ALBERT

Description: Background: Bortezomib (Bz, VELCADE®) is a novel proteasome inhibitor that has demonstrated efficacy and safety in patients (pts) with relapsed and/or refractory multiple myeloma (MM) in clinical trials. In a large randomized phase 3 trial, Bz achieved superior response rates (RR), TTP, and survival compared with high-dose dexamethasone in relapsed MM pts after 1–3 prior therapies. Peripheral neuropathy (PN) has been dose limiting, especially in pts with pre-existing PN. The objective of this multicenter phase 2 trial was to evaluate the activity and safety of Bz as monotherapy in previously untreated MM pts and prospectively examine PN at baseline (BL) and across treatment. Methods: Pts received Bz 1.3 mg/m2 on d 1, 4, 8, 11 of a 21-d cycle, with RR (CR + PR; EBMT) assessed every 2 cycles. Eligible pts had symptomatic MM with measurable disease and no prior chemotherapy. Concomitant steroids (〉 10 mg/d), plt count 〈 30 x 109/L within 14 d of enrollment or 〉 grade (G) 2 PN were exclusion criteria. RR, TTP, safety and incidence/severity of PN were evaluated. The effect on PN of dose modification (DM) and supplements with specified interventions for symptoms was assessed. Neurologic evaluation involving a pt questionnaire and neurologist’s exam was performed before and after treatment. A 33 pt subset had BL nerve conduction studies (NCS), quantitative sensory testing (QST), autonomic testing, and skin biopsy for quantitation of small-diameter neurite densities (ND). Toxicities were assessed by NCI-CTC, v3.0. Results: 63 pts (median age 60 y) have been treated. Data are available for 50 pts; 27 pts had IgG kappa MM and 21 pts had stage III disease. BL small-fiber neuropathy (SFN; normal NCS but abnormal QST or autonomic studies) was seen in 16/33 pts (48%). BL skin biopsy showed that 18/30 pts (60%) had small-fiber ND at the 15th percentile of age-adjusted norms; global mean density was at the 23rd percentile. NCS revealed BL axonal PN in 3/33 pts (9%). Among 46 pts evaluable for response, best response after ≥ 2 cycles was CR in 5 pts (11%) and PR in 9 pts (20%) for a RR of 30%. 13 pts (28%) achieved MR, 17 (37%) had SD, and 2 had PD (4%). The most common adverse events were PN, fatigue and rash. 24 pts (48%) developed PN, including 18 with G1 and 5 with G2. One pt had G3 PN and was discontinued. DM (dose reduction or discontinuation) was required in 9 pts. 23 pts have been studied after treatment. New large-fiber neuropathy (LFN) was seen in 4/23 pts (17%) and new SFN in 8 pts. Of 3 pts with BL LFN, 1 was not restudied, 1 had worsening SFN and 1 was unchanged. Small-fiber neurite counts were increased to the 35th percentile. Of 16 pts with BL SFN, 7 had worsening SFN (by QST). Bz was otherwise generally well tolerated. Conclusion: Bz as monotherapy has shown activity in newly diagnosed MM pts, with a CR of 11% and manageable toxicity. Underlying SFN appears more common in MM than appreciated. Treatment-emergent ≥ G2 PN was observed in 12% of pts and was G3 in 1 pt. Assessment of PN, the effect of interventions and skin biopsy analyses are ongoing.

Description: Key Points Complete genome sequence analysis of 40 DLBCL tumors and 13 cell lines reveals novel somatic point mutations, rearrangements, and fusions. Recurrence of mutations in genes involved in B-cell homing were identified in germinal center B-cell DLBCLs.

Description: Background: Patients with severe and prolonged neutropenia are at high-risk of developing life-threatening IFIs. Despite current treatment option, IFIs are difficult to treat and frequently associated with high mortality rates. Antifungal prophylaxis has shown benefits in high-risk populations and is a standard practice in many institutions. We compared Posaconazole (POS), a new broad-spectrum triazole, vs Standard Azoles for the prevention of IFIs in patients with a new diagnosis or first relapse of acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) being treated with intensive chemotherapy. Methods: Patients in this randomized, evaluator-blinded, active controlled, multi-center study received oral POS (200mg tid) or oral Standard Azoles (either Fluconazole [FLU 400mg qd] or Itraconazole solution [ITRA 200mg bid]) with each cycle of chemotherapy until complete remission or for up to a maximum of 12 weeks. The primary efficacy end point was the comparison of the incidence of IFIs during the treatment phase (7 days after last dose) as adjudicated by the blinded expert panel based on EORTC/MSG criteria. The incidence of IFIs 100 days after randomization and the clinical outcome at the end of the treatment phase were also compared. Treatment failure was defined as presence of proven or probable IFIs, use of 〉3 days of empirical systemic antifungal treatment, use of 〉3 consecutive days or a total of 10 or more days of IV study drug (or alternative formulation for POS), discontinuation due to an adverse event related to study drug or lost to follow-up during treatment. Results: 602 patients were enrolled (304 POS, 298 Standard Azole [240 FLU, 58 ITRA]). The incidence of proven/probable IFIs was lower with POS prophylaxis (see table). The overall mortality was 49 (16%) vs 67 (22%) for patients in the POS and Standard Azoles arms respectively. Analysis of time to death (all cause mortality) within 100 days post randomization yielded a P-value of 0.035, indicating a significant survival benefit in favor of POS. Twenty-one (21) deaths due to IFIs occurred during the study (POS 5, Standard Azoles 16, P = 0.012). Treatment failures were fewer in the POS arm vs Standard Azoles arm (36% vs 46%, P = 0.0091). Safety and tolerability were comparable. Conclusions: In this study, POS was superior to the use of a Standard Azoles (FLU or ITRA) in preventing IFIs and in preventing aspergillosis, in high-risk neutropenic patients with AML or MDS undergoing intensive chemotherapy. Patients receiving POS experienced a significant survival benefit. POS N=304 FLU/ITRA N=298 Proven/Probable IFIs n (%) n (%) P-value All IFIs during Treatment Phase 7 (2) 25 (8) 0.0009 Aspergillus during Treatment Phase 2 (1) 20 (7) 0.0001 All IFIs within 100 days post-randomization 14 (5) 33 (11) 0.0031

Description: Abstract 404 To characterize the genomic events associated with distinct subtypes of AML, we used whole genome sequencing to compare 24 tumor/normal sample pairs from patients with normal karyotype (NK) M1-AML (12 cases) and t(15;17)-positive M3-AML (12 cases). All single nucleotide variants (SNVs), small insertions and deletions (indels), and cryptic structural variants (SVs) identified by whole genome sequencing (average coverage 28x) were validated using sample-specific custom Nimblegen capture arrays, followed by Illumina sequencing; an average coverage of 972 reads per somatic variant yielded 10,597 validated somatic variants (average 421/genome). Of these somatic mutations, 308 occurred in 286 unique genes; on average, 9.4 somatic mutations per genome had translational consequences. Several important themes emerged: 1) AML genomes contain a diverse range of recurrent mutations. We assessed the 286 mutated genes for recurrency in an additional 34 NK M1-AML cases and 9 M3-AML cases. We identified 51 recurrently mutated genes, including 37 that had not previously been described in AML; on average, each genome had 3 recurrently mutated genes (M1 = 3.2; M3 = 2.8, p = 0.32). 2) Many recurring mutations cluster in mutually exclusive pathways, suggesting pathophysiologic importance. The most commonly mutated genes were: FLT3 (36%), NPM1 (25%), DNMT3A (21%), IDH1 (18%), IDH2 (10%), TET2 (10%), ASXL1 (6%), NRAS (6%), TTN (6%), and WT1 (6%). In total, 3 genes (excluding PML-RARA) were mutated exclusively in M3 cases. 22 genes were found only in M1 cases (suggestive of alternative initiating mutations which occurred in methylation, signal transduction, and cohesin complex genes). 25 genes were mutated in both M1 and M3 genomes (suggestive of common progression mutations relevant for both subtypes). A single mutation in a cell growth/signaling gene occurred in 38 of 67 cases (FLT3, NRAS, RUNX1, KIT, CACNA1E, CADM2, CSMD1); these mutations were mutually exclusive of one another, and many of them occurred in genomes with PML-RARA, suggesting that they are progression mutations. We also identified a new leukemic pathway: mutations were observed in all four genes that encode members of the cohesin complex (STAG2, SMC1A, SMC3, RAD21), which is involved in mitotic checkpoints and chromatid separation. The cohesin mutations were mutually exclusive of each other, and collectively occur in 10% of non-M3 AML patients. 3) AML genomes also contain hundreds of benign “passenger” mutations. On average 412 somatic mutations per genome were translationally silent or occurred outside of annotated genes. Both M1 and M3 cases had similar total numbers of mutations per genome, similar mutation types (which favored C〉T/G〉A transitions), and a similar random distribution of variants throughout the genome (which was affected neither by coding regions nor expression levels). This is consistent with our recent observations of random “passenger” mutations in hematopoietic stem cell (HSC) clones derived from normal patients (Ley et al manuscript in preparation), and suggests that most AML-associated mutations are not pathologic, but pre-existed in the HSC at the time of initial transformation. In both studies, the total number of SNVs per genome correlated positively with the age of the patient (R2 = 0.48, p = 0.001), providing a possible explanation for the increasing incidence of AML in elderly patients. 4) NK M1 and M3 AML samples are mono- or oligo-clonal. By comparing the frequency of all somatic mutations within each sample, we could identify clusters of mutations with similar frequencies (leukemic clones) and determined that the average number of clones per genome was 1.8 (M1 = 1.5; M3 = 2.2; p = 0.04). 5) t(15;17) is resolved by a non-homologous end-joining repair pathway, since nucleotide resolution of all 12 t(15;17) breakpoints revealed inconsistent micro-homologies (0 – 7 bp). Summary: These data provide a genome-wide overview of NK and t(15;17) AML and provide important new insights into AML pathogenesis. AML genomes typically contain hundreds of random, non-genic mutations, but only a handful of recurring mutated genes that are likely to be pathogenic because they cluster in mutually exclusive pathways; specific combinations of recurring mutations, as well as rare and private mutations, shape the leukemia phenotype in an individual patient, and help to explain the clinical heterogeneity of this disease. Disclosures: Westervelt: Novartis: Speakers Bureau.

Description: Introduction: Bortezomib (Bz, VELCADE) is a proteasome inhibitor proven safe and effective for patients (pts) with relapsed and/or refractory multiple myeloma (MM). Pts in SUMMIT (NEJM2003;348:2609) and CREST (BJH2004;127:165) underwent rigorous testing (neurologic evaluations, FACT/GOG-Ntx questionnaires, nerve conduction studies) to determine the incidence, characteristics and reversibility of PN. In these phase 2 trials, the PN rate was 31–41% (grade [G] ≥ 3 in 11–12%). Specific dose modification (DM) guidelines for PN were not incorporated into the phase 2 trials but were developed for subsequent studies. The objective of this report was to evaluate the frequency, characteristics and reversibility of PN in an updated analysis of the randomized phase 3 APEX trial (NEJM2005;352:2487), in which specific DM guidelines for PN were implemented. Methods: Pts with relapsed MM were randomized to Bz 1.3 mg/m2 IV on d 1, 4, 8, 11 q3wk for 8 cycles then 3 cycles on d 1, 8, 15, 22 q5wk, or dexamethasone (Dex) 40 mg PO on d 1–4, 9–12, 17–20 q5wk for 4 cycles, then 5 cycles on d 1–4 q4wk. Pts with G ≥ 2 PN were excluded. Data were collected on incidence, severity and reversibility of PN. Results: Of 331 pts safety evaluable enrolled on Bz, 120 (36%) developed PN, including 30 (9%) with G ≥ 3. PN was classified as sensory or not specified in the vast majority of cases; motor neuropathies were rare. Of 91 pts with G ≥ 2 PN, 68 had DM, including 37 with doses reduced, held or schedule modification and 31 with Bz discontinuation (DC); 23 pts not following DM protocol. The majority (58 pts; 64%) of the 91 pts improved (9%) or had complete resolution (55%) of symptoms. Of 37 pts with DM without Bz DC, the majority (26 pts; 70%) had improvement, all with complete resolution of PN to baseline, with a median time to resolution of 78 d. Of 31 pts requiring Bz DC, 2 (6%) improved and 17 (55%) had complete resolution of PN with a median time to improvement/resolution of 121 d. Of 23 pts who did not follow the DM protocol, 12 (52%) had complete resolution with a median time to resolution of 106 d. The rates of PN (any G, G ≥ 3) were similar regardless of pt age or number/type of prior therapies (Table). The median TTP of 91 pts with G ≥ 2 PN, 68 pts with DM, and the Bz arm overall were 6.2, 6.9 and 6.2 months, respectively. Conclusion: The overall rate of PN in APEX was similar to that of SUMMIT and CREST, but the rate of G ≥ 3 PN was lower, perhaps because of specific DM guidelines. PN was reversible in the majority of pts, and DM did not compromise efficacy. Pt age or number/type of prior therapies did not appear to affect the rate or severity of PN. PN Bz Subgroup Any G*, % G ≥3, % *Bz related ge; 65 y 35 9 〈 65 y 37 9 〉 1 prior therapy 36 10 1 prior therapy 37 8 Steroids 38 7 Alkylating agent 35 7 Anthracycline 39 7 Vinca alkaloid 38 8 Thalidomide 44 8 Transplantation 37 7

Description: Introduction: In the international, multicenter phase 3 APEX trial, 669 patients (pts) with multiple myeloma (MM) who had relapsed after 1–3 prior therapies were randomized to receive bortezomib (VELCADE®) 1.3 mg/m2 IV d 1, 4, 8, 11 q3wk for 8 cycles followed by 3 cycles on d 1, 8, 15, 22 q5wk, or dexamethasone (Dex) 40 mg PO d 1–4, 9–12, 17–20 q5wk for 4 cycles followed by 5 cycles on d 1–4 q4wk. Pts refractory to Dex were excluded, and those with progressive disease on Dex were eligible to cross over to bortezomib. Pts receiving bortezomib achieved significant improvement in time to progression (TTP, primary end point), response rate (CR + PR using EBMT criteria), and survival (Richardson. NEJM.2005;352:2487), which resulted in early closure of the trial. The duration of response (DOR) was longer with bortezomib, and infections ≥ grade 3, time to skeletal events, grade 4 adverse events (AE), serious AE, and discontinuations due to AE were similar in the 2 treatment arms. Methods: In this analysis, updated response rates, time to response (TTR), DOR, survival, and TTP are presented after extended follow-up. A matched-pairs analysis comparing survival and TTP of pts on bortezomib in APEX with those in another trial of MM pts who received bortezomib after Dex will also be presented. Results: 669 pts received a median of 7 cycles of therapy. Based on a median follow-up of 15.8 months, the median TTP, 1-year and overall survival (OS), response rates, median TTR, and median DOR for pts receiving bortezomib are shown in the table. Median duration of therapy for responders (CR + PR) was 7.2 months. Improved response with longer therapy (after cycle 6) was observed in 76 pts (56% of responders) in the bortezomib arm (20 pts improved from MR or PR to CR, and 56 pts improved from MR to PR). Furthermore, 28 of 135 responders (21%) achieved first response (CR/PR) after cycle 4, including 18 pts (13%) on or after cycle 6, and 10 pts (7%) on or after cycle 8. OS increased substantially with more follow-up. Median TTR was more rapid, and median DOR was longer in pts achieving CR and near CR than in those with PR. Conclusion: Updated TTP, response rates, survival, TTR, and DOR for the bortezomib group continue to support the findings of the original analysis. Thus, the clinical benefits of single-agent bortezomib in pts with relapsed MM remain robust after extended follow-up, supporting its early use in relapsed MM and its further study in the treatment of newly diagnosed disease. Efficacy Bortezomib (n = 333) Median TTP, mo 6.2 1-year survival, % 80 Median OS, mo 25.4 Response rate, % (n/N) 43 (135/315) CR 9 (27/315) PR 34 (108/315) -near CR 7 (21/315) Median TTR, mo (range) 1.4 (0.5–6.0) CR 0.8 (0.5–4.0) PR 1.4 (0.5–6.0) -near CR 0.8 (0.6–2.4) Median DOR, mo 7.8 CR 9.9 PR 7.6 -near CR 11.5

Description: Abstract 99 Whole genome sequencing with next generation technologies represents a new, unbiased approach for discovering somatic variations in cancer genomes. Our group recently reported the DNA sequence and analysis of the genomes of two patients with normal karyotype acute myeloid leukemia (AML). Improvements in next generation sequencing technologies (principally, paired-end sequencing) led us to reevaluate the first case (Ley et al, Nature 456:66–72, 2008) with deeper sequence coverage. We discovered a novel frameshift mutation in DNMT3A, one of the three genes in humans (DNMT1, DNMT3A, and DNMT3B) that encodes a DNA methyltransferase that catalyzes the addition of methyl groups to cytosine within CpG dinucleotides. We then sequenced all the coding exons of this gene in 280 additional de novo cases of AML to define recurring mutations. 62/281 de novo AML cases (22%) had mutations with translational effects in the DNMT3A gene. 18 different missense mutations were identified, the most common of which was at amino acid R882 (37 cases). Frameshifts (n=6), nonsense mutations (n=6), splice site mutations (n=3), and a 1.5 Mbp deletion that included the DNMT3A gene were also identified. DNMT3A mutations were highly enriched in cases with intermediate risk cytogenetics (56/166=33.7%; p

Description: Background: Therapy for patients (pts) with high risk and/or relapsed or refractory AML remains unsatisfactory. Retrospective studies have demonstrated activity of fludarabine, cytarabine, granulocyte colony stimulating factor and idarubicin (FLAG-IDA) as salvage therapy in pts with relapsed or refractory AML. Furthermore, a recent randomized trial has indicated high complete remission (CR) rates with improved relapsed-free survival when FLAG-IDA is administered as frontline induction chemotherapy (Burnett et al. J Clin Oncol 2013). Therefore, since January 2011, we have employed FLAG-IDA as first line therapy in pts with high risk AML (i.e. poor risk cytogenetics, antecedent myeloproliferative neoplasm or myelodysplastic syndrome, or therapy-related AML), or as first salvage in pts with primary refractory or relapsed AML, in an attempt to improve CR rates and permit more patients with AML to advance to allogeneic hematopoietic stem cell transplantation (alloSCT). Methods: A retrospective review was conducted of the 62 consecutive patients with high risk AML or primary refractory or relapsed AML treated with FLAG-IDA between January 2011 to December 2013 at the Princess Margaret Cancer Centre to determine the CR rate and overall survival (OS) associated with FLAG-IDA remission induction chemotherapy. Results: Baseline characteristics of the patients are listed in Table 1. Fourteen pts received FLAG-IDA as first induction, whereas 48 pts received FLAG-IDA as salvage (39 as first salvage and 9 as second salvage). The overall CR rate (i.e. CR + CR with incomplete platelet recovery [CRi]) using FLAG-IDA as frontline therapy was assessed in 13 patients, as one pt died during induction therapy and therefore, was not evaluable. Of the 13 evaluable patients, all achieved CR or CRi. The overall CR rate for the salvage induction group was 73% (i.e. 31% CR and 42% CRi). The CR duration was censored at time of transplant. The CR duration for pts receiving FLAG-IDA as first induction was 3 mos (range, 0-15 mos). For pts receiving FLAG-IDA as salvage therapy, the CR1 duration for primary refractory AML pts was 6 mos (range, 2-58 mos) and CR2 duration for relapsed AML pts was 4 mos (range, 1-12 mos). 76% of patients (n=10) who received frontline FLAG-IDA induction chemotherapy, and achieved CR/CRi, had a donor identified, but only 40% of those pts underwent alloSCT. 85% of pts (n=30) who received salvage FLAG-IDA, and achieved CR/CRi, had a donor identified, but only 53% of those pts proceeded to alloSCT. The length of hospital stay during the first FLAG-IDA induction was 33 days (range, 17-96 days), whereas the length of hospital stay for salvage FLAG-IDA induction was 43 days (range, 10-305 days). Fourteen percent of pts in the first induction group were admitted to the ICU during their induction, compared to 17% of pts in the salvage induction group. The median ICU stay was 39.5 days and 14 days, respectively. There was a 14% death rate during FLAG-IDA induction for both groups. The median follow up time from diagnosis for both groups was 15.28 mos (range, 2-70.4 mos). Overall survival at 1 and 2 years in the upfront FLAG-IDA induction group was 65% and 41%, respectively, while OS at 1 and 2 years for the salvage FLAG-IDA group was 60% and 35%, respectively. Conclusions: The toxicities associated with FLAG-IDA induction, including induction death rates and ICU admission rates, are acceptable and similar in the untreated and heavily pre-treated groups. FLAG-IDA induction can result in durable CR rates, permitting patients with high risk AML or patients with primary refractory or relapsed AML to proceed to allogeneic transplantation. Table 1: Patient Characteristics Front-LineN=14 SalvageN=48 Median age, y (range) ≥70y (%) ≥60y (%) 65.5 (21-76) 2 (14%) 10 (71%) 50 (18-76) 2 (4%) 10 (21%) Gender 7M : 7F 22M : 26F Secondary/Therapy-related Prior MDS Prior MPN 14 (100%) 2 (14%) 2 (14%) 17 (35%) 8 (17%) 2 (4%) Cytogenetic risk group Good Intermediate Poor 0 4 (28%) 10 (71%) 3 (6%) 28 (58%) 17 (35%) Molecular abnormalities cKit mutated FLT3-ITD mutated 0 1 (7%) 2 (4%) 5 (10%) Median no. prior treatment regimens (range) 0 1 (1-2) Prior chemotherapy regimen Daunorubicin + cytarabine NOVE-HiDAc Other NA NA NA 43 (90%) 11 (23%) 3 (6%) Disease status Primary refractory Relapsed CR1 duration

Description: Introduction: Bortezomib (Bz, VELCADE®) is a novel proteasome inhibitor that has demonstrated safety and efficacy for patients (pts) with relapsed and/or refractory multiple myeloma (MM) in phase 2 and 3 trials. Bz was associated with transient, cyclical thrombocytopenia in SUMMIT (NEJM.2003;348:2609) and CREST (BJH.2004;127:165). This analysis evaluated the hematologic profiles in pts treated with Bz or high-dose dexamethasone (Dex) in APEX, the largest phase 3 MM trial in relapsed pts. (NEJM.2005;352:2487). Methods:669 pts with relapsed MM were randomized to Bz 1.3 mg/m2 d 1, 4, 8, 11 q3wk for 8 cycles, then 3 cycles on d 1, 8, 15, 22 q5wk, or Dex 40 mg d 1–4, 9–12, 17–20 q5wk for 4 cycles, then 5 cycles on d 1–4 q28d. Data were collected at baseline and regularly through therapy for adverse events, laboratory values, and transfusion (tf) experience. Results:Thrombocytopenia, anemia, and neutropenia reported as adverse events, all grades (G) and those ≥ G3 in pts are shown (Table). Bz-associated thrombocytopenia was cyclical, with recovery toward baseline during the rest period of each cycle. Overall, 15% of pts on Bz and 1% of those on Dex received platelet (plt) tfs and 33% of pts on Bz and 20% of those on Dex received blood tfs. The majority of plt and blood tfs were completed in the first 2 cycles in both treatment arms. Although the number of plt tfs was higher with Bz, the number of clinically significant bleeding events was similar in the 2 arms. Pts on Bz with complete or partial response experienced an increase in mean hemoglobin over time and the requirement for blood tfs decreased from 21% in cycle 1 to 0% after cycle 4. Median duration of therapy for plt transfused pts was 3.8 and 3.4 mo in the Bz and Dex arms, respectively. Bz-associated neutropenia was also transient and cyclical, and febrile neutropenia was rare. Bz was otherwise not associated with severe myelosuppression. Conclusions:Bz is associated with transient, reversible thrombocytopenia and neutropenia—both with a periodicity related to drug administration—but with rapid recovery and few complications compared with Dex. When clinically indicated, plt tf support, rather than dose reduction, particularly in the first 2 cycles, may be warranted to maximize the benefit of Bz therapy. Impact on plts does not appear to be cumulative, and the mechanism of the cyclic variation in neutrophil counts needs further study. Event Bz (n = 331) Dex (n = 332) *Tfs between baseline and last dose of treatment. Thrombocytopenia, n (%) 116 (35) 36 (11) G3/4 98 (30) 22 (7) Neutropenia, n (%) 63 (19) 6 (2) G3/4 49 (15) 5 (2) Anemia, n (%) 92 (28) 77 (23) G3/4 33 (10) 39 (12) Median plt count, x 109/L (range) Baseline count in blood transfused pts 132 146 Baseline count in plt transfused pts 99 74.5 Responders Baseline 205 162.5 First response 195 175 Last assessment 199 198

Description: Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B cell-like (ABC), and primary mediastinal (PM) DLBCL. The BCL6 gene on chromosome 3q27 is a transcriptional repressor and is required for GC formation and function. Two molecular alterations involving the BCL6 gene are commonly observed in DLBCL: chromosomal translocations and mutations in the 5′ non-coding region. The functional consequences of BCL6 translocation and mutation have not been studied in the context of DLBCL subgroups. Therefore, we examined the frequency of translocations and the spectrum of BCL6 mutations in exon 1 and intron 1 in different DLBCL subgroups. We correlated these findings with BCL6 mRNA and protein expression, as well as the expression of BCL6 target genes. Fluorescence in situ hybridization (FISH) using a break-apart probe detected BCL6 translocations in 24 of 112 (21%) DLBCL cases. Surprisingly, the frequency of BCL6 translocation was higher in the ABC subgroup than the GCB subgroup (10 of 40; 25% vs 5 of 44; 11%, respectively) and the PM subgroup had the highest incidence (4 of 8; 50%). Expression of BCL6 protein was detected by immunohistochemistry in 75 of 138 cases (54%), including 15 of 44 (34%) in the ABC subgroup, 46 of 57 (80%) in the GCB subgroup and 4 of 10 (40%) in the PM subgroup. A good correlation of protein and mRNA expression, as assessed by cDNA microarrays (NEJM346:1937–47; 2002) was observed in the GCB subgroup but not in the other subgroups. BCL6 mutations were detected in 71 of 128 cases (55%) of DLBCL with a higher frequency in the GCB subgroup (34 of 50; 68%) and PM subgroup (10 of 12; 90%) than the ABC subgroup (14 of 43; 32%). Interestingly, there was a distinctive pattern of distribution of BCL6 mutations in the DLBCL subgroups. For example, 11 of 100 (11%) of the mutations targeted exon 1 and 8 of these 11 mutants were observed in the GCB subgroup. They preferentially affected the BCL6, STAT1 or predicted CEBP binding sites. High BCL6 mRNA and protein expression was generally associated with mutants affecting the 3′ BCL6 binding sites. We also examined the expression of 25 known BCL6 target genes in the different subgroups of DLBCL and observed the repression of this set of genes mainly in the GCB but not in the ABC subgroup, and the presence of a translocation did not correlate with target gene suppression. In conclusion, the frequency of BCL6 translocation and mutation is distinctly different in the DLBCL subgroups and BCL6 expression has different regulatory influences on target genes in different subgroups of DLBCL. Exon 1 mutations preferentially occur in the GCB subgroup and affect transcription factor binding sites. However, BCL6 translocations did not show good correlation with BCL6 expression or functional repression of target genes. The influence of the reciprocal partner genes in the translocations on the pathogenesis of DLBCL warrants further investigation.