ALBERT

Description: Introduction: Immune reconstitution after stem cell transplantation (SCT) plays a crucial role in host defense against microbial agents. The thymus atrophy occurring with ageing represents a limit for de novo T-cell reconstitution, although the reactivation of thymic function after BMT is documented. The proper strategy to improve the thymic output is currently matter of debate. Pre-clinical and clinical evidences suggest that Zinc oral supplementation may contribute to thymic reactivation and to improve the T-mediated cellular defense against pathogens. We tested the role of Zinc oral supplementation in immune reconstitution, using as model the autologous SCT in multiple myeloma. Methods: From January 2014 to May 2016, we prospectively enrolled 18 patients (12 male, 6 female; average age: 58 years, range 43-72) undergoing single MEL 100 or 200 auto-SCT after one or two lines of therapy (VTD, lyposomal anthracycline and lenalidomide) and stem cell collection (mobilizing therapy: cyclophosphamide 3 g/sqm and G-CSF). The trial, prospective and randomized, has been approved by local ethics committee (EUDRACT: 2014-004499-47). All patients undersigned the informed consent. The clinical trial was carried out in accordance with Helsinki declaration. Randomization was effected at day 0 (day of PBSC infusion). Results: nine patients were treated from day +5 to day +100 after transplant with 600 mg/day of Zinc sulfate (uncoated tablets), whereas nine patients received only standard antimicrobial prophylaxis. Peripheral blood samples were collected in both groups at day +30 (t2) and day +100 (t3). Eight-colour flow cytometry was performed for CD3, CD4, CD8, CD45RA, CD45R0, CD27, CD28, CD25, CD127, with specific gates to identify specific lymphocyte populations (T naïve, T central memory, T effector memory, T terminal memory). The lymphocyte's population's count was statistically analysed intra- and inter-group with Wilcoxon test. Droplet-digital PCR for TRECs was performed on lymphocytes isolated by peripheral blood. A qPCR for viral load of Torquetenovirus (TTV), a harmless virus whose viral load is related to immunodepression, was performed. Zinc serum level was measured. Results: The only significant difference in the clinical features between the 2 groups was the mean age (58 years in the control group, 63 years in the sample). The recovery of naïve CD4 cells was visible in both groups from day +30 to day +100, but a significant increase was detected only in the Zinc group. Similarly, TRECs showed a significant increase in the treated group. CD8+ T cells showed a notable decrease until day 100 in the control group, specifically the CD8 naïve, memory, and effector memory populations. TTV load in the control group increased in inverse proportion to the decrease of CD8 population. In the Zinc group there were no differences between the TTV loads at the two time points. As a result, the viral load of TTV was higher in the control group than in the Zinc group at day +100. Zinc serum levels were normal in all patients. Conclusions: data show that Zinc plays a role in a faster recovery of TRECs and CD4 naïve T cells after autologous transplantation. Furthermore, Zinc seems to prevent the expected CD8 decrease of the control group, thus probably explaining the TTV reactivation. Zinc-deficiency was not observed in any of the patient; however, a Zinc supplementation may contribute to a better T-cell reconstitution. To the best of our knowledge, this is the first study describing the role of Zinc after stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Radioimmunotherapy (RIT) is a new treatment for B non Hodgkin’s lymphoma (NHL) patients. 90Y ibritumomab tiuxetan (Zevalin®) consists of a murine monoclonal antibody to CD20, conjugated to a metal chelator tiuxetan for retention of the beta emitter 90Y. Thus Zevalin® delivers radiation to B-NHL, combining the tumor targeting attributes of a monoclonal antibody and the beta radiation of 90Y. Zevalin® is approved for the treatment of follicular lymphoma (FL) refractory to or relapsed after rituximab, on the bases of clinical trials where it achieved a response rate as high as 83%. Several ongoing registrational trials are evaluating the efficacy of Zevalin® in other NHL, as diffuse large B cell (DLCL) and mantle cell lymphoma (MCL). We are here evaluating the effect of Zevalin® as consolidation therapy in NHL patients that achieved a complete clinical response (CCR) with chemotherapy. Methods: In B cell NHL patients that achieved a CCR after 1st or multiple lines anthracyclines based chemotherapy +/− Rituximab, minimal residual disease was evaluated by PCR on bone marrow samples, for the following rearrangements: JH, Bcl-1, Bcl-2. Patients received Zevalin® 6-9 weeks post chemotherapy. Evaluation of molecular response was assessed after a follow up period at 12 weeks. The aim of the study was the role of Zevalin® in inducing a complete molecular response (CMR). Results: 23 B-NHL patients (13 FL, 6 MCL, 4 DLCL; male:female 13:10, median age 63, range 42–73. See table) in a CCR after chemotherapy (documented by TC scan and/or PET-scan negative for abnormal lesions or glucose captation) have been enrolled. 10 patients had a pathological rearrangement before RIT, while 13 were already in a CMR condition. Zevalin® was completed in all 23 patients and the post infusion evaluation was performed after 12 weeks. In the follow-up period thrombocitopenia was commonly documented, but it was not associated to bleeding or need of platelet transfusion, but in one singular case. After 12 weeks from RIT a new molecular evaluation was performed on bone marrow samples. All the 23 patients have completed the 12 weeks follow-up: 8 of 10 (80%) patients positive before RIT achieved a CMR with Zevalin® administration. The 13 PCR negative patients maintained the CMR. The 21 PCR negative patients are now under follow-up to evaluate the molecular disease free survival after Zevalin® RIT. Conclusion: Zevalin® is an efficient consolidation therapy in B cell NHL patients after chemotherapy. In this series of patients Zevalin® administration allowed to convert 8 of 10 CCR to CMR. In the remaining 13 patients Zevalin® maintained the CMR. Zevalin® addition to medication treatment is feasible and associated with manageable hematological toxicity. Pts disease sex age previous chemotherapy lines molecular response before RIT molecular response after RIT 1 FL M 68 1 POS NEG 2 FL F 53 1 NEG NEG 3 FL M 54 1 NEG NEG 4 FL M 51 4 NEG NEG 5 DLCL F 66 2 POS NEG 6 DLCL F 67 1 NEG NEG 7 FL F 42 1 POS POS 8 FL M 52 1 POS NEG 9 FL F 54 3 NEG NEG 10 FL M 57 2 POS NEG 11 FL F 62 2 POS NEG 12 FL M 58 2 POS NEG 13 FL F 69 2 NEG NEG 14 MCL M 62 1 POS NEG 15 MCL M 66 1 POS POS 16 MCL M 66 2 NEG NEG 17 MCL M 67 1 POS NEG 18 FL F 67 2 NEG NEG 19 DLCL F 67 3 NEG NEG 20 MCL M 70 2 NEG NEG 21 FL M 61 4 NEG NEG 22 DLCL M 43 2 NEG NEG 23 MCL F 73 2 NEG NEG

Description: Background and rationale In chronic myeloid leukemia (CML) about half of patients (pts) achieving a deep and stable molecular response (MR) with tyrosine kinase inhibitors (TKIs) may discontinue TKI treatment without disease recurrence. As such, treatment free remission (TFR) has become an ambitious goal of treatment. Given the evidence that deepness and duration of molecular response are necessary but not sufficient requisites for a successful TFR, additional biological criteria to possibly identify more and better CML patients suitable for an efficacious discontinuation are today focus of research in CML. Leukemia stem cells (LSCs) are supposed to be the reservoir of disease. We first showed in a cross-sectional study including 112 pts in TFR for a median of 31 months (mos) that residual circulating CD34+/CD38-/CD26+ CML-specific LSCs were still detectable in the majority of CML pts despite stable and deep molecular response. This evidence suggested that the level of BCR-ABL transcript only may not reflect the actual residual CML LSCs burden and that there could be a "threshold" of LSCs predicting a successful TFR. Aims To further study the behavior of residual LSCs during TKI discontinuation we designed a prospective multicentered study (AIRC IG 20133 study) in which we monitored circulating CD26+ LSCs in CML pts from the time of TKI discontinuation until molecular relapse. Methods CML pts meeting the current molecular criteria for TKI withdrawal entered this multicenter study. At TKI stop (baseline) and at +1, +2, +3, +6, + 12 mos after discontinuation and at any time if molecular relapse, CML pts were evaluated for peripheral blood number of CD34+/CD38-/CD26+ LSCs by centralized flow-cytometry analysis and for BCR-ABL transcript level by standard (IS) quantitative RT-PCR assay. Results 49 consecutive CML pts were enrolled in the study so far. Pts characteristics at diagnosis, type of TKI, disease response and treatment duration before discontinuation are shown in Tab. 1. After a median time of 7 mos since TKI stop (range 1-24), 13/49 (26.5%) pts lost their molecular response and restarted TKI treatment. Median time to relapse after discontinuation was 4 mos (range 2-7). 36/49 (73.4%) pts are still in TFR after a median time of 7.5 mos (range 1-24). If considering a cut-off of 6 mos from discontinuation as the period with higher risk of relapse, 14/36 pts actually in TFR have discontinued treatment for ≤ 6 mos (range 1-6) while 22/36 pts are in TFR for a median of 10 mos (range 7-24). Regarding residual CML LSCs evaluation, at baseline 23/49 (46%) pts had still measurable circulating CD26+LSCs with a median number of 0.0204µ/L (range 0.0077-0.1197), while 26/49 (54%) had no detectable CD26+ LCSs. Considering the small number of molecular relapses no statistical difference in number of residual CD26+ LSCs at time of discontinuation was shown between pts losing vs maintaining TFR (13 pts median CD26+ LSC 0.0237/µ/L, range 0-0.1197 and 36 pts median CD26+ LSCs 0.0204/µ/L, range 0-0.1039, respectively). However, the number of pts with undetectable CD26+ LSCs at baseline was 6/13 (45%) and 20/36 (55%) in the two subgroups, respectively. Considering subsequent time points, the 13 relapsed pts showed a small yet progressive increase of residual CD26+ LSCs number until molecular relapse, while the 36 pts in TFR showed a fluctuation of CD26+ cells number. However, Kendall rank correlation coefficient, Mood test and bi-linear relation model of the whole cohort showed no correlation between BCR-ABL/ABLIS ratio and number of residual CD26+ LSCs either at baseline or at each time points after discontinuation, thus confirming our previous observations. Conclusions Yet very preliminary our results showed that CD26+ LSCs are detectable at time of TKI discontinuation and during TFR. Moreover, at least for the observation median time of the study (7.5 mos) the persistence of "fluctuating" values of residual CD26+ LSCs do not hamper the possibility to maintain a stable TFR. Due to the short follow up and the small number of molecular relapsed pts we could not find a threshold of CD26+ LSCs predictive of TFR loss. Our data may suggest other factors then LSCs "burden" to play an active role in controlling disease recurrence. Additional studies evaluating CD26+ LSCs ability to modulate the immune system through a variable expression of immune response inhibitory molecules and through their interactions with effectors cells are ongoing. Table Disclosures Bocchia: Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Abruzzese:Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Iurlo:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Galimberti:Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Liberati:Bristol & Mayer: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria.

Description: Abstract 3945 Poster Board III-881 Introduction Non Hodgkin Lymphoma could be clinically divided as low grade/indolent NHL (LG NHL) and high grade/aggressive NHL (HG NHL). These diseases are chemo and radio-sensitive and improvements have been achieved by immunotherapeutic approaches. However some patients will relapse and a follow up strategy has to be planned in order to detect and treat them. Several aspects should be considered in planning a follow up including safety, specificity, sensitivity, costs and impact on the patient's psychology: an optimal follow up should mediate between these ones. The more diffuse follow up have been planned years before the introduction of innovative methods and imaging techniques, suggesting the opportunity to revise these programs. Methods We collected data about 418 NHL patients -both low and high grade- treated at our institution from 1990 to 2005 who achieved a complete remission according to Cheson criteria and who entered a follow up program which schedule is planned for 5 years divided in two periods: first two years evaluation every 3 months and in the following three years every sixth month. At each visit physical examinations, blood testing (blood count, chemistry) are performed; for imaging techniques we alternate a whole body CT scans to ultrasounds and chest X-ray coupled. Analyzing time to relapse (TTR), we tried to optimize our follow up schedule trough a computation application known as multi-objective analysis. The first step of this method has been to choose and try to quantify the costs of a follow up which reflect its effectiveness. We considered as costs the expected time between relapse and its detection (Ca) and the expected number of performed examinations before failure or censoring occurs (Cb). The total follow up costs could be summarized in a vector Cref: (Ca,Cb). After doing that we described survival analysis, relapse rate and their onset time in order to be suitable for the informatic analysis. We used a log logistic parametric model to do that. Next we shaped our ideal follow up as a structure based one, which is currently the most used in medical practice: a first period where examinations are more tightly spaced, followed by a second period where they are performed further apart. We describe such follow-up schedule “S” as (d1, k, d2) : d1 is the time between the first k examinations; d2 is the time between the remaining examinations. Applying the log-logistic model we calculated the Cref and schedule for our follow up: Cref was (Ca,Cb)=(8.5,6.2); this means that, on average, every patient entering the follow-up will perform 6.2 examinations, and among those who incur in a relapse, the relapse will be detected 8.5 units of time (eg. weeks) after its onset. The follow up structure was (13,8,26). We then calculated all the possible combination of (d1, k, d2) values from 1 to d1 = 25, k= 24 and d2 = 60. Results 360000 follow up schedules had been detected after searching all the possible combinations for d1, k and d2. For each one Ca e Cb costs have been calculated and than compared by multi-objective analysis to those of the current schedule, We look for both follow up structured and “free” follow up, where “free” means that intervals between examination is continuously variable. When comparing follow-up schedules, we apply the rule of Pareto-dominance: the schedule S1 is superior to the S2 schedule if and only if the cost values for S1 are both lower than the cost values for S2. The method then takes into account only those schedules which improve the current one, with respect to both the Ca and the Cb costs. After multi-objective analysis six follow up were detected. These were as following: (16,10,22), (15,8,20), (15,6,19), (16,8,19), (16,5,18), (16,4,18) and are shown in Fig. 1. Conclusions no differences in follow up schedules emerged when we considered separately LG and HG lymphoma patients. Maximum improving of our follow was just 4% and all the new schedules had wide time frequency visit in the first period and a narrow one in the second period: this was a consequence of the higher relapse distribution in the first period and this follow up organization lead to a lower total number of visit without risk of lose any relapse. This is the first application of this analysis to hematological patients confirming the validity of a follow up schedule should be shaped in two different period. Disclosures: No relevant conflicts of interest to declare.

Description: TNF-a is a potent mediator of inflammation and bone resorption that seems to be involved in the malignant transformation of plasma cells, since mononuclear cells, obtained from Multiple Myeloma (MM) patients and exposed in vitro to TNF-a and interleukin-4, produced monoclonal plasma cells. In addition, TNF-a can stimulate plasma cell proliferation, by triggering interleukin-6 secretion, and shows proangiogenic properties in vitro (1). In addition, TNF-a determination was reported to be a good parameter for estimating tumour mass and for monitoring therapy outcome during treatment with different protocols (2). In particular, TNF-a is able to activate NF-kB, which is the main target of bortezomib (3). Variation in TNF-a levels can be related to gene expression, which is regulated at transcriptional level, as well as to genetic polymorphism. Single nucleotide polymorphisms (SNPs) have been identified at position -308 and -238 in the gene promoter. In particular, a G to A substitution at position -308 is associated with higher levels of TNF-a (1). The aim of this study was to investigate the effect of this TNF-a polymorphism on the outcome of refractory and relapsed MM patients (pts), receiving Bortezomib containing regimen as second line therapy. From September 2005 to April 2008 we selected 90 MM pts, who received at least one cycle of chemotherapy before treatment with Bortezomib and at least one cycle of high dose chemotherapy with peripheral blood stem cell transplantation in 25 pts. TNF a polymorphism at position -308 was determined on genomic DNA extracted from blood samples. The genotype frequencies obtained (71/90 G/G; 19/90 G/A) were in agreement with Hardy–Weinberg equilibrium. Patients were categorized as responders (Complete + Partial response =R) or non responders (Stable + Progression disease=NR) to treatment. The overall response in our patients was about 74% (67/90). In MM pts carrying the rarest A allele the overall response was reduced to 58% (11/19 pts), whilst GG carriers showed a better response rate of 79% (56/71 pts) (OR = 0.37). Our data indicate that the SNP at position -308 of the TNF-a gene promoter may be associated with different outcome in patients affected by MM and treated with bortezomib containing regimen. Similar conclusion was reached in a previous study where TNF-a gene promoter could predict the outcome after thalidomide therapy (1). To investigate whether TNF-a SNP is an independent predictor for response to MM therapy, larger studies are necessary, however if these preliminary results will be confirmed, they may represent a rationale for targeting TNF-a in novel therapeutic approaches to MM (4).

Description: Background: Several studies have suggested that genetic variability related with single nucleotide polymorphisms (SNPs) of the BER system, DNA synthesis and folate-metabolizing pathway genes could modulate DNA repair capacity. Moreover, these genes are supposed to be related to cancer risk. However, the prognostic impact of the association of individual and/or combined genetic variants in patients with myelodysplastic syndromes (MDS) remains undetermined. Methods: We genotyped 113 MDS patients, 54 with IPSS low/int-1 receiving only best supportive care (BSC group) and 59 with IPSS int-2/high treated with azacitidine (AZA-group), for the following polymorphisms: XRCC1 194 and 399, APE1 148, XRCC3 241, TS5'-UTR (2R/3R and G/C) and 3'-UTR (6bp+/6bp-), MTHFR 677 and 1298. Genomic DNA was analyzed by High Resolution Melting assay and restriction digests of PCR products. Overall survival (OS) was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analyzed by the log-rank test. Multivariate analyses were performed using the Cox method. Results: For all the target genes, the distribution of genotypes was consistent with the Hardy-Weinberg equilibrium. Among the baseline characteristics analyzed (age, sex, diagnosis according to WHO, hemoglobin) there was no statistically significant difference in the genotype distribution of studied polymorphisms. In the BSC group, the variants XRCC1 399 GG [Hazard ratio (HR)=7.07; p=0.02], -6/-6 of TS3'-UTR (HR=4.65; p=0.05), 2R/3G, 3C/3G, 3G/3G of TS5'-UTR (HR=11.44; p=0.02) and TT of MTHFR 677 (HR=67.12; p

Description: Abstract 5033 The pharmacology of pegylated liposomal doxorubicin (PLD) gives rise to a compound with major advantages that could potentially improve response and decrease toxicity. PLD comprises doxorubicin incorporated into polyethylene-glycol-coated liposomes, resulting in a longer circulation time in the body compared with the conventional formulation and a unique toxicity profile. The lower toxicity, especially less cardiotoxicity, is also related to the encapsulation of doxorubicin into microscopic liposomes, which preferentially penetrate and accumulate in tumour vasculature. Because increased angiogenic activity occurs in the bone marrow of patients with multiple myeloma, this pegylated formulation can enhance the delivery of doxorubicin to the tumour site. In addition, because myeloma cells divide slowly, the increased exposure of these cells to doxorubicin has the potential of overcoming resistance and increasing tumour cell killing capacity, theoretically resulting in improved response rates. Between December 2008 and May 2010, we treated a total of 39 MM resistant/refractory patients with two different scheme including PLD. Twenty-five patients were resistant/relapsed after at least one prior therapy including conventional doxorubicin. The scheme therapy was based on VMD: bortezomib (Velcade®: 1.3 mg/m2) as intravenous bolus twice a week for 2 weeks (on days 1, 4, 8 and 11 of each cycle) in a 28-day cycle for a total of 4 cycles; oral dexamethasone (40 mg) on the day of each bortezomib dose and the day after, and PLD (Myocet® 30 mg/m2) on day 4 of each cycle, 1 h after bortezomib infusion. Baseline characteristics are shown in table 1. All 25 patients were evaluable for response. Response rates following VMD showed: 2 patients in nCR (10%), 4 in VGPR (20%), 14 in PR (70%) resulting in an overall response rate (ORR) of 80% (20 patients). Global toxicities were lower and included neutropenia (12.5%), fatigue (25%), thrombocytopenia (25%) and neuropathy (37.5%). In the other group, fourteen patients were treated with RMD a combination regimen of lenalidomide (Revlimid®), PLD (Myocet®) and dexamethasone and 11 patients were eligible for evaluation. All the patients had multiple myeloma resistant or progressed after 1 to 5 previous anti-myeloma regimens including at least one doxorubicin containing scheme. RMD was administered for six 28-day cycle. Lenalidomide 25mg d1-21, Liposomal doxorubicin 30 mg/m2 d4, Dex 40 mg d1-4 and d17-20. Eight of eleven patients (73%) achieved an objective response to the therapy. Respectively, 2 patients (25%) obtained a VGPR and additional 6 patients (75%) a PR. The most common side effects was haematological toxicity with grade neutropenia (32%), thrombocytopenia (32%) and anaemia (18%). Under thrombosis prophylaxis with aspirin 100 mg per day we observed thrombembolic complications in only in one patients. Other non haematological side effects were pain (9%), diarrhoea (9%). Neither neurotoxicity nor constitutional symptoms of grade 3/4 were found. In both of groups no cardiovascular events were reported. Additionally 12 patients of RVM group and 4 patients of RMD group underwent to high dose chemotherapy and successfully collected an adequate number of peripherals stem cells at the first attempt. The introduction of pegylated liposomal doxorubicin, in bortezomib or lenalidomide based regimens, can play a key role in overcoming anthracycline resistance and improving the quality of response without limiting toxicity, especially in patients who have already received at least one prior therapy. We believe that there is a rational basis for the use of PLD as a second-, third-, or fourth-line therapy in patients with advanced MM. Table I. Characteristics of MM Patients undergoing VMD or RMD therapy Characteristics Cases Age at diagnosis (median and range) 65 (44–76) Number of patients     VMD 25 (14M, 11F)     RMD 14 (10M, 4F) Stage at diagnosis     Durie-Salmon (II/III) 10/29 Prognostic Markers     b2-microglobulin (m/L.) 2.2 (1.1–35)a     Creatinin (mg/dl.) 0.9 (0.5–4.4)a     Albumin (g/dl)) 4.0 (2.1–4.9)a     Hemoglobin (mg/dl) 11.3 (5.7–16.4)a a Median (Range) Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (PC). Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification but the clinical and prognostic value of immunophenotyping in MM remains questionable. We have analyzed the prognostic impact of a relatively new marker as CD69. CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous laboratoristic data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. In a previous abstract, we described for the first time in a clinical report, the CD69 expression on pathological PCs of MM patients. Our preliminary data also suggested an intriguing role of CD69 in patients treated with chemotherapy in different stages of disease. In this study, we report a larger setting of 97 patients where we confirmed the expression of CD 69 in 48 of them (49%) (see table I). Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was 〉 20%. After an induction regimen of treatment with four cycles of VDT (bortezomib, dexametasone, thalidomide), 69 patients were evaluable. 40/69 (65%) of patients obtained at least of a very good partial response or better (Responding pts). In this subgroup of patients 30/45 (66.6%) showed the expression of CD 69. On the contrary only in a little part of partial or less responding patients (NR pts) 9/24 (37.5%) CD69 was detected (see table II) (p=0.02 using a chi squared test and p=0.019 using a Fisher's exact test). Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in 〉90% of patients. Clinical data were available in all patients and CD69 maintained its association with different response, independently of other prognostic variables. In conclusion CD69 is often expressed in PCM cases, and the expression of this marker is useful to reveal poor prognostic categories and delineate a risk stratification. This molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table I Pts Characteristics Total CD69+ 97 48/97 Sex Male 51(52%) Female 46(48%) Clinical status MM non evaluableMM after VTD 28/9769/97 in VGPR/CR 45/69 in PR/SD/PD 24/69 Table II Pts treated with VTD Responding pts NRpts 45 24 CD69+ 39/69 30/45 9/24 CD69- 30/69 15/45 15/24 Disclosures No relevant conflicts of interest to declare.

Description: Imatinib (Glivec, Novartis) is a tyrosine kinase specific inhibitor used for the treatment of CML. The occurrence of cytogenetic abnormalities in Ph-negative cells emerging after suppression of the Ph-positive clone has been described; however the origin as well as the biological and clinical significance are unknown. We collected data on 32 patients through the GWP in CML registry. Bone marrow cell segregation, cell culture and morphologic features in a subgroup of these patients were studied to acquire insights into the origin of the Ph-negative clone. Patient characteristics and clinical follow up (up to 49 months) are presented together with hypothesis regarding their biological significance. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 32 patients after a median of 16.2 months after starting Imatinib. Median age was 51 years, F:M=18:14, median time from CML diagnosis 35 months. All patients but one have started Imatinib while in chronic phase and were in chronic phase at detection of the abnormal Ph-negative clone. Eight patients were treated with Imatinb at onset. At diagnosis no additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 21 complete, 5 major and 6 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 14 patients, −Y in 5 patients,, three del(20q), two del(5q) and del(7q), one −7, del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), with one patient presenting with both +8 and +21, and one three clones with +8, double +8, and double +8 and −X. Retrospective analyses of stored pellet using FISH did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 7 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. Two patients that lost response to Glivec were treated with a second generation tyrosine kinase inhibitor Dasatinib (Sprycel, Bristol-Myers Squibb) and, at reduction of Ph+ clone, the del (7q) reappeared in one patient, while the +8 clone of the second patient was not further evidenced. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone was present into the CD34+ compartment suggesting the stem cells involvement. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. We performed cell culture on a subgroup of 6 patients demonstrating normal growth in five patients and an abnormal growth pattern in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. While a longer follow up observation and laboratory analyses are required, we remark that after 〉4 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS/AML, nor it seems to be associated with CML clonal evolution and disease progression.

Description: Allogeneic stem-cell transplantation is a potential curative option in multiple myeloma (MM). Reduced-intensity conditioning regimens (allo-RIC) result in a lower transplant related mortality (TRM) compared to conventional conditioning, despite of a higher relapse rate. Several prospective studies compared single or tandem autologous stem cell transplantation (SCT) with planned tandem autologous-reduced intensity allogeneic SCT, with discordant results in overall and progression-free survival (OS and PFS). Many studies were conducted using a “mini-allo-SCT”, a regimen containing a low-dose total body irradiation (TBI) and Fludarabine (Flu). Moreover, introduction of new drugs (bortezomib, thalidomide or lenalidomide) in the first decade of 2000 changed the biological history of MM. We analyzed the results of ten-year experience with mini-allo-SCT in patients with MM in our institution. Patients, materials and methods: Between June 2000 and December 2010, 21 patients (9 M, 12 F, median age 54 – range 36-66) received a mini-allo-SCT, 17 from an HLA identical sibling donor, 4 from a MUD full-matched. The source of stem cell was the peripheral blood in all patients. All grafts were not manipulated. At the time of diagnosis, Durie-Salmon (DS) stage was I in 5 patients (23.8%), II in 3 patients and III in 13 patients (61.9%). Disease status at the time of transplant was partial response (PR) in 17 patients (81%), 13 of them in first PR, 4 in second or more PR; 4 patients received allo-SCT as salvage therapy in active ore refractory disease. Eleven patients (52.4%) underwent to transplant after one line of treatment, 5 patients after 2 lines, 5 patients after 3 or more lines. Five patients (23.8%) were treated with new drugs. Auto-SCT is included in previous lines. Nine patients received one auto-SCT before the mini-allo-SCT; ten patients (47.6%) underwent transplantation after two auto-SCT. Two patients were allo-grafted frontline. Conditioning regimen was Flu-TBI in all patients. Graft versus Host Disease (GvHD) prophylaxis consisted on cyclosporine and MMF in all. Results: Overall response rate was 76%, 5 PR and 11 complete remission (CR). One patient developed progression next allo-SCT. Four patients died in the first 100 days after allo-SCT, and they are censored for OS and PFS analyses. Of 17 pre-transplant PR, 11 achieved CR (64%), 4 maintained PR, 2 died before response evaluation. Of 4 patients who underwent allo-SCT in active disease, only 1 obtained a PR, whereas the other 3 patients developed progression or were not-valuable. Six patients (28.6%) developed acute GvHD, but no one died for complicated acute GvHD. Eleven patients (52.4%) had chronic GvHD. Follow up range was from 4 to 96 months. The median time was 19 months. The relapse/progression rate in course of follow up was 29%. Two patients progressed after PR (40%), 3 after CR (5.9%). At the time of the last follow-up 8 patients died (47%), 3 of them for progression of MM. Survival analyses: TRM at 1 and 3 years was respectively 24% and 31%. OS at five years was 51%, with a plateau trend after 3 years (Fig. 1). In univariate statistical analysis, early DS stage at diagnosis (I-II), double auto-transplant, development of chronic GvHD have a significant impact (p value .05 ) Fig. 3a:. OS in patients previously treated or not with new drugs ( p value 〉 .05 ); Fig. 3b: PFS in patients previously treated or not with new drugs ( p value 〉 .05 ) Disclosures No relevant conflicts of interest to declare.