 53 years (range, 19-70). The indications for transplant were acute myeloid (n=19) or lymphoblastic (n=10) leukemia, myelodysplastic syndrome (n=10), myelofibrosis (n=4), chronic myeloid (n=2) or lymphocytic (n=1) leukemia, and T-prolymphocytic leukemia (n=1). Myeloablative conditioning was mainly utilized (n=36), with the exception of certain patients who received reduced-intensity (n=10) or non-myeloablative (n=1) regimens. The graft source was peripheral blood in 29 and bone marrow in 18 cases. Tacrolimus in combination with mycophenolate mofetil was administered for prevention of graft-versus-host disease. Recipient/donor cytomegalovirus (CMV) serostatus was -/- (n=2), -/+ (n= 5), +/- (n=11), or +/+ (n=29). CMV, Epstein-Barr virus (EBV), and human herpesvirus-6 (HHV-6) reactivation was monitored by real-time quantitative PCR (RQ-PCR) in plasma and/or leukocytes weekly for at least 6 months post haploSCT. BK virus (BKV) reactivation was assessed by RQ-PCR in urine and/or blood specimens in cases with symptoms suggestive of hemorrhagic cystitis (HC). Prophylaxis with letermovir was available in 1 patient only, and preemptive antiviral therapy was the principal modality for the management of CMV infection. Cellular immunity reconstitution was assessed by flow cytometry at 3, 6, and 12 months after transplant. Results: With a median follow-up time of 30 months (range, 2-64), the cumulative incidences (CIN) of relapse and non-relapse mortality (NRM) were 13.4% (95% confidence interval [CI], 5.4-25.1%) and 31.4% (95% CI, 18.3-45.4%) at 2 years, respectively. Disease-free (DFS) and overall survival (OS) were 55.2% (95% CI, 42.3-72.1%) and 61.8% (95% CI, 48.9-78.1%) at 2 years, respectively. The CIN of CMV reactivation (>100 copies/ml) plateaued at 75.6% (95% CI, 60.1-85.7%) at 3 months. CMV infection developed in 34 out of 45 patients who were at risk, whereas recurrent CMV reactivation was observed in 17 patients with a median number of 1.5 episodes (range, 1-6) per patient. The median total duration of antiviral therapy for CMV infection was 27 days (range, 14-199). CMV disease (pneumonia) was documented in 2 patients. The CIN of EBV reactivation (>1,000 copies/ml) was 47.1% (95% CI, 34.2-63.9%) at 12 months. No case of EBV-related post-transplant lymphoproliferative disorder was observed, however preemptive therapy with rituximab was required in 2 patients with rapidly increasing EBV viral load. HHV-6 reactivation (>1,000 copies/ml) was observed in 6 patients (CIN, 10.6% at 6 months; 95% CI, 3.8-21.4%), but only one required therapy with foscarnet due to high viral load (>10,000 copies/ml). The CIN of BKV-related HC reached 27.7% (95% CI, 15.7-40.9%) at 3 months. Cystoscopy for bladder hemostasis was required in 5/13 and nephrostomy in 1/11 patients with HC. Reconstitution of helper T cell immunity was considerably delayed, with median absolute CD4+ cell counts of 83/uL (range, 7-337), 216/uL (range, 80-509), and 236/uL (range, 97-586) at 3, 6 and 12 months, respectively. Recurrent CMV infection was significantly associated with the recovery of CD4+ cells at 3 months (Figure; median CD4+ count of 191/uL versus 62/uL in patients with 1 and 2 or more episodes of CMV reactivation, respectively; p=0.009). Conclusions: HaploSCT with PTCY is associated with substantial rates of viral reactivation (especially CMV and BKV) resulting in the need for prolonged antiviral therapy and considerable morbidity. Strategies to prevent viral infection are strongly warranted in haploidentical stem cell transplantation. The timing and duration of such interventions (like letermovir or adoptive immunotherapy) may be guided by the tempo of immune reconstitution following haploSCT. Figure Disclosures Tsonis: Gilead: Other: Travel Grant; Astellas: Other: Travel Grants; Gilead: Other: Travel Grant; Aenorasis: Other: Travel Grant; Takeda: Other: Travel Grant; Pfizer: Other: Travel Grant; Innovis: Other: Travel Grant.

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset # 1, # 2 and # 8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset # 2 (44%) versus subset # 1 and # 8 (4.6% and 0%, respectively; P

We performedIGHclonotypic sequence analysis in WM in order to determine whether a preferentialIGHgene rearrangement was observed and to assessIGHVmutational status in blood and/or bone marrow samples from 36 WM patients. In addition we investigated the presence ofMYD88L265P somatic mutation. AfterIGHVDJ locus amplification, monoclonal VDJ rearranged fragments were sequenced and analyzed.MYD88L265P mutation was detected by AS-PCR. The most frequent family usage wasIGHV3(74%);IGHV3-23andIGHV3-74segments were used in 26% and 17%, respectively. Somatic hypermutation was seen in 91% of cases.MYD88L265P mutation was found in 65,5% of patients and absent in the 3 unmutated. These findings did not correlate with clinical findings and outcome. Conclusion.IGH genes’repertoire differed in WM from those observed in other B-cell disorders with a recurrentIGHV3-23andIGHV3-74usage; monoclonalIGHVwas mutated in most cases, and a high but not omnipresent prevalence ofMYD88L265P mutation was observed. In addition, the identification of 3 patients with unmutatedIGHVgene segments, negative for theMYD88L265P mutation, could support the hypothesis that an extra-germinal B-cell may represent the originating malignant cell in this minority of WM patients.

BLyS is involved in CLL biology and its low soluble serum levels related to a shorter time to first treatment (TFT). TACI is a BLyS receptor and can be shed from cells’ surface and circulate in soluble form (sTACI). We investigated the impact of serum BLyS and sTACI levels at diagnosis in CLL patients and their relationship with disease parameters and patients’ outcome. Serum BLyS was determined in 73 patients, while sTACI in 60. Frozen sera drawn at diagnosis were tested by ELISA. sTACI concentrations correlated with BLyS (P=-0.000021), b2-microglobulin (P=0.005), anemia (P=-0.03), thrombocytopenia (P=0.04), Binet stage (P=0.02), and free light chains ratio (P=0.0003). Soluble BLyS levels below median and sTACI values above median were related to shorter TFT (P=0.0003and 0.007). During a ten-year followup, sTACI levels, but not BLyS, correlated with survival (P=0.048). In conclusion, we confirmed the prognostic significance of soluble BLyS levels with regard to TFT in CLL patients, and, more importantly, we showed for the first time that sTACI is a powerful prognostic marker, related to parameters of disease activity and staging and, more importantly, to TFT and OS.

Introduction: Monoclonal Gammopathy of undetermined significance (MGUS) is an asymptomatic premalignant plasma cell disorder occurring mainly in the elderly population. Its evolution, association with various diseases and behavior is an interesting study field in an attempt to understand its pathogenesis and disease course. Aim: To study the grade of coexistence of non-malignant and malignant diseases along with disease evolution and behavior in patients with MGUS, diagnosed in a single center. Patients and methods:We studied 138 MGUS-patients that were diagnosed in our center and then followed up to a median of 36 months (6months - 22 years). Median age was 66 years (27-92 years). 57% were of female sex. Monoclonal heavy chain was IgG in 76%, IgA in 14% and IgM in 10% of the patients while 63% presented k-chain clonality. Non-malignant and malignant preexisting diseases were documented at the time point of MGUS-Diagnosis. Patients with B-NHL expressing monoclonal Protein were not classified as MGUS since malignant B-Lymphocytes can be responsible for its production. Results: 10.9% of the patients presented solid tumors. The most common malignancy was Prostate-Cancer in 8.5% of the male patients followed by Thyroid-Cancer which was present in 2.2% of the whole patient group.Hematological malignancies were existent in 10.9% of the patients. 4.3% presented myeloproliferative neoplasms while myelodysplastic syndromes were represented in 5% of the patients.18.1% of the patients presented with diverse benign tumors, 8% had been diagnosed with Diabetes Mellitus while 32.6% presented cardiovascular disease, mainly hypertension (23.2%). Hyperlipidemia was present in 8.7%. Finally 18.1% of the patients presented non-malignant thyroid disease, mainly hypothyroidism (10.9%) which is increased compared to the general population.17 MGUS-Patients (12%) presented disease evolution. 3 Patients evolved directly to multiple myeloma while 3 more evolved initially to smoldering myeloma (SMM) before developing overt myeloma. 8 patients evolved to SMM without any further progression. 2 patients with IgM-MGUS presented Waldenström's maroglobulinemia in the follow up while one patient developed a B-NHL. We performed a statistical analysis, where only abnormal serum free light chain ratio (sFLCR) was found to have a prognostic impact on MGUS-progression (p=0.03).Within this group of evolving MGUS-patients two of them presented a very remarkable course. The first one was diagnosed with MGUS while she was in remission after Hodgkin's Lymphoma. She evolved then to SMM confirmed by bone marrow biopsy with more than 10% plasma cell infiltration by immunohistochemistry. After being stable for several months, monoclonal protein was no longer detectable and plasma cells in the bone marrow were normal without any treatment. The second patient was initially diagnosed with MGUS with a high sFLCR of 60. She then evolved to SMM with further sFLCR-increase up to 100 but remained without treatment according to the guidelines at that time. Four years later she developed anemia and the final diagnosis was B-NHL. Conclusion: In our study group MGUS was associated with numerous malignant and non-malignant disorders. Hypothyroidism was a common finding, increased compared to the general population. MGUS-evolution was also observed however disease course was unexpected in some patients showing the heterogeneity of the disease. sFLCR was confirmed as a prognostic factor. Further study is necessary to investigate any possible implication of the above findings in the disease pathogenesis and course. Disclosures Kyrtsonis: Genesis: Honoraria; Millenium: Research Funding; Lilly: Research Funding; Amgen: Research Funding.

The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with >90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p Disclosures Tausch: Gilead: Other: Travel support. Shanafelt:Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Cephalon: Research Funding. Niemann:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy; Novartis: Other: Travel grant. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:Janssen Pharmaceuticals: Research Funding.

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.

Background and aim Waldenström’s Macroglobulinemia (WM) cells express a unique clonotypic rearrangement of heavy chain immunoglobulin (IgH) genes in each individual patient. Recent studies demonstrate high frequency of the MYD88 L265P somatic mutation in patients with WM, implying a new pathway for the pathogenesis of this disease. In this study we characterized clonal IgH genes rearrangements, IGHV gene use, somatic hypemutations (SHM) and presence of the ΜΥD88 L265P mutation in a cohort of WM patients and we investigated any eventual correlation with patients’ clinical features. Patients and methods 36 patients with WM were studied (median age 64y, 19 males and 17 females, symptomatic 72%, asymptomatic 28%) of whom 45%, 39% and 16% were stage IPSSWM 1, 2 and 3, respectively. Median time to treatment initiation was 13 months and median overall survival was 61 months. DNA was extracted from patients’ bone marrow/blood samples and clonal IgH VDJ rearrangements were amplified by PCR using the Biomed-2 Concerted Action protocol. Monoclonal VDJ rearranged PCR fragments were sequenced and analyzed using the IMGT database (www.imgt.org). Thirty-one patients’ DNA samples were also analyzed for the presence of the ΜΥD88 L265P mutation by using a mutation specific PCR assay. Results The most frequent clonal rearrangements observed in our patients were in the IGVH3 family (73% of cases); the IGVH3-23 gene and the IGVH3-74 gene were used in 24.4% and 18,9% of cases, respectively. Somatic hypermutation (>2%) was seen in all but three cases (92%). Mean percentage of mutations in all cases, IGVH3 family, IGVH3-23 and IGVH3-74 genes was 7,5%, 8%, 9,4% and 7,5%, respectively. Sixty-two percent of the patients were positive for the ΜΥD88 L265P mutation and notably all three unmutated WM cases were negative for this MYD88 point mutation. No correlation between VH or DH gene usage, CDR3 length, and mutational status with clinical parameters, time to treatment initiation, or survival was found. Conclusions WM IgH genes repertoire, as expected, differs from that observed in normal B-cells and other B-cell diseases such as MZL, MCL and B-CLL/SLL. In addition to the known hyper-representation of the IGVH3-23 gene, another member of the VH3 family, the IGVH3-74 gene is also over-represented in WM. The high IGVH mutation rate supports a derivation of WM cells from postgerminal center memory B cells in the majority (92%) of WM patients. However, the identification of a minority of WM patients (3 of 36) with unmutated IGHV gene segments, negative for the ΜΥD88 L265P mutation, supports the hypothesis that they represent a subgroup of WM not arising from post-germinal B cells with a different disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

The negative prognostic consequences of extramedullary plasmacytomas in the context of symptomatic MM is well known. On the contrary, the clinical impact of bone plasmacytomas (BP) has not been extensively studied, although their presence is one of Duries’ major MM diagnostic criteria. The aim of the present study was to study eventual differences between MM patients presenting at diagnosis with or without BP. Patients and Methods Two hundred and twelve symptomatic MM patients were studied from diagnosis to last follow-up. Their median age was 66 years and 60% were men. Twenty-three percent, 27% and 50% of patients were in ISS stage 1, 2 and 3 respectively; MM type was IgG in 59% , IgA in 26%, light chain only in 12%, IgD in 1,5% and non- or oligo-secretory in 1,5%. All patients required treatment and BP ones were additionally given radiotherapy. The median follow-up time of the whole cohort was 46,4 months. Results Forty patients (19%) had a BP at the time of their diagnosis. BP was located in the spine (BP-S) in 19 out of 40 and in other sites (BP-O: skull, pelvis, long bones, and ribs) in 21 patients. BP patients had equivalent demographics, stage and MM type as the others. Non-BP patients tended to have a better survival than BP ones but the difference was not statistically significant (p=0.1). However, BP-S patients had a significantly worse outcome than BP-O patients (24±6 versus 48±11 months, p In conclusion, MM patients presenting spine plasmacytoma at diagnosis constitute a distinct subgroup with adverse outcome that is difficultly predictable, given that the current staging system does not separate them. Further confirmation of this finding in larger cohorts is needed, as well as exploration of the biologic background. Disclosures: No relevant conflicts of interest to declare.

Contribution of new immunoglobulin-derived biomarkers in plasma cell dyscrasias and lymphoproliferative disorders

Abstract 4976 Background and Aims: TGF-beta1 normally down-regulates B-cell proliferation. Resistance to its effects is reported in malignant cells of B-lineage such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells. There are however very few studies on serum TGF-beta1 levels in patients with B-cell lymphoproliferative diseases and their eventual correlations with parameters of disease activity and survival. The purpose of our study was to assess any eventual relationship between serum TGF-beta1 levels and disease parameters, as well as time to first treatment (TFT) and overall survival (OS) in a series of MM, Waldenstrom's macroglobulinemia (WM) and CLL patients at diagnosis. Patients and Methods: 92 MM, 64 WM and 110 CLL patients were studied. In the MM group, paraprotein was IgG in 64, IgA in 18, LC in 8 and IgD in 2 patients while 32, 25 and 43% of patients were in ISS stage 1, 2 and 3 respectively. MM patients' median follow-up was 51 months. All patients required treatment a some point, 55% immediately. In the WM group, 44, 27 and 29% of patients were in WM – IPSS stage 1, 2 and 3 respectively. WM patients' median follow-up was 49 months; 75% of them required treatment during follow – up. In the CLL group, 54% and 74%, 25% and 20%, 14% και 6%, were in stage 0 and A, 1 and β, 2 and C according to Rai and Binet respectively. Median follow up was 43 months; 14 patients needed immediate treatment, 30 more required treatment during follow up while the others are still just followed. Patients' frozen sera, drawn at diagnosis, were retrospectively analyzed while sera from 20 healthy individuals (HI) were tested as controls. Serum TGF-beta1 measurements were done by ELISA according to the manufacturer's instructions. Statistical analysis was performed by SPSS software, version 15. 0. Results: Median serum TGF-beta1 levels were 36330 pg/ml (range 4. 6 – 77976) in MM patients, 33965 pg/ml (range 1665–615000 pg/ml) in WM, 12207 pg/ml in CLL (range 0 – 42970) and 32902 pg/ml in HI (range 1941 – 58123). CLL patients presented significantly lower TGF-beta1 levels than those with MM and WM (p Conclusions: Our results of longer TFT in MM and CLL and improved OS in MM and WM patients with increased serum TGF-beta1 levels, suggest that a degree of sensitivity to the suppressive effect of TGF-beta1 may remain in neoplastic B-cells in the majority of B-cell lymphoproliferative disorders. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 5019 A new method for the quantification of IgGκ, IgGλ, IgAκ and IgAλ individually and of deriving IgGκ/IgGλ and IgAκ/IgAλ ratios (HLCR) has been recently made available, allowing measurements of the monoclonal component alone as well as of the polyclonal, non-tumor immunoglobulins (Ig) in plasma cell dyscrasias. The purpose of the present study was to retrospectively determine HLCR and to investigate its contribution in a series of 98 multiple myeloma patients at presentation and in 38 of them, during their disease's course. There were 45 women and 53 men (median age 67 years). Disease stages were evenly distributed (36%, 30%, and 34% in ISS stage 1, 2, and 3 respectively). Paraprotein was IgG in 74 patients (50 IgGκ, 24 IgGλ) and IgA in 24 (12 IgAκ, 12 IgAλ) of them. Seventy-one symptomatic patients received conventional treatment while the other 27 were asymptomatic and were regularly followed only. Of the 38 patients also studied during disease course, 28 had IgG- and 10 IgA-MM; the median number of disease fluctuation events studied (remision-relapse) was 3 (range 2–9). Patients' median follow-up was 31 months. HLC analyses were performed on a Dade Behring BN™II nephelometer using polyclonal sheep antibodies (Hevylite™, Binding Site, UK) and HLCR was calculated with the involved Ig (either G or A) as numerator. HLC and HLCR values were then compared with disease parameters and survival. Statistical analysis was performed by standard methods. At patients' presentation, median monoclonal HLC IgG and IgA were 25 g/L and 32 g/L respectively while in patients with IgG-MM median IgG HLCR was 24.5 and IgA HLCR 60 in IgA-MM. HLCR was not correlated to b2-microglobulin, hypercalcemia, LDH or bone disease, FLCs or FLCR. It strongly correlated with time to treatment (p=0.0000093) and with a shorter overall survival (p=0.03) using as cut-0ff HLCR values in the upper tertile of symptomatic patients (>56 for IgG and >228 for IgA). Of patients also studied during their disease course, 25 out of 38 patients responded at least partially (PR) to first line treatment and 9 to a subsequent one; 20 relapsed and then responded again. In most patients HLCR followed disease fluctuations. It normalized in 9 patients and was below normal in 1 patient that achieved a complete (CR) or stringent response (sCR). In some patients HLCR allowed a better evaluation of disease status. In 5 thought to be responding, ηLCR remained stable or increased and disease course confirmed the fragility of the believed response. Interestingly, more than 50% of patients that received VAD showed greater reduction of the polyclonal Ig than the tumor Ig during treatment and sometimes thereafter. In conclusion, we provide evidence that HLCR correlates with survival in MM and allows, in some patients, better disease monitoring. Disclosures: Mirbahai: Binding Site Group Ltd: Employment. Bradwell:Binding Site: Equity Ownership, Patents & Royalties. Harding:Binding Site Group Ltd: Employment.

Abstract 3004 Resolution of IgM monoclonal proteins (M-proteins) on serum protein electrophoresis (SPE) can make accurate quantification difficult. The same applies to the determination of total IgM by nephelometry which inherently includes monoclonal and polyclonal immunoglobulins. Specific polyclonal antibodies, which recognise epitopes spanning the junction of the heavy and light chains of the individual immunoglobulin isotypes, have been produced and used to develop nephelometric assays (Hevylite™). The purpose of the present study is to evaluate the prognostic value IgMκ/IgMλ ratios (HLCR) at diagnosis and the role of HLCR in disease monitoring. Retrospective sera from 31 WM patients, 4 IgM-MGUS, 2 IgM-like syndrome at diagnosis and for 9 of them during disease course, were included in the study. Analysis of IgMκ and IgMλ was performed on a Siemens Dade-Behring BN™II nephelometer and results were compared with disease parameters such as blood count, beta2-microglobulin (β2M), serum albumin, lactate dehydrogenase (LDH), and lymphoplasmacytic bone marrow infiltration. Abnormal HLCR were reported in 30/31 WM patients. There was a good correlation between summated IgMκ + IgMλ and total IgM (r=0.832 p0.01). These 12 patients presented polyclonal hypogammaglobulinemia (with regard to their IgG and IgA levels). Median IgM HLCR (expressed as IgMκ/IgMλ or IgMλ/IgMκ with the involved Ig as numerator) were 90.88 (range 1.91–1000) in WM v 17.65 (range 1.08–34.57) in IgM-MGUS (p=0.06). Median IgM HLCR was significantly higher in WM patients requiring treatment (n=24) at presentation than in patients not (n=7) requiring treatment (185.7 v 13.45 p=0.023). IgM HLCR correlated with bone marrow infiltration (p=0.029) and time to first treatment (p=0.003). A simple risk stratification model utilising: IgM HLCR>median, β2M>5 and abnormal LDH, identified 3 prognostic groups with respect to survival (p Disclosures: Mirbahai: Binding Site Group Ltd: Employment. Bradwell:Binding Site: Equity Ownership, Patents & Royalties. Harding:Binding Site Group Ltd: Employment.

Abstract 5008 Introduction Differentiating Waldenström's Macroglobulinemia (WM) from other lymphomas and especially Splenic Marginal Zone lymphoma (SMZL), which presents common features with WM, consists a challenge. There is no characteristic molecular abnormality neither for WM nor for SMZL, while in addition, serum IgM cut off is no longer used for the diagnosis of WM. Moreover, while paratrabecular and intrasinusoidal pattern of bone marrow infiltration are described as typical of WM and SMZL respectively, there is often overlap between them. B-lymphocytes' immunophenotype is usually CD5 and CD23 negative in both entities, although deviations from this pattern may be observed; CD38, a marker of lymphocyte activation, theoretically related to lymphoplasmacytic differentiation and adverse prognosis in Chronic Lymphocytic Leukemia, may be expressed. Both WM and SMZL are included among the least reproducible diagnoses of the WHO classification when the diagnosis is based only on clinical grounds, routine laboratory tests and bone marrow biopsy (absence of lymph node or spleen biopsy or typical leukemic picture). CD138 (Syndecan) is expressed in immature as well as in terminally differentiated B-lymphocytes, especially in plasma cells. A strong CD138 expression is observed in myeloma plasma cells. Aim To evaluate firstly whether CD138 is expressed in WM and SMZL and secondly if its expression may help in the differential diagnosis of these entities. Patients and methods 61 patients were studied at presentation, 40 with WM and 21 with SMZL. Among WM patients, 16 were females and 24 males with a median age of 64 years. 57% presented anemia with hemoglobin Results 62% of WM patients presented CD138 expression in contrast with 14% of SMZL patients. Median percentage of total CD138 expression in neoplastic cells in WM was 26.5% (range 2-100%) while in SMZL it was 8% (range 0-30%). These differences were statistically significant (p Conclusions Patients with WM presented increased CD138 expression when compared to SMZL patients. If confirmed in larger series, CD138 expression could help differentiating the two entities. Disclosures No relevant conflicts of interest to declare.

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

MM is characterized by the presence of a monoclonal immunoglobulin (Ig) in the serum, produced by neoplastic plasma cells infiltrating the bone marrow (BM). Small amounts of free light chains (FLC) are secreted in normal individuals. In case of monoclonality, the FLC ratio increased. The detection and quantification of serum FLCs and of their ratio, was recently introduced as a useful method for the diagnosis and monitoring of light chain MM, amyloidosis, non-secretory MM and MGUS. However, the possible role of FLCR as a prognostic variable has not been investigated yet. We therefore studied the relationship of baseline serum FLCR with disease characteristics and prognosis in MM patients. Eighty-six MM patients (55 kappa-, 31 lambda- ), 16 individuals with MGUS (8 kappa-, 8 lambda-) and 29 healthy individuals (HI) were studied. Serum free light chain levels were measured in frozen sera drawn at diagnosis, before treatment, using a latex-enhanced immunoassay (The Binding Site, Birmingham, UK) on a Behring BN II nephelometric analyzer. Then, the FLCR was calculated, accordingly as kappa/lambda or lambda/kappa, depending on the monoclonal light chain type of the patient. Forty-nine percent of MM patients were males and 65% were ≥ 65 years old. The Ig type was IgG in 61%, IgA in 22%, BJ in 16% and IgD in 1%. Thirty-five percent of patients were in stage I, 31% in stage II and 34% in stage III according to Durie and Salmon while 36% were in stage 1, 22% in stage 2 and 42% in stage 3 of the ISS staging system. Twenty-five percent of patients had Hb < 10g/dL, 13% creatinin ≥2 mg/dl, 10% elevated LDH, 19% serum albumin < 3.5 g/dL, 45% CRP ≥4 mg/L, 41% beta-2-microglobulin ≥5.5 mg/L, 17% presented spontaneous bone fractures and 41% had ≥50% plasma cell BM infiltration. The median serum FLCR at diagnosis was 3.68 in the 55 kappa-MM patients, 38.49 in the 31 lambda-MM patients, 1.24 in kappa-MGUS, 2.14 in lambda-MGUS and 0.43 in HI (κ/λ). Differences between MM and HI and MM and MGUS were significant (p < 0.001). Baseline serum FLCR correlated with creatinin (p=0.03), LDH (p=0.003), the percentage of BM infiltration (p=0.001), the presence of spontaneous fractures (p=0.002) and Durie and Salmon staging (p=0.003). In patients with BJ MM, FLCR was higher then in the ones with other Ig types (p=0.001). Serum FLCR did not correlate with age, gender, hemoglobin, CRP, serum albumin, beta-2-microglobulin, paraprotein level nor with the ISS staging. With a median follow-up of 27.2 months (3.2–89.8), MM patients with serum FLCR < median, had a 3- and 5- year survival of 92±6 and 79±10 respectively, while the corresponding 3- and 5-year survival of patients with FLCR ≥ median were 67±8 and 32±13 (p=0.001). In multivariate analysis, serum FLCR above the median provided prognostic information independent of the value of ISS. In conclusion, baseline serum FLCR appears to be a powerful prognostic factor of survival in MM.

Syndecan-1 (CD138) is a heparin sulphate proteoglycan overexpressed on the surface of both normal and malignant plasma cells. Its role in multiple myeloma (MM) pathogenesis remains unclear. It may be shed from plasma cell surface and released in its soluble form (soluble syndecan-1: s-synd-1). Serum s-synd-1 levels have been shown to be a powerful prognostic factor of survival in myeloma patients. The role of s-synd-1 in the current prognostic classification systems requires further investigation. We have retrospectively determined soluble syndecan-1 in frozen sera of 99 MM patients, 18 MGUS patients, and 25 healthy individuals (HI). In all patients sera samples were collected at diagnosis. Serum s-synd-1 levels were determined by ELISA (Diaclone Research, Besanson, France) according to the manufacturer’s instructions. MM patients median age was 69 y (44–82), 51 were female, 48 male, 26 were Durie-Salmon (DS) stage I, 41 stage II, and 32 stage III while, according to the International Scoring System for MM (ISS), 32 were in stage 1, 25 in stage 2 and 42 in stage 3. Median serum s-synd-1 was 15 ng/ml (7–40) in HI, 16.5 ng/ml (8–220) in MGUS and 64 ng/ml (7–3500) in MM. Differences between HI and MM patients levels and between MGUS and MM patients levels were both significant (0.01 and 0.05 respectively). Overall survival was decreased in patients with elevated (mean + 2SD) serum s-synd-1 (5-year overall survival 35±9 vs 80±13, p=0.004). S-synd-1 levels were an independent prognostic factor of survival compared to DS and ISS staging systems. In multivariate analysis, only ISS and s-synd-1 were significant parameters of prognosis (the prognostic significance of DS, ISS and s-synd-1 was 0.06, 0.006 and 0.02 respectively). Further analysis of survival of patients stratified according to the ISS system showed a difference in survival within groups according to s-synd-1 levels. The 5-year overall survival was 100 vs 58±16 in ISS stage 1 patients with low or elevated s-synd-1 respectively, 100 vs 18±16 in stage 2 and 50±25 vs 14±13 in stage 3. The difference was statistically significant for stage 2 only (p=0.04) and there was only a trend due to the low number of patients in stages 1 and 3. In conclusion serum s-synd-1 levels at diagnosis constitute an independent prognostic factor of survival, able to further differentiate MM patients within the ISS stages.

Bortezomib is a reversible proteasome inhibitor that was recently approved for the treatment of relapsed-refractory MM. However, dosage, schedule, management of adverse events and duration of response are still under investigation. We present our experience with bortezomib. 27 relapsed-refractory MM patients were studied. They were scheduled to receive bortezomib 1.3 mg/m2 IV days 1, 4, 8, 11 every three weeks for eight cycles and dexamethazone was planned to be added at 20 mg every other day of bortezomib (2,5,9,12) if no signs of response were observed after the 1st cycle. There were 20 males and 7 females (median age 71y old). Immunoglobulin type was IgG in 16, IgA in 7, BJ in 3 and IgD in 1. All patients had received at least 2 previous treatment lines (median 4, thalidomide in 52%). At treatment initiation, 59% of patients were Durie-Salmon stage III and 63% ISS stage 3. B2Microglobulin was increased in 81%, CRP in 33%, LDH in 22%, creatinin in 7%, plasma cell bone marrow infiltration> 60% in 100%, hemoglobin< 10g/L in 59%, platelet count>100x109/L in 92% and no patient had peripheral neuropathy> grade 1. Free light chains ratio (FLCR, FREELITETM) was determined in 16 patients at baseline (increased in all) and during treatment. With the treatment scheme described above, 55% of patients received additional dexamethasone. Two in resistant relapse died during the 2nd course. Treatment was stopped or reduced in the majority of patients because of severe neuropathy after 4–6 cycles and discontinued in 3 patients because of no response (4 patients completed 3 courses, 17 completed 11, 1 completed 5, 3 terminated 6). Hematology toxicity, fatigue, gastrointestinal toxicity and fever were mild and manageable. Severe side effects were neuropathy in 63% (sensory, instability, burning pains- median time of onset 85 days after bortezomib initiation and median duration 2 months) hypotension in 7% and hypoglycemia in 3%. 21/25 (84%) patients responded (2 complete remission [CR], 4 near CR, 10 partial response, 5 minimal response. Median time to response was 42 days but with median follow-up of 6 months 6/21 (28%) patients have relapsed and 3/25 (12%) died. Relapse was aggressive (onset with acute renal failure in 3, spine plasmacytoma in 1, automatic bone fracture in 1). B2M, CRP and stage were not predictors of response or early relapse but of 8/16 with FLCR>200 at baseline, 4 were resistant and 4 relapsed. In 2 of the 4 resistant patients FLCR decreased after the 1st course and increased after the 2nd; in the same way in responders with an early relapse, FLCR started to increase during the last cycles of bortezomib. It is possible that some patients develop quickly resistance to the drug. In conclusion, Bortezomib, in combination with low-dose dexamethasone produce rapid responses but neuropathy represent a frequent and severe side effect and the duration of response is short. An adequate or palliative treatment for neuropathy is needed as well as a maintenance schedule overcoming resistance. FLCR seems to predict response and early relapse.

Cytokines, such as MIP-1a (macrophage inhibiting factor) and OPG (osteoprotegerin) are assumed to play a role in MM disease biology and bone disease, by mechanisms that are still under investigation. MIP-1a is constitutively secreted by myeloma cells, plays a possible role in the development of MM bone lesions, enhance MM cell adhesion to stromal cells and its serum levels have been recently related to survival in MM. OPG is a soluble decoy receptor which acts as a soluble receptor antagonist that prevents osteoclasts activation and the development of bone disease. Reported findings on serum OPG levels in MM patients are controversial as well as its possible role in the biology of the disease. In order to investigate the possible relationship of MIP-1a and OPG levels in MM patients with prognosis and bone disease, we determined by ELISA serum MIP-1a and OPG levels in 20 MGUS, 82 MM patients and 27 healthy individuals (HI). Both cytokines were determined by ELISA (R&D, Quantikine, USA) in frozen sera collected at dignosis, before treatment. The median age of MM patients was 69 years (44–84) and 50% were males. MM patients’ stage was as follows: 23 stage I, 28 stage II, 31 stage III according to Durie-Salmon staging system and 27 stage I, 17 stage II, 35 stage III according to the International Scoring System (ISS). In HI median MIP-1a was 28 pg/ml (15–54) and median OPG 1600 pg/ml (450–4600). In subjects with MGUS, median MIP-1a was 34 pg/ml (17–58) and median OPG 2300 pg/ml (820–6200). In MM patients, median pretreatment serum MIP-1a was 32 pg/ml (16–100) and OPG 3000 pg/ml (820–25000). No statistical significant difference was observed between HI, MGUS and MM patients with regard to MIP-1a levels but for OPG levels differences between HI and MM patients and between MGUS and MM patients were both significant (0.01 and 0.05 respectively). No relationship was found between MIP-1a or OPG levels and bone disease. However, there was a trend for longer survival in patients with MIP-1a or OPG levels lower than median (5-year overall survival 60 ± 12 vs 38 ± 14, p=0.08 and 66 ± 13 vs 29 ± 13, p=0.07 respectively). In addition MIP-1a levels were correlated with ISS stage: MIP-1a levels were 28.3±11.3 in ISS stage 1, 29.8±11.1 in ISS stage 2, 39±19.2 in ISS stage 3 (p=0.02). In conclusion, in our experience serum OPG levels are higher in MM patients than in MGUS or HI, MIP-1a levels are correlated with the ISS stage and both high serum MIP-1a and OPG levels at diagnosis are related with a shorter survival.

Extranodal marginal zone B-cell lymphomas of mucosa associated lymphoid tissue (MALT) are indolent lymphomas with specific clinical and pathologic features. MALT lymphomas can arise from any extranodal site. Non-gastrointestinal (non-GI) MALT lymphomas are rarer (

Primary cutaneous lymphoma comprise a wide range of rare diseases of which the commonest T- cell subtypes include mycosis fungoides (MF), Sezary syndrome (SS) and peripheral T-cell lymphoma (PTCL) while follicle center cell lymphoma (FCL), diffuse large B-cell lymphoma (DLBL) and extranodal marginal zone B-cell lymphoma (MZL) represent the B-cell subtypes. The behavior of these diseases is different from nodal NHL and it has been shown that IFN-a can produce long-lasting remissions in early stages MF. On the contrary, there are no reports on the efficacy of IFN- a for primary cutaneous B-cell lymphomas (CBCL). The purpose of this study was to present our experience on IFN -a treatment for patients with both primary T-cell and B-cell cutaneous NHL. Forty-one patients, 25 with primary cutaneous T-cell NHL (CTCL) (16 MF, 4 SS, 5 PTCL) and 16 with CBCL (4 MZL, 3 FCL, 8 DLBL, 1 lymphoblastic NHL) were included in this study. Of the 25 CTCL patients, 20 were males (median age 48y); 16 received IFN-a as first line treatment, 5 after PUVA and 4 after failure of other treatment modalities. Of the 16 CBCL patients, 8 were males (median age 57y); 14 received IFN-a frontline and 2 after treatment failure. Three to 5 MU IFN-a were administered subcutaneously daily with paracetamol prophylactically 1 hour before injection and two hours after. 17 out of 24 CTCL patients responded (overall response rate 71%, 42% CR and 29% PR) (12/16 MF, 1/4 SS and 3/5 PTCL). Median time to response was 4 months (1–17). Median duration of response was 15 months (8–38). 11 patients relapsed, 1 responder rapidly discontinued treatment because of side effects (cataract), the others are still under treatment with IFN-a. Among patients with CBCL 12/14 responded (overall response rate 86%, 64% CR and 22% PR). Median time to response was 2 months (1–4) and median response duration 23 months (13–30); 4 patients relapsed, 1 discontinued treatment because of side effects (depression), another continued with Peg- IFN because of chronic fatigue and the others are still under IFN-a treatment. With respect to histologic subtype, all 4 MZL patients responded; 2/3 FCL and 4/8 DLBL also responded. Flu-like syndrome was observed in the majority of patients at treatment initiation and 60% of patients presented mild leukopenia. In conclusion IFN-a is an effective treatment in primary cutaneous NHL and is usually well tolerated. As already reported response rate is satisfactory in MF. The high response rate observed in CBCL, especially in low-grade subtypes, but also in half of DLBL patients has not been reported previously.

Lymphomas of the oral cavity consist 1 involved sites. Thirty six patients (pts) (78.3%) had clinical stage I or II and 10 (21.7%) advanced stage disease. A monoclonal lymphocytic population was detected in the blood of 7 pts (15.2%) by PCR (IgH or TCR rearrangement), 4 of them with early stage lymphoma. 56.5% of the lesions were diagnosed as B-DLCL and only 10.8% as peripheral T-cell lymphomas. Nineteen pts with B-DLCL received CHOP or equivalent chemotherapy regimens and 11 CHOP combined with anti-CD20. Chlorambucil was administered in 4 pts with MALT lymphoma and local radiotherapy as the sole treatment in 1. The pts with T-cell lymphoma received CHOP (3) or interferon alfa-2b (2). Thirty eight pts are in complete remission. There were 3 deaths due to the disease (1 MALT, 1 predominantly small cleaved cell and 1 B-DLCL). Three additional pts relapsed and achieved complete response with a second chemotherapy course and 2 pts developed a lymphoma of different histology. In conclusion, oral lymphoma is a rare disease mainly of a diffuse large B-cell histology and a prolonged survival, when treated with chemotherapy (10-year Event Free Survival 82.5%).

Impaired erythropoiesis with inappropriate erythropoietin (EPO) production is seen in about 40% of symptomatic multiple myeloma (MM) patients at diagnosis and worsens during terminal refractory disease. Up-regulation of proinflammatory and proapoptotic factors, namely TNF-alpha, INF-gamma, TGF-beta1, FAS-Ligand and TRAIL are involved in the pathogenesis of defective erythroid maturation. The involvement of these factors in the progression of MM has been studied and no correlation was found for TNF-alpha, INF-gamma and TGF-beta1, while it was shown that the up-regulation of FAS-L/TRAIL in MM cells contributes to MM aggressiveness. Increased soluble syndecan-1 (s-synd-1) serum levels have been shown to constitute an adverse prognostic factor for survival in MM. Furthermore at least 3 studies, including ours, also found a negative correlation of s-synd-1 levels with hemoglobin. The purpose of this study was to investigate the correlations between s-synd-1, hemoglobin (Hb), endogenous EPO levels and other parameters of disease in anaemic MM patients before any treatment including rHu-EPO administration. 41 MM patients (19 males and 22 females) with variable degree of anaemia (Hb

In chronic lymphocytic leukemia (CLL), the molecular features of the clonotypic B-cell receptor immunoglobulins (BcR IG) are set from the birth of the clone and in contrast to genetic aberrations, remain stable overtime, rendering the BcR IG a reference biomarker that is usually not significantly affected by clonal evolution. Approximately 30% of CLL cases carry quasi-identical BcR IGs and can be assigned to distinct stereotyped subsets. While preliminary evidence alludes to BcR IG stereotypy being relevant from a clinical viewpoint, this aspect has never been explored systematically or in a cohort of adequate size to enable meaningful conclusions. In order to assess the clinical implications of BcR IG stereotypy, we evaluated clinicobiological data from 8593 CLL patients, particularly focusing on 14 major stereotyped subsets of cases with unmutated (U-CLL) or mutated IGHV genes (M-CLL). The largest subset was #2 (n=254, 3%), within which 156 and 98 cases were M-CLL and U-CLL, respectively, followed by U-CLL subsets #1 (n=173, 2%) and #7 (n=123, 1.4%). Amongst M-CLL, the largest subsets were #4 (n=94, 1.1%) and #148 (n=92, 1%). Stereotyped subsets, even of the same mutational category i.e. U-CLL or M-CLL, exhibited significant clinicobiological differences regarding: age at diagnosis (median age ranging from 53-68 years, p75% in M-CLL subsets #77 and #148 (p Disclosures Montillo: Janssen: Honoraria.

Summary Background About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptor immunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggests that B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never been explored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn. Methods For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogenetic data were available. These patients were followed up in 15 academic institutions throughout Europe (in Czech Republic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collected between June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptor immunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressing unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome of our analysis was time to first treatment, defined as the time between diagnosis and date of first treatment. Findings 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 major subsets. Stereotyped subsets showed significant differences in terms of age, sex, disease burden at diagnosis, CD38 expression, and cytogenetic aberrations of prognostic significance. Patients within a specific subset generally followed the same clinical course, whereas patients in different stereotyped subsets—despite having the same immunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status—showed substantially different times to first treatment. By integrating B-cell receptor immunoglobulin stereotypy (for subsets 1, 2, and 4) into the well established Dohner cytogenetic prognostic model, we showed these, which collectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separate clinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2). Interpretation The molecular classification of chronic lymphocytic leukaemia based on B-cell receptor immunoglobulin stereotypy improves the Dohner hierarchical model and refines prognostication beyond immunoglobulin mutational status, with potential implications for clinical decision making, especially within prospective clinical trials. Funding European Union; General Secretariat for Research and Technology of Greece; AIRC; Italian Ministry of Health; AIRC Regional Project with Fondazione CARIPARO and CARIVERONA; Regione Veneto on Chronic Lymphocytic Leukemia; Nordic Cancer Union; Swedish Cancer Society; Swedish Research Council; and National Cancer Institute (NIH).