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Introduction: High dose chemotherapy followed by autologous stem cell transplantation (ASCT) cures a subset of patients with chemosensitive relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL). Several factors associated with post-ASCT outcome have been identified, including pre-ASCT PET status, but better biomarkers are needed in order to optimally select candidates for the procedure. In other lymphoma subtypes with defining chromosomal translocations, PCR detection of pre- and post-ASCT minimal residual disease (MRD) in peripheral blood and of tumor contamination in the stem cell product is associated with inferior outcome. Until recently, MRD detection in DLBCL was limited by the rarity of detectable circulating disease using conventional techniques. The immunosequencing platform (Adaptive Biotechnologies, Corp.) is a next-generation-sequencing (NGS)-based MRD assay that detects small amounts of circulating tumor DNA (CTD) in patients with lymphoid malignancies. The assay detects CTD at diagnosis in most DLBCL patients and CTD levels track with response to induction therapy (Armand, 2013). Persistence of CTD or recurrence of CTD after completion of therapy is highly associated with DLBCL relapse (Kurtz, 2015; Roschewski, 2015). We evaluated whether CTD in autologous stem cell grafts was predictive of outcome in patients with rel/ref DLBCL undergoing ASCT. Methods: We retrospectively studied patients with rel/ref DLBCL, including transformed indolent lymphoma (TIL), who had paired archival tumor and autologous stem cell specimens and underwent ASCT at Brigham and Women's Hospital/Dana-Farber Cancer Institute from 2003-2013. Genomic tumor DNA was extracted from archival formalin-fixed paraffin-embedded (FFPE) tissue and analyzed using the NGS-based MRD assay. PCR amplification of IGH-VDJ, IGH-DJ,and IGK regions using universal consensus primers was performed followed by NGS to determine the tumor clonotype(s), defined as having a frequency > 5% in the tumor specimen. DNA from all available autologous peripheral blood or bone marrow stem cell specimens from each patient was amplified using universal consensus primers and sequenced to determine the level of CTD, defined as the number of lymphoma molecules per diploid genome. Results: We identified 98 eligible patients with rel/ref DLBCL/TIL. The median age was 60 (range 22-77) years; 63% were male; 65% had DLBCL, 29% had TIL, and 5% had primary mediastinal DLBCL; the median number of prior lines of therapy was 2 (range 2-5); all had received prior rituximab; 38% had primary refractory disease; 60% were in complete remission at ASCT; 96% received CBV conditioning. Median follow-up was 56 (range 19-123) months. The 4y progression-free survival (PFS) and overall survival (OS) in the entire cohort were 46% and 64%, respectively. Among 83 patients (85%) with sufficient DNA for clonotype determination, a clonotype was identified in 59 (71%). CTD data was complete in 53 patients (52 received peripheral blood stem cells (PBSC) and 1 received bone marrow). Eight patients (15%) had detectable CTD (CTD+) in the stem cell autografts (all PBSC) and 6/8 relapsed after ASCT. One CTD+ patient had early non-relapse mortality less than 1 month after ASCT and was never restaged. Seven of 8 CTD+ patients had TIL histology, 5 of whom relapsed (4 with aggressive lymphoma). The 4y PFS and OS in CTD+ v CTD- patients were 13% v 48% (p=0.01), and 38% v 67% (p=0.013), respectively [Figure 1]. In multivariable models including CTD status and pre-ASCT characteristics, CTD+ was the only factor associated with OS (HR 3.1, p=0.018), but was not significantly associated with PFS. Discussion: In patients with rel/ref DLBCL undergoing ASCT, the presence of CTD in the autologous stem cell graft is associated with inferior survival. CTD detection in the autograft may be more common in patients with TIL. In studies evaluating CTD detection in DLBCL, the plasma compartment has been more sensitive for detecting CTD than mononuclear cells. The use of concentrated cell specimens in this study may have decreased the sensitivity of the assay. Nevertheless, if the present findings are confirmed in a larger population, CTD detection may permit the identification of a subgroup of patients with a particularly poor outcome after ASCT, for whom alternative approaches could be considered. Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 2. Figure 2. Disclosures Herrera: Sequenta, Inc.: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding. Kong:Adaptive Biotechnologies, Corp.: Employment, Other: Stockholder. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Armand:BMS: Research Funding; Infinity: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Sequenta, Inc.: Research Funding.

Background: Double-hit lymphomas (DHL) are a subset of diffuse large B-cell lymphoma (DLBCL) with concurrent chromosomal rearrangements involving the MYC and BCL2 or BCL6 genes, and are associated with dismal outcomes with standard upfront therapy. Double expressing lymphomas (DEL) are a subset of DLBCL with co-expression of MYC and BCL2 by immunohistochemistry (IHC), and also have a poor prognosis with standard therapy. While DHL status may be associated with inferior outcomes in patients with relapsed or refractory (rel/ref) disease (Cuccuini, Blood, 2012), little is known about the outcome of DHL patients with rel/ref disease who proceed to autologous stem cell transplantation (ASCT), and no study to date has examined the outcome of patients with DEL following ASCT. We evaluated the prognostic impact of DHL and DEL status in patients with rel/ref DLBCL who underwent ASCT at 2 centers. Methods: We retrospectively studied patients with rel/ref DLBCL, including transformed indolent lymphoma (TIL), who underwent ASCT at Brigham and Women's Hospital/Dana-Farber Cancer Institute (DFCI) and City of Hope (COH) between 1/2000 and 7/2013. The most recent biopsy prior to ASCT was used for testing when possible. IHC for MYC and BCL2 were performed using the Ventana (MYC: DFCI) and Leica BOND III (MYC: COH; BCL2: DFCI, COH) platforms according to standard protocols. For DEL, IHC cutoffs of ≥ 40% MYC-positive and ≥ 50% BCL2-positive cells were used (Johnson, JCO, 2012). Fluorescence in situ hybridization (FISH) for MYC was performed using LSI MYC dual-color break-apart probes (Abbott Molecular, Des Plaines, IL). MYC -rearranged cases had FISH for BCL2 and BCL6 performed using LSI BCL2 and BCL6 dual-color break-apart probes. DHL was defined as > 20% nuclei with break-apart signals for MYC and BCL-2 and/or BCL-6. Results: 201 patients with available archival tissue and clinical data were included. The median age was 60 (range 30-77) years; 60% were male; 26% had TIL; the median number of prior lines of therapy was 2 (range 2-5); 99% had prior rituximab; 53% had primary refractory disease or early (< 6 mo) relapse; 60% were in CR by PET at ASCT; and conditioning regimens were: 66% CBV v 16% BEAM v 10% rituximab and/or ibritumomab tiuxetan-BEAM v 8% other. Overall, the 4y progression-free survival (PFS) and overall survival (OS) were 44% and 61%, respectively. Among 185 patients with complete IHC data, 38% were DEL. The 4y PFS and OS in patients with DEL compared to non-DEL patients were 37% v 52% (p =0.001), and 51% v 69% (p =0.005), respectively [Figure 1]. Results were similar using other reported IHC cutoffs for DEL (e.g. MYC ≥ 40%/BCL2 ≥ 70%, Green, JCO, 2012). Among 93 patients with complete FISH and IHC data available, 13% had MYC rearrangement: 4% were MYC/BCL2 DHL, 3% were MYC/BCL6 DHL, and 2% had rearrangements of all 3 loci. The 4y PFS and OS in DHL v non-DHL were 30% v 42% (p =0.042), and 40% v 57% (p =0.026), respectively. Patients with DEL (excluding DHL) and patients with DHL had similar PFS, which was inferior to non-DEL/non-DHL patients (4y PFS 35% v 30% v 45%, respectively, p =0.026) [Figure 2]. In multivariable models testing pre-ASCT variables, including PET response to salvage, DEL (HR 2.1, p=0.0002), TIL histology (HR 1.8, p=0.009), and SD/PD at ASCT (HR 2.9, p=0.025) were associated with poorer PFS, while DEL (HR 2.0, p=0.004) and SD/PD (HR 3.1, p=0.021) were associated with poorer OS. Neither MYC (≥ 40%) nor BCL2 (≥ 50%) expression alone was independently associated with PFS or OS. When analysis was restricted to the subset of patients with complete IHC and FISH data, DEL (HR 1.9, p=0.023), DHL (HR 2.4, p =0.048), and SD/PD at ASCT (HR 7.6, p =0.009) were associated with inferior PFS. No center effect was observed. Conclusions: DEL and DHL status are both associated with inferior PFS in patients with rel/ref DLBCL who undergo ASCT, regardless of remission status. Although ASCT remains a potentially curative approach, these patients should be targeted for study of pre- or post-ASCT relapse risk reduction strategies. Figure 1. (A) Overall survival and (B) Progression-Free Survival after ASCT in DEL vs non-DEL Patients Figure 1. (A) Overall survival and (B) Progression-Free Survival after ASCT in DEL vs non-DEL Patients Figure 2. Progression-Free Survival after ASCT in Patients with DEL vs DHL vs non-DEL/DHL Figure 2. Progression-Free Survival after ASCT in Patients with DEL vs DHL vs non-DEL/DHL Disclosures Herrera: Genentech: Research Funding; Pharmacyclics: Research Funding; Sequenta, Inc.: Research Funding. Budde:Atara Biotherapeutics: Consultancy; Seattle Genetics, Inc.: Research Funding; Merck: Research Funding; Ikara Inc: Patents & Royalties. Chen:Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau. Davids:Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Other: ad board. Nademanee:Seattle Genetics Inc.: Research Funding; Celgene: Consultancy; Gilead: Consultancy; Spectrum: Research Funding. Siddiqi:Pharmacyclics: Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Forman:Mustang: Research Funding; Amgen: Consultancy. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding. Krishnan:Onyx: Speakers Bureau; Janssen: Consultancy; Millenium: Speakers Bureau; BMS: Consultancy; Jazz: Consultancy; Celgene: Consultancy, Speakers Bureau. Armand:Merck: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; BMS: Research Funding; Sequenta, Inc.: Research Funding.

Polymerase chain reaction (PCR) amplification of the t(14;18) has been shown to be a highly sensitive method to detect minimal residual disease in patients with non-Hodgkin's lymphoma (NHL) whose tumors bear this translocation. The ideal tissue source to detect residual lymphoma would be from a previously involved lymph node. However, lymphoid tissue is rarely available once patients achieve complete remission. Although PCR amplification has been used to detect residual lymphoma cells in both bone marrow (BM) and peripheral blood (PB) of patients in complete remission, it is presently unknown whether BM and PB are equivalent tissue sources to detect residual disease. In the present study, we compared the clinical utility of the detection of residual lymphoma in both the BM and the PB of patients with advanced-stage non- Hodgkin's lymphoma before, at the time of, and after high-dose therapy and autologous BM transplantation (ABMT). The detection of residual lymphoma in either the BM or PB was associated with decreased disease- free survival. However, in the present study, 44% of patients who relapsed had no evidence of circulating lymphoma cells in their PB. At the time of BM harvest, PCR-detectable residual lymphoma cells were detected in 211 of 212 patients; although, in a subset of these patients analyzed, lymphoma cells were detected in the peripheral blood of only 49% of patients. When residual lymphoma cells within the autologous BM are infused into the patient these cells are rapidly detectable circulating in the PB in the patient. These cells continue to circulate during the immediate posttransplant period and be detectable in the PB in the majority of patients who are infused with marrow containing residual lymphoma. We conclude that BM is a more informative tissue source than PB in detecting minimal residual disease at the time of and after ABMT, and that contamination of PB early after ABMT appears to be the consequence of reinfusion of lymphoma cells within autologous marrow.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) clearly hastens myeloid recovery in patients with relapsed hematologic malignancies undergoing autologous bone marrow transplantation (ABMT). In efforts to further improve neutrophil engraftment and shorten hospital stay in ABMT patients, rhGM-CSF was administered by a potentially more potent route (continuous infusion) to non-Hodgkin's lymphoma (NHL) patients with better BM reserve (first remission). Time to myeloid engraftment was compared with that of NHL patients treated in first remission at our institution on a similar ABMT protocol but without growth factor support (controls). Median neutrophil engraftment (absolute neutrophil count, 500 cells/microL) in first remission patients treated with rhGM-CSF was 14 days, compared with 22 days in controls (P = .0001). Hospital stays were also significantly reduced for rhGM-CSF patients (P = .0003). Platelet engraftment did not differ between the two groups. Persistent fever and generalized serositis were the primary toxicities. rhGM-CSF, delivered by this route, was efficacious but more toxic than 2-hour rhGM-CSF infusions previously reported by other investigators. Future alterations in both dose and schedule may retain comparable efficacy yet diminish toxicity.

The B-lymphocyte/accessory-cell activation antigen B7 (BB1) has been shown in vitro to stimulate T-lymphocyte proliferation and cytokine production via CD28 present on the latter cells. In this study, benign lymphoid tissues, lymphomas, and extralymphoid inflammatory sites were examined immunohistochemically using anti-B7 and other relevant monoclonal antibodies. B7 was expressed by benign transformed germinal center B cells, as it was by B cells of follicular lymphomas. B7 was also expressed by a subpopulation (a mean of 31% to 65%) of macrophages and dendritic cells in a variety of lymphoid tissues. It was present in abundance on all macrophages constituting sarcoid granulomas in lymph nodes. In extralymphoid inflammation, 17% to 35% of macrophages expressed B7 only weakly. Cases of Hodgkin's disease showed expression of B7 by the majority of Reed-Sternberg cells or malignant mononuclear variants, a phenomenon that potentially contributes to the lymphocytic accumulation that is a feature of this condition. CD28+ T cells were seen in all areas where T cells were present. B7+ and CD28+ cells colocalized in, for example, lymphoid follicles, lymph node paracortex, sarcoid granulomas, and Hodgkin's disease tissue, indicating a potential for cellular interaction via these molecules at these sites.

Data from several recent studies have shown that the overall survival for patients with low grade follicular lymphoma has improved over the last two decades [1–3 ]. Although some studies have attri...

Background The prognosis for patients with most forms of T-cell lymphoma is poor. Allogeneic hematopoietic stem-cell transplantation (HSCT) may improve the outcome. Patients and methods This study examines the outcome of 52 patients who underwent ablative or nonablative allogeneic HSCT for peripheral T-cell lymphoma (PTCL) or advanced mycosis fungoides/Sezary syndrome over a 12-year period at a single institution. We divided the patients into those with predominantly nodal histologies: peripheral T-cell not otherwise specified (PTCL NOS), angioimmunoblastic (AITL), or anaplastic large cell lymphoma, T/null type (systemic) (ALCL), and predominantly extranodal histologies: natural killer (NK)/T cell, enteropathy type, hepatosplenic, subcutaneous panniculitic, mycosis fungoides, or T cell or NK cell other. Results Median follow-up of survivors is 49 months. Non-relapse mortality and relapse at 3 years was 27% and 43%, respectively. The incidence of grade II–IV acute graft-versus-host disease (GVHD) was 21%. The incidence of extensive chronic GVHD at 2 years was 27%. The 3-year progression-free survival was 30%: 45% in patients with predominantly nodal histologies (PTCL NOS, AITL, and ALCL) and 6% in patients with predominantly extranodal histologies (P = 0.016). Overall survival at 3 years was 41% for all patients. Conclusion Allogeneic HSCT can produce long-term remissions in relapsed/refractory T-cell lymphoma, especially those with nodal histologies.

Forty patients with plasma cell dyscrasias underwent high-dose chemoradiotherapy and either anti-B-cell monoclonal antibody (MoAb)- treated autologous, anti-T-cell MoAb-treated HLA-matched sibling allogeneic or syngeneic bone marrow transplantation (BMT). The majority of patients had advanced Durie-Salmon stage myeloma at diagnosis, all were pretreated with chemotherapy, and 17 had received prior radiotherapy. At the time of BMT, all patients demonstrated good performance status with Karnofsky score of 80% or greater and had less than 10% marrow tumor cells; 34 patients had residual monoclonal marrow plasma cells and 38 patients had paraprotein. Following high-dose chemoradiotherapy, there were 18 complete responses (CR), 18 partial responses, one non-responder, and three toxic deaths. Granulocytes greater than 500/microL and untransfused platelets greater than 20,000/microL were noted at a median of 23 (range, 12 to 46) and 25 (range, 10 to 175) days posttransplant (PT), respectively, in 24 of the 26 patients who underwent autografting. In the 14 patients who received allogeneic or syngeneic grafts, granulocytes greater than 500/microL and untransfused platelets greater than 20,000/microL were noted at a median of 19 (range, 12 to 24) and 16 (range, 5 to 32) days PT, respectively. With 24 months median follow-up for survival after autologous BMT, 16 of 26 patients are alive free from progression at 2+ to 55+ months PT; of these, 5 patients remain in CR at 6+ to 55+ months PT. With 24 months median follow-up for survival after allogeneic and syngeneic BMT, 8 of 14 patients are alive free from progression at 8+ to 34+ months PT; of these, 5 patients remain in CR at 8+ to 34+ months PT. This therapy has achieved high response rates and prolonged progression-free survival in some patients and proven to have acceptable toxicity. However, relapses post-BMT, coupled with slow engraftment post-BMT in heavily pretreated patients, suggest that such treatment strategies should be used earlier in the disease course. To define the role of BMT in the treatment of myeloma, its efficacy should be compared with that of conventional chemotherapy in a randomized trial.

Twenty patients with poor prognosis B-cell chronic lymphocytic leukemia (B-CLL) underwent uniform high-dose chemoradiotherapy followed by rescue with multiple monoclonal antibody-purged autologous bone marrow (BM) (12 patients) or T-cell-depleted allogeneic BM from HLA-identical siblings (8 patients) in a pilot study to assess the feasibility of BM transplantation (BMT) in this disease. All had poor prognosis disease by either staging, BM pattern, tumor doubling time criteria, or cytogenetics. All patients achieved remission criteria (defined as < or = 2 adenopathy, absence of splenomegaly, < or = 20% of the intertrabecular space involved on BM biopsy) before BMT. Despite the use of fludarabine, a median of three treatment regimens were required to achieve BMT eligibility. After BMT, all patients achieved complete hematologic engraftment. Toxicities were not significantly different between autologous versus allogeneic BMT. Two toxic deaths were observed. Of 19 evaluable patients, 17 clinical complete clinical remissions (89%) were observed, with 2 patients (1 allogeneic and 1 autologous) exhibiting persistent BM disease. Complete clinical remissions were documented at the phenotypic and molecular level for the majority of patients in whom dual fluorescence for CD5 and CD20 (15 of 15; 100%) and Ig gene rearrangements (11 of 14; 79%) were performed. Although long-term follow-up is needed to assess any potential impact on the disease-free and overall survival of these patients, this study shows the feasibility of using high-dose chemoradiotherapy and BMT in patients with poor prognosis B-CLL.

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl- 2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR- detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR- detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.

Autologous bone marrow transplantation is a curative treatment for some hematologic malignancies and potentially some solid tumors. Sensitive laboratory studies have demonstrated that histologically uninvolved bone marrow is often infiltrated with tumor cells, providing at least a theoretical reason to postulate that reinfused tumor cells may contribute to disease recurrence. Several recent studies support the hypothesis that effective marrow purging may improve the disease-free survival in patients with leukemia and lymphoma.

PURPOSE Using high-dose therapy and autologous bone marrow transplantation (ABMT) to overcome cellular resistance and eradicate minimal disease, we initiated a pilot study during first remission in patients with non-Hodgkin's lymphoma (NHL) to examine whether the long-term disease-free survival (DFS) rate can be improved for patients with poor-prognosis intermediate/high-grade NHL. PATIENTS AND METHODS Twenty-six patients with advanced-stage diffuse intermediate/high-grade B-cell NHL (including 16 patients with diffuse small cleaved-cell [DSC]) were selected at presentation by histologic and clinical characteristics to have less than a 25% probability of long-term DFS with conventional treatment. After induction chemotherapy, 16 patients were in complete remission (CR) and 10 were in a minimal disease state. Patients were then treated with high-dose cyclophosphamide, total-body irradiation (TBI), and anti-B-cell monoclonal antibody-purged ABMT. RESULTS Following ABMT, no acute in-hospital treatment deaths occurred, and engraftment of granulocytes and platelets was significantly faster than for patients undergoing ABMT who were in second or subsequent remission. Of 26 patients, 21 remain in CR maintained without continued therapy, three relapsed in sites of prior nodal disease (4.8, 5.4, and 28 months post-ABMT), and two died in remission. The DFS rate is estimated to be 85% at 28 months and thereafter. The median follow-up period for the 21 patients who are alive and disease-free is 32 months. CONCLUSION This pilot study suggests that consolidation of first remission with ABMT may improve the long-term DFS rate for diffuse intermediate/high-grade NHL patients at high risk for relapse.

Anti-B-blocked ricin (anti-B4-bR) combines the specificity of the anti- B4 (CD19) monoclonal antibody with the protein toxin “blocked ricin.” In blocked ricin, affinity ligands are attached to the ricin B-chain to attenuate its lectin binding capacity. In a phase I trial, Anti-B4-bR was administered by 7-day continuous infusion to 12 patients in complete remission after autologous bone marrow transplantation (ABMT) for relapsed B-cell non-Hodgkin's lymphoma (NHL). Patients were treated at 20, 40, and 50 micrograms/kg/d for 7 days. Potentially therapeutic serum levels could be sustained for 3 to 4 days. The maximum tolerated dose was 40 micrograms/kg/d for 7 days (total 280 micrograms/kg). The dose-limiting toxicities were reversible grade IV thrombocytopenia and elevation of hepatic transaminases. Mild capillary leak syndrome was manifested by hypoalbuminemia, peripheral edema (4 patients), and dyspnea (1 patient). Anti-immunotoxin antibodies developed in 7 patients. Eleven patients remain in complete remission between 13 and 26 months post-ABMT (median 17 months). These results show that Anti-B4- bR can be administered with tolerable, reversible toxicities to patients with B-cell NHL in complete remission following ABMT.

One hundred and twenty-eight patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and acute lymphoblastic leukemia (ALL) previously reported from a phase III trial of rhGM-CSF or placebo following autologous bone marrow transplantation (ABMT) were investigated for the development of late toxicities. Median follow-up is 36 months. No apparent long-term deleterious effects on BM function were observed. Moreover, disease-free survival and overall survival were similar for patients on both treatment arms, arguing for the long- term safety of recombinant human granulocyte macrophage-colony- stimulating factor (rhGM-CSF). The only factors predictive for both a high risk of relapse over time and mortality were having the diagnosis of ALL and/or undergoing ABMT in resistant relapse. We attempted to identify clinical variables before BM harvest, at the time of marrow infusion, or events within the first 100 days posttransplant, which might predict speed of neutrophil recovery in the setting of placebo or rhGM-CSF administration after ABMT. Only previous exposure to agents that deplete stem cells led to a significant delay in neutrophil recovery, suggesting their avoidance in patients who may undergo ABMT. Nevertheless, even those patients benefited from rhGM-CSF. For all patients, rhGM-CSF and agents that deplete stem cells were the strongest independent predictors for neutrophil engraftment. With the increasing use of newer hematopoietic growth factors both alone and in combination, long-term follow-up is essential to confirm the same safety that we report with rhGM-CSF.

Lenalidomide is an immunomodulatory drug related to thalidomide that has recently been reported to have significant single agent activity in relapsed CLL, with response rates of 35-50% including some complete responses1,2. The mechanism of action is unknown but appears to be immune-mediated given that lenalidomide alters cytokine levels and stimulates T and NK cell function, and lacks cytotoxicity against CLL in vitro3. In CLL patients, the administration of lenalidomide can be associated with tumor flare, a syndrome of painful enlarging lymphadenopathy, increased white count, fever, and rash1,2. This tumor flare can potentially escalate to become life-threatening, with renal insufficiency, tumor lysis or a systemic inflammatory response4,5. The mechanism of tumor flare remains unknown.

Approximately 10% of patients with chronic lymphocytic leukaemia (CLL) have a family history of the disease or a related lymphoproliferative disorder, yet the relationship of familial CLL to genomic abnormalities has not been characterized in detail. We therefore studied 75 CLL patients, half familial and half sporadic, using high-resolution array comparative genomic hybridization (CGH), in order to better define the relationship of genomic abnormalities to familial disease and other biological prognostic factors. Our results showed that the most common high-risk deletion in CLL, deletion 11q, was significantly associated with sporadic disease. Comparison of familial to sporadic disease additionally identified a copy number variant region near the centromere on 14q, proximal to IGH@, in which gains were associated both with familial CLL, and with mutated IGHV and homozygous deletion of 13q. Homozygous deletion of 13q was also found to be associated with mutated IGHV and low expression of ZAP-70, and a significantly longer time to first treatment compared to heterozygous deletion or lack of alteration. This study is the first high resolution effort to investigate and report somatic genetic differences between familial and sporadic CLL.

In germinal centers, B lymphocytes are intimately associated with follicular dendritic cells (FDCs). It has been hypothesized that FDCs are involved in the regulation of B-cell growth and differentiation through cell-cell interactions. In this study, highly enriched preparations of FDCs were isolated by cell sorting using the FDC restricted monoclonal antibody DRC-1. When irradiated FDCs were cultured with mitogen stimulated B cells, B cell 3H-TdR uptake was inhibited by up to 80%. This inhibitory effect was not seen when paraformaldehyde fixed FDCs were added to B-cell cultures, suggesting that the FDCs needed to be metabolically active. Moreover, supernatants from cultured FDCs were similarly able to inhibit B-cell proliferation. These results demonstrate that FDCs may downregulate the clonal expansion of B cells that occurs within lymphoid follicles as part of the normal physiologic immune response. Potentially, the loss of the inhibitory role of FDCs in vivo may be of importance in certain infectious and neoplastic processes in which germinal centers are affected.

Using the extremely sensitive technique of polymerase chain reaction (PCR) to detect the bcl-2 translocation, only 50% of bone marrows could be purged of PCR-detectable lymphoma cells using a cocktail of three anti-B-cell monoclonal antibodies (MoAbs) and complement-mediated lysis. This observation is of clinical importance because those patients whose reinfused marrows harbored residual lymphoma cells showed a significantly increased incidence of relapse. To improve purging, we used PCR detection of the bcl-2 translocation to compare the efficiency of complement-mediated lysis with immunomagnetic bead depletion. Using either a three or a four MoAb cocktail followed by immunomagnetic bead depletion, all PCR-detectable cells were purged after three cycles of treatment. In these same patient samples, treatment with three MoAbs and complement purged only 11 of the 25 (44%) samples. The addition of a fourth MoAb followed by complement lysis purged the marrows of only an additional five patients. Immunomagnetic bead depletion was specific because there was no loss of committed myeloid progenitor cells. The above results suggest that immunomagnetic bead depletion of the harvested marrow will likely be superior to our previous method of purging and the lack of nonspecific toxicity to myeloid progenitor cells predicts that it will not impair engraftment. This methodology will now be used to determine whether the reinfusion of lymphoma free marrow affects the incidence of relapse after autologous bone marrow transplantation.

PURPOSE Acute and chronic graft-versus-host disease (GVHD) continues to be the major causes of morbidity and mortality after allogeneic bone marrow transplantation (BMT). In this study, we have evaluated the clinical effects of selective in vitro T-cell depletion of donor allogeneic bone marrow by using a single monoclonal antibody ([MoAb] anti-T12, CD6) and rabbit complement. This antibody recognizes mature T cells, but not other cellular elements such as natural-killer (NK) cells, B cells, and myeloid precursors. PATIENTS AND METHODS From August 1983 to April 1991, 112 consecutive adult patients with hematologic malignancies underwent BMT with bone marrow from HLA-identical sibling donors. Marrow was harvested and depleted of mature T lymphocytes ex vivo by the use of three rounds of incubation with an anti-T12 antibody and rabbit complement. The preparative regimen consisted of cyclophosphamide and fractionated total body irradiation (TBI) in 108 patients. No patients received prophylactic immune suppression post-BMT. Purgation by anti-T12 was used as the only method for the prevention of GVHD. RESULTS Twenty patients (18%) developed acute GVHD (grade 2 to 4); only eight patients developed chronic GVHD. The incidence of GVHD did not increase significantly with age. Only three of 112 patients (2.7%) exhibited acute graft failure. One patient developed late graft failure that was associated with cytomegalovirus (CMV) infection. Within the subset of 50 patients who had not previously undergone unsuccessful conventional therapy (acute leukemia in first remission or chronic myelogenous leukemia [CML] in stable phase), we estimated by the Kaplan-Meier method that the probability of disease-free survival was 50% at 3 years post-BMT, with a median follow-up of 44 months. The treatment-related mortality rate in this group was only 14% and was independent of patient age. CONCLUSIONS We conclude that selective in vitro T-cell depletion with an anti-T12 monoclonal antibody effectively reduces the incidence of both acute and chronic GVHD after allogeneic BMT without compromising engraftment. Moreover, depletion of CD6-positive cells from donor marrow obviates the need to administer immune suppressive medications to the majority of patients. This approach reduces the morbidity and mortality of allogeneic BMT and permits the BMT of older patients.

The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte- macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA- compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin “blocked ricin.” The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.

B7 is an activation antigen expressed on activated B cells and gamma- interferon-stimulated monocytes. The B7 antigen is the natural ligand for CD28 on T cells. After engagement of T-cell receptor with antigen in association with major histocompatibility complex class II, a second signal mediated through the binding of B7 to CD28 greatly upregulates the production of multiple lymphokines. We have now mapped the B7 gene to human chromosome 3 using the technique of polymerase chain reaction on a panel of hamster x human somatic cell hybrid DNAs. We have further localized the gene to 3q13.3–3q21 using in situ hybridization on human metaphase chromosomes. Trisomy of chromosome 3 is a recurrent chromosome change seen in various lymphomas and lymphoproliferative diseases, particularly diffuse, mixed, small, and large cell lymphomas, human T-cell lymphotropic virus type I-induced adult T-cell leukemia, and angioimmunoblastic lymphadenopathy. A number of chromosomal defects involving 3q21 have been described in acute myeloid leukemia and also in myelodysplastic and myeloproliferative syndromes. The mapping of B7 may permit further insight into disease states associated with aberrant lymphocyte activation and lymphokine synthesis.

Follicular lymphomas recapitulate the architecture of germinal centers (GCs) of normal secondary lymphoid follicles. Using an in vitro binding assay, it has recently been demonstrated that the normal B lymphocytes bind to GCs. This interaction is mediated by a receptor-ligand pair consisting of the beta 1 integrin very late antigen 4 (VLA-4) on the B cell, and the vascular cell adhesion molecule-1 (VCAM-1) expressed on follicular dendritic cells (FDC). Considering the similarities between follicular lymphomas and normal GCs, the adhesive interaction of follicular non-Hodgkin's lymphoma (NHL) cells and GCs was examined. Cells isolated from 16 of 24 cases of follicular NHL bound to normal GCs. Neoplastic follicles could similarly support the binding of follicular NHL cells. This adhesion was inhibited by monoclonal antibodies (MoAbs) directed against VLA-4 and VCAM-1. This supports the hypothesis that the neoplastic follicles used the identical adhesive interactions responsible, at least in part, for the localization of normal B cells to GCs. Adhesion receptors have an important role in the regulation of normal lymphoid cell proliferation, differentiation, and localization. Therefore, an understanding of the adhesive interaction of follicular NHL cells with GCs may provide insight into the clinical and biologic behavior of these diseases.

CrkL, a cellular homologue of the v-crk oncogene, belongs to the family of adaptor proteins, containing SH2 and SH3 domains, but no catalytic domain. Stimulation of normal B-cells and B-cell lines through beta1 integrin or - cell antigen receptor (BCR) promoted the association of CrkL with a set of 105-130 kD tyrosine phosphorylated substrates. The principal substrate is a recently identified molecule known as p105HEF1 (HEF1), which is highly homologous to p130Cas (Cas), the major tyrosine-phosphorylated protein detected in fibroblasts after transformation by v-crk. Immunodepletion studies indicated that all the tyrosine phosphorylated HEF1 or Cas was complexed with CrkL. Furthermore, the guanine nucleotide exchange factor C3G, which is thought to be involved in the regulation of the ras pathway and constitutively binds to the C-terminal SH3 domain of CrkL, could be detected in HEF1 immunoprecipitates. Therefore, CrkL is involved in the formation of a HEF1-CrkL-C3G ternary complex in B-cells, suggesting that it is likely to play an important role, allowing the propagation of the stimulation initiated by both BCR and beta1 integrin ligation.

Toll-like receptor-9 (TLR-9) agonists have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and T helper cell type 1 shift in the immune response. We combined a TLR-9 agonist (1018 ISS, 0.2 mg/kg sc weekly x 4 beginning day 8) with standard rituximab (375 mg/m(2) weekly x 4) in patients (n = 23) with relapsed/refractory, histologically confirmed follicular lymphoma, and evaluated immunological changes following the combination. Treatment was well-tolerated with no significant adverse events attributable to therapy. Clinical responses were observed in 48% of patients; the overall median progression-free survival was 9 months. Biologically relevant increases in ADCC and circulating CD-3 positive T cells were observed in 35% and 39% of patients, respectively. Forty-five percent of patients had increased T cells and dendritic cells in skin biopsies of 1018 ISS injection sites 24 h post-therapy. Pre- and post-biopsies of tumour tissue demonstrated an infiltration of CD8(+) T cells and macrophages following treatment. This group of patients had favourable clinical outcome despite adverse prognostic factors. This study is the first to histologically confirm perturbation of the local immune microenvironment following systemic biological therapy of follicular lymphoma.

The marginal zone lymphomas (MZLs) are a recently defined group of related diseases that probably arise from a common cell of origin, the marginal zone B cell. Data on therapy for subtypes other than gastric mucosa-associated lymphoid tissue (MALT) lymphoma has been largely limited to retrospective case series. This prospective phase 2 study of fludarabine and rituximab for the treatment of marginal zone lymphomas enrolled 26 patients, 14 with nodal MZL, eight with MALT lymphomas and four with splenic MZL; 81% were receiving initial systemic therapy. Only 58% [95% confidence interval (CI) 37-77%] of patients completed the planned six cycles, due to significant haematological, infectious and allergic toxicity. Four late toxic deaths occurred due to infections [15% (95% CI 4.3-35%)], two related to delayed bone marrow aplasia and two related to myelodysplastic syndrome. Nonetheless, the overall response rate was 85% (95% CI 65-96%), with 54% complete responses. The progression-free survival at 3.1 years of follow-up is 79.5% (95% CI 63-96%). We conclude that, although concurrent fludarabine and rituximab given at this dose and schedule is a highly effective regimen in the treatment of MZLs, the significant haematological and infectious toxicity observed both during and after therapy is prohibitive in this patient population, emphasizing the need to study MZLs as a separate entity.

Polymerase chain reaction (PCR) of bcl-2 provides an extremely sensitive method to detect minimal disease in approximately 50% of patients with non-Hodgkin's lymphomas (NHL). In an attempt to determine the clinical usefulness of this technique, we examined the bone marrow (BM) of 152 patients with advanced-stage NHL at the time of evaluation and after induction or salvage chemotherapy before autologous BM transplantation. The BM proved to be an accessible and reproducible tissue source to determine PCR positivity because all of the 102 patients examined had the same PCR-amplifiable breakpoint in their BM and lymph node. At the time of evaluation, PCR analysis in advanced- stage NHL patients added little additional information to morphologic analysis because each technique identified BM infiltration in approximately 70% of patients. PCR was significantly more useful in determining BM infiltration after induction or salvage therapy. At that time, approximately 50% of patients had morphologically normal BM, whereas PCR analysis remained positive in 100% of those with an amplifiable breakpoint. These observations were confirmed in a clinical trial attempting to induce remission in previously untreated low-grade advanced-stage NHL patients. In this series, PCR was positive in all patients after treatment although the BM was histologically uninvolved in 50% of cases, showing that conventional therapy did not eradicate bcl-2-positive cells.

We reviewed the medical records of 97 patients undergoing T cell-depleted allogeneic bone marrow transplantation at our institution from 1984 to 1990 to determine the incidence of hepatic dysfunction, including venoocclusive disease of the liver following BMT. All patients received allogeneic marrow that had been purged with monoclonal antibody to the CD6 surface antigen (T12) and rabbit complement as the sole method of graft-versus-host disease prophylaxis. No additional immunosuppressive agents were routinely administered to these patients. Overall, 55% of patients in our series developed two-fold elevations in serum bilirubin, SGOT, or alkaline phosphatase within the first 30 days following BMT. A five-fold elevation in any liver function test was noted in only 19% of patients. Logistic regression analysis revealed that the presence of GVHD, female sex, and administration of amphotericin B all were independently associated with laboratory evidence of hepatic dysfunction. While LFT abnormalities were common in our series, they were generally mild, and the development of VOD was rare. Only three patients (3.1%) fulfilled clinical criteria sufficient to establish a diagnosis of VOD. Among the 86 patients whose ablative regimen consisted of cyclophosphamide (60 mg/kg x2) and total-body irradiation (1200-1400 cGy in 200 cGy fractions), only 1 patient (1.2%) developed VOD. Our experience suggests that patients undergoing allogeneic BMT are at low risk for VOD and other serious hepatic complications when they receive high-dose cyclophosphamide, fractionated TBI, and T cell-depleted marrow without hepatotoxic medications for GVHD prophylaxis.

The use of autologous bone marrow transplantation is increasing in the management of advanced cancers. Many investigators have attempted to "purge" autologous marrow of residual tumor cells because of concern that reinfused tumor cells might contribute to relapse. The efficacy of purging remains unproved.We performed clonogenic assays in a tumor cell line in culture to determine the efficiency of immunologic purging. Amplification by the polymerase chain reaction (PCR) was used to detect residual lymphoma cells before and after purging of bone marrow from 114 patients with B-cell non-Hodgkin's lymphoma in whom a translocation (t(14;18] that could be amplified by PCR was detected at the time of their initial evaluation.Immunologic purging in vitro resulted in a 3-to-6-log destruction of cells in the tumor cell line. Residual lymphoma cells were detected by PCR in the bone marrow of all patients before purging. No lymphoma cells could be detected in the marrow of 57 patients after purging. Disease-free survival was increased in these 57 patients as compared with those whose marrow contained detectable residual lymphoma (P less than 0.00001). The ability to purge residual lymphoma cells was not associated with the degree of bone marrow involvement (P = 0.4494) or the previous response to therapy (P = 0.1298).The inability to purge residual lymphoma cells was the most important prognostic indicator in predicting relapse. These results provide evidence of the clinical usefulness of ex vivo purging of autologous bone marrow in the treatment of patients with lymphoma and suggest that the reinfusion of malignant cells in autologous marrow contributes to relapse

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4 + T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4 + T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50–70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4 + T cells; and that there is a delay of the EC effect on CD4 + T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.

The majority of non-Hodgkin's lymphomas (NHLs) are of B-cell lineage, with less than 20% of cases being of T-cell lineage. The B-cell NHLs phenotypically correspond to normal cells in the mid stages of normal differentiation. More specifically, by their expression of B-cell activation antigens, these tumors are the neoplastic counterparts of normal activated B cells. The follicular lymphomas--including the small cleaved, mixed small and large cell, and large cell types, as well as the small noncleaved cell (Burkitt's) lymphomas--represent malignant expansions of normal germinal center B cells by their expression of pan-B cell antigens, B-cell activation antigens, and CD10 (CALLA). The diffuse lymphomas also correspond to normal activated B cells. The small lymphocytic lymphomas express the low-affinity IL-2 receptor and CD5, both of which are induced on normal B cells following mitogen stimulation. The other diffuse B-cell NHLs similarly express activation antigens and resemble "transformed" B cells. The T-cell NHLs generally correspond to normal activated CD4+ T cells. These tumors--which include most peripheral T-cell lymphomas, cutaneous T-cell lymphomas, and HTLV-I-associated adult T-cell leukemias/lymphomas--express antigens induced on activated T cells, including IL-2 and transferrin receptors (CD25 and CD71, respectively), as well as HLA-DR. The lymphoblastic lymphomas, which are generally of T-cell lineage, phenotypically correspond to stages of intrathymic differentiation, often by their coexpression of CD4 and CD8, as well as expression of CD1. It remains controversial whether the immunophenotype of lymphoblastic lymphoma differs significantly from T-cell acute lymphoblastic leukemia. Since immunologic heterogeneity of NHL was first observed, attempts have been made to employ the data as a prognostic variable. Early studies suggested that lineage derivation or expression of markers of proliferating cells affected outcome in NHL. However, these reports were often retrospective, included various histologies, and did not treat patients uniformly. More recent prospective studies with relatively uniformly treated patients, predominantly involving DLCL, suggest that certain immunologically defined subgroups may have significantly different clinical outcomes. However, additional clinical studies will be necessary before treatment options are based upon immunologic markers.

Occupancy of the T-cell receptor complex does not appear to be a sufficient stimulus to induce a T-cell-mediated immune response. Increasing evidence suggests that cognate cell-cell interaction between an activated T cell and an antigen-presenting cell may provide such a stimulus. A candidate T-cell surface molecule for this costimulatory signal is the T-cell-restricted CD28 antigen. Following crosslinking with anti-CD28 mAb, suboptimally stimulated CD28+ T cells show increased proliferation and markedly increased secretion of a subset of lymphokines. Recently, the B-cell surface activation antigen B7 was shown to be a natural ligand for the CD28 molecule, and both B7 and CD28 are members of the immunoglobulin superfamily. Here we report that B7-transfected CHO cells can induce suboptimally activated CD28+ T cells to proliferate and secrete high levels of interleukin 2. The response is identical whether T cells are submitogenically stimulated with either phorbol myristate acetate or anti-CD3 to activate the T cells. This response is specific and can be totally abrogated with anti-B7 monoclonal antibody. As has previously been observed for anti-CD28 monoclonal antibody, B7 ligation induced secretion of interleukin 2 but not interleukin 4. We have previously demonstrated that B7 expression is restricted to activated B lymphocytes and interferon gamma-activated monocytes. Since these two cellular populations are involved in antigen presentation as well as cognate interaction with T lymphocytes, B7 is likely to represent a central constimulatory signal that is capable of amplifying an immune response.

The period of neutropenia after autologous bone marrow transplantation results in substantial morbidity and mortality. The results of previous phase I-II clinical trials suggest that recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) may accelerate neutrophil recovery and thereby reduce complications in patients after autologous bone marrow transplantation.We conducted a randomized, double-blind, placebo-controlled trial at three institutions. The study design and treatment schedules were identical, and the results were pooled for analysis. One hundred twenty-eight patients were enrolled. Sixty-five patients received rhGM-CSF in a two-hour intravenous infusion daily for 21 days, starting within four hours of the marrow infusion, and 63 patients received placebo.No toxic effects specifically ascribed to rhGM-CSF were observed. The patients given rhGM-CSF had a recovery of the neutrophil count to 500 x 10(6) per liter 7 days earlier than the patients who received placebo (19 vs. 26 days, P less than 0.001), had fewer infections, required 3 fewer days of antibiotic administration (24 vs. 27 days, P = 0.009), and required 6 fewer days of initial hospitalization (median, 27 vs. 33 days; P = 0.01). There was no difference in the survival rate at day 100.In patients undergoing autologous bone marrow transplantation for lymphoid neoplasia, rhGM-CSF significantly lessens morbidity. Further studies will be required to establish its optimal dosage and schedule of administration.

Sixty-nine patients with a history of low-grade B-cell non-Hodgkin's lymphoma (NHL) in sensitive relapse or incomplete first remission underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-treated autologous bone marrow transplantation (ABMT). At ABMT, 51 patients had low-grade histology and 18 patients had a history of low-grade NHL that had undergone histologic transformation to a higher-grade NHL. Before ABMT, only 20 of the 51 low-grade patients and 10 of the 18 patients with transformed histologies were in complete remission. Moreover, at the time of marrow harvest, 24 of the low-grade and eight of the transformed histology patients had histologic evidence of lymphoma cells infiltrating the marrow. Following high-dose therapy, only one acute, in-hospital death was observed. There was no significant difference in the disease-free survival (DFS) between patients with low-grade and patients with transformed histologies. Among patients with low-grade NHL, the patients in complete remission before ABMT experienced significantly longer DFS than those in partial remission (P less than .05). This preliminary study suggests that some patients with relapsed low-grade NHL may experience prolonged DFS following high-dose ablative therapy.

One hundred and sixty eight adult patients with B-cell non-Hodgkin's lymphoma (NHL) and other hematologic malignancies who underwent autologous or allogeneic bone marrow transplantation (BMT) were investigated for the subsequent development of hemolytic-uremic syndrome (HUS). All patients were conditioned with cyclophosphamide and total body irradiation. When examined at 3-month intervals for the first year post-BMT, all patients had uniform measurements of hematocrit (Hct) and serum creatinine. Sixteen patients who initially exhibited Hct and creatinine values that were normal range for the BMT populations developed a sudden decrease in Hct and increase in creatinine between 3 and 11 months post-BMT and fulfilled the clinical and laboratory criteria for HUS. None of these patients had known active cytomegalovirus infection, graft-versus-host disease, or cyclosporine administration. The degree of decrease in Hct and creatinine elevation ranged from solely laboratory abnormalities to a clinically significant syndrome. Twelve of the 16 patients developed acute clinical complications of congestive heart failure, hypertension (HTN), or peripheral edema. Twelve patients required red blood cell support, whereas only four patients required platelet transfusions. Both hemolytic anemia and thrombocytopenia have resolved in virtually all cases. At a mean follow up of 18 months postdiagnosis, creatinine elevations have persisted along with HTN. All patients have survived without life-threatening long-term sequelae. With the increasing use of BMT as a curative modality for patients with hematologic malignancies, it becomes important to prospectively monitor patients for the development of HUS and its potential long-term impact on renal function.

Eleven patients with plasma cell dyscrasias underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-treated autologous bone marrow transplantation (ABMT). The majority of patients had advanced Durie-Salmon stage myeloma at diagnosis, all were pretreated with chemotherapy, and six had received prior radiotherapy. At the time of ABMT, all patients demonstrated good performance status with Karnofsky score of 80% or greater and had less than 10% marrow tumor cells. Eight patients had residual monoclonal marrow plasma cells and 10 patients had paraprotein. Following high-dose melphalan and total body irradiation (TBI) there were seven complete responses, three partial responses, and one toxic death. Granulocytes greater than 500/mm3 were noted at a median of 21 (range 12 to 46) days posttransplant (PT) and untransfused platelets greater than 20,000/mm3 were noted at a median of 23 (12 to 53) days PT in 10 of the 11 patients. Natural killer cells and cytotoxic/suppressor T cells predominated early PT, with return of B cells at 3 months PT and normalization of T4:T8 ratio at 1 year PT. Less than 5% polyclonal marrow plasma cells were noted in all patients after transplant. Three of the seven complete responders have had return of paraprotein, two with myeloma, and have subsequently responded to alpha 2 interferon therapy. Eight patients are alive at 18.9 (8.9 to 43.1) months PT and four remain disease-free at 12.3, 17.5, 18.9, and 29 months PT. This preliminary study confirms that high-dose melphalan and TBI can achieve high response rates without unexpected toxicity in patients who have sensitive disease, and that MoAb-based purging techniques do not inhibit engraftment. Although the follow-up is short- and long-term outcome to be determined, relapses post-ABMT in these heavily pretreated patients suggest that ABMT or alternative treatment strategies should be evaluated earlier in the disease course.

Increasing data suggest that activating kinase mutations play a significant causative role in chronic and acute hematologic malignancies. The prototypical example is the BCR-ABL kinase that is activated in CML and Philadelphia chromosome positive ALL, but activating kinase mutations have also been identified in chronic myelomonocytic leukemia, chronic eosinophilic leukemia, and systemic mastocytosis. Circumstantial evidence suggests that gain-of-function tyrosine kinase alleles could also contribute to the pathogenesis of chronic lymphocytic leukemia (CLL). Microarray studies of gene expression differences between steadily progressive CLLs that lack somatic hypermutation of the immunoglobulin heavy chain variable gene region (IgVH unmutated) and more indolent IgVH mutated CLLs have found that many of the differentially expressed genes are involved in B cell receptor signaling, most notably the ZAP-70 tyrosine kinase. Other kinases found to be overexpressed in CLL by gene expression studies include BLK, JAK2, LYN, and NTRK3, although mutational analysis of these genes in CLL has been limited. The goal of this project was therefore to search systematically for activating mutations in protein tyrosine kinases in CLL using high-throughput DNA resequencing. A total of 178 exons were sequenced from each of 95 patients. The patients were selected to encompass the range of prognostic groups defined by IgVH homology, ZAP-70 expression, and FISH cytogenetics. IgVH homology (unmutated defined as greater than or equal to 98% homology to the closest germline match) and ZAP-70 expression (positive defined as >20%) were determined by the CLL Research Consortium tissue core as previously described1, and cytogenetics were evaluated by FISH for the most common abnormalities (del 13q, trisomy 12, del 11q, del 17p, chromosome 14 rearrangements) at DFCI2. Although the CLL cases studied showed some bias toward lower risk features (IgVH mutated 56%, unmutated 28%, ND 16%; ZAP70 negative 52%, positive 24%, ND 24%; CD38 negative (

Increasing evidence points to a heritable contribution in the development of lymphoma. The goal of this study was to determine the rate of familial lymphoproliferative malignancy among consecutive lymphoma patients presenting to a tertiary care center and to enroll families with multiple affected first-degree relatives on a data and tissue collection study. Beginning in 2004 all new patients presenting to the Dana-Farber Cancer Institute with non-Hodgkin (NHL) or Hodgkin lymphoma (HL) or chronic lymphocytic leukaemia (CLL) were asked to complete a one-page self-administered family history questionnaire. 55.4% of 1948 evaluable patients reported a first-degree relative with a malignancy, with the highest rate among CLL probands. Lymphoid malignancies were particularly common, with 9.4% of all probands reporting a first-degree relative with a related lymphoproliferative disorder (LPD). This frequency was again highest for CLL, at 13.3% of CLL probands, compared to 8.8% of NHL probands and 5.9% of HL probands (P = 0.002). The prevalence of CLL was significantly increased in parents of CLL probands (P < 0.05), and a greater risk of NHL was seen in fathers of NHL probands than in mothers (P = 0.026). We conclude that familial aggregation of LPDs is common among newly diagnosed patients, varies significantly by diagnosis and contributes meaningfully to the population disease burden.

Allogeneic stem cell transplantation (SCT) with reduced-intensity conditioning (RIC) has the potential to lead to long-term remissions for patients with lymphoma. However, the role of RIC SCT in the treatment of lymphoma is still unclear. Specifically, the relative benefit of RIC SCT across lymphoma histologies and the prognostic factors in this population are incompletely defined. We retrospectively analyzed the outcomes of 87 patients with advanced lymphoma who underwent RIC SCT at the Dana-Farber Cancer Institute over a 6-year period with a homogeneous conditioning regimen consisting of fludarabine and low-dose busulfan. Thirty-six patients had Hodgkin disease (HD) and 51 had non-Hodgkin lymphoma (NHL). Sixty-eight percent had undergone prior autologous transplantation. The 1-year cumulative incidence of nonrelapse mortality was 13%, and the 3-year cumulative incidence of progression was 49%. The incidence of grade 3-4 acute GVHD was 11%. The 2-year cumulative incidence of chronic GVHD was 68%, and its development was associated with a decreased risk of progression and an improved progression-free survival (PFS). Three-year overall survival (OS) was 56% for patients with HD, 81% for indolent NHL, 42% for aggressive NHL, and 40% for mantle cell lymphoma. The corresponding figures for 3-year PFS were 22%, 59%, 22%, and 30%, respectively. Multivariate analysis identified elevated pretransplantation lactate dehydrogenase (LDH) as an adverse factor for PFS, while indolent NHL histology was favorable. For OS, advanced age and elevated pretransplantation LDH were adverse factors, whereas indolent NHL histology was favorable. Low early donor chimerism was not predictive of poor outcome in univariate or multivariate analyses. Moreover, progression was not associated with loss of chimerism. These results emphasize the importance of lymphoma histology for patients undergoing RIC SCT, as well as the lack of relevance of donor chimerism for outcome in this patient population.

Peripheral blood mononuclear cells from 24 patients with prolymphocytic leukemia (PLL) were isolated using a Ficoll-Hypaque gradient and stained by indirect immunofluorescence using a wide panel of monoclonal antibodies against B cell restricted and associated antigens, including HLA DR (la), CD19, CD21 (C3dR) surface membrane immunoglobulin (Slg), CD10 (CALLA), C3b, B5, CD25 (TAC), PCA1, T9, and T10. The cells were also tested for the FMC7, defined previously on PLL cells and the RAB1, a newly described hairy cell leukemia antigen. Thirteen out of the 24 samples expressed with variable intensity all the above antigens. While la, CD19, CD20, FMC7, and RAB1 were strongly or moderately expressed in all, the complement receptors (CD21 and C3b) were only weakly expressed in 12 cases; and the activation antigens B5, TAC, T9, T10, and PCA1 were found with variable intensity in two-thirds of the cases. In 50% of the cases tested, the CD5 antigen (usually strongly expressed on B CLL cells) was weakly to moderately expressed. These findings (absence or weak expression of complement receptors with variable expression of activation antigens) suggest that the PLL cells are activated B cells. When stimulated in vitro by anti-mu and TPA, (phorbol ester) tumor cells showed a decrease in CD21 and Slg and a stronger expression of CD25, T9, T10, and PCA1, with evidence of lg secretion in four out of the seven cases studied. This confirms that the PLL cells arrested at an advanced stage of differentiation progressed narrowly to more differentiated cells. In view of our findings, we believe that the term prolymphocytic leukemia is inaccurate to define the stage of cell differentiation, and we suggest calling the disease preplasmacytic leukemia.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a disease with a variable clinical course, despite the fact that the neoplastic cells in this disorder are homogeneous with respect to morphology, immunophenotype, and cell cycle stage. To further investigate the heterogeneity observed in the clinical behavior of B-CLL, we determined the phenotype and growth requirements of clonogenic cells from 28 patients with B-CLL from low-, intermediate-, and high-risk groups as defined by the Rai staging system. Using methyl-cellulose as a semi- solid media with feeder cells and/or growth factors, colonies were observed with one or more of the culture conditions tested in 25 of 28 CLLs. Phenotypic analysis of colonies demonstrated that the clonogenic cells uniformly expressed la, CD19, CD20, CD5, and the identical light chain as the original CLL cell cultured. However, heterogeneity was observed in clonogenic B-CLL cell growth among the three different CLL risk groups. Clonogenic cells from patients with low-risk CLL required either irradiated unstimulated T cells, with or without conditioned media (CM) or irradiated activated T cells alone for colony formation. Both the number of colonies (227 +/- 15) as well as the number of cells per colony (220 +/- 82) were large, with a mean cloning efficiency of 0.39%. In contrast, clonogenic cells from patients with intermediate- and high-risk CLL required the combination of both irradiated activated T cells and CM. As compared with the low-risk CLLs, both the number and size of the colonies formed by the intermediate- (74 +/- 17, 70 +/- 39) and high- (83 +/- 28, 40 +/- 14) risk groups were significantly lower (P less than .0001). Similarly, the mean cloning efficiency was significantly reduced to 0.15% and 0.14%, respectively. None of the recombinant cytokines (interleukin 1 [IL-1] to IL-7, tumor necrosis factor, alpha and gamma-interferon, B-cell growth factor, and granulocyte macrophage colony-stimulating factor) alone or in combination with each other could entirely replace the stimulatory effect of the activated T cells. These data suggest that clinical progression of B-CLL is associated with a loss of clonogenic potential in the circulating pool of neoplastic cells, which require as yet undefined factors provided by activated T cells and CM.

Fourteen patients with T-cell-derived leukemia and lymphoma underwent high-dose chemoradiotherapy and anti-T-cell monoclonal antibody-treated autologous bone marrow transplantation (ABMT). All patients were either in sensitive relapse or had adverse prognostic features, and five patients had a history of bone marrow involvement with disease. Patients received a median of 2 (1 to 3) prior chemotherapy regimens; 10 patients received local radiotherapy. After high-dose ablative therapy, greater than 500/mm3 granulocytes and greater than 20,000 untransfused platelets/mm3 were noted at a median of 23 (13 to 48) and 26 (15 to 43) days post-ABMT, respectively. Natural killer (NK) cells, T cells (predominantly T8+), and monocytes were noted within the first 1 to 2 months post-AMBT, as seen in other series. Disease-free survival was a median of 10.1 months, 5.9 months for patients with T acute lymphoblastic leukemia or lymphoblastic lymphoma and 25.6 months for patients with T non-Hodgkin's lymphoma (NHL). Toxicities were common and severe. Thirty-six percent of patients developed bacteremias early post-BMT. Late complications included a skin rash consistent with graft versus host disease; infections with Herpes zoster, hepatitis, and Pneumocystis carinii; and the development of Epstein-Barr virus associated lymphoproliferative syndrome. Our findings suggest that patients who have undergone T-depleted ABMT have a profound immunodeficiency not reflected in the phenotypic reconstitution of the T and NK cells. Characterization of the functional deficiency may facilitate the development of methods to reduce the long-term toxicity of AMBT in these patients.

One hundred patients with B-cell non-Hodgkin's lymphoma (NHL) in sensitive relapse or incomplete first remission underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MAb)-treated autologous bone marrow transplantation (ABMT). These patients demonstrated good performance status with a Karnofsky score of 80% or greater. The majority of these patients had one or more adverse prognostic features including a failure to achieve a complete remission (CR) with conventional combination chemotherapy (37 patients), bone marrow infiltration (69 patients), a history of extranodal disease other than bone marrow infiltration (42 patients), and histologic conversion (18 patients). At the time of ABMT, only 52 patients were in CR; however, all patients achieved a minimal disease state following conventional intensive therapy. Moreover, at the time of marrow harvest, 37 of these patients had histologic evidence of lymphoma cells infiltrating the marrow. Following high-dose ablative therapy, two acute in-hospital treatment-related deaths were observed. Two late deaths were observed, not due to recurrent lymphoma. Of the remaining 96 patients, 61 are in unmaintained CR with a median follow-up of 13 months. Kaplan-Meier actuarial analysis predicts 50% probability of disease-free survival (DFS) at 37.8 months. This very low treatment-related mortality provides the rationale to apply high-dose therapy and ABMT as consolidative therapy for patients in first remission who are at high risk for relapse following conventional therapy.

The majority of CLLs are of B lineage derivation with about 5 per cent of cases of T lineage. Although morphologically resembling the small peripheral blood B cell, by virtue of the expression of B cell restricted and associated cell surface antigens, B-CLLs are not the neoplastic counterparts of normal resting B cells. Similar to the peripheral blood B cell, B-CLLs express CD19, CD20, CD21, CD24, CD40, CD44, CD45R, and sIgM/D. However, unlike peripheral blood B cells, B-CLLs generally do not express C3b complement receptor, LFA-1, or CD22. In addition, B-CLLs express the T cell associated antigen CD5, and a number of antigens induced on normal B cells following in vitro activation (B5, Blast-1, CD23). These findings support the hypothesis that B-CLLs are the neoplastic counterparts of one or more unique subpopulations of normal B cells. Normal CD5+ B cells, which phenotypically resemble B-CLL, are present in fetal lymphoid tissues and in small numbers in adults. Moreover, normal CD5+ B cells are present in increased numbers in patients with autoimmune diseases and a subset of normal in vitro activated B cells phenotypically resemble B-CLL. Similar studies into the state of differentiation of T-CLL cells suggest that although most cases resemble normal activated T helper cells, a significant number are the neoplastic counterparts of natural killer cells. Recent studies have examined the function of B and T cells in B-CLL. Although controversial, these studies suggest that the in vitro response to mitogens and cytokines of B-CLL cells is abnormal. T cell proliferation in B-CLL is depressed due to an inability to produce sufficient T cell growth factor (IL-2) as well as a poor response to exogenous IL-2 possibly from ineffective IL-2 receptor expression. Purified populations of T helper and T suppressor cells demonstrate insufficient support of Ig production by normal B cells as well as excess suppression, respectively. These studies have further supported the previous hypothesis that the depressed cellular and humoral immunity in CLL is multifactorial with both abnormal B and T cell function.

Mixed hematopoietic chimerism (MC) is a common finding after allogeneic bone marrow transplantation (BMT), but the natural history of this phenomenon remains unclear. To understand the evolution and the implications of this finding, we performed a prospective analysis of the development of mixed chimerism in 43 patients with hematologic malignancies who received bone marrow (BM) from human leukocyte antigen (HLA)-identical sibling donors. T-cell depletion in vitro with anti-T12 (CD6) monoclonal antibody and rabbit complement was used as the only method of graft-versus-host disease (GVHD) prophylaxis. Overall, MC was identified in peripheral blood (PB) and BM in 22 of 43 (51%) patients evaluated. MC was found by restriction fragment length polymorphism (RFLP) analysis in 21 of 40 (53%) patients, by cytogenetic analysis in 6 of 29 (21%) patients, and by red blood cell phenotyping in 4 of 9 (44%) patients. RFLP studies were performed at 0.5, 1, 3, 6, 9, and 12 months post-BMT and then every 6 months, and showed a high probability of developing MC in the first 6 months after BMT followed by stabilization after 12 months. Cytogenetic analysis was less sensitive in detecting MC. Once MC was detected after BMT, the percentage of recipient cells increased very slowly over more than 3 years of follow- up, and no patient reverted to complete donor hematopoiesis (CDH). Thus, recipient and donor cells remained in a relative state of equilibrium for prolonged periods that seemed to favor recipient cells over donor cells. Patient's disease, remission status, or intensity of the transplant preparative regimen did not influence the subsequent development of mixed chimerism. Early immunologic reconstitution was the only factor that correlated with the subsequent chimeric status of the patients. The percentage and absolute number of T3 (CD3) and T4 (CD4) positive cells at day 14 after BMT were significantly higher in the patients who maintained CDH but NK cell reconstitution was similar in both groups, suggesting that early reconstitution with T cells may play a role in preventing recovery of recipient cells after BMT. GVHD was also associated with maintenance of CDH, but the probability of relapse, survival, and disease-free survival was identical in patients with MC and CDH.

Prior studies have demonstrated that relatively immature hematopoietic stem cells, including CD34(+) CD38(-) and CD34(+) HLA-DR(-) subsets, correlate with short-term hematopoietic reconstruction (SHR) after transplantation. The aim of this study was to investigate whether these immature CD34(+) subsets also correlate with long-term hematopoietic reconstitution (LHR) in recipients of ABMT.We examined stem cell grafts from 58 patients with B-cell lymphoma or CLL who underwent ABMT after myeloablative conditioning. We determined whether total mononuclear cell dose (MNC), colony-forming unit-granulocyte-monocyte (CFU-GM), CD34(+) cell dose and CD34(+) cell subsets (CD34(+) CD38(-) and CD34(+) HLA-DR(-) were associated with SHR and/or LHR. Time to neutrophil engraftment (TNE) and time to platelet engraftment (TPE) were used to measure SHR, while platelet counts at day 100 and 1 year post-ABMT were used as indicators for LHR.CD34(+) cell dose and CD34(+) cell subsets were significantly associated with SHR. However, at day 100 and 1 year post-transplant only total CD34(+) cell dose was associated with LHR. The association of total CD34(+) cell dose with LHR persisted after adjusting for age, sex and disease. None of the CD34(+) cell subsets analyzed showed evidence of significant association with LHR.

Background: While CD37 is widely expressed on malignant B cells in NHL and chronic lymphocytic leukemia (CLL), few therapies are in development exploiting this target. In normal tissues, high CD37 expression is restricted to blood cells and lymphoid tissues, making CD37 well suited to an ADC approach. IMGN529 is a CD37-targeting ADC consisting of a CD37-binding antibody with intrinsic pro-apoptotic and immune effector activities conjugated to the maytansinoid anti-mitotic, DM1. Its unique profile enables IMGN529 to potentially kill CD37-positive B cells via multiple mechanisms of action. In preclinical studies, IMGN529 exhibits targeted, potent activity against NHL cells via direct inhibition of cell survival and effector function by its antibody and tubulin-disruption by DM1. Methods: A Phase I study is being conducted to evaluate safety, pharmacokinetics, pharmacodynamics, exploratory biomarkers and preliminary evidence of activity of IMGN529 and to determine the maximum tolerated dose/recommended phase 2 dose of IMGN529 in adult patients (pts) with relapsed or refractory NHL. IMGN529 is given intravenously on Day (d) 1 of each 21d cycle (C). Efficacy is evaluated based on Cheson response criteria. CD37 is being evaluated by IHC in available tumor samples to assess expression of CD37 among different NHL subtypes. Results: To date, 31 pts have been enrolled (66% male), median age of 65 years: Diffuse large B-cell (DLBCL, n = 14), Follicular lymphoma (FL, n = 10), MCL (n = 5), MZL (n = 2). Dose escalation began at 0.1 mg/kg. Early onset, transient grade 3-4 neutropenia was reported in 6 patients receiving doses at or below 0.8 mg/kg, potentially attributed to cytokine release. Peri-infusional steroid administration was added to the study protocol, and the incidence and severity of this neutropenia was significantly reduced. Additional patients have been enrolled and increasingly higher dose levels evaluated. To date the highest dose evaluated is 1.0 mg/kg. Cytokine levels are currently being evaluated in trial patients to gain a better understanding of the mechanisms underlying the early onset neutropenia. At the 1.0 mg/kg dose cohort grade 3-4 neutropenia was reported (1 DLT of febrile neutropenia among 6 patients) around d10 of the cycle and granulocyte colony stimulating factor (G-CSF) was added as primary prophylaxis. The most common treatment-emergent adverse events (AEs) occurring in ≥ 20% of the 31 pts enrolled were neutropenia (30%), fever (27%), asthenia (20%) and fatigue (20%). A reduction in lymphocyte count seen early after dosing (d2) in the majority of pts suggests a CD37-mediated reduction in lymphocytes, consistent with the mechanism of action of a CD37-targeted therapy. Four objective responses have been reported in patients who had received multiple lines of prior therapy. At the 1.0 mg/kg dose level, two patients with DLBCL who were heavily pretreated and who relapsed following autologous transplant have achieved an objective response that is ongoing. One pt has achieved a PR and one patient has achieved a CR. As previously reported, at doses of 0.2 mg/kg and 0.4 mg/kg one pt with transformed FL, who progressed following an autologous transplant, and one pt with DLBCL achieved a PR. The maximum tolerated dose has not yet been achieved and dose escalation is ongoing with additional data expected. Conclusions: IMGN529, a CD37-targeting ADC, demonstrates clinical activity in patients with NHL and has the potential to be a novel therapeutic for B-cell lymphoproliferative malignancies. Disclosures Stathis: ImmunoGen, Inc: Travel assistance Other; Pfizer: Research Funding; Oncoethix SA: Research Funding. Flinn:ImmunoGen, Inc: Research Funding. Maddocks:Pharmacyclics, Seattle Genetics, MorphoSys: Advisory Board Other, Research Funding. Zucca:Roche, Mundipharma, Novartis, Jannsen, Celgene: Consultancy, Honoraria, Research Funding, travel assistance Other. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Zildjian:ImmunoGen, Inc: Employment. Ruiz-Soto:ImmunoGen, Inc: Employment; Sanofi: Past employment within 1 year, Past employment within 1 year Other.

Background: Patients with diffuse large B cell lymphoma (DLBCL) that are primarily refractory to, or relapse after initial therapy with R-CHOP have a poor outcome and novel therapies are needed in these clinical scenarios. Pre-clinical data suggests that inhibition of the histone deacetylases HDAC-2 and HDAC-6 causes decreased proliferation in DLBCL cell lines. We sought to investigate the activity of panobinostat, an oral pan-HDAC inhibitor, combined with rituximab in this population Methods: We conducted a phase II study employing a Simon’s optimal two-stage design. Eligible subjects had previously received ≥1 prior line of chemotherapy and had either relapsed after autologous stem cell transplantation or were not candidates for transplant. Panobinostat was administered orally once daily at 40 mg on three times a week every other week on a 28-day cycle. Rituximab 375mg/m2 was administered IV weekly during the first cycle, then on Day 1 of cycles 2-6. Subjects received up to 6 cycles of treatment in the absence of disease progression. Patients without disease progression after 6 cycles continued panobinostat monotherapy for up to 6 additional cycles in the absence of disease progression. The primary end point was overall response rate (ORR) as defined by the Revised International Workshop Response Criteria (2007). Secondary endpoints included progression free survival (PFS) at 1 year, duration of response, toxicity, and correlation of response with baseline tissue AKT, pAKT, and BCL6 expression. The study was designed with 90% power to show a 30% ORR, with a 10% ORR considered unworthy for further study. Twenty-five patients were required but the study was terminated at 18 due to slow accrual. Results: Between June 2011 and March 2013, 18 eligible subjects were enrolled. The median age was 68 (range 37-83). The median number of prior therapies was 5 (range 1-11, median time from prior therapy was 4 months, and 5 (28%) of subjects had undergone prior autologous stem cell transplant. Seventeen (94%) patients presented with advanced stage at relapse, 9 (50%) had an elevated LDH and 14 (78%) had greater than 1 extranodal site at relapse. The median number of cycles completed was 2 (range 1-12 and 2 (11%) subjects received more than 5 cycles. The ORR was 11% (90% CI [2%-31%] with 2 subjects having a PR. The duration of response in these subjects was 51 and 60 days. One subject with SD remained on therapy a total of 12 cycles. The median PFS was 54.5 days. Six-month progression-free survival was 6% (90% CI 0.7-21%). At the time of last follow-up, seven subjects were alive and eleven were dead. The most common toxicities while on study were thrombocytopenia (14 patients, 78%); fatigue (11, 61%), ; anemia (10, 56%); diarrhea (8, 44%); and nausea, lymphopenia, anorexia, and hypophosphatemia (5 each, 28% of patients) the majority of which was grade 2 or less. Thrombocytopenia was significant with 8 episodes of grade 3-4 thrombocytopenia but no episodes of serious bleeding. Conclusions: These data indicate that the combination of panobinostat with rituximab is able to induce responses in a limited number of subjects with relapsed DLBCL. Ongoing correlative studies are assessing predictive biomarkers of response. Disclosures No relevant conflicts of interest to declare.

The evolution of indolent lymphomas to aggressive histologies, known as histologic transformation (HT), is a frequent occurrence for all subtypes of low grade B cell lymphoproliferative disorders. The risk of developing HT is approximately 3% per year for patients with indolent lymphoma. Clinically these present with a rapid change in the behavior of the disease, with evidence of a highly proliferative malignancy with a propensity to involve extranodal sites. The prognosis of patients following transformation is generally poor, with median survival of about 12 months. Recent studies suggest that the development of HT is very complex with the acquisition of multiple cytogenetic abnormalities in the low-grade lymphoma cells prior to HT. To date, there are no biologic or genetic parameters predictive of the development of HT. A myriad of genetic lesions have been identified in HT, and provide insight into its pathogenesis. These include genes regulating proliferation (C-MYC and C-MYC-regulated genes); control of the cell cycle (CDKN2a and CDKN2B); and programmed cell death (TP53, C-MYC, and BCL2). Gene expression profiling has been applied to the study of HT and has increased our understanding of the transformation process. There has been limited progress in the treatment of patients with HT. Conventional chemotherapy is generally of limited benefit, although a subset of patients are long-term survivors following high-dose therapy and autologous stem cell transplantation. The use of radioimmunotherapy and new agents targeting specific lesions or aberrant pathways may impact on the management of these aggressive diseases.

Testicular involvement with indolent lymphoma is extremely rare, particularly in the absence of transformation to an aggressive histology. We report a case of a 64-year-old man who presented with cervical lymphadenopathy. Staging CT scans revealed extensive lymphadenopathy as well as bilateral testicular and epididymal masses. Histologic examination of lymph node, bone marrow, and testicular/epididymal biopsies revealed involvement with grade I follicular lymphoma. The patient was started on chemotherapy with cyclophosphamide, vincristine, prednisone, and rituximab in addition to intrathecal methotrexate and testicular radiation. He is now 6 months into therapy and responding well. A review of the literature demonstrated this to be the first confirmed case of testicular and epididymal involvement with grade I follicular lymphoma.