Search

In the S0313 trial, we evaluated the impact of adding ibritumomab tiuxetan consolidation to 3 cycles of standard cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy plus involved field radiotherapy (IFRT) in patients with limited-stage aggressive B-cell non-Hodgkin lymphoma (LD-NHL). Patients with at least 1 stage-modified adverse risk factor (nonbulky stage II, age >60 years, elevated lactate dehydrogenase, or World Health Organization performance status of 2) were treated with CHOP on days 1, 22, and 43, followed 3 weeks later by 40 to 50 Gy of IFRT. An ibritumomab tiuxetan regimen was initiated 3 to 6 weeks following IFRT. Forty-six patients were registered and eligible, with median follow-up of 7.3 years. The progression-free survival estimate is 89% at 2 years, 82% at 5 years, and 75% at 7 years. The overall survival estimate is 91% at 2 years, 87% at 5 years, and 82% at 7 years. Grade 4 adverse events occurring more than once included neutropenia (8), leukopenia (5), and lymphopenia (2). Febrile neutropenia was observed in 4 patients. No cases of treatment-related myeloid neoplasms were noted. In conclusion, patients with high-risk LD-NHL treated with 3 cycles of CHOP plus IFRT followed by ibritumomab tiuxetan consolidation had outcomes that compare favorably to our historical experience. The clinical trial was registered at www.clinicaltrials.gov as #NCT00070018.

Epstein-Barr virus (EBV) is associated with Hodgkin lymphoma (HL) and can be detected by in situ hybridization (ISH) of viral nucleic acid (EBER) in tumor cells. We sought to determine whether plasma EBV-DNA could serve as a surrogate for EBER-ISH and to explore its prognostic utility in HL. Specimens from the Cancer Cooperative Intergroup Trial E2496 were used to compare pretreatment plasma EBV-DNA quantification with EBV tumor status by EBER-ISH. A cutoff of >60 viral copies/100 µL plasma yielded 96% concordance with EBER-ISH. Pretreatment and month 6 plasma specimens were designated EBV(-) or EBV(+) by this cutoff. Patients with pretreatment EBV(+) plasma (n = 54) had inferior failure-free survival (FFS) compared with those with pretreatment EBV(-) plasma (n = 274), log-rank P = .009. By contrast, no difference in FFS was observed when patients were stratified by EBER-ISH. Pretreatment plasma EBV positivity was an independent predictor of treatment failure on multivariate analyses. At month 6, plasma EBV(+) patients (n = 7) had inferior FFS compared with plasma EBV(-) patients (n = 125), log-rank P = .007. These results confirm that plasma EBV-DNA is highly concordant with EBER-ISH in HL and suggest that it may have prognostic utility both at baseline and after therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003389.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which approximately 40% of the patients respond well to current chemotherapy, but the prognosis for the other 60% is poor. The Leukemia/Lymphoma Molecular Profiling Project (LLMPP) used microarray technology to define a molecular profile for each of 240 patients with DLBCL and develop a molecular outcome predictor score that accurately predicted patient survival. Data from our laboratory and others suggest that alterations in antioxidant defense enzyme levels and redox environment can be oncogenic and affect the response to glucocorticoid treatment, one of the components of combination chemotherapy regimens for lymphoma. The goal of the current study was to reanalyze the LLMPP microarray data to determine whether the levels of antioxidant defense enzymes and redox proteins were correlated with prognosis in DLBCL. We found that patients with DLBCL with the worst prognosis, according to the outcome predictor score, had decreased expression of catalase, glutathione peroxidase, manganese superoxide dismutase, and VDUP1, a protein that inhibits thioredoxin activity. The data suggest that the patients with the worst prognosis combine a decrease in antioxidant defense enzyme expression with an increase in thioredoxin system function (the redox signature score).

Decreased major histocompatibility class II (MHCII) expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL). Immune-privileged site DLBCL (IP-DLBCL) patients reportedly have frequent large deletions at the MHCII locus whereas the mechanism of decreased expression in non-IP-DLBCL is unknown. Gene expression profiling data were used for correlation analyses between expression levels of MHCII genes with each other and their transcriptional regulator, CIITA. Comparative genomic hybridization (CGH) assessed chromosomal alterations at MHCII-related loci. Finally, a map was created of expression of genes that are telomeric, within, or centromeric to the MHCII locus. Correlation coefficients among MHCII genes ranged from 0.73 to 0.92, whereas those between adjacent and intervening genes were lower (-0.12 to 0.49). Correlations between MHCII and CIITA expression were higher (0.53 to 0.60) than between CIITA and neighboring genes (-0.05 to 0.22). In 23 MHCII(-) cases, CGH detected 2 losses and 2 gains at MHCII loci. Expression of genes telomeric, within, and centromeric to MHCII loci were near normal in most MHCII(-) cases. Large deletions of the MHCII locus are uncommon in non-IP-DLBCL, implicating altered transcription as the operative mechanism for decreased expression.

The Leukemia and Lymphoma Molecular Profiling Project recently published results from DNA microarray analyses of 240 diffuse large B-cell lymphomas (DLBCLs). Four gene expression "signatures" were identified as correlated with patient outcome, including the major histocompatibility complex (MHC) class II genes (eg, HLA-DRA) which correlated with better survival. We further analyzed the effects of HLA-DRA on survival and correlated gene expression with protein status and tumor-infiltrating lymphocytes. The 5-year overall survival was 24% in the lowest 10% of HLA-DRA expression, 37% in the 10% to 25% group, 50% in the 25% to 50% group, and 55% for patients in the highest 50%. Further analysis demonstrated that the hazard ratio of death was a nonlinear function of HLA-DRA expression. Adjustment for the International Prognostic Index did not alter the impact of HLA-DRA on survival. Other MHC class II genes were found to predict survival similarly. Microarray HLA-DRA expression correlated with the presence or absence of human leukocyte antigen-DR (HLA-DR) protein in 20 of 22 cases assessed. Fewer tumor-infiltrating CD8(+) T cells were detected in MHC class II-negative cases compared with positive cases (2.8% versus 11.0%; P =.001), supporting the hypothesis that loss of tumor immunosurveillance has a devastating effect on patient outcome in DLBCL.

Advanced follicular lymphoma is incurable with conventional chemotherapy and radiotherapy. The Southwest Oncology Group (SWOG) conducted a phase 2 trial (S9911) of a novel regimen consisting of 6 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy followed 4 to 8 weeks later by tositumomab/iodine I 131 tositumomab (anti-CD20 antibody) in 90 eligible patients with previously untreated, advanced stage follicular lymphoma. Treatment was well tolerated. Reversible myelosuppression was the main adverse event and was more severe during CHOP chemotherapy than following radioimmunotherapy. The overall response rate to the entire treatment regimen was 90%, including 67% complete remissions (CRs plus unconfirmed CRs [CRu's]) and 23% partial remissions (PRs). Twenty-seven (57%) of the 47 fully evaluable patients who achieved less than a CR with CHOP improved their remission status after tositumomab/iodine I 131 tositumomab. With a median follow-up of 2.3 years, the 2-year progression-free survival (PFS) was estimated to be 81%, with a 2-year overall survival of 97%. This study has established the feasibility, tolerability, and efficacy of this regimen for patients with advanced follicular lymphoma. This novel treatment appears promising compared with the SWOG's historical experience using CHOP alone and is currently being compared with CHOP plus rituximab in a randomized phase 3 trial (S0016).

The Revised European-American Lymphoma classification gives Burkitt-like lymphoma (BLL) provisional status, leaving unresolved the differential diagnosis with Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). This study compared the biologic features of adult BLL and DLBCL. The phenotypic distinction between BLL and DLBCL was determined by immunohistochemical staining of frozen tissue from 13 patients with BLL and 55 patients with DLBCL by using an extensive antibody panel including Ki-67, CD10, CD11a/lymphocyte function-associated antigen 1alpha (LFA-1alpha), CD18/LFA-1beta, CD58/LFA-3, and CD54/intercellular adhesion molecule, CD8 for tumor-infiltrating cytotoxic T cells (T-TILs), CD44 homing receptor, and p53 and Bcl-2 oncogenic proteins. Compared with DLBCL, BLL had a higher proliferative rate (mean Ki-67, 88% versus 53%), greater expression of CD10 and p53 antigens, and decreased expression of Bcl-2. BLL cases had a consistent absence of one or more cell adhesion molecules (92% versus 27%), low T-TIL numbers, and absence of CD44 homing receptor (92% versus 14%). The t(8;14) translocation was identified in 80% of BLL cases, but no patients with BLL had the t(14;18) translocation. In a 10-year analysis, median survival of patients with BLL was 1.2 years, and that of patients with DLBCL was 2.5 years. Although the proportion of patients cured was similar in the 2 groups, BLL patients had an increased risk of early death. We conclude that BLL can be recognized by its combined morphologic and phenotypic features and that it represents a high-grade lymphoma much closer to BL than DLBCL. Retention of the BLL category or inclusion of BLL as a variant of BL is biologically and clinically more appropriate than absorbing the category of BLL into DLBCL. (Blood. 2001;97:3713-3720)

The objectives of this study were (1) to determine the clinical presentation and natural history associated with two newly recognized pathologic entities termed mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), including the mucosa-associated lymphoid tissue (MALT) and monocytoid B-cell subcategories, and (2) to determine whether these entities differ clinically from the other relatively indolent non- Hodgkin's lymphomas with which they have been previously classified. We reviewed the conventional pathology and clinical course of 376 patients who had no prior therapy; had stage III/IV disease; were classified as Working Formulation categories A, B, C, D, or E; and received cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) on Southwest Oncology Group (SWOG) studies no. 7204, 7426, or 7713. All slides were reviewed by the three pathologists who reached a consensus diagnosis. Age, sex, performance status, bone marrow and/or gastrointestinal involvement, failure-free survival, and overall survival were compared among all the categories. We found that (1) MCL and MZL each represent approximately 10% of stage III or IV patients previously classified as Working Formulation categories A through E and treated with CHOP on SWOG clinical trials; (2) the failure-free survival and overall survival of patients with MZL is the same as that of patients with Working Formulation categories A through E, but the failure-free survival and overall survival of the monocytoid B-cell patients were higher than that of the MALT lymphoma patients (P = .009 and .007, respectively); and (3) the failure-free survival and overall survival of patients with MCL is significantly worse than that of patients with Working Formulation categories A through E (P = .0002 and .0001, respectively). In conclusion, patients with advanced stage MALT lymphomas may have a more aggressive course than previously recognized. Patients with MCL do not have an indolent lymphoma and are candidates for innovative therapy.

The growth fraction of tumors from patients with non-Hodgkin's lymphomas (NHL) has been shown to correlate with survival in retrospective studies. The growth fraction can be evaluated using immunohistochemical techniques employing the Ki-67 monoclonal antibody (MoAb) that marks a nuclear protein present in cycling cells. The purpose of this study was to evaluate the clinical utility of the Ki-67 MoAb for predicting survival. Using a prospective trial design in a multi-institutional cooperative trials group, the proliferative index, clinical outcome, and statistical correlations were independently assessed for previously untreated patients with advanced stages of intermediate- and high-grade histologies of NHL treated on Southwest Oncology Group study (SWOG 8516, Intergroup 0067). The proportion of Ki- 67-positive cells was determined on snap-frozen thin tissue sections. A proliferative index of 80% or greater, as determined from prior retrospective studies, identified a group of patients (18%) who had a poor outcome. Overall survival was significantly reduced in these patients with a high Ki-67-associated proliferative index compared with those with a low proliferative index (P = .001). One-year survival estimates were 82% (low proliferative index) versus 18% (high proliferative index). A multivariate regression analysis incorporating commonly used clinical prognostic features confirmed the independent effect of proliferation on survival (relative risk estimate 5.9; 95% confidence interval, 2.2, 16.1). The Ki-67 MoAb identifies a group of patients with rapidly fatal NHL for whom currently available chemotherapy is inadequate.

A distinct subset of patients with peripheral T-cell lymphoma (PTCL) is described which reacts with Leu-19 (CD56), an antibody that has been shown to identify the neural cell adhesion molecule (NCAM). These NCAM- positive PTCL patients (11 of a series of 46 PTCL; 24%) exhibited a striking predilection for unusual anatomic sites of involvement: central nervous system (36%), muscle (18%), gastrointestinal tract, and nasopharynx (27% each). Additional extranodal sites of involvement included the pituitary, thyroid, parathyroids, adrenals, and pancreas. The NCAM-positive subset also exhibited a characteristic phenotypic profile, with significantly lower expression of CD3 and CD5 compared with the NCAM-negative group. RNA transcripts consistent with the NCAM gene were detected in tissue samples from five Leu-19-positive cases using a reverse transcriptase-polymerase chain reaction assay, supporting the idea that Leu-19 recognizes NCAM in these patient samples. This suggests that the expression of the NCAM plays a role in the behavior and localization of lymphomas. Because of the unique clinical and phenotypic characteristics of this group it may be designated as “NCAM-positive peripheral T-cell lymphoma.”

Verapamil was evaluated as a chemosensitizer for reversing multidrug resistance in multiple myeloma both in vitro and in clinical trials. Bone marrows from 59 myeloma patients in relapse were evaluated for several resistance parameters: expression of p-glycoprotein (MDR1), doxorubicin (Adriamycin) and vincristine sensitivity, and the ability of added verapamil to reduce resistance to the cytotoxic agents. We found that verapamil was capable of sensitizing myeloma cells that exhibited resistance to doxorubicin and vincristine in vitro, but did not enhance sensitivity of cells that were drug sensitive (P less than .001). Myeloma cells expressing MDR1 immunohistochemically tended to be more doxorubicin resistant in vitro than MDR1-negative cells. In the clinical trials, 22 patients with myeloma refractory to vincristine- Adriamycin-dexamethasone (VAD) were treated with VAD plus high-dose intravenous verapamil (Ve). Among the 22 patients treated with VAD/Ve, five achieved a partial remission (23%). The median relapse-free survival for the VAD/Ve responders was 5.4 months and their overall survival from the start of VAD/Ve was better than that of the nonresponders. Among the subset of 10 patients whose myeloma cells were MDR1 positive, four responded clinically (40%), whereas none of five patients with MDR1-negative myeloma cells achieved remission with VAD/Ve. We also observed that myeloma cells from three of four VAD/Ve clinical responders exhibited in vitro chemosensitization with verapamil, whereas in vitro verapamil chemosensitization was seen in only one of six clinical nonresponders. Our observations demonstrate that clinical reversal of multidrug resistance can be achieved in some patients with VAD-refractory myeloma with the use of verapamil. In addition to their value in drug development, in vitro tests of MDR1 expression and of chemosensitizers plus cytotoxic drugs on the patients' bone marrow myeloma cells may identify patients who will respond clinically to chemosensitizer-containing regimens. We anticipate that chemosensitizer regimens capable of inhibiting multidrug resistance will play an increasing role in the treatment of hematologic malignancies, including B-cell neoplasms such as multiple myeloma and the non-Hodgkin's lymphomas.

A gene expression signature of tumor proliferation rate in mantle cell lymphoma (MCL) is an overriding molecular predictor of the length of survival following diagnosis. Many strongly proliferative MCL tumors have exceptionally high cyclin D1 mRNA levels and preferentially express short cyclin D1 mRNA isoforms. We demonstrate here that these short mRNAs are cyclin D1a isoforms with truncated 3′UTRs, not alternatively spliced cyclin D1b mRNA isoforms. Among 15 MCL tumors with truncated cyclin D1 mRNAs, 7 had genomic deletions in the CCND1 3′UTR region. In 3 others, CCND1 contained point mutations that created premature polyadenylation signals, giving rise to 1.5-kb mRNAs lacking most of the 3′UTR. Both types of genomic alteration created transcripts lacking mRNA destabilization elements present in the wild-type cyclin D1a mRNA. Premature polyadenylation due to a 3′UTR mutation also was present in the Z-138 MCL cell line, which expressed both truncated and full-length cyclin D1a mRNAs. In these cells, the half-life of the short cyclin D1a mRNA was much longer than that of the full-length mRNA. We conclude that alterations of CCND1 3′UTR structure can significantly increase its oncogenic effect and worsen the clinical course of MCL patients.

Introduction: Patients with limited stage aggressive B-cell non-Hodgkin lymphoma (LS-NHL) and at least one stage-modified adverse risk factor have an excessive relapse rate leading to a 5-year overall survival (OS) of 50-77% and 10-year OS of 0-50%. In SWOG S0014 we have shown that the addition of rituximab to 3 cycles of CHOP plus involved field radiation therapy (IFRT) resulted in an improved estimated 4-year progression-free survival (PFS) of 88% and OS of 92%. Relapses were largely systemic (5 of 6 evaluable) and continued to be seen with longer follow-up. Ibritumomab tiuxetan (Zevalin ®) is a radiolabeled anti-CD20 antibody that has excellent single agent activity in diffuse large B-cell lymphoma and could prevent systemic relapse of disease. We now report long term results of SWOG S0313, a phase II study of ibritumomab tiuxetan consolidation after 3 cycles of CHOP plus IFRT in patients with LS-NHL. Methods: Patients with LS-NHL and at least one stage-modified adverse risk factor (non-bulky stage II, age > 60 years, elevated LDH, or WHO performance status of 2) were treated with CHOP on days 1, 22, and 43, followed 3 weeks later by 40-50 Gy of IFRT. Ibritumomab tiuxetan regimen was initiated 3 – 6 weeks following IFRT. Results: Forty-six patients were registered and eligible, with median follow-up of 7.3 years. Median age was 61, 37% of patients had elevated LDH, and 20% had systemic symptoms. Grade 4 adverse events occurring more than once included neutropenia (8 patients), leukopenia (5), and lymphopenia (2). Febrile neutropenia was observed in 4 patients. No cases of treatment-related myeloid neoplasms were noted. Eleven patients progressed and 8 patients died. The PFS estimate is 89% at 2 years, 82% at 5 years, and 75% at 7 years. OS estimate is 91% at 2 years, 87% at 5 years, and 82% at 7 years. These outcomes compare favorably to matched cohorts on prior SWOG trials, with 7-year PFS estimate of 68% on S0014 and 65% on S8736 (original pre-Rituximab trial); and 7-year OS estimate of 80% on S0014 and 73% on S8736 cohorts. Conclusions: Patients with high-risk LS-NHL treated with 3 cycles of CHOP plus IFRT followed by ibritumomab tiuxetan consolidation had outcomes that compare favorably to our historical experience. A US cooperative group study of R-CHOP and response-adapted IFRT followed by consolidative ibritumomab tiuxetan is ongoing. Disclosures Off Label Use: ibritumomab tiuxetan in diffuse large B-cell lymphoma.

Background Imexon is a 1-carboxamido-2-cyan-aziridine isomer investigated as an anti-cancer agent given its pro-oxidant properties. By binding reduced sulfhydryls leading to the accumulation of reactive oxygen species, imexon interferes with the endoplasmic reticulum and mitochondrial reduction-oxidation (redox) balance, inhibiting protein translation and cell growth and inducing apoptosis. Pre-clinical studies demonstrated activity across an array of tumor cells in vitro and increased activity amongst B-cell non-Hodgkin lymphomas (NHL). A partial response in a follicular lymphoma (FL) patient was observed in a previous phase I study. This phase II trial was initiated to further investigate the clinical activity of imexon in patients with relapsed or refractory NHL. Methods Histologically confirmed NHL, > 1 prior therapy, age≥18, ECOG performance status 0–2, measurable disease, signed informed consent, creatinine and bilirubin < 2.0 x IULN as well as G6PD > IULN were required. Patients were treated with imexon 1000 mg/m2 IV daily on days 1-5 of a 21 day cycle for up to 1 year. Messenger RNA analysis was performed on pre-treatment tumor specimens, evaluating 22 genes important for antioxidant enzyme expression, 16 genes previously associated with outcome in NHL as well as 4 immune cell surface markers. Included were 13 genes used to generate a redox signature score, previously demonstrated to correlate with NHL prognosis (Tome, Blood 2005). Results Twenty-two NHL patients [9 FL, 5 diffuse large B cell (DLBCL), 3 mantle cell, 2 transformed follicular, 2 chronic lymphocytic leukemia and 1 Burkitt] with a median age of 64 (range 43-92) completed a median of 2.5 (range 1-13) cycles of therapy. With a median number of 4 prior therapies, 9 patients had undergone a prior stem cell transplant, 10 had stage IV disease and 6 were refractory to prior therapy. Twenty patients were evaluable for response, 2 pts discontinued therapy during cycle 1 due to progressive disease and grade 5 sepsis respectively. Of the 20 evaluable patients, the overall response rate was 30% (6/20) with another 35% achieving stable disease. Responses were observed in 4 FL and 2 DLBCL pts. After a median follow-up of 7 months, the median progression free survival (PFS) was 2.4 mos (range, 0.6 to 19.1 mos) with a median PFS of 6.7 mos (range, 1.2 to 9.0 mos) in FL patients. The median overall survival has not been reached. Grade 3 and 4 toxicities consisted of anemia (7 pts), thrombocytopenia (2 pts), neutropenia (2 pts), sepsis (2 pts), vomiting (2 pts), pneumonia (2 pts), fatigue (2 pts), dehydration (2 pts) as well as hypokalemia, hyperuricemia, transient ischemic attack, increased creatinine, rash and urinary tract infection in 1 pt each. 13 pts had available pre-treatment tumor biopsies, 2 of which attained a partial response with therapy. Patients with a higher redox score were more likely to achieve an objective response (p=0.03). Further, individual genes most predictive of response included CD68, GPX1 and SOD2. Conclusions This is the first trial to demonstrate that targeting the cellular redox environment is a viable therapeutic strategy in NHL and may be particularly effective in FL. The side effect profile may lend imexon to rational combination studies. Lymphomas reliant on antioxidant defense enzymes for proliferation and survival may be more susceptible to redox directed therapy. Evaluation of antioxidant related gene expression as a predictive biomarker is warranted in future investigations of imexon and similar targeted agents. (NCT01314014) Disclosures: Barr: Seattle Genetics: Consultancy; Celgene: Consultancy. Off Label Use: Imexon; being investigated for use in Non-Hodgkin lymphoma. Schwartz:HTG Molecular Diagnostics: Employment. Dorr:Amplimed Corporation: Employment.

Background Radioimmunoconjugates are effective treatment in relapsed/refractory follicular lymphoma (FL) when used as single agents, and can result in effective disease control (Kaminski et al, NEJM 1993; Witzig, JCO 2002). When used as consolidative treatment following a course of initial chemotherapy for patients with newly diagnosed FL, durable remissions have been noted (Gordon LI et al, Blood 2004). As a pure high energy beta emitting isotope, Zevalin has several advantages and has been well studied in relapsed refractory indolent NHL (Witzig TE et al, JCO 2002). Therefore, our hypothesis is that radioimmunoconjugates significantly change outcome for patients with FL when given in a situation of minimal residual disease, and number of long term remissions would increase with initial cytoreduction. For cytoreduction, we chose to use an outpatient formulation of ESHAP (etoposide, methylprednisolone, cytarabine, cisplatin): ESHAP has excellent efficacy as a salvage regimen in treating relapsed low-grade lymphomas (Rodriguez-Monge EJ et al, Hem Oncol Clin N Am, 1997). Further, it is not utilized by community oncology practices fostering chemo sensitivity in a relapsed setting. Methods Histologically confirmed CD 20 + relapsed FL, ≥1< 4 prior therapies, age 18, ECOG performance status 0–2, measurable disease, signed informed consent, creatinine, bilirubin< 2.0 x ULN , platelet counts ≥150,000 able to receive 0.4 mCi/kg of Zevalin; patients with platelet counts 100,000-150,000 received 0.3mCi/kg dose of Zevalin. Patients were treated with 2 cycles of ESHAP every 28 days. At subsequent restaging, if bone marrow aspirate, biopsy showed25 % was noted, patients were taken off study secondary to treatment failure. Results Twenty-eight patients with FL were enrolled with total 8 year follow-up. 6 patients did not complete the study: one patient was ineligible secondary to re-review of path showing DLBCL. Three patients were ineligible for study completion secondary to bone marrow showing residual involvement. Two patients withdrew secondary to toxicities: one from a perforated duodenal ulcer (SAE), and one from side effects of ESHAP chemotherapy including nausea, progressive functional decline. Other Grade 3, 4 adverse events included myelosuppression. Twenty-two patients were evaluable for response. Of the 22 evaluable patients, the overall response rate was 72% (17/22) with another 13 % achieving stable disease. After follow-up of 8 years, the median progression free survival (PFS) was 10 months for both the intent to treat analysis and responders (p=0.14). The median overall survival (OS) in the intent to treat analysis was 63 months, and the median OS in the 8 year follow-up of responders has not been reached (p=0.02). When analyzed by median number of prior therapies, the median PFS for patients with more than one prior chemotherapy regimen was 9, whereas the median PFS for patients with one prior regimen was 22 months. Similarly, for patients with more than one prior therapy, the median OS was 54 months, whereas the median OS for patients with one prior regimen has not been reached at 8 year follow-up. Conclusions In prior long term follow-up data(7 years) of a phase I/II study of Zevalin (Gordon et al, Blood 2004) in NHL including FL, the median time to progression in responders was 12.6 months, and durable responses were noted in 5/51 patients with FL. In a phase II study of relapsed FL treated with Zevalin (Witzig et al, JCO 2002) the median time to progression was 6.8 months, and in a subsequent Phase III study comparing Zevalin to rituximab, the median time to progression was 11.2 months. In comparison to above studies, our study has shown that outpatient ESHAP is an effective cytoreductive regimen. Zevalin is active when administered in a setting of minimal residual disease early in the disease course as evidence by the excellent overall survival of the responders. Disclosures: Off Label Use: Use of the investigational agent MLN8237 in combination in patients with aggressive B-cell NHL. Persky:Millennium: The Takeda Oncology Company: Research Funding.

Background Amplification and/or over-expression of the mitotic Aurora A kinase (AAK) have been reported in a variety of tumors. The investigational, selective, AAK inhibitor MLN8237 (alisertib) has shown signs of anti-tumor activity in pts with hematologic malignancies including aggressive non-Hodgkin lymphoma (NHL). Over-expression of AAK leads to resistance to microtubule targeted agents such as vincristine (V) and inhibiting AAK leads to synergy in the presence of these agents (Mahadevan D et al. CCR 2012). The combination of MLN8237 (M) + rituximab (R) ± V has shown synthetic lethality in pre-clinical B-NHL mouse models (ibid) supporting clinical evaluation of this combination; we report the safety and recommended phase 2 dose (RP2D) from the phase 1 clinical data of this MR ± V combination in pts with aggressive B-NHL. Methods Adults with CD20+ B-NHL after 1–4 prior regimens (including ASCT) and ECOG PS 0–2 were eligible. In part 1 (MR), pts received one dose level of M 50 mg BID days 1–7 + R 375 mg/m2 IV day 1 in 21-day cycles (up to 8) in combination, followed by single agent M. In part 2 (MRV), M dose escalation followed a 3+3 design (M starting dose: ∼50% of MR RP2D [30mg BID] days 1–7 + R 375 mg/m2 IV on day 1 + V 1.4 mg/m2 [max, 2 mg] IV on days 1 and 8 of 21-day cycle). The RP2D was determined as the dose level at which Results As of 16 July 2013, 35 pts were enrolled (28 DLBCL, 1 transformed follicular, 1 Burkitt, and 5 mantle cell) with data entered for 32 pts (21 male) at time of data cut-off (part 1, n=13; part 2, n=19 [MLN8237: 30 mg, n=4; 40 mg n=12; 50 mg, n=3]). 1 pt in the MR cohort and 8 pts in the MRV cohorts continue on treatment. Pt demographics, exposure, and safety data are shown in the table. MR was well tolerated with 1 DLT; therefore M 50 mg BID x 7 d + R 375mg/m2 was the recommended regimen for this doublet. For the MRV triplet the RP2D was M 40 mg BID. Common grade (G) ≥3 drug-related AEs across the cohorts included: thrombocytopenia, leukopenia, neutropenia, fatigue and anemia. To date, 26 pts have discontinued treatment (15 progressive disease [PD], 5 AEs [including 2 unrelated deaths, unrelated G4 T7 spinal cord mass and compression, G1 related pulmonary fibrosis, G2 unrelated cough and dysphagia], 3 symptomatic deterioration, 2 other, 1 withdrawal by subject). An additional unrelated, on-study death occurred in a pt who had discontinued treatment due to PD. At the MR RP2D combination, MLN8237 median Tmax was 2 hr and geometric mean steady-state AUC0-12hr was 18,960 nM*hr (n=11, CV: 61.2%), which is consistent with what was achieved in M single agent at 50 mg BID. Assessment of the effect of MLN8237 on vincristine PK is ongoing. 12 pts administered MR were response evaluable; 1 50 y male pt with DLBCL had a complete response (CR), 2 pts had partial response (PR) including one 79 y male pt with DLBCL ongoing at 29 cycles, and 5 pts had stable disease (SD) for up to 13 cycles. 8 MRV pts are currently response evaluable (M 30 mg, n=3; 40 mg, n=5); one 78 y male pt with DLBCL in the 30 mg cohort had CR; at the RP2D (40 mg BID cohort) one 71 y male with non- germinal center B-cell-like DLBCL and having progressed following prior transplant had CR; one 66 y male pt with large B-cell had PR, 5 pts had SD. Response assessments are pending for 5 pts. Enrollment continues to the MRV RP2D to complete the required number of PK-evaluable pts. Conclusions MR ± V is overall well tolerated and has shown preliminary anti-tumor activity in pts with relapsed/refractory B-cell lymphomas. Disclosures: Off Label Use: Use of the investigational agent MLN8237 in combination in patients with aggressive B-cell NHL. Persky:Millennium: The Takeda Oncology Company: Research Funding. Mahadevan:Novartis: Honoraria; Millennium: The Takeda Oncology Company: Honoraria. Miller:Spectrum: Research Funding; Abbott Laboratories: Research Funding. Hayslip:Celgene: Research Funding. Park:TEVA: Research Funding; Seattle Genetics, Inc.: Research Funding. Ruan:Seattle Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Rosen:Disney Family Cancer Center: Research Funding. Padmanabhan Iyer:Millennium: The Takeda Oncology Company: Research Funding. Zhang:Takeda Pharmaceutical Company Ltd.: Equity Ownership; Millennium: The Takeda Oncology Company: Employment. Zhou:Millennium: The Takeda Oncology Company: Employment. Dansky Ullmann:Millennium: The Takeda Oncology Company: Employment. Leonard:Millennium: The Takeda Oncology Company: Employment, Equity Ownership.

Introduction CC-292, an oral, highly selective, small-molecule irreversible-inhibitor of Btk is under investigation for the treatment of CLL and other B-cell malignancies. This phase 1 trial investigated the safety, dose limiting toxicities (DLT), and clinical activity of CC-292 monotherapy in subjects with relapsed or refractory (R/R) CLL or non-Hodgkin's lymphoma. This interim analysis focused on the safety and clinical activity in subjects with CLL and small cell lymphocytic leukemia (SLL). Methods Eligible subjects with R/R (≥ 1 prior therapy) CLL/SLL were treated with monotherapy CC-292 in a dose-escalation study with doses ranging from 125 mg to 1000 mg QD and BID dose levels of 375 mg and 500 mg. As a maximum tolerated dose was not established, CLL patients have been enrolled in an early dose expansion cohort of 750 mg QD and preliminary recommended phase 2 dose expansion cohort at 500 mg BID. All subjects received continuous dosing in 28-day cycles until progressive disease or intolerable toxicity. Clinical activity was investigator assessed per the 2008 iwCLL criteria. Results 83 subjects with R/R CLL/SLL have been enrolled as of June 30, 2013. Baseline characteristics include median age of 67 years (34-89), 52% Rai stage 3/4 disease, median 3 prior therapies (1-12), and 34% refractory to last treatment. Poor-risk factors were present in 67% of subjects, including del11q (22%), del17p (24%), and unmutated IgVH (54%). 67% of subjects remain on study. Subjects have received a median of 6 treatment cycles (0.2-21.6). The most frequent grade 3/4 adverse events (AEs) included neutropenia (21%), thrombocytopenia (15%), pneumonia (10%), and anemia (8%). Rates of febrile neutropenia were low (4%). The most common treatment-emergent AEs (≥ 10% of subjects) were diarrhea (59.7%), fatigue (37.5%), neutropenia (26.4%), thrombocytopenia (26.4%), nausea (26.4%), pyrexia (22.2%), headache (19.4%), cough (19.4%), upper respiratory infection (16.7%), peripheral edema (15.3%), abdominal pain (15.3%), dizziness (13.9%), muscle spasms (13.9%), contusion (13.9%), anemia (12.5%), pneumonia (12.5%), sinusitis (12.5%), and urinary tract infection (11.1%). One CLL patient at the 500 mg BID dose level has experienced a drug-related adverse event of grade 4 thrombocytopenia during Cycle 1 that was assessed as a DLT. The number of patients discontinuing study treatment due to AEs was low (2.4%). Results are summarized for efficacy-evaluable patients treated at the 4 dose levels where at least a partial response (PR) was achieved (750 mg, 1000 mg, 375 mg bid and 500 mg bid) (n = 55). During cycle 1, 89% experienced a ≥ 25% increase in absolute lymphocyte count (ALC), which usually resolved with continued treatment. The table below details the best responses achieved to date. In subjects with del11q, del17p, unmutated IgVH, and no high-risk genomic factors, the PR rate was 42% (5/12), 25% (3/12), 44% (7/16), and 47% (7/15), respectively. Importantly, nodal responses were induced in the majority of subjects receiving BID dosing (375 mg: 67%; 500 mg: 62%) with an overall PR rate of 40%. When achieved, nodal responses were typically observed by cycle 2 and were sustained with continued treatment. Although the sample size is small, subjects treated at 375 mg or 500 mg BID showed continued lymph node size reduction over time from cycle 2 (mean reduction of 42% and 45%, respectively) to cycle 7 (mean reduction of 60% and 71%, respectively). Conclusion CC-292 is well tolerated as an oral daily therapy. Single-agent therapy with CC-292 is sufficient to achieve high nodal and partial response rates in relapsed/refractory CLL subjects, including those with high-risk genomic features. These results support continued development of CC-292 for the treatment of patients with CLL/SLL. Disclosures: Brown: Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding. Off Label Use: The abstract shows scientific information on CC292 which is an investigational product developed by Celgene. This investigational product is not approved by any health authority for any indication. Harb:Celgene: Research Funding. Hill:Celgene: Honoraria, Research Funding. Sharman:Celgene: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Cylene Pharmaceuticals: Consultancy, Research Funding; Avila Pharmaceuticals: Consultancy, Research Funding. Barr:Celgene: Consultancy. Foran:Celgene: Research Funding. Burger:Pharmalytics: Research Funding; Gilead: Research Funding. Mahadevan:Novartis: Speakers Bureau; Millennium: Speakers Bureau. Ma:Genentech: Consultancy; Abbvie: Consultancy. Barnett:Celgene: Employment, Equity Ownership. Marine:Celgene: Employment, Equity Ownership. Nava-Parada:Celgene: Employment, Equity Ownership. Azaryan:Celgene: Employment, Equity Ownership. Mei:Celgene: Employment, Equity Ownership. Kipps:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 3923 Background: BCL-2 is highly expressed in indolent non-Hodgkin lymphomas (NHL), mantle cell lymphoma (MCL) and other selected aggressive lymphomas, and is a promising target for therapeutic intervention. The first-generation BCL-2 inhibitor navitoclax showed some activity in indolent lymphoma, but its co-inhibition of BCL-xL resulted in dose-limiting thrombocytopenia, precluding the full exploration of the potential of BCL-2 inhibition with this drug in NHL. ABT-199 is an orally bioavailable, second-generation BH3-mimetic that inhibits BCL-2 (Ki Methods: This is a phase-I dose-escalation trial using a modified Fibonacci design in patients with relapsed/refractory NHL. The primary objectives of this study are to determine the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of ABT-199; to recommend a phase-2 dose; and to assess efficacy and biomarkers in patients with relapsed/refractory NHL. Adult patients requiring therapy, with ECOG performance status £1, and adequate marrow function received ABT-199 on Week 1 Day −7 (W1D-7), followed by continuous once-daily dosing from W1D1, until progressive disease (PD) or unacceptable toxicity. Due to concerns of potential tumor lysis, a strategy of commencing with a 2 to 3 week lead-in period with step-wise increases to the target cohort dose is being evaluated. In the first four cohorts, the starting dose increased from 50 to 200 mg (50, 100, 200, and 200 mg, respectively), with target cohort doses of 200 mg [n=3], 300 mg [n=3], 400 mg [n=4], and 600 mg [n=7]. Evaluations include: adverse events (AE; NCI-CTCAE-V4) and tumor response (IWG 2007 criteria). Results: To date, 17 patients (median age, 71 [35–85]) have been treated with ABT-199. Median prior therapies were 3 (range, 1–7) and 6 patients had bulky adenopathy (>5cm). Most common AEs (experienced by >2 patients) were nausea (41%), diarrhea (24%), dyspepsia (24%), fatigue (24%), extremity pain (24%); and anemia, constipation, upper respiratory tract infection and cough (18% each). Grade 3 or 4 AEs occurring in >1 patient were anemia (18%) and neutropenia (12%). Treatment-related thrombocytopenia has not been reported and no dose-limiting toxicities (DLTs) have been identified to date. After a single dose administration with a high-fat meal, ABT-199 reached Cmax at approximately 7 hrs with a terminal half-life of about 15 hrs. Food increased ABT-199 exposure by approximately 3-fold. With a median follow-up of 2.8 months (range, 1.2 to 10.8), 14 patients remain on study and 3 have discontinued due to PD. In patients who have completed at least a W6 assessment, reductions of >50% in target lesions have been observed in 8/15 patients (53%); 6/6 patients with MCL, 1/2 patients with WM and 1/2 patients with DLBCL. Additionally, 5 FL patients have been evaluated (3 with rituxan-refractory disease) with a median time on study of 6.4 months (range, 3.5 to 10.8). 4/5 FL patients had nodal disease reductions ranging from 18% to 40%. Conclusions: ABT-199 shows single agent anti-tumor activity in patients with NHL; particularly in MCL. Activity is also observed in DLBCL and WM. To date, no DLTs have been identified and tumor lysis syndrome related to ABT-199 has not been reported. Dose escalation is continuing to identify the optimal dosing regimen and MTD of ABT-199 in NHL. Updated results will be presented. Disclosures: Seymour: Roche: Advisory Board member Other, Consultancy; Genentech: Advisory Board member, Advisory Board member Other, Consultancy. Anderson:Abbott: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Kipps:Abbott: Consultancy, Research Funding. Wierda:Abbott: Research Funding; Genentech: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding; AmGen: Research Funding; Merck: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; Genzyme: Consultancy. Kahl:Abbott: Research Funding; Genentech: Consultancy, Research Funding. Miller:Abbott: Research Funding; Genentech: Research Funding. Darden:Abbott: Employment, owner of Abbott stock Other. Nolan:Abbott: Employment, own Abbott stock Other. Gressick:Abbott: Employment, stock owner Other. Xiong:Abbott: Employment, own Abbott stock Other. Huang:Genentech: Research Funding; Abbott: Research Funding; Walter and Eliza Hall Institiute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Chyla:Abbott: Employment, Stock owner Other. Busman:Abbott: Employment, Stock owner Other. Graham:Abbott: Employment, Stock owner Other. Cerri:Abbott: Employment, Stock owner Other. Enschede:Abbott: Employment, own Abbott stocks Other. Humerickhouse:Abbott: Employment, own Abbott stocks Other. Roberts:Abbott: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, Receives commercial income related to ABT-199, Receives commercial income related to ABT-199 Other.

Abstract 98 Background: Advanced follicular lymphomas (FL) are incurable with conventional chemotherapy and there is no consensus on the best treatment approach. The SWOG cancer research cooperative group and Cancer and Leukemia Group B (CALGB) compared the safety and efficacy of two immunochemotherapy regimens for FL in a Phase III randomized intergroup protocol (S0016) that enrolled 554 patients with previously untreated, advanced stage FL between 3/1/2001 and 9/15/2008. Methods: Pts were eligible if they had advanced stage (bulky stage II, III or IV) evaluable FL of any grade (1, 2, or 3) and had not received any prior therapy. In one arm (CHOP-R), patients received 6 cycles of CHOP chemotherapy (750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, 1.4 mg/m2 vincristine, and 100 mg prednisone daily for 5 days) at 3 week intervals with 6 doses of rituximab anti-CD20 antibody (375 mg/m2 on days 1, 6, 48, 90, 134 and 141 according to the schedule described by Czuczman et al.[J.Clin.Oncol 17:268, 1999]). In the second arm of the protocol, patients received 6 cycles of CHOP, followed by a dosimetric infusion of tositumomab/iodine I-131 tositumomab and then 1–2 weeks later a therapeutic infusion of I-131-tositumomab labeled with sufficient I-131 (median 85 mCi) to deliver a total body dose of 75 cGy (CHOP-RIT). The study was designed to have 86% power to detect a hazard ratio (HR) of CHOP-RIT to CHOP-R of 0.67 for 2 yr PFS based on a one-sided.021 level test (accounting for 3 interim analyses). Results: Of the 554 enrolled pts, 532 were eligible and 526 were evaluable for toxicity (263 on each arm of the protocol). Pt characteristics (age, sex, race, stage, beta 2 microglobulin level, tumor bulk, B symptoms) were well-balanced in the two arms of the protocol. In general, both regimens were well-tolerated (Table I). Median follow-up time among patients still alive is 4.9 years. One hundred and six of 267 eligible pts on the CHOP-R arm have progressed or died compared to 86 of 265 eligible pts on the CHOP-RIT arm. The 2 year estimate of PFS was 76% on the CHOP-R arm and 80% on the CHOP-RIT arm (Figure 1). In multivariate Cox regression adjusting for the stratification factor (serum beta-2 microglobulin level), the hazard ratio for CHOP-RIT vs. CHOP-R was 0.79 (95% CI: 0.60–1.05, p=.11 [2-sided] or p=.06 [1-sided]). Twenty-six of 267 pts on the CHOP-R arm have died compared to 40 of 265 eligible pts on the CHOP-RIT arm. The 2-year estimate of overall survival was 97% on the CHOP-R arm and 93% on the CHOP-RIT arm. In multivariate Cox regression adjusting for the stratification factor serum beta-2 microglobulin, the hazard ratio for CHOP-RIT vs. CHOP-R was 1.55 (95% CI: 0.95–2.54, p=.08 [2-sided]). Conclusion: No statistically significant differences in PFS, OS, or serious toxicities are yet demonstrable with either regimen administered in this trial. However, PFS and OS are outstanding with either of the two regimens with median times to progression not yet reached for either treatment. Future studies will be needed to assess whether combining CHOP-R with RIT consolidation and with maintenance rituximab will confer additive benefit, as being evaluated in a follow-up trial (SWOG protocol S0801) that has recently completed accrual. Disclosures: Press: Spectrum: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Off Label Use: Front-line use of I-131-tositumomab for consolidation therapy in 1st remission of follicular lymphoma. Friedberg:Genentech: Consultancy, Honoraria. Czuczman:Glaxo Smith Kline: Consultancy, Research Funding; Genentech: Consultancy, Honoraria. Kaminski:Glaxo Smith Kline: Patents & Royalties. Maloney:Genentech/Roche: Consultancy, Honoraria, Speakers Bureau; Glaxo Smith Kline: Consultancy, Honoraria, Speakers Bureau. Cheson:Glaxo Smith Kline: Research Funding. Fisher:Roche: Consultancy, Honoraria.

Abstract 2721 Most aggressive B-cell non-Hodgkin lymphomas (B-NHL) are not curable with current chemo-immunotherapy combinations. Synthetic lethal interactions with novel agents may yield effective combination therapies with minimal toxicity for aggressive B-NHL. Auroras (A and B) are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. Aberrant over-expression of Auroras leads to genetic instability, polyploidy, tumor initiation and progression. Over-expression of Auroras in aggressive B-NHL promotes resistance to microtubule targeted agents (MTA, taxanes and vinca alkaloids). Si-RNA knockdown or pharmacologic inhibition of Aurora with MLN8237 [M], an ATP-site small molecule inhibitor, leads to enhanced sensitivity of B-NHL cells to MTAs. We hypothesized that promotion of microtubule de-polymerization with vincristine [V] would be more effective in synergizing with M than a microtubule polymerizing agent docetaxel [D]. We demonstrated that M plus V is synergistic while M plus D is additive in B-NHL cell culture models. Further, the addition of rituximab [R] enhanced apoptosis of B-NHL cells treated with MV or MD therapy. Mouse xenograft models of mantle cell lymphoma show modest single agent activity for M, R, D and V with tumor growth inhibition (TGI) of ∼10–15% (p=0.01). Of the doublets, MV caused tumor regression while MD and MR caused TGI (∼55–60%) and (∼20–25% (p=0.001) respectively. Although MV caused tumor regression, mice relapsed after 2 weeks of stopping therapy. In contrast, MVR had no relapses 120 days after stopping therapy, while MDR led to TGI of ∼85% (p=0.001). Kaplan-Meier analysis of overall survival showed that the mice treated with MV and MVR had a statistically significant improvement in overall survival when compared with the control (p Disclosures: No relevant conflicts of interest to declare.

Abstract 321 Gene expression profiling of newly diagnosed diffuse large B-cell lymphomas shows a correlation between the expression of oxidative stress-related genes and patient prognosis following chemotherapy. Anthracyclines and cyclophosphamide used in lymphoma therapy are known to work, at least in part, through oxidative stress. We hypothesized that single nucleotide polymorphisms (SNPs) in oxidative stress-related genes may contribute to clinical outcomes and/or the development of hematologic toxicities for patients treated with curative intent anthracycline-based therapies for aggressive B-cell Non-Hodgkin lymphomas. Our study involved 473 patients enrolled in 7 prior Phase II or Phase III Southwest Oncology Group clinical trials. DNA for the SNP analyses was obtained from banked, paraffin-embedded diagnostic tissue. Genotyping was performed for 72 SNPs in 33 oxidative stress-related genes. After excluding pathologically or clinically ineligible patients and those with SNP call rates Overall Survival Progression-Free Survival Gene Genotype N Adjusted HR 95% CI P value* Adjusted HR 95% CI P value* AKR1C3 CC 25 1.0 (ref) 1.0 (ref) CT 75 0.40 0.23-0.72 0.0021 0.36 0.21-0.64 0.00042 TT 237 0.45 0.27-0.74 0.0018 0.48 0.29-0.78 0.0028 NOS2 AA 32 1.0 (ref) 1.0 (ref) AG 16 0.54 0.24-1.23 0.14 0.52 0.24-1.13 0.098 GG 284 0.52 0.32-0.86 0.01 0.55 0.35-0.87 0.011 MPO AA 205 1.0 (ref) 1.0 (ref) AG 113 1.31 0.93-1.84 0.12 1.17 0.86-1.61 0.32 GG 22 2.54 1.50-4.30 0.0005 1.96 1.18-3.24 0.0093 * P values not adjusted for multiple comparisons. Disclosures: No relevant conflicts of interest to declare.

Abstract 3933 Oncogenic Auroras (A and B) are serine/threonine kinases that play key roles in mitotic initiation/progression and spindle assembly checkpoint (SAC) activity during the mammalian cell cycle. Over-expression of Auroras overrides the SAC that leads to aneuploidy in human malignancies including non-Hodgkin lymphomas (NHL). Our previous study utilizing gene expression profiling, tissue microarray and siRNA knockdown in aggressive B- NHL demonstrated Auroras to be markers of poor prognosis and validated therapeutic targets respectively (ASH 2009). Hence we hypothesized that (1) targeting mitosis/SAC with MLN8237 an Aurora inhibitor and a microtubule targeting agent [MTA] (vincristine or docetaxel) are effective in promoting apoptosis and tumor growth inhibition (TGI) in mouse xenograft model(s) of mantle cell lymphoma (MCL) and (2) targeting the proliferative marker CD20 with rituximab when combined with MLN8237 plus vincristine or docetaxel would further enhance apoptosis and manifest synergy of triplet therapy. In several B-NHL cell culture models single agent pro-apoptotic activity of MLN8237, vincristine, docetaxel and rituximab were quantified across multiple exposures (5 to 50 nM) by utilizing flow cytometry. Next, pro-apoptotic activity was investigated after exposure to multiple doublet and triplets, including MLN8237 + vincristine + rituximab and MLN8237 + docetaxel + rituximab. Triplet therapy was significantly superior to doublet combinations (MLN8237 + rituximab, MLN8237 + docetaxel, MLN8237 + vincristine). PARP cleavage studies of the doublets and the triplets confirmed enhanced apoptosis observed by flow cytometry. Anti-tumor activity of rituximab in doublet and triplet combinations were then evaluated in vivo in mouse xenograft models of MCL (Granta-519). The first mouse model evaluated MLN8237 (10 or 30 mg/kg, QD for 3 weeks) alone, rituximab (10 mg/kg, Q1W x3) alone, rituximab (10 mg/kg, Q1Wx3) + MLN8237 (10 or 30 mg/kg, QD for 3 weeks) combination versus control. Tumor growth inhibition (TGI) was 0% for MLN8237, 25% for rituximab and 35% for MLN8237 + rituximab with an overall survival (OS) ∼15 days longer for the combination over control (p=0.009). The second mouse model evaluated treatment with MLN8237 (30 mg/kg, QD for 3 weeks) + rituximab (10 mg/kg, Q1W x3), MLN8237 (30 mg/kg, QD for 3 weeks) + docetaxel (10 mg/kg, Q1W x3), MLN8237 (30 mg/kg, QD for 3 weeks) + docetaxel (10 mg/kg, Q1W x3) + rituximab (10 mg/kg, Q1W x3) which showed TGI of 25%, 62% and 85% over control (p=0.005) respectively. The overall survival of >40 days, increased apoptosis (cleaved caspase 3) and decreased proliferation (Ki-67) significantly favoured triplet therapy. Gene expression profiling studies of harvested tumors at the end of treatment are ongoing to gain mechanistic insights into the synergy observed with triplet therapy. Also a mouse xenograft model evaluating anti-lymphoma activity of MLN8237 + vincristine + rituximab is ongoing. In conclusion, our data indicate that MLN8237 plus rituximab is equally effective when combined with either vincristine or docetaxel. Therefore, targeting two hallmarks of cancer, limitless replicative potential and proliferation using MLN8237 + MTA and rituximab respectively promotes enhanced apoptosis and may offer a promising treatment strategy for patients with aggressive B-cell NHL [Funded by the Lymphoma SPORE, P50 CA130805501A1]. Disclosures: Persky: Millennium, Takeda: Consultancy, Research Funding.

Abstract 3930 Aurora kinases (A and B) are oncogenic serine/threonine kinases that play a central role in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are over-expressed in numerous tumors including aggressive B-cell non-Hodgkin's lymphomas (B-NHL) and are considered validated oncology targets. AT9283 a pan-Aurora/JAK-2 inhibitor has undergone early phase trials in acute and chronic myeloid leukemia with promising anti-tumor activity. Hence, we hypothesized that 1) targeting mitosis with AT9283 would be effective in promoting apoptosis in aggressive B-NHL cell lines, and 2) addition of a microtubule targeting agent activating the spindle assembly checkpoint would lead to enhanced anti-tumor activity in a mouse xenograft model of mantle cell lymphoma (MCL). AT9283 inhibited Aurora A and B activity as demonstrated by reducing phosphorylation of Aurora A and histone H3 as well as inhibition of cell proliferation (IC50 of 0.02–1.6 μM) in a variety of aggressive B-NHL cell lines (diffuse large B-cell lymphoma, MCL and transformed follicular lymphoma). B-NHL cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora kinase inhibitor. Treatment of B-NHL cells with AT9283 induced apoptosis demonstrated by flow cytometry and PARP-cleavage in a dose and time dependent manner. Aurora A over-expression has been demonstrated to override the spindle assembly checkpoint (SAC) and result in resistance to microtubule targeting agent (e.g. taxols and vinca alkaloids) induced apoptosis. At very low doses (5-50 nM) apoptosis was almost doubled in the combination (30%) compared to AT9283 or docetaxel alone (15%) compared to controls. A mouse xenograft MCL model (Granta-519) demonstrated that AT9283 (10 and 15 mg/kg) or docetaxel (10 mg/kg) alone had modest anti-tumor activity (10-20% tumor growth inhibition). However, AT9283 plus docetaxel demonstrated a statistically significant tumor growth inhibition of >60% over control. The body weights of all mice in all cohorts did not change significantly (within 10%) during the study and mice appeared to tolerate treatment(s) well. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in aggressive B-cell NHL and warrant early phase clinical trial evaluation [Funded by the Lymphoma SPORE, P50 CA130805501A1]. Disclosures: No relevant conflicts of interest to declare.

Abstract 591 Introduction: VEGF and VEGF receptors (VEGFR) are frequently over-expressed in DLBCL. Elevated levels of circulating VEGF have also been associated with a worse prognosis and resistance to chemotherapy. This trial was initiated to determine if the addition of anti-VEGF targeted therapy to standard R-CHOP would improve PFS without adding significant toxicity in previously untreated patients with stage III, IV or bulky stage II DLBCL. Methods: 64 eligible patients were treated with standard dose R-CHOP plus bevacizumab administered at 15mg/kg on day1. Patients received therapy every 21 days for a maximum of 8 cycles. Both biologics were administered prior to chemotherapy. Pretreatment tumor biopsies from 40 patients were submitted for construction of tissue microarrays for VEGF/VEGFR immunohistochemical analysis. Two 1 mm tissue cores were assessed for each DLBCL specimen and expression of VEGF and VEGFR scored on a scale of 0 (no expression) to +3 (strong expression) by 2 pathologists. Both lymphoma and endothelial cell (blood vessel) staining was assessed with the higher score being recorded. Results: 73 patients were enrolled with 9 patients found ineligible, the majority (n = 7) secondary to incorrect histology. Patient characteristics for the 64 eligible patients included: median age 68 years with 77% of the patients over 60 years (age range: 22–85), 44 males, IPI scores: 3 low, 22 low-intermediate, 29 high-intermediate, and 8 high. Median follow-up is 2.0 years. Based on prior studies, and adjusted for the observed IPI factor frequencies seen in this study, the projected 1 year historical PFS rate in this population would be 71%, while the observed 1 year PFS estimate was 77% (95% CI: 66 – 87%). The PFS estimate at 2-years was 69% (95% CI: 57 – 80%). The 1 and 2 year overall survival estimates were 86% and 79%, respectively. Among all patients (including ineligibles), a total of 30 patients experienced 40 serious adverse events (SAE) while on study. The majority of SAE were hematologic, but also included 2 patients with sudden death, 5 patients with GI perforations (4 occurring after cycle 1), and 5 patients with grade 3 or 4 thrombotic events. Additional adverse events of interest included 7 patients who developed hypertension (4 patients with grade 3), and 13 patients who developed grade 2 or 3 left ventricular dysfunction. VEGF expression was observed in 98% (IHC scoring: +1 in 18%, +2 in 38%, and +3 in 43%) of lymphoma cells and VEGFR was expressed in 95% (IHC scoring:+1 in 28%, +2 in 40%, and +3 in 28%) of blood vessels by immunohistochemistry analysis. Conclusions: Despite high protein expression of VEGF and VEGFR in newly diagnosed DLBCL specimens, the addition of bevacizumab to standard R-CHOP chemotherapy did not significantly improve PFS from expected results with R-CHOP alone. Although the observed 1-year PFS estimate trended higher than the historical estimate, a target PFS of 81% was pre-specified as warranting further investigation. Significant toxicities were associated with the addition of bevacizumab, including an increased incidence of cardiac dysfunction and GI perforation. Our results do not support additional studies of this regimen in this patient population. Disclosures: Stopeck: Genentech: Consultancy, Honoraria, Speakers Bureau. Off Label Use: Bevacizumab therapy in NHL. Fisher:Roche: Consultancy.

Abstract 1768 Background: Although advances in therapy have improved outcomes for patients (pts) with FL, no optimal patient management strategy for pts with FL grade 3 has been identified largely due to debate regarding the benefits of anthracyclines for this subtype. Methods: The NLCS is a multi-center, longitudinal, observational study that collects data on treatment and outcomes for pts with FL diagnosed at 265 community (80%) and academic practices in the United States from 2004–2007. Pts enrolled in NLCS with a diagnosis of FL grade 3 as recorded by the treating physicians were examined to compare patterns of presentation, treatment strategies, and outcomes. No central pathology review was required. Data from pts who received watchful waiting (WW), single agent rituximab (R), R with cyclophosphamide, adriamycin, vincristine, and prednisone (R-CHOP), or R with cyclophosphamide, vincristine, and prednisone (R-CVP) as initial therapy were summarized using median and range for continuous variables and frequencies for categorical variables. Univariate associations between demographic, baseline disease characteristics, treatment setting, and initial treatment strategy, were tested using a standard χ2 test where sample size allowed. Relationship and hazard ratios for progression free survival (PFS) were estimated using Cox regression models adjusted for FL International Prognostic Index (FLIPI). Results: Of 2736 pts enrolled in NLCS, 500 had grade 3 FL. FL grade 3 pts had a median age of 64 years (range 26–97 years), 53% were female, and 67% had stage 3/4 disease. Pts were evenly distributed across FLIPI categories (30% good, 33% intermediate and 37% poor risk). Initial therapies for pts with FL grade 3 were WW (n=48), R alone (n=41), R-CVP (n=30), R-CHOP (n=245), R with another anthracycline (n=26), R with other chemotherapy (n=28), radiation alone (n=14), combined modality therapy (n=26), and regimens without R (n=42). Pts with FL grade 3 who received R-CHOP were younger than those who received R-CVP but similar in other FLIPI factors (Table). Events, defined as death or disease progression, occurred for 58% of patients who watched and waited and 33% of those who received treatment. Treated patients had improved PFS (median not reached [NR] 95% CI 66 months-NR) versus WW (median 29 months; 95% CI 19–43 months). Pts who received R-CHOP had improved PFS compared with WW (hazard ratio [HR] 3.09 95% CI 1.97– 4.86) but not R-CVP (HR 1.06 95% CI 0.52–2.14; Figure) Conclusions: In this large observational dataset of FL grade 3 patients, R-CHOP was the most commonly used first-line regimen. These results suggest that R with chemotherapy provides meaningful PFS benefits over WW for this population. Prospective randomized trials in pathologically identified pts are needed to evaluate the benefit of R-CHOP versus non-anthracycline regimens. Disclosures: Flowers: Millenium: Research Funding; Prescription Solutions: Consultancy; Genentech: Consultancy; Biogen/Idec: Consultancy; Celgene: Consultancy. Taylor:Genentech: Consultancy; Roche: Stock Ownership. Byrtek:Genetech: Consultancy; Roche: Stock Ownership. Hirata:Genentech: Employment; Roche: Stock Ownership. Cerhan:Genentech: Consultancy, Honoraria, Research Funding. Hainsworth:Genentech: Consultancy, Research Funding. Link:Genentech: Consultancy, Research Funding; Biogen Idec: Consultancy, Research Funding; Millennium Pharm: Consultancy, Research Funding; GlaxoSmithKline: Research Funding. Friedberg:Genentech: Consultancy.

Abstract 620 Classification of DLBCL into cell-of-origin (COO) subtypes based on gene expression profiles has well-established prognostic value. These subtypes, termed Germinal Center B cell (GCB) and Activated B cell (ABC) also have different genetic alterations and over-expression of different pathways that may serve as therapeutic targets. Thus, accurate classification is essential for analysis of clinical trial results and planning new trials using targeted agents. The gold standard for COO classification uses gene expression profiling (GEP) of snap frozen tissues, and a Bayesian predictor algorithm utilizing the expression levels of 14 key genes (G. Wright et al PNAS 2003). An immunohistochemistry (IHC) classification scheme by C. Hans et al (Blood 2004), based on 3 antibodies, is widely used as a substitute for GEP classification, however does not completely correlate with GEP. We recently described a qNPA assay (ArrayPlateR, High ThroughPut Genomics, Tucson, AZ) with excellent correlation between frozen and formalin fixed paraffin embedded (FFPE) tissues (R. Roberts et al, Lab Invest 2007). In this study, we investigated whether this technique could be used for accurate classification of COO using FFPE tissues. We expanded the previous gene probe repertoire of the DLBCL-ArrayPlateR assay to include the 14 genes (represented by 17 probe sets) most pertinent to COO classification. 52 cases of R-CHOP treated DLBCL that had undergone GEP using the Affymetrix U133 Plus 2.0 microarray and had matching FFPE blocks were analyzed with qNPA in duplicate. The genes included CD10, LRMP, CCND2, ITPKB, PIM1, IL16, IRF4, FUT8, BCL6, PTPN1, LM02, CD39, MYBL1, IGHM. Results were evaluated using the previously published algorithm with a leave-one-out cross validation scheme to classify cases into GCB or ABC subtypes. These results were compared to COO classification based on frozen tissue GEP profiles. All 14 genes in all 52 cases were successfully analyzed with no missing data points. For each case, a probability statistic was generated indicating the likelihood that the classification using qNPA was accurate. Of the 54 cases, 25 were GCB, 27 were ABC and 4 were unclassifiable by GEP. Of the GCB cases, 23/25 (92%) were classified correctly by qNPA with a confidence cut-off of >0.9 and 25/25 (100%) classified correctly with a confidence cut-off of >0.8. Of the ABC cases, 25/27 (93%) were correctly classified as ABC using qNPA with a confidence cut-off of >0.9 and 27/27 (100%) classified correctly with a confidence cut-off of >0.8. In summary, the qNPA technique accurately categorized DLBCL into GCB and ABC subtypes, as defined by GEP. There were no technical difficulties with any of the pathological materials although they were collected retrospectively from a variety of institutions and countries with different fixation methods. This approach represents a substantial improvement over previously published IHC methods and is applicable to FFPE tissues, therefore overcoming the need for snap frozen materials. This technically robust classification method has potential to have a significant impact on future DLBCL research and clinical trial development. Disclosures: Rimsza: High Throughput Genomics: HTG provided the assays at no charge to Dr. Rimsza's lab. Schwartz:High Throughput Genomics: Employment. Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 111 Background: Fc gamma receptor (FCGR) polymorphisms have previously been linked to outcome in follicular lymphoma (FL) patients treated with the anti-CD20 monoclonal antibody (mAB), rituximab, as a monotherapy. This is thought to be due to differences in binding affinity of host effector cells for the therapeutic antibody between individuals with different genotypes. In particular, the R/R genotype of FCGR2A and the F/F genotype of FCGR3A have been correlated with a less favorable outcome compared to the higher affinity FCGR genotypes H/H and V/V, respectively. Previous studies have had relatively low statistical power and have used consecutive series of patients and the prognostic significance remains largely untested. Therefore, we questioned whether this association would hold up in the context of a chemotherapy regimen combined with a monoclonal antibody in a larger set of patients treated on Southwest Oncology Group (SWOG) clinical trials. Specifically, we wished to test whether the genotypes were predictive of survival following all treatment regimens, or just the regimens containing mAB. Methods: Excess paraffin embedded tissue from 146 patients previously treated on SWOG clinical trials with chemotherapy alone (protocol S8809, N=70) or combined chemotherapy and mAB (anti-CD20 antibodies rituximab or I-131 tositumomab on protocols S9800 and S9911, N=33 and 43 respectively) were analyzed. DNA was extracted using the Qiagen RNA/DNA kit, then assayed for the R131H polymorphism (rs1801274) in FCGR2A and the V158F polymorphism (rs396991) for FCGR3A using a TaqMan SNP assay. Stratified Cox regression was used to assess marker associations with overall survival (OS). Results: Patients in each clinical trial had similar IPI and FLIPI scores. Overall frequencies of the FCGR2A genotypes were 27% H/H, 54% R/H, 16% R/R. Frequencies of the FCGR3A genotypes were 36% FF, 41% VF, and 8% VV. There were no differences in genotype frequencies between trials and no associations between genotypes. Neither FCGR 2A nor 3A were associated with survival in S8809 (chemotherapy only). In S9800 and S9911 (chemotherapy + mAb), having at least 1 FCGR2A H allele was weakly associated with improved OS vs R/R (hazard ratio [HR] 0.42, 95% confidence interval [CI] 0.16–1.12, p=0.06), and having at least 1 FCGR3A V allele was more strongly associated with improved OS vs F/F (HR 0.28, 95% CI 0.10–0.80, p=0.02), with 5 year OS of 72% for F/F, 97% for V/F, and 100% for V/V. The interaction term between antibody usage and FCGR3A genotype was significant (p=.01), suggesting that the effect of this marker may be truly different in 9800/9911 than in 8809. All survival analyses held up after adjustment for IPI score. Summary: Overall, we found an association of FCGR3A genotype with overall survival in patients treated with chemo + mAB (rituximab or I-131 tositumomab). An association with survival was not identified in patients treated with chemotherapy alone. Interestingly, in the analysis of the overall dataset we found significant evidence of an interaction between mAb therapy (S9800/S9911 vs. S8809) and FCGR3A. This study suggests that FCGR3A polymorphism status may be predictive of survival in FL patients given mAB-containing treatment as opposed to any treatment in general. Therefore, FCGR3A polymorphism status may be an important factor to consider in designing new FL trials with mAB-containing regimens. Disclosures: Persky: Millennium, The Takeda Oncology Company: Consultancy, Research Funding. Dornan:Genentech, Inc.: Employment, Equity Ownership. Fisher:Genentech: Honoraria, Speakers Bureau. Maloney:Genentech, Biogen Idec, GSK: Membership on an entity's Board of Directors or advisory committees. Miller:Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Research Funding; Milleneum: Consultancy. Rimsza:Genentech: Research Funding.

Abstract 284 Aurora kinases (A and B) are oncogenic serine/threonine (S/T) kinases that play central roles in the mitotic phase of the eukaryotic cell cycle. Over-expression of Aurora kinases during the cell cycle over-rides mitotic and spindle check points leading to aneuploidy in many human cancers; Aurora kinases are therefore attractive therapeutic targets. Gene expression profiling in aggressive B- and T-cell non-Hodgkin's lymphoma (NHL) has shown the Aurora kinases to be over-expressed and they may be key component genes of the ‘proliferative' signature. We hypothesized (1) Aurora kinases are over-expressed in human aggressive B-cell NHL (mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (TFL)), (2) Aurora ATP-binding site small molecule inhibitor (SMI) is effective in promoting apoptosis in cell culture and tumor growth inhibition (TGI) in mouse xenograft model(s) of NHL, and (3) Aurora SMI will be safe and effective in treating patients with relapsed aggressive B-cell NHL in early phase clinical trials. To analyze Aurora expression, tissue microarrays (TMA) were constructed from 43 patients with DLBCL and 40 with MCL, and Aurora A and B expression optimized with commercially available antibodies by immunohistochemistry (IHC). The IHC was rated as a staining intensity on a scale of 0 to 3+. The NHL TMAs demonstrated intense staining (2+ to 3+) for Aurora A (nucleus) and Aurora B (nucleus) in >60% compared to normal lymph nodes. We also analyzed the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) publicly available database for Aurora A and B expression in MCL and found a worse survival in those with A > B over-expression (p Western blotting analysis of 13 B-cell NHL cell lines (DLBCL, MCL and TFL) for Aurora A and B expression showed significant over-expression compared to B-cells isolated from normal lymph nodes. Aurora A knockdown by shRNA in the B-NHL cell lines showed inhibition of mitosis with a polyploid phenotype (4n, 8n) that ends in apoptosis as shown by PARP-cleavage. The Aurora A specific inhibitor (MLN8237) evaluated in the 13 NHL cell lines phenocopies shRNA knockdown with associated inhibition of proliferation (IC50=0.05 mM) and promotes apoptosis (flow cytometry, PARP-cleavage) in a dose-dependent manner. Combination of MLN8237 with a microtubule targeting agent (docetaxel) to abrogate the spindle checkpoint is synergistic in a sequence specific manner. Moreover, MLN8237 effectively inhibits Aurora A auto-phosphorylation and eliminates phospho-histone H3 (Ser10) phosphorylation. Currently, 2 mouse MCL (Granta 519) xenograft models are underway evaluating tumor growth inhibition (TGI), safety and survival of MLN8237 alone and in combination with docetaxel or rituximab respectively. Preliminary data show that MLN8237 is synergistic with docetaxel in TGI in the MCL xenograft model. Together the data suggest inhibition of Aurora kinases may offer a promising treatment strategy for patients with aggressive B-cell NHL [Funded by the Lymphoma SPORE, P50 CA130805501A1, PI: Richard Fisher, MD.]. Disclosures: Rimsza: High Throughput Genomics:.

Background: The addition of Rituximab to CHOP chemotherapy (R-CHOP) has significantly improved the overall survival of patients with diffuse large B cell lymphoma (DLBCL) by 10–15%. To evaluate the biological basis of survival for DLBCL, we profiled gene expression in biopsy samples from patients treated with R-CHOP. Methods: Whole genome Affymetrix U133 2.0 plus arrays were used to profile gene expression in pre-treatment biopsies from patients with de novo DLBCL who received CHOP (n=181) or R-CHOP (n=233). Samples were classified as germinal center B cell-like (GCB), activated B cell-like (ABC) or unclassified DLBCL. A multivariate gene expression-based survival predictor was created using CHOP cases as training and R-CHOP cases as validation set. Results: R-CHOP treated patients with GCB DLBCL had a more favorable survival than those with ABC DLBCL, with 3-year overall survival rates of 84% and 56%, respectively (p Conclusion: Survival in DLBCL is critically influenced by the composition of the tumor microenvironment, suggesting that therapeutic interference with microenvironmental interactions might further improve outcome in DLBCL. Specifically, inhibition of angiogenesis may be beneficial for a subset of patients with high relative expression of the stromal-2 signature and increased tumor blood vessel density.

Galiximab is an anti-CD80 monoclonal antibody with human IgG1 constant regions and macaque variable regions. CD80 is an immune costimulatory molecule that is constitutively expressed on the surface of follicular lymphomas. Modest single-agent clinical activity (ORR 11%) was demonstrated in a single agent Phase I study of galiximab in relapsed/refractory, follicular NHL with the observation of late and prolonged responses. We previously conducted a phase II multicenter study evaluating the combination of galiximab and rituximab for relapsed/refractory follicular NHL, and demonstrated an ORR of 64% (17% CR, 14% CRu, and 33% PR). Here, we analyze long-term safety and efficacy data from this trial. Patients received galiximab (500 mg/m2 qwk x 4 weeks) concurrently with a standard course of rituximab (375 mg/m2 qwk x 4 weeks). No maintenance therapy was allowed. Sixty-four patients received treatment. Mean age at study entry was 59 yrs. The majority of patients (88%) were Stage III/IV, and FLIPI risk groups were distributed as low (27%), intermediate (39%), and high (34%). All patients had received at least 1 prior lymphoma therapy; 42% were rituximab naïve; rituximab-refractory patients (no response or a response with TTP 2 years, or TTNT duration of > 2 years (p=NS, chi-square analysis). Obtaining a CR predicted for PFS and TTNT duration >2 years (p=0.001). Response duration to prior therapy was not correlated to outcome following rituximab/galiximab therapy (p=0.18). Quantitative immunoglobulin levels (IgA, IgM and IgG) did not differ between prolonged responders and other patients, and did not significantly change over the first year of follow-up. Median Cmax, half-life, AUC and Cl were similar between prolonged responders and other patients. No late opportunistic infections, secondary malignancies, or infusion-associated deaths have been reported. We conclude that the combination of rituximab and galiximab is well-tolerated in long-term follow-up, with a substantial number of durable responses. Almost one-third of patients treated with only 4 weeks of this combination do not require additional lymphoma therapy for more than three years. There is no clear prognostic marker that predicts for prolonged benefit from the combination. These durable responses provide strong rationale for ongoing phase III clinical trials of the galiximab/rituximab combination.

The 5-year survival rate of patients with limited disease (LD) and aggressive histologies of non-Hodgkin lymphoma (NHL) who have at least one adverse risk factor is about 70% after treatment with three cycles of CHOP followed by involved-field radiotherapy (CHOP(3) plus IFRT). We have previously reported the effect of adding four infusions of rituximab to CHOP(3) plus IFRT demonstrating an improved estimated overall 5-year survival (OS) of 82% (Persky et al. J Clin Oncol26:2258, 2008). In this study we test the effect of adding ibratumomab tiuxetan consolidation to CHOP(3) plus IFRT in 40 evaluable patients. Patients had a biopsy diagnosis (no FNA allowed) of diffuse aggressive B-cell NHL without any follicular component and high risk LD. High risk LD is defined, using the stage-modified International Prognostic Index, as stage I with at least one adverse risk factor (age > 60 years, elevated LDH and a performance status of 2) OR non-bulky stage II disease. Patients with stage I and no risk factors were excluded because their survival exceeds 90% at 10 years using standard therapy. Patients with bulky stage II disease were not eligible because their prognosis is similar to advanced disease. CHOP chemotherapy was given at standard doses on days 1, 22, and 43. IFRT was given as previously described (above) starting on day 64 to include 40 – 46 Gy. The rituximab plus ibritumomab tiuxetan regimen was given 3 – 6 weeks following completion of IFRT over a period of 7 – 9 days. Rituximab, 250 mg/m2 was given on day 1 followed within 4 hours by 5 mCi of In-111 ibritumomab tiuxetan followed by whole body gamma camera images to assess biodistribution of the ibritumomab tiuxetan. Scans were obtained at 2 – 24 hours and at 48 – 72 hours. Following acceptable biodistribution, rituximab is repeated on days 7, 8, or 9 followed within 4 hours by Y-90 ibritumomab tiuxetan, 0.4 mCi/kg, not to exceed 32 mCi total dose. Forty patients are eligible. Patients have been followed for a median of 24 months (range 1 – 50 months). During this time there have been only three events recorded (progression or death from any cause). Estimated 2-year progression-free survival is 91% (95% CI: 81%-100%) and 2-year OS is 95% (95% CI: 87%-100%). Toxicity has been reversible with no treatment related deaths. There were 9 grade 3 and 10 grade 4 hematologic events, but only 3 episodes of febrile neutropenia and only one patient requiring platelet support. Other grade 3 toxicities were mostly gastrointestinal symptoms including mucositis and nausea/vomiting or flu-like symptoms. We conclude that patients with aggressive histologies of NHL and high-risk LD treated with CHOP(3) plus IFRT followed by ibritumomab tiuxetan consolidation result in outcome that compares favorably to our historical experience in the short term. Longer follow-up will determine whether these patients exhibit a pattern of late recurrences as seen in our prior experiences.

Large studies using snap frozen tissue for gene expression profiling (GEP) have identified a limited number of genes predicting outcome in DLBCL treated with CHOP alone (17 genes from Rosenwald et al NEJM 2002, 13 from Shipp et al, Nat Med 2002, 6 from Lossos et al, NEJM 2004, 2 from Tome et al, Blood 2005). Our objective was to test the prognostic value of these genes (38 total, 36 different genes) using FFPE tissues and compare results from patients treated with CHOP or CHOP-like regimen alone (C) (N=93) to those treated with CHOP + rituximab (R) (N=101). We used a multiplexed quantitative nuclease protection assay, the ArrayPlate (High Throughput Genomics), to measure mRNA levels reliably in FFPE samples as recently described. (Roberts et al, Lab Invest, ePub, 13 Aug 2007). Of 194 cases attempted, there was only 1 that did not result in adequately detectable signal. Univariate associations between gene levels and patient OS were performed using Cox proportional hazard regression models. For C alone cases, gene expression levels were significantly correlated with overall survival (OS) at p < 0.05 for 15/36 prognostic genes including the MHCII genes HLA-DR and HLA-DP, as well as C-MYC, BCL-6, BCL-2 and ten other genes. These genes represented all 4 Rosenwald prognostic signatures, 3/6 of Lossos genes, and 4/13 Shipp genes. In the R-treated cases, 8/36 genes correlated with OS including HLA-DR and HLA-DQ, the germinal center B-cell (GCB) genes BCL-6, and SERPINA9 as well as C-MYC, PLAU, PDCD4, and MnSOD. Again, all 4 Rosenwald signatures were represented. In our assay, genes that represent key gene expression signatures previously identified as the most prognostically important in large discovery-oriented GEP experiments were significantly associated with patient OS using FFPE tissues and retained significance in the R-treated patients, suggesting that previous findings remain relevant in the R-CHOP era. For most genes in both treatment groups, the estimated hazard ratios tended in the direction originally predicted, suggesting these genes would be significantly related to survival in a larger, more powered study. Previous studies have relied on unfixed, snap-frozen tissues which are available only in a minority of patients for GEP or quantitative RT-PCR measurements of mRNA expression. The ArrayPlate assay can assess prognostic genes in patients for which only FFPE material is available, is highly correlated with discovery microarray results, and should be easily adaptable as more prognostic genes are identified. Importantly, previously identified prognostically important genes such as MHCII and GCB genes remain correlated with survival in the R-CHOP treatment era.

Background: Chronic lymphocytic leukemia (CLL) is a common leukemia that is incurable. Survival curves for Rai stage 0/I and III/IV are distinct and a specific gene expression signature may be associated with stage progression (SP). However, distinct oncogenic pathways that differentiate stage progression are not well defined. Methods: Mononuclear cells from Rai stage 0/I (N=8) and III/IV (N=8) CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array after isolating and purifying total RNA (Qiagen, RNAeasy). Control RNA samples are from 4 normal volunteers’ peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Statistical significance of individual genes and pathways was analyzed using ‘BioConductor’. Differential gene expression was analyzed using ‘Limma’. Pathways and cytogenetics regions were analyzed for enrichment using ‘Category’. Real time RT-PCR on specific genes was performed to validate the GEP based on stage. A serum cytokine profile (120 cytokines, RayBiotech) on 8 CLL patients (N=4 low risk; N=4 high risk) and 4 normal volunteers was evaluated to identify a signature for diagnosis and prognosis. Results: Data are presented as “robust” increases or decreases of relative gene expression common to 8 patients in the low or high risk stages. The Wnt/Ror-1 non-canonical, and TGFb pathways are over-expressed in Rai stage 0/I, implicating a program of cell survival and dysregulated migration. The Hedgehog, Proteasome, TNF/FAS/NF-kB, and MAPK pathways are over-expressed in Rai stage III/IV, suggestive of a tumor microenvironment driven program in SP. Genes located on chromosome 3q27.1 are also dysregulated in Rai stage III/IV. A distinct serum cytokine profile also differentiates low from high stage CLL. Conclusions: GEP of CLL with RT-PCR validation has identified distinct oncogenic pathways that distinguish Rai stage 0/I from III/IV. A serum cytokine profile also differentiates between low and high risk CLL. These data together provide a ‘molecular signature’ for SP in CLL.

High-dose chemotherapy with autologous HSCT is potentially curative in patients (pts) with relapsed or refractory lymphoma. However, the optimal pre-HSCT conditioning regimen is not known. Mitoxantrone, an antineoplastic agent that inhibits DNA synthesis by inhibition of topoisomerase II and by intercalation into the DNA molecule, has shown activity in several hematological malignancies, including NHL and HD. Recent evidence suggests that mitoxantrone may be more cardiotoxic than previously recognized. We evaluated a novel mitoxantrone-containing preparative regimen for autologous HSCT in pts with relapsed or refractory NHL or HD. Between 1989 and 1996, 59 pts (31 NHL, 28 HD) with chemotherapy refractory or relapsed lymphoma were enrolled. The median age was 36 yr (range, 10–60); 38 pts were male, and 21 were female. Cohorts of pts received escalating doses of mitoxantrone (24–60 mg/m2) on day -8, etoposide 1800 mg/m2 on day -8, cyclophosphamide 1800 mg/m2 on days -7 through -3, and carmustine 300 mg/m2 on day -3. Autologous stem cells were infused on day 0. G-CSF was begun day +3. Patients with areas of bulky disease pre-transplant underwent consolidative involved field radiotherapy, but no earlier than day +14. Results: Patients received mitoxantrone according to the following dose escalation: 24 mg/m2 (n=3), 30 mg/m2 (n=1), 36 mg/m2 (n=3), 42 mg/m2 (n=3), 48 mg/m2 (n=48), 54 mg/m2 (n=1). Median time to neutrophil engraftment was 13 days (range, 8–60 days) and median time to platelet engraftment was 17 days (range, 5–51 days). Non-hematologic toxicities were measured according to the NCI CTCAE Version 3.0. Toxicities greater than grade 2 included mucositis in 35 patients (24 grade 3, 11 grade 4), nausea and vomiting in 9 (6 grade 3, 3 grade 4), diarrhea in 5 (all grade 3), hepatotoxicity in 5 (all grade 3), and nephrotoxicity in 14 (12 grade 3, 2 grade 4). The dose limiting toxicity for mitoxantrone was cardiac toxicity, and the maximum tolerated dose was 48 mg/m2. Early cardiac toxicity was observed in 2 patients: one was withdrawn from the clinical trial before HSCT due to cardiotoxicity, and one patient died on day +8 of cardiotoxicity. Late (≥ 100 days after HSCT) nonfatal cardiac toxicity included cardiomyopathy (n=1), and cardiac tamponade (n=1). For all patients, the 5-year overall survival post HSCT was 51%, and 5-year disease free survival was 37%. Median time to relapse was 24 mo (range, 3.6–71.9 mo) after HSCT. For the 31 patients with NHL, the 5-year overall survival was 48% and 5-year disease-free survival was 35%. For the 28 patients with HD, the 5-year overall survival was 50% and the 5-year disease-free survival was 39%. Conclusion: The addition of mitoxantrone to standard preparative regimens may improve outcomes in patients with relapsed or refractory lymphoma, however further investigations of this agent in pre-HSCT conditioning regimens must carefully consider strategies that minimize the risks of cardiac toxicity.

Low stage DLBCL patients have better prognosis than those with advanced disease. We searched for differences in gene expression between disease stages to find correlations with tumor stage as possible therapeutic targets. We used 2 microarray datasets from the Leukemia/Lymphoma Molecular Profiling Project containing nodal and extra-nodal de novo DLBCL samples on Lymphochip (LC), spotted arrays of ~7400 and Affymetrix arrays (Affy) of ~45,000 elements. The Affy dataset was a subset of the LC samples. LC elements were median-centered, log2 transformed; Affy elements were array-centered to a 500 average. We compared average gene expression in stage I vs. III/IV, looking for elements with >2-fold differences between low and high stage, with p3-fold. A subset of these genes, CTGF (connective tissue growth factor), FN1 (fibronectin), INHBA (activin), POSTN (periostin), THBS1 (thrombospondin), and BCL2, are coregulated and/or coregulatory. Many are expressed in the microenvironment and associated with fibrosis, wound healing and Th2 immune response. The presence of these genes at higher levels and their known associations implied a pathway of TGF-β signaling present mainly in the low stage DLBCL. Although TGF-β levels were not different between stages, differences in localization/activation of TGF-β could account for observed differences. In conclusion, there are a small number of stage-specific gene expression differences in DLBCL, and many of these relate to TGF-β signaling in the microenvironment. CTGF and FN1 were previously identified as key prognostic molecules in DLBCL (Rosenwald et al, NEJM 2002). These differences may relate to different prognoses between DLBCL stages and constitute therapeutic targets. Genes Differentially Expressed by Stage higher in stage I higher in stage III/IV LC Affy LC Affy CTGF CTGF IGHM IBRDC2 (2 elements) SERPINA1 MYBPC1 HTR3A POSTN NCR3 BCL2 FN1 INHBA (2 elements) CRYM THBS1 EMP1 SIX1 KLHL14 MGC23911 PP1665 GPM6A (2 elements) FLJ33069 TUBB IgM rheum. factor RF-TT9 CD1C transcribed locus LOC150568 LOC346887 LOC283454 MGC17624 IMAGE:5311619 transcribed locus (2x)

As previously demonstrated by gene expression profiling, MHCII is an independent gene expression signature in DLBCL, which when expressed at low levels, is associated with poor patient survival. The mechanism of decreased survival in low- expressing DLBCL patients is presumably loss of tumor immunosurveillance. However, the possibility remains that MHCII expression is merely a marker of another aspect of tumor biology such as chemosensitvity, radiation sensitivity, or proliferation rate. MHCII status could also reflect a change in the cellular redox environment since we have previously found an association between decreased antioxidant defense enzyme expression, increased thioredoxin system function and poor prognosis in DLBCL. We therefore investigated whether there are alterations in these other aspects of tumor biology by using a MHCII (−) DLBCL cell line model in which we restored MHCII expression by transfection with CIITA, the master transactivator of MHCII. To do this, we stably transfected the MHCII (−) DLBCL cell line, DB, (American Tissue Culture Corp), with a CIITA expression vector pcDNA 3.1 (InVitrogen) and isolated MHCII (+) clones. The phenotype was confirmed by RT-PCR and flow cytometry using HLA-DR as the representative molecule of MHCII expression (reported as MnX or mean channel of fluorescence). The parental line DB, clones 1–5, 4–3, 6-1, vector only control (pcDNA 3.1), and a positive cell line (Raji), were then subjected to in vitro chemosensitivity assays with the individual components of the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and dexamethasone, the latter was used in the place of prednisone). Chemosensitivity was measured with MTS cell survival assay by calculating the EC 50 (molar concentration of drug required to kill 50% of cells). Radiation sensitivity was measured as percent cell survival 48 hours after exposure to 5 Gy. Proliferation rate was assessed by cell doubling time (in hours) using manual cell counts. The potential of the cells to reduce oxidants was assessed using the AOP-490 kit, which measured anti-oxidant biomarkers as uM uric acid EQ/ug protein (Oxis Research Inc, Portland, OR). No significant differences were detected in sensitivity to any of the tested treatment modalities, including the 4 different chemotherapy agents or radiation, between the parental or transfected clones. Nor were reducing potential or doubling times significantly altered with induction of MHCII expression. Representative data for the DB cell line, vector only control, and a representative clone are listed in Table1. Results for the other clones were similar. Our results help to exclude altered chemosensitivity, radiosensitivity, anti-oxidant potential, or proliferation rates as factors contributing to the unfavorable prognosis in patients with MHCII(−) DLBCL. Therefore, diminished tumor immunosurveillance remains the most likely explanation for the poor outcome in these patients. Table 1 DB parental Vector only Clone 1–5 HLA-DR (MnX) 3 2 500 Dox (EC 50 uM) 4.72 3.61 4.19 Radiation (% ctrl) 49.6 49.14 39.41 Redox potential 33.3 31.2 34.8 Doubling time (hr) 7.19 7.68 7.66

Background Burkitt lymphoma(BL) is a potentially curable, aggressive lymphoma. The distinction between BL and diffuse large B-cell lymphoma (DLBCL) is important because they differ significantly in clinical management. The distinction can be difficult because DLBCL can resemble BL in morphology, immunophenotype and cytogenetics. We investigated whether gene expression profiling (GEP) could create a molecular definition of BL that can reliably distinguish it from DLBCL. Methods Biopsy samples were collected from 312 patients with a diagnosis of sporadic BL or Burkitt-like lymphoma, or DLBCL. All cases were reviewed by a panel of expert hematopathologists. GEP of all the samples was carried out using a specialized oligonucleotide microarray. We constructed a predictor using 197 genes to distinguish BL from each molecular subtype of DLBCL. Leave-one-out cross-validation was used to evaluate the predictor’s performance. Chemotherapy treatments were grouped into either CHOP-like(CHOP, CNOP) or intensive(BFM, CODOX-M IVAC, regimens requiring stem cell rescue). Results After pathology review, the samples were reclassified as:classic BL(25 cases), atypical BL(19), DLBCL(261), and unclassifiable lymphoma(7). All classic BL and 18/19 cases of atypical BL shared a profile that was strikingly different from that of all the molecular subtypes of DLBCL, including those DLBCL cases that have a c-myc translocation. C-myc and its target genes, and genes related to germinal center differentiation were expressed at high levels in BL. NF-kB and its target genes and MHC class-I genes were expressed at very low levels in BL. Interestingly, 10 cases that were DLBCL by pathology were classified as BL by the predictor. The diagnosis of BL was supported by FISH analysis indicating a c-myc translocation. Among adults identified as having BL by the predictor (with full clinical data in N=15), overall survival was markedly superior for those receiving intensive regimens compared to CHOP-like regimens(Fig 1). The groups were similar with regard to age, stage, performance status and sites of involvement. Conclusion This study demonstrates that the molecular characteristics of BL can be used to accurately distinguish it from DLBCL. Importantly, a subgroup of BL was identified by the predictor that could not be diagnosed as BL by conventional criteria. The ability of the predictor to identify patients who benefit from aggressive therapies suggests that it will be useful in the diagnosis and management of patients with Burkitt lymphoma. Figure Figure

Galiximab is a PRIMATIZED®, anti-CD80, monoclonal antibody with human IgG1 constant regions and macaque variable regions. CD80 is an immune costimulatory molecule that is constitutively expressed on the surface of follicular lymphomas. Modest single-agent clinical activity was demonstrated in a Phase I study in relapsed or refractory, follicular NHL (ORR=11%). Preclinical data show that galiximab + rituximab may be more effective than either antibody alone. Here we report updated results from the Phase II part of a multicenter study (Study 114–21) evaluating galiximab + rituximab for relapsed or refractory, follicular NHL. Patients (pts) were administered galiximab (500 mg/m2 qwk x 4) concurrently with a standard course of rituximab (375 mg/m2 qwk x 4). Rituximab-refractory pts (no response or a response with TTP

Clinical management differs significantly for the various types of non-Hodgkin lymphoma (NHL), and the diagnosis of these lymphomas can be challenging in some cases. Further, existing NHL categories include subgroups that can differ substantially in gene expression, response to therapy and overall survival. We have created a custom oligonucleotide microarray, named LymphDx, which could prove clinically useful for molecular diagnosis and outcome prediction in NHL. Biopsy specimens were obtained from 559 patients with a variety of lymphomas and lymphoproliferative conditions. Gene expression profiles of these samples were obtained using Affymetrix U133 A and B microarrays. The 2653 genes on LymphDx were chosen to include:(1)Genes most differentially expressed among NHL types based on Affymetrix U133 or Lymphochip microarrays (2)Genes predicting length of survival in diffuse large B cell lymphoma(DLBCL), follicular lymphoma(FL) and mantle cell lymphoma(MCL) (3)Genes encoded in the EBV and HHV-8 viral genomes (4)Genes encoding all known surface markers, kinases, cytokines and their receptors, as well as oncogenes, tumor suppressors, and other genes relevant to lymphoma. The LymphDx microarray was used to profile gene expression in 434 biopsy samples. These data were used to create a diagnostic algorithm that can distinguish various NHL types and benign follicular hyperplasia(FH) based on gene expression. The algorithm classifies a sample into one of the following categories: Burkitt’s lymphoma(BL), DLBCL, FL, MCL, small lymphocytic lymphoma(SLL) or FH. The algorithm further distinguishes the 3 recognized DLBCL subgroups: germinal center B cell-like, activated B cell-like or primary mediastinal lymphoma. Using a leave one out, cross validation strategy, the algorithm was found to agree well with the pathology diagnosis (see Figure). Some samples were deemed unclassified when their gene expression did not adequately match with that of any of the NHL categories. For a few samples, the gene expression-based diagnosis and the pathology diagnosis were discordant. Pathology review showed that two NHL types coexisted (eg FL and DLBCL) in many of these cases, potentially explaining the results of the diagnostic algorithm. LymphDx could also reliably predict the overall survival of patients with DLBCL, FL and MCL. Prospective evaluation of the LymphDx microarray is warranted since it could be used to provide objective molecular diagnostic, and prognostic information for patients with NHL. Figure Figure

The natural history of follicular lymphoma has not changed over the last 30 years (Horning, S.J., Seminars in Oncology 20: 1993, 75–88). Median survivals have ranged from 7 – 10 years and the disease is generally considered incurable as there has been no plateau in the survival curve. However, multiple new treatment options, including biologic agents, have been developed in the last decade and their impact on the natural history of follicular lymphoma remains unknown. In order to determine the cumulative effects of all these new treatment options, we identified all previously untreated, advanced stage, follicular lymphoma patients treated with three sequential treatment approaches: CHOP chemotherapy +/− nonspecific immunostimulants (SWOG 7426 and 7713: 1974 – 1978), ProMACE-MOPP +/− interferon (SWOG 8809: 1988 – 1994), and CHOP followed by monoclonal antibody therapy (SWOG 9800 and 9911: 1998 – 2000) and determined their Progression-Free Survival (PFS) and Overall Survival (OS). More specificially, the monoclonal antibody trials included CHOP followed by rituximab (SWOG 9800) and CHOP followed by 131I-Tositumomab (SWOG 9911). The PFS are shown below: Progression-Free Survival by Treatment Strategy | TREATMENT | N | DEATH/PROGRESSION | 4-YR PFS | |:----------- | --- | ----------------- | -------- | | CHOP + MoAb | 179 | 75 | 61% | | ProMACE | 425 | 290 | 48% | | CHOP | 356 | 257 | 46% | The results demonstrate that the PFS remained unchanged until the recent studies that utilized CHOP followed by a monoclonal antibody for initial treatment. The results of OS from these three groups are shown below. Overall Survival by Treatment Strategy | TREATMENT | N | DEATH | 4-YR OS | |:----------- | --- | ----- | ------- | | CHOP + MoAb | 179 | 18 | 91% | | ProMACE | 425 | 189 | 79% | | CHOP | 356 | 226 | 69% | In contrast to the PFS, OS has increased with each subsequent study. These data are consistent with the hypotheses that initial therapy with chemotherapy followed by a monoclonal antibody has a significant impact on PFS (p= .005) and OS (p < .0001) and that, even in earlier studies where we could not demonstrate improved initial treatment, sequential new treatment options have also changed the OS (p < .0001) and thus the natural history of follicular lymphoma.

Most MCLs harbor the t(11;14) translocation resulting in overexpression of cyclin D1 that promotes tumor proliferation. Gene expression profiling of 92 cyclin D1 positive MCL biopsy samples defined a gene expression signature of tumor proliferation that provided strong prognostic information: the quartile of patients with the highest proliferation gene expression signature had a 0.8 year median survival whereas the quartile with the lowest expression had a 6.7 year median survival (Cancer Cell 2003, 3:185). Sixteen of the 23 MCL samples in the highest proliferation quartile displayed exceptionally high cyclin D1 levels and preferentially expressed short cyclin D1 mRNA forms that lacked parts of the 3′UTR known to harbor RNA-destabilizing elements. To test whether expression of short cyclin D1 mRNA is due to genomic deletions, we screened DNA from all samples in the highest proliferation quartile by quantitative genomic PCR. We found genomic deletions of the 3′UTR region in 6 of the 10 samples that expressed short mRNAs. We next sequenced the proximal 400bp region of the 3′UTR in those samples with high expression of short mRNAs that did not have genomic deletions. In 2 samples, we found 2 distinct mutations: a 3bp deletion and an A insertion. Both of these mutations created a premature polyadenylation signal (AATAAA) about 300bp 3′ of the stop codon, giving rise to a predicted mRNA of 1.5kb that lacks the destabilizing elements present in the full-length form. We identified two MCL cell lines, Jeko-1 and Z138, that also showed high-level expression of the short cyclin D1 form. In Jeko-1 we found a deletion of the 3′UTR similar to that observed in the MCL patient samples, whereas the mechanism responsible for expression of the short cyclin D1 mRNA in Z138 remains unclear. In both lines, we confirmed that the half-life of the short cyclin D1 mRNA is longer than that of the full length mRNA. Our results demonstrate that MCLs increase cyclin D1 expression not only through the t(11;14), but also by the additional mechanisms of genomic deletions and mutations that create alternative polyadenylation sites that yield more stable mRNAs. Higher cyclin D1 expression increases proliferation and decreases survival, suggesting that strategies aimed at reducing cyclin D1 expression in MCL could have therapeutic promise.

The 5-year survival rate of patients with limited disease (LD) and aggressive histologies of non-Hodgkin lymphoma (NHL) who have at least one adverse risk factor is about 70% after treatment with three cycles of CHOP followed by involved-field radiotherapy (CHOP(3) plus RT). We tested the effect of adding four infusions of rituximab to CHOP(3) plus RT in 62 evaluable patients. Patients had an aggressive histology of diffuse B-cell NHL (diffuse large cell or Burkitt’s-like) and had LD. LD is defined as stage I with at least one adverse risk factor (age > 60 years, elevated serum LDH, or a performance status of 2) OR non-bulky stage II disease. Adverse risk factors are defined using a stage-modified IPI system and include: non-bulky stage II, age >60 years, elevated serum LDH and a performance status of 2. Patients with bulky stage II disease were excluded as their prognosis is similar to advanced disease, and patients with stage I disease and no risk factors were excluded as their prognosis exceeds 90% survival at 10 years with CHOP(3) plus RT. Rituximab was infused at 375 mg/m2 on days −7, 1, 22, and 43, and CHOP was given at standard doses on days 3, 24, and 45 (CHOP(3) plus R plus RT). Involved-field RT was given as previously described (N Engl J Med1998;339:21–26). With a median follow-up of 2.4 years, the progression-free survival (PFS) and overall survival (OS) measured at 2 years was 94% and 95%, respectively. In order to estimate whether the addition of rituximab adds benefit to CHOP(3) plus RT, and therefore, would be worth testing in a comparative trial, we identified 68 patients from SWOG study 8736 treated with CHOP(3) plus RT using the same criteria to include LD and aggressive diffuse B-cell histologies. These previously reported patients treated with CHOP(3) plus RT (ref above) have a median follow-up of 12.0 years. The PFS and OS for this group measured at 2 years was 85% and 93%, respectively. Four patients have relapsed in the rituximab-treated group and 10 patients had relapsed in the CHOP(3) plus RT alone treated group within the first 2 years. Three patients have died in the rituximab- treated group and five patients had died on the CHOP(3) plus RT alone treated group within the first 2 years (see Table). Short-term toxicity appeared similar. We conclude that the addition of rituximab to standard treatment with CHOP(3) plus RT results in outcome which compares favorably to our historical experience and merits further study. Effect of Rituximab in Treating Limited Disease MEASUREMENT CHOP(3) + R + RT (0014) CHOP(3) + RT (8736) Results of SWOG study 0014 compared to SWOG study 8736 No. patients 62 68 2-year PFS 94% 85% 2-year OS 95% 93% No. relapses 2 yrs 4 10 No. deaths 2 yrs 3 5

INTRODUCTION: The expression of valine (V/V or V/F) at position 158 of the FCγIIIA (CD16) in contrast to phenylalanine alone (F/F) has been associated with higher response rates and longer PFS to single agent Rituximab. A polymorphism in position 131 FCγ RIIA (CD32) encodes histidine (H) or arginine (R) and has also been associated with improved outcome. These polymorphisms have been associated with higher affinity binding of human IgG1 and greater in vitro ADCC using effector cells and Rituximab, and suggest that the major mechanism of Rituximab is through the interaction with FC receptors on cells capable of mediating ADCC. There is no data on the effect of these polymorphisms on the outcome of Rituximab following or combined with chemotherapy. METHODS: We evaluated these polymorhisms using DNA from blinded samples collected from patients on SWOG 9800, a phase II trial evaluating the sequential use of CHOP followed by single agent Rituximab (375 mg/m2 x 4 weekly doses). We developed a SSCP method to rapidly determine these polymorphisms from DNA samples and confirmed this method through DNA sequencing. Briefly, DNA was amplified using specifically designed PCR primers that amplified the relevant portions of CD16 and CD32. PCR products were then run on SSCP gels under controlled conditions that allowed identification of the patterns associated with each of the polymorphisms. These results were then analyzed with respect to outcome based on the hypothesis that the presence of high affinity FC receptors would be associated with an improved outcome to CHOP followed by Rituximab in newly diagnosed patients with advanced stage follicular NHL. RESULTS: DNA samples were successfully evaluated on 87/89 eligible patients (no DNA on 2 patients). The freqeuncy of CD16 V/V, V/F and F/F was 13%, 51%, and 37% and CD32 H/H, H/R and R/R was 36%, 44%, and 21% respectively. There was no significant association between any combination of phenotypes and the two year PFS following sequential treatment with CHOP followed by Rituximab. The two-year PFS for patients with V/V, V/F or F/F in CD16 was 55%, 80% and 75% and for patients with H/H, H/R and R/R was 71%, 76% and 78% respectively. For CD16 the presence of one V vs no V resulted in identical estimates of 2 year PFS of 75% (p=.56). Likewise the presence of one H vs no H in the CD32 polymorphism did not result in significantly different PFS (78% vs 74%. p=.88). We also determined the effect of the polymorphism’s on the clearance of PCR detectable disease by analysis for t(14:18). There was no impact on the clearance of molecular disease following CHOP alone. Twenty-two of 32 patients who were still PCR positive for t(14:18) after CHOP converted to PCR negative following Rituximab. There was a suggestion of a trend toward a higher rate of molecular remission following Rituximab for patients with CD16 with at least one V phenotype (V/V or V/F: 12/15 cleared vs 10/17 with F/F, p=.26). In conclusion, we found no evidence that the encouraging efficacy of CHOP followed by Rituximab antibody therapy for patients with advanced follicular NHL is dependent on the presence of high-affinity FC gamma receptor polymorphisms.

The clinical significance of immunophenotyping of the non-Hodgkin's lymphomas is controversial. Therefore, we conducted the present study of 103 consecutively accrued diffuse large-cell lymphoma (DLCL) patients to define, independently of histologic subtypes, the prognostic importance of phenotyping. We used an extensive panel of monoclonal antibodies to T- and B-cell antigens to assign all patients immunologically into the T-cell (20 patients) or B-cell group (83 patients). The only significant differences in pretreatment clinical variables between the two patient groups were the higher frequency of bulky disease (greater than 10 cm) in B-cell patients (P = .008) and more frequent skin involvement in the T-cell group (P less than or equal to .001). Multiagent doxorubicin-containing chemotherapy regimens were employed as initial therapy in over 83% of the patients in each group. Our study revealed that disease-free survival (DFS) was significantly shorter in the T-cell patients than in the B-cell DLCL patients (median DFS, 10.8 months for T-cell and 42.7 months for B- cell; P = .01, log rank). No patient with T-cell DLCL remained disease free for longer than 2 years, whereas 55% of the B-cell group were disease free at 2 years. Univariate and multivariate analyses of all major prognostic factors of DFS suggest that the T-cell phenotype indicates an incurable subset of DLCL patients. Although the B-cell group had a twofold advantage in median survival (35 months v 18 months), actuarial overall survival was not significantly different between the two patient groups (P = .23). Our results indicate the need for new approaches in the search for a curative treatment for T-cell DLCL.

To assess the prognostic significance of the growth fraction in diffuse large cell lymphoma (DLCL), we studied 105 DLCL patients with the monoclonal antibody Ki-67 applied to frozen tissue sections. Ki-67 detects a nuclear antigen associated with cell proliferation not found in resting cells. Ki-67 findings and other clinical prognostic factors were correlated with outcome using univariate and multivariate analyses in the proportional hazards model. High proliferative activity, defined as nuclear Ki-67 expression in greater than 60% of malignant cells (Ki- 67 greater than 60), was found to be a strong predictor of poor survival among these patients (P = .003, log-rank). The 19 patients with Ki-67 greater than 60% had a median survival of 8 months compared with a median survival of 39 months for the 86 patients with Ki-67 less than or equal to 60%. Examination of pretreatment clinical variables indicated the patient groups were similar with regard to age, sex, stage, B symptoms, tumor bulk, and lactate dehydrogenase (LDH). Both patient groups received comparable curative intent therapy and showed comparable complete response rate precluding treatment differences as modifying outcome. Multivariate analysis indicated Ki-67 is an independent predictor of survival (multivariate P = .006). Further statistical analysis using only B-cell DLCL patients treated with CHOP (63 patients) indicated that Ki-67 greater than 60 retained strong prediction of poor outcome (P = .002, log-rank) among this homogeneous group. We conclude that high proliferative activity (Ki-67 greater than 60) is an independent factor allowing laboratory prediction of probable poor outcome of DLCL.

Forty-five patients with localized non-Hodgkin's lymphoma of unfavorable histologic type (41 patients had diffuse histiocytic, 2 had nodular mixed, and 2 had minimally nodular histiocytic lymphoma) were treated with initial chemotherapy (28 patients), including cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP), or with initial CHOP followed by involved field radiotherapy (17 patients). Patients were clinically staged and found to have stage I (7 patients), stage IE (8), stage II (12), and stage IIE (18). Despite the presence of potentially adverse prognostic factors, including age older than 65 yr (11 patients), bulky disease (17), gastrointestinal involvement (9), and “B” symptoms (4), 44 of 45 patients (98%) achieved a complete response. Forty-two patients (93%) are alive with a median follow-up time of 41 mo (range 3–128 mo). Thirty-eight patients (84%) remain continuously free of disease. Neither the pretreatment clinical features nor the type of treatment significantly influenced the outcome for patients treated with initial chemotherapy. Patients who failed treatment relapsed at distant sites or in initially involved sites whether or not they received radiotherapy. Initial treatment for localized lymphomas of unfavorable histology with chemotherapy regimens of proven curative potential in advanced disease is a successful treatment strategy and obviates the need for extensive staging. The role of involved field radiotherapy following initial chemotherapy needs to be defined.