ALBERT

Description: Introduction: Secondary CNS dissemination (SCNSL) is a rare but lethal event in pts with diffuse large B-cell lymphoma (DLBCL). It can occur both at presentation, in pts with systemic disease, or at relapse, during or after primary therapy. Following the experience from primary CNS lymphoma, pts with SCNSL are currently treated with high-dose-methotrexate-based chemo and autologous transplant (ASCT). However, this strategy is associated with poor control of extra-CNS disease, and only 1/3 of pts proceed to ASCT and recover from this event. Thus, we designed a multicenter phase II trial addressing an intensified chemoimmunotherapy consolidated by ASCT in HIV-neg pts with SCNSL (NCT02329080). Methods: Inclusion criteria were: histologically confirmed DLBCL; CNS involvement at presentation (concomitant to systemic disease) or relapse (isolated or concomitant to systemic lymphoma); age 18-70 ys; ECOG-PS ≤3; no prior treatment with high-dose methotrexate. Registered pts received 3 courses of MATRIX followed by 3 courses of RICE combined with intrathecal chemo and consolidated by BCNU-thiotepa/ASCT. The primary endpoint was 1-yr PFS. The Fleming design was used; to detect a difference in 1-yr PFS from 50% (P0) to 65% (P1), 69 pts were required (one-sided, type I error 5%, power 80%), with a dropout of 10%, 76 pts were needed. If ≥41 pts were progression-free at 1 yr, the strategy would be considered effective. Results: Between 3/2015 and 8/2018, 79 pts were enrolled at 24 centers in 4 countries; 75 pts (median age 58, range 23-70; 38 males) were assessable. CNS involvement was recorded at presentation in 32 (43%) pts and at relapse in 43 (isolated site in 15, concomitant to systemic relapse in 28). CNS sites were brain parenchyma in 34 (45%) pts, brain + eyes in 10 (13%), brain + CSF in 13 (17%), brain + CSF + eyes in 6 (8%), CSF/meninges in 8 (11%), spinal cord in 2 (3%), and eyes in 2 (3%). Median time to CNS involvement was 5 months (range 1-61) in the 43 pts registered at relapse; 20 (47%) of them had refractory disease. 320 (71%) of the 450 planned chemo courses were delivered; 64 (85%) pts received intrathecal chemo. 78 SAEs were recorded in 42 pts, mostly due to FN and infections (64) or bleeding (5); 74 (95%) SAEs were followed by recovery. The 4 lethal SAEs (TRM= 5%) and the 5 transient interruptions occurred during MATRIX. Dose reductions were indicated in 33 (10%) courses. Most common g4 toxicities were thrombocytopenia in 118 (37%) courses, neutropenia in 113 (35%) and infections in 9 (3%). Stem cells collection was successful (median of 6.75M/kg; range: 2.4 - 45) in 42 (88%) of the 48 pts referred for leukapheresis. 55 (73%; 95%CI 63-83%) pts achieved a response after 2 courses of MATRIX; 19 (95%) of the 20 pts who had a CR after 2 MATRIX maintained the response after RICE; 9 (26%) of the 35 pts who had a PR after 2 MATRIX achieved a CR after RICE. Conversely, only 3 of 16 pts with PD/SD after 2 MATRIX achieved a response from RICE. 49 pts (65%; 95%CI 54-76%) achieved a response after MATRIX-RICE induction, and 36 responders received ASCT; 13 responders did not receive ASCT due to insufficient mobilization (n=4), PD due to treatment delay (5), frailty (2), neurological decline (1), and consent withdrawal (1). 45 pts (60%; 95%CI 50-70%) had responsive disease after the whole treatment. At 1 year from registration, 41 pts were progression free (efficacy threshold ≥41). At a median follow-up of 25 (12-47) months, 31 pts are progression free, with a 2-yr PFS of 42 ± 6% for the whole series and 75 ± 7% for the 36 transplanted pts (Fig. A & B). Sites of relapse/progression were CNS in 10 pts, extra-CNS organs in 9 and both in 18. Overall, 33 pts are alive, with a 2-yr OS of 42 ± 6% for the whole series and 82 ± 7% for transplanted pts. Causes of death were lymphoma (35) and toxicity (4); 3 pts died without evidence of disease due to neurological decline, PTE and sudden death. Pts with CNS disease at presentation had the best outcome (Fig. C), whereas CSF/meningeal disease (Fig. D) and age 〉60 ys were independently associated with poor outcome. Conclusions: MATRIX-RICE followed by ASCT achieved the primary endpoint in this very-poor-prognosis population, without major safety concerns. Survival figures of transplanted pts seem a little better than reported in prior trials, whereas pts with MATRIX-refractory disease had no benefit from crossing to RICE. The best survival figures were recorded in chemo-naïve pts treated at presentation and in pts without CSF/meningeal disease. Figure Disclosures Ferreri: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Doorduijn:Roche: Honoraria, Research Funding. Nassi:Merck: Consultancy; Takeda: Consultancy; Janssen: Consultancy. McKay:Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Epizyme: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Davies:ADCT Therapeutics: Honoraria, Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Janssen: Honoraria, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BioInvent: Research Funding. Fox:Celgene: Consultancy; Gilead: Consultancy; AbbVie: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Takeda Pharmaceuticals: Consultancy; Atara Biotherapeutics: Consultancy; Adienne: Other: Travel Support. Osborne:Gilead: Membership on an entity's Board of Directors or advisory committees; NIL: Employment; NIL: Other: leadership; NIL: Other: Stock & other ownership interests; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees. Liberati:Incyte: Consultancy; Novartis: Other: Clinical trial support; Janssen: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Roche: Other: Clinical trial support; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Celgene: Honoraria, Other: Clinical trial support; Bristol-Myers Squibb: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees. Zambello:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Zucca:Celltrion Helathcare: Membership on an entity's Board of Directors or advisory committees; AstraZenaca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Merck: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant, Research Funding; Kite, A Gilead Company: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grant. Cwynarski:Adienne: Consultancy; Takeda: Consultancy, Other: conference and travel support , Speakers Bureau; Roche,: Consultancy, Other: conference and travel support, Speakers Bureau; Autolus: Consultancy; KITE: Consultancy; Gilead: Consultancy, Other: conference and travel support, Speakers Bureau; Celgene: Consultancy; Atara: Consultancy; Janssen: Other: conference and travel support, Speakers Bureau.

Description: Chromosome 7 translocations, deletions, or monosomy are associated with myelodysplasia (MDS) and acute myeloid leukemia both in children and adults. These chromosomal anomalies represent one of the most common cytogenetic abnormalities associated with these diseases and usually herald a poor prognosis. In this study two cosmid DNA probes that mapped to 7q22.1 and were known to be separated by approximately 500 kb were identified to flank the proximal inversion breakpoint in a patient carrying a constitutional inversion (7q22.1–34) associated with MDS. A yeast artificial chromosome (YAC) clone that encompassed the two cosmids was identified and shown to span the breakpoint. Fluorescence in situ hybridization was then used to analyze six additional patients with myelodysplasia and chromosomal rearrangements of the 7q22 region (three patients had translocations and three carried deletions). The breakpoint in one of the patients was found to be contained within the same YAC clone that spanned the inversion breakpoint. Moreover, this same interval was determined to be absent in all three patients with chromosomal deletions. These results suggest that this segment of DNA on chromosome 7q22.1 may contain specific gene(s) that have a significant role in myeloid malignancies.

Description: To approach the goal of consistent long-term erythropoietin (Epo) expression in vivo, we developed an implantation procedure in which transduced autologous vascular smooth muscle was introduced into rats in a chamber created from a polytetrafluoroethylene (PTFE) ring placed under the serosa of the stomach. The implant became vascularized and permitted the long-term survival of smooth muscle cells expressing Epo. Hematocrits of treated animals increased rapidly and monitored over 12 months gave a mean value of 56.0 ± 4.0% (P 〈 .001; n = 9), increased from a presurgery mean of 42.3 ± 1.6%. Hemoglobin levels rose from a presurgery mean of 15.2 ± 0.4 g/dL and for 12 months were significantly elevated with a mean value of 19.5 ± 1.3 g/dL (P 〈 .001; n = 9). The hematocrit and hemoglobin levels of control animals receiving human adenosine deaminase (ADA)–expressing cells were not significantly different from baseline (P 〉 .05; n = 5). In response to tissue oxygenation, kidney, and (to a lesser extent) liver are specific organs that synthesize Epo. Treated animals showed downregulation of endogenous Epo mRNA in kidney over a 12-month period. The PTFE implant provides sustained gene delivery, is safe, and is minimally invasive. It allows easy engraftment of transduced cells and may be applied generally to the systemic delivery of therapeutic proteins such as hormones and clotting factors. © 1998 by The American Society of Hematology.

Description: Regulatory factors integral to the differentiation of mesenchymal stem cells (MSC) and to their maintenance of pluripotency are not yet well defined. Characterizing the repertoire of such factors present within phenotypically similar MSC isolated from different tissues would provide pivotal insight into the developmental and regenerative capabilities of the MSC present within each of these tissues and could also reveal the ideal MSC sources to use for tissue-specific transdifferentiation in future stem cell therapies. In previous studies, we showed that Stro-1 + MSC isolated from various human tissues exhibit a predisposition to differentiate into cells of their tissue of origin following transplantation. Here, we examined these Stro-1 + human MSC from fetal brain, liver, lung, and adult bone marrow (BM) to identify genes common to MSC from all 4 tissues as well as differentially expressed genes to define the mechanism responsible for the in vivo differentiative bias of these MSC. We compared the transcriptomes of Stro-1 + MSC from each tissue with Affymetrix GeneChip microarrays, and analyzed the resultant data with GeneSifter software. Quantitative RT-PCR was used to confirm altered transcription levels of representative genes which the microarray indicated were differentially expressed. We identified 256 transcripts that were specifically upregulated in the BM-derived MSC relative to MSC from the other tissues, including Early B-cell factor 1, a Notch regulated transcription factor, and HoxA10 which is involved in hematopoietic lineage commitment. We also identified 145 transcripts that were downregulated in BM-derived MSC compared to MSC from the other tissues. In the liver-derived MSC, we identified 298 downregulated transcripts and 168 upregulated transcripts, including the putative lymphocyte G0/G1 switch gene 2 (G0S2), which is induced in the liver in response to fasting and plays a key role in adipogenesis. The lung-derived MSC displayed 316 downregulated transcripts and 173 upregulated transcripts, including secreted frizzled-related protein 1, a mediator between the WNT and hedgehog pathways, and SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), which plays a critical role in the regulation of TGF-b-Smad signaling. Brain-derived MSC exhibited 169 downregulated transcripts and 328 upregulated transcripts including Jagged 1, a Notch ligand involved in maintenance of an undifferentiated state; TAFA5, a brain-specific regulator of immune and nervous cells; and Growth-arrest specific 1, a potential tumor suppressor growth regulatory protein. Interestingly, MSC derived from liver and lung possessed similar gene expression profiles, exhibiting upregulation of similar genes when compared to MSC from brain and BM, perhaps due to their shared endodermal derivation. It is also of note that many of the Hox genes were found to be differentially regulated between the MSC from the four tissues, suggesting this gene family may play a role in the MSCs’ tissue identity. In conclusion, our studies show that MSC derived from 4 distinct tissues express many common genes, but also possess unique molecular signatures that tie them to their tissue of origin. These results provide a possible explanation for the propensity of MSC to give rise to cells of their native tissue upon transplantation and will also pave the way towards elucidating the precise genes that allow MSC to give rise to seemingly unrelated tissues in vivo.

Description: Introduction: Combined modality treatment with Adriamycin, Bleomycin, Vinblastine and Dacarbazine (ABVD) chemotherapy followed by consolidative radiation to start within 3-4 weeks is the current accepted approach in the treatment of patients with early stage Hodgkin lymphoma (HL). Bleomycin pulmonary toxicity (BPT) is a well-known complication of treatment in HL patients. We undertook this study to investigate the risk of radiation pneumonitis (RP) in the setting of BPT and to determine the need for delay or omission of radiation in these patients. Methods: We reviewed the records of all HL patients treated with ABVD followed by radiation therapy (RT) to the chest between January 2009 and December 2014. We defined bleomycin toxicity as: the occurrence of clinical respiratory symptoms leading to discontinuation of bleomycin and/or bilateral opacities noted on computed tomography (CT) imaging and/or drop in diffusing capacity of the lung for carbon monoxide (DLCO) by 25%, in the absence of infection. We identified 129 patients, 100 of which received consolidation RT as part of combined modality and are the subject of this report, 29 patients were excluded because they developed relapse before getting RT. We compared patients with and without bleomycin toxicity for the following outcomes:Frequency of RP using the Pearson chi-square test.Interval between BPT and Radiation using Mann-Whitney U test (MWT)Interval between end of chemotherapy and radiation using MWT. We used univariate Cox regression analysis to assess the risk of RP by looking at the time-interval in weeks from end of bleomycin to start of RT. Results: Median follow up was 23 months (6 - 69), Median age was 31 years (18-77), and 60% were females. Per our criteria, 28 patients developed BPT (25.5%). All patients received intensity modulated radiation therapy, radiation dose median was 30.60 Gy (20-42Gy). Mean lung dose (MLD) was a median of 9.4 Gy (2.6- 13.9 Gy). The median interval between chemotherapy and RT was 3 weeks (1- 8 weeks). Median interval from stopping bleomycin, either as a precaution or because of toxicity, to the start of RT was 5 weeks (1-20 weeks). Interval between documented bleomycin toxicity to start of radiation was a median of 8.5 weeks (2-20 weeks). We had 10 cases of RP (10%), 5 of which were ≥ Grade 2. There was no significant difference in RP risk in patients with or without BPT; 10.7% (3/28) versus 9.6% (7/72) respectively, P= 0.82. Patients with BPT versus those without BPT had no significant difference in baseline characteristics. The interval time from chemotherapy to radiation was a median of 3 weeks in both groups with or without BPT showing no difference; P= 0.83. However, Patients with BPT had a significantly longer interval from last bleomycin cycle to start of radiation compared to those without BPT (median 8.5 vs. 5 weeks, p =0.014). The intervals from chemotherapy to radiation treatment and from bleomycin to radiation treatment showed no significant correlation with RP on univariate Cox regression analysis (P= 0.41 and P= 0.12, respectively). This was maintained when adjusted for the number of bleomycin cycles. Treatment of BPT Of the 28 patients, 17 were managed by stopping bleomycin and observation only; 10 patients required a 2 week course of steroids. One patient went into severe respiratory compromise, was started on continuous oxygen and eventually recovered 48 hours later and went on to receive RT beginning 2 weeks after completing his steroid treatment. This patient did not have pulmonary complications after RT. All 28 BPT patients eventually completed their planned course of radiation. At last follow up, all 28 patients were alive and free of respiratory symptoms. Conclusion: In our cohort of Hodgkin lymphoma patients, those patients with bleomycin toxicity who received standard RT had no excess risk of subsequent RP. Moreover, patients were able to receive complete courses of RT to intended conventional radiation doses. Our findings suggest that RT does not need to be delayed following chemotherapy, except to allow for the completion of steroids or clinical recovery from BPT. Table 1. BPT_clinical BPT _imaging BPT_DLCO≥25% Clinical+(CTorDLCO≥25%) BPT per criteria All patients n=100 25 17 10 13 28 RP No n=90 22 15 9 12 25 Yes n=10 3 2 1 1 3 Disclosures No relevant conflicts of interest to declare.

Description: Flow cytometric analyses of cellular DNA, RNA, and double-stranded RNA content were performed on lymph nodes and extranodal tissue from 177 patients with non-Hodgkin's lymphoma. With increasing histologic grade, a higher incidence of aneuploidy, higher proliferative activity, and higher total and double-stranded RNA content were found. Despite considerable cytometric heterogeneity within histologic grades and morphologic subdivisions, conformity between cytometric and morphologic classifications was observed in 85% of cases. Among intermediate-grade and high-grade lymphomas, increased proliferative activity and diploidy were associated with more frequent responses to treatment. Thus, nucleic acid-derived parameters relate to morphologic subtypes and permit an objective approach to lymphoma classification based on ploidy, proliferation, and RNA characteristics that also had prognostic implications.

Description: Abstract 1636 Background: Radioimmunotherapy (RIT) is effective treatment for relapsed and refractory indolent lymphomas. Results in aggressive lymphomas such as diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) have been less impressive, with lower response rates and short duration of response. We hypothesized that administration of the proteasome inhibitor bortezomib as a radiosensitizer in patients receiving RIT would be tolerable and potentially improve efficacy in both aggressive and indolent lymphomas. Methods: We performed a Phase 1 dose escalation study to evaluate the maximum tolerated dose (MTD) of bortezomib in combination with 131I-tositumomab. The study underwent review and was approved by each institution's Institutional Review Board. Dose escalation proceeded using a Time to Event-Continuous Reassessment Model (TiTE-CRM). Patients with relapsed or refractory DLBCL, MCL or indolent B cell non-Hodgkin's lymphoma were eligible if they had not undergone prior stem cell transplant, organ function was preserved, and bone marrow involvement by lymphoma was less than 25% of the intertrabecular space. Neutrophil count at least 1500/uL and platelet count at least 150 × 103/uL were required. A dosimetric dose of 131I-tositumomab was administered on day 1, followed by three whole body gamma camera scans for purposes of dose calculation and evaluation of biodistribution. After an initial cohort received a reduced whole-body dose of 50 cGy 131I-tositumomab, patients received RIT at the standard dose of 75 cGy on day 8. Patients were treated with escalating doses of bortezomib (0.3 to 1.2 mg/m2) on days 6, 10, 13, 16 and 20. Dose limiting toxicity (DLT) was defined as any grade 3 or 4 non-hematologic toxicity, or grade 4 hematologic toxicity. Results: The study has completed enrollment, with 25 patients having received study treatment. These include 8 patients with DLBCL, 5 with MCL, 11 with follicular lymphoma (FL), and one with marginal zone lymphoma (MZL). Median age is 68 (range 40–81). Median number of prior therapies is 2 (range 1–4), and all but one had received prior rituximab. Twenty-three patients are evaluable for response, while two patients have not yet undergone restaging. Twenty-four patients are evaluable for the primary endpoint of MTD. One treated patient was not evaluable for toxicity due to early progression of disease and need for further therapy prior to the end of the observation period. Of 24 patients evaluable, 4 experienced DLT (Table), all at dose level 5 (1.2 mg/m2). These events included grade 3 hyponatremia, herpes zoster, and grade 4 thrombocytopenia. Grade 3 hematologic toxicities included two patients with leukopenia, three with neutropenia, one with anemia, and five with thrombocytopenia. Dose level 4 (0.9 mg/m2 bortezomib, 75 cGy 131I-tositumomab) was well tolerated, and this dose was identified as the MTD. Fourteen of 23 (61%) patients evaluable for response have responded, including 3/8 with DLBCL, 3/5 with MCL, and 8/9 with FL. Ten patients have achieved complete remission, including one patient with DLBCL, one with MCL, and 8 with FL. At median follow up of 8 months, median progression free survival is 6 months, and seven patients (50% of responders) remain in remission at 2 to 28 months. Conclusions: Bortezomib can be safely administered in combination with 131I-tositumomab. Responses were seen in a majority of patients, including those with aggressive histology. The MTD has been defined as 0.9 mg/m2 bortezomib plus 75 cGy 131I-tositumomab. This strategy of radiosensitization using bortezomib shows promise, and efficacy should be further evaluated in a Phase 2 trial. Disclosures: Off Label Use: Azacitidine is approved for use in MDS. Discussion here is off label. Leonard:Glaxo SmithKline: Consultancy, Honoraria. Martin:Cephalon: Consultancy; Celgene: Consultancy; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lebovic:Genentech: Speakers Bureau. Coleman:Celgene Corp: Speakers Bureau. Kaminski:GlaxoSmithKline: Patents & Royalties, Research Funding.

Description: Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC 〉 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: The impact on patients’ quality of life (QoL) is an important consideration in both decisions of individual patient care, and in allocating health care resources between competing treatments. This study aims to better inform those decisions by investigating the differential impact on patients’ QoL of two equally safe and effective iron chelators: one a subcutaneous infusion, the other a dispersible tablet. The current standard of care in patients with chronic iron overload secondary to haematological conditions such as the thalassaemias, myelodysplastic syndrome and sickle cell disease is the subcutaneous administration of desferrioxamine (DFO), for 8 to 12 hours per day, 5 to 7 days a week. The burden of this treatment regimen in some cases leads not only to poor compliance, and hence a reduction in the extent of effective iron chelation, but also to a reduction in patients’ QoL. Novartis Pharmaceuticals AG has developed a once daily oral iron chelator (deferasirox, DFX) shown to be as safe and effective as DFO in treating iron overload. To investigate the differential impact of DFO and DFX on patients’ QoL, a community sample of 110 individuals were asked to participate in a time trade off (TTO) exercise to elicit community utility values, or preferences, for their different modes of administration. Following standard TTO methodology, participants were asked how they value ten years of full health compared with time in each of three health states for patients with thalassaemia: an anchor/base state that described a patient who has iron chelation without describing the treatment itself; the anchor state plus iron chelation via a subcutaneous infusion; the anchor state plus iron chelation via a once daily oral medication. To avoid ordering bias, respondents were randomised to respond to either scenario (2) or (3) first after the anchor state. Scenarios were not labelled in terms of the iron chelator included and differed only in their description of the mode of treatment administration. Maximum possible utility values are 1 for perfect health and 0 for death. Preliminary results based on the first 32 respondents showed an average utility value of 0.73 for the anchor state, 0.57 for the subcutaneously infused iron chelator and 0.82 for the once a day oral iron chelator. The difference of 0.25 in the utility value for the two treatments was statistically significant (Wilcoxon-signed rank test, p=0.002). Treatment with the subcutaneously infused agent was associated with a lower QoL value, being 0.16 below that for the anchor state (p=0.025). In contrast, treatment with a once daily oral iron chelator had a utility value that was 0.08 (p=0.002) higher than the anchor state. While data collection is ongoing (completion date late Sept), these preliminary results indicate that society associates oral administration of an iron chelator such as DFX with an improvement in the QoL of patients requiring such treatment. Assuming equal safety and efficacy, a once daily oral treatment may therefore be preferred to a burdensome subcutaneous infusion due to the benefits it confers on patients’ QoL.