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Abstract Background: Germinal center B cell-like (GCB) DLBCL, as determined by gene expression profiling or immunohistochemistry, is more likely to be cured by initial conventional chemoimmunotherapy than non-germinal center B cell-like (non-GCB) DLBCL. For patients with relapsed or refractory chemosensitive DLBCL, high-dose chemotherapy and autologous hematopoietic stem cell (HSC) transplant is considered standard-of-care treatment, but it is unknown whether the outcome of these patients is similarly influenced by the subtype of DLBCL. We therefore explored the differences between patients with GCB and non-GCB DLBCL as regards their clinical features at relapse after first line therapy and their outcome following salvage autologous HSC transplant. Methods: Patients undergoing BEAM conditioning and autologous HSC transplantation for relapsed or refractory chemosensitive DLBCL at Mayo Clinic, Rochester, MN between 2001 and 2006 were included. Immunohistochemical analysis was performed for CD10, BCL-6 and MUM1 allowing classification in GCB and non-GCB-like DLBCL, as well as for BCL-2. GCB and non-GCB groups were compared in terms of known prognostic factors at time of progression and outcome after HSC transplant Results: Fifty-nine patients were included and had retrievable tumor samples to allow immunohistochemical analysis. Median follow-up of survivors was 25 months; median age at the time of transplant was 60 years (range 17–77); All patients had failed at least one previous anthracycline-based regimen (15 had refractory disease). Overall, 25/59 cases (42%) were positive for CD10, 32/58 (55%) for BCL-6, and 19/58 (32%) for MUM1. Thirty-two patients (54%) were classified as having GCB and 27 (46%) non-GCB-like DLBCL. Patients in the GCB and non-GCB group had similar time to progression (TTP) (median 12.5 months vs. 11 months, Wilcoxon P=0.81) after first line therapy and similar IPI-R scores (Chi-square, P=0.38). In univariate analysis, GCB and non-GCB did not differ in time to relapse after HSC transplant (log rank test P=0.77) or survival (log rank test P=0.48; figure). The lack of demonstrable difference in survival persisted even after correction for IPI-R and TTP, factors know to affect transplant outcome (Cox regression, RR=0.80 for GCB; P=0.28). BCL-2 was highly expressed in both GCB (81%) and non-GCB (96%) and did not correlate with outcome in the entire population nor in any of the two groups. Conclusion: Patients with chemosensitive relapsed or refractory GCB and non-GCB-like DLBCL derive similar benefit from autologous HSC transplant. Figure Figure

Abstract MRC UKALLXII/ECOG2993 accrued 739 ECOG pts over 13 years. ECOG’s reference laboratories centrally characterized 613 (91%) and 505 were B-Lin ALLs. Expression of 32 antigens (Ags), including 9 myeloid-associated Ags, was determined by multiparameter flow cytometry on gated blasts, both as percentage of Ag expressing blasts and intensity of antibody staining (equivalent of Ag density). To test for the potential therapeutic efficacy of monoclonal antibodies, such as rituximab, epratuzumab, alemtuzumab, or gemtuzumab in future trials, expression of Ags for these antibodies (CD20, CD22, CD52, or CD33, respectively) was determined. In E2993, 14% of pts typed as Pro-B (CD10 negative), 71% as Early Pre-B (CD10 positive), 13% as Pre-B (intracytoplasmic mu positive), and 2% as Mature B-ALL (surface mu chains positive). CD20 expression correlated with the stage of B-lymphoid maturation (p=5.4E-13) (see table). Median CD20 intensity of fluorescence was lowest in Pro-B and highest in Mature B-ALL. Membrane expression of CD22 was lower in Pro-B compared with the other three groups, Early Pre-B, Pre-B and Mature B (p=1.0E-5) but indistinguishable among the latter three (p=0.21). While levels of CD20 and CD22 had no effect on complete remission (CR) and overall survival (OS) univariatly, higher CD22 expression was significantly correlated with better OS (p=0.02) in a bivariate Cox model of CD20 and CD22. With respect to myeloid Ags, CD33/CD13 expression was associated with Early Pre-B (p=0.02), and CD65(s)/CD15(s) with Pro-B ALL (p<0.0001). While CD65(s) and CD15(s) did not affect outcome, the combined expression of CD33/ CD13 conferred an inferior prognosis; this effect was due to the association of CD33/ CD13 with BCR/ABL positivity (p<0.0001). BCR/ABL negative B-Lin ALL (N=350) expressed CD33 on a median of only 4% of blasts, compared with a median of 54% in BCR/ABL positive ALL (N=152) (p=3.5E-09). However, intensity of CD33 staining was significantly lower in both BCR/ABL negative and positive blasts (median 3.1) compared with leukemic myeloblasts (median 56) (p=<0.001). Median CD52 expression was higher in BCR/ABL positive versus negative B-Lin ALL (p=3.5E-06); in neither group was CD52 associated with outcome. We conclude that antibodies to CD52 are better suited for BCR/ ABL positive than negative B-Lin ALL. Since expression of CD33 in ALL is weak, CD33- independent effects of gemtuzumab must be investigated. Low CD20 combined with high CD22 expression confers superior OS. The expression of these Ags in most B-Lin ALL pts justifies the incorporation of antibodies into frontline chemotherapy regimens. B-Lin ALL Stage CD20 Percentage Median Intensity CD22 Percentage Median Intensity ≥20% ≥50% ≥75% ≥20% ≥50% ≥75% Pro-B 21 10 3 2 88 72 55 12 Early PreB 66 48 37 21 96 90 84 28 Pre-B 61 46 37 21 100 90 81 37 Mature B 87 80 64 106 92 84 80 9

Abstract Results from clinical trials may change with time. Data presented in initial abstracts or early reports are often different from what appears in subsequent publications, sometimes due to a more rigorous analysis (as in initial response rates) or to maturity of data (as in reports of survival). Survival curves usually provide projected data based on Kaplan-Meier analyses. Such data may change with time when unexpected events occur (as, for example, relapse after T-cell depleted allogeneic transplants) or more patients advance to the initially projected time point. For AML, several groups have reported that there are very few relapses beyond 3 years. At issue is the optimal time for reporting these survival data. Table 1 reports data from 6 consecutive clinical trials addressing induction and post-remission questions in AML patients performed by ECOG. These data compare the 3-year overall survival (OS) and disease-free survival (DFS) for the identical cohorts of patients as presented at 1 year and at 3 years after completion of the study accrual. PC 486 was a phase II study of induction therapy followed by allogeneic or autologous transplant without prior consolidation. E 3489 was a US intergroup study in which patients received post-induction therapy using an allogeneic transplant (allo), an autologous transplant (auto) or chemotherapy (chemo). E1490 was a study in older adults of induction and consolidation with GM-CSF or placebo. E2491 was an intergroup study comparing ATRA induction versus standard chemotherapy in patients with APL. After consolidation, patients were randomized to maintenance with ATRA versus observation (obs). E3993 was a study in older adults comparing priming with GMCSF or placebo. E4995 was a phase II study of intensified induction and consolidation followed by an autologous transplant. For the same cohorts of patients the 3-year OS tends to decrease as the time from completion of study accrual increases. When looking at the DFS, while a decrease may also occur, in several studies there was an improved DFS with time (for example, in E2491, chemo to ATRA, or in E3993 with GM-CSF priming). There were no changes in either OS or DFS beyond 3 years (data not shown). Table 1: Three-year OS and DFS Presented at Different Time Points OS (%) DFS (%) Study One year Three years One year Three years PC 486 auto or allo HSCT 42 → 38 45 → 39 E 3489 allo HSCT 50 → 50 48 → 47 auto HSCT 47 → 46 39 → 39 chemo 56 → 51 36 → 36 E 1490 Induction / consolidation GM-CSF 23 → 22 23 → 24 placebo 15 → 12 16 → 15 E2491 APL induction+ maintenance ATRA + ATRA 87 → 84 72 → 73 ATRA + obs 83 → 80 61 → 60 chemo + ATRA 81 → 75 43 → 58 chemo + obs 54 → 45 16 → 18 E 3993 Induction/consolidation GM-CSF priming 13 → 11 5 → 10 placebo 14 → 14 9 → 9 E 4995 Induction / consolidation / auto HSCT 54 → 48 63 → 53 In conclusion: For large cooperative group studies, treatment results in AML presented one year from conclusion of study accrual are unlikely to dramatically alter the overall results. Nonetheless, some differences may occur, both in OS and DFS, warranting caution in interpreting the data and comparing with other published reports. Such differences are likely to be even greater if data are presented upon completion of study accrual, prior to one year - although, in a cooperative group setting, this should be balanced against the need to release data for future planning. Publication of interim data, before conclusion of the study accrual, may be misleading and are to be strongly discouraged. Three years appear to be the appropriate time for the publication of definitive survival data.

Abstract This phase 1 clinical trial evaluated the safety and effects of autologous dendritic cells (DC) injected to patients in late chronic or accelerated phase of chronic myelogenous leukemia (CML). Seven patients with a median age of 63 years (range 55–78) entered the trial. Median time from diagnosis to registration was 38 months (range 16–94). Two patients were in the accelerated phase of disease based on the presence of additional bone marrow chromosomal abnormalities, all other patients were in chronic phase. Prior therapies included hydroxyurea and alpha interferon with or without cytosine arabinoside. Four patients were on therapy with imatinib mesylate during the course of the trial, two were treated before imatinib became available and one was previously intolerant of imatinib. CD14+ monocytes were selected from a leukapheresis product by clinical grade immunoadsorption (Miltenyi Biotec). The monocytes were matured to CD83+ and bcr-abl+ DC after 10 days in culture with X-VIVO 15 medium with 1% human AB serum, GM-CSF, IL-4, as the base culture media followed by maturation mediated by TNF-alpha, IL-6 and prostaglandin E2. The first patient entered had a positive mycoplasma reaction by polymerase chain reaction at the time of his first DC injection, the cells were not given and he was withdrawn from the study. The other six patients were treated in two cohorts of three patients and received four injections each of 3x106 DC and 15x106 DC per injection, respectively. CML-DC injections were generally well tolerated and all non-hematologic toxicities were ≤ grade 2. All injections were given intra-nodally into a cervical lymph node except in the first patient where the DCs were injected subcutaneously. We observed no major clinical responses, but the white blood cell counts normalized or were maintained at normal level in four patients during therapy. Three patients experienced a transient reduction in the percentage of bcr-abl+ cells suggesting that the treatment may have affected the cells in a leukemia specific manner. We detected vascular endothelial growth factor (VEGF) in the plasma prior to therapy. In four of five patients, VEGF levels decreased by more than 75 percent during therapy. T cells isolated from patients’ blood and incubated with autologous CML DC before, during and after therapy showed increased proliferation and secretion of interferon gamma during the course of therapy. DCs from three patients were transfected with a recombinant replication-defective adenovirus carrying the gene for IL-2 under a constitutively active promoter. IL-2 secreted by transfected dendritic cells enhanced in vitro T cell proliferation and interferon gamma release and these effects increased with increasing numbers of DC injections. This study demonstrates that CML-DC can be prepared from the blood of patients treated with imatinib mesylate, that they can be administered safely and that progress of therapy is accompanied by an increase in DC-specific T cell reactivity and a decrease in circulating VEGF.

Abstract Background: In childhood acute lymphoblastic leukemia (ALL), a rapid decline of circulating blasts in response to induction chemotherapy or prednisone is one of the most important prognostic factors for achieving remission and for relapse-free survival (RFS). However, in AML parameters of chemosensitivity have been restricted to the assessment of residual bone marrow (BM) blasts during aplasia. We hypothesized that time to peripheral blood (PB) blast clearance would have prognostic relevance with respect to RFS in AML. Methods: From 1994 to 2006 outcomes of 86 adults (47 males; median age 52 y) with previously untreated AML (non- acute promyelocytic leukemia) achieving CR and receiving high-dose cytarabine (HDAC)-based consolidation have been included in this retrospective analysis. The median and range of white blood cell count (WBC), platelet count and PB blast percent were 5.3 × 109/L (0.9–267), 59 ×109/L (5–361) 22.5% (0–97), respectively. A 100 cell manual slide differential count was done in all with PB blasts unless the WBC was less than 0.5 ×109/L. Karyotype was classified as favorable, intermediate and unfavorable in 10, 63 and 12 cases, respectively and failed in one. Induction consisted of idarubicin at 12 mg/m2/ d (n=70) or daunorubicin at 45 mg/m2/d (n=16) on days 1–3 with infusional cytarabine at 100 mg/m2/ d on days 1–7 and repeated in those with persistent BM blasts on day 14 (n=17). The first consolidation was the same as that used to achieve CR. Thereafter, 3 courses of HDAC at 3g/m2 (1.5 g/m2 if ≥ 60 y) q12 h on days 1, 3 and 5 were planned. Results: At the time of analyses, 43% had died, primarily of relapse (97%). Median overall survival (OS) and RFS were 30.2 m (5.5–133) and 14 m (2–131.5), respectively. The median time to PB blast clearance was five days (range: 2–10). For this analysis, only those with PB blasts at initiation of induction (n=73) were included. We defined the day of PB blast clearance as the first day after commencing induction that PB blasts were absent. Separation according to blast clearance on or before day 5 resulted in the most balanced distribution and strongest significant difference between each subgroup of 45 and 28 patients, with significantly different rates of relapse of 33% and 79%, respectively (p<0.0001). We also defined three “blast risk groups” as good, intermediate and poor, according to PB blast clearance on or before day 3, on days 4 or 5, or on day 6 or beyond, respectively. This provided 3 well-balanced groups (good, intermediate and poor) of 16, 30 and 27 patients with significantly different and escalating relapse rates of 12.5%, 47% and 78%, respectively (Fig 1). Univariate analysis of several variables identified PB blast clearance day (p<0.0001), number of inductions to CR (p =0.0043) and cytogenetic risk group (p=0.028) as being significantly associated with RFS. On multivariable analysis, only the first sustained its significance. The results were similar for OS. Conclusion: In adults who achieve CR after induction chemotherapy for non-APL AML, early PB blast clearance predicts superior RFS and OS. Figure Figure

Abstract INTRODUCTION The bone marrow plasma cell labeling index (PCLI) and the beta 2 microglobulin (β2M) when combined provide a powerful staging system highly predictive of outcomes in myeloma patients (Greipp et. al., Blood, 81:3382–87, 1993). This staging system lacks information on cytogenetics and it has been demonstrated that deletion 13 (Δ13) by conventional cytogenetics is associated with poor outcomes. In conventionally treated patients Δ13 by FISH is an independent prognostic variable. We analyzed the importance of Δ13 by FISH in a cohort of patients receiving high dose therapy and assessed its value relative to known prognostic parameters, PCLI and β2M. PATIENTS AND METHODS 238 patients undergoing high dose therapy between January 1990 and September 2001 were studied. The minimum follow-up of patients was 34 months. The median age of the cohort 56 years. Interphase fluorescence in situ hybridization with Probes LSI13/D13S319 was performed with simultaneous immunofluorescent detection of bone marrow plasma cells by the presence of cytoplasmic immunoglobulin. PCLI was done by a slide-based immunofluorescence method with antibodies to bromodeoxyuridine. RESULTS Of the 238 patients studied 215 had a successful FISH analysis and 212 of these had a bone marrow labeling index. Deletion 13 was absent in 104 patients (48%) and present in 111 patients. The overall survival and time to progression for patients lacking and having deletion 13 was 47.8 vs 26.4 (OS) and 19.0 vs 11.7 (TTP)months respectively. A Cox model was constructed with the PCLI, the presence or absence of deletion 13 and serum β2M. Although all three variables were independently predictive of survival and time to progression, the β2M was the weakest of these three variables. The hazard function for survival for PCLI, deletion 13 and β2M was 1.4, 1.6 and 1.1 respectively. A staging system was constructed assigning 1 point for the presence of deletion 13 or a PCLI ≥ 1%. Patients fell into 3 categories of 0 points (N=69), 1 point (N=104) and 2 points (N=39). The overall survival for the three groups respectively were 56.5, 36.6 and 13.7 months (figure). The time to progression for the three groups were 22.3, 15.4 and 8 months (P<0.001). When analysis was limited to those patients transplanted within 12 months of diagnosis the results remained significant (P<0.003) with median survivals of stage 0, 1 and 2 of 72.5, 59.1 and 15.4 months respectively. TTP for stage 0, 1 & 2 in this same cohort of patients transplanted < 12 mos after diagnosis is 30.6, 21.5 & 8.2 months respectively. CONCLUSION Deletion 13 by FISH is an independent predictor of survival and time to progression providing information independent of the β2M and the PCLI. Combining the PCLI with FISH del 13 identifies patients who following transplant have a median time to progression of 8 months and a median OS of 13.7 months. This group may not benefit from autologous transplant and are candidates for novel therapeutic approaches. Figure Figure

Abstract The infused autograft absolute lymphocyte count (A-ALC) is an independent prognostic factor for survival after autologous hematopoietic stem cell transplant (AHSCT) in non-Hodgkin’s lymphoma (NHL). Previous studies have shown that A-ALC directly correlates with the peripheral blood absolute lymphocyte count (PC-ALC) at the time of apheresis collection. However, factors affecting the PC-ALC at apheresis remain undefined. We hypothesized that one possible factor impacting PC-ALC may be the time interval from last chemotherapy to stem cell collection (TILC). Data from 160 patients who underwent AHSCT for treatment of relapsed NHL at Mayo Clinic between 1993 and 2001 were collected and analyzed. The primary end point of this study was correlation between TILC and PC-ALC, and our analysis revealed a strong correlation (r = 0.67, p < 0.0001). Further analysis revealed higher PC-ALC numbers in patients with TILC ≥ 60 days versus TILC < 60 days (median of 7.44 x 109/L in the ≥ 60 day group versus 3.87 x 109/L in the < 60 day group, p < 0.0001). Both the median overall survival (figure below) and the progression-free survival were longer in the TILC ≥ 60 days group versus the TILC < 60 days group (76 versus 21 months, p < 0.0037; 76 versus 11 months, p < 0.018, respectively). These findings were independent of other prognostic indicators for relapsed NHL patients undergoing APHSCT. The data supports our hypothesis that TILC affects PC-ALC and survival post-AHSCT in NHL. Figure Figure

Summary Late relapse [>3 years from complete remission (CR)] in acute lymphoblastic leukaemia (ALL), is unusual. Data from the MRC UKALLXII/ECOG E2993 trial are presented to evaluate the incidence and characteristics of late relapse in adult ALL. Of 1,909 patients, 1,752 (92%) achieved CR and among these 757 (43·2%) relapsed; 691 (91·3%) within three years and 66 (8·7%) beyond. Among these 66 patients, median time to relapse was 47 (37–144) months. Relapse beyond three years occurred in 3·8% of all who achieved CR. The cumulative risk of relapse was 40%, 43% and 45% at three, five and ten years respectively. Out of the 1 752 patients who achieved CR, 11·7% underwent autologous and 40·6% allogeneic transplant, while in CR1. Of the autologous patients, 43·2% relapsed early and 3·4% relapsed late. However, among the allogeneic patients, 13·2% relapsed early and only 1·3% late. The five‐year overall survival from relapse was 5·8% and 20% in the early and late relapse patients respectively. In conclusion, late relapse in adults with ALL is not uncommon, and is associated with better outcome after relapse compared to early relapse.