We performed immunophenotypic and IgVH mutation analyses on the chronic lymphocytic leukemia (CLL) cells of 307 patients for expression of CD38, ZAP-70, and IgVH mutation status to determine the ...relative strength of each in predicting the need for early treatment. For this we assessed the time from diagnosis to initial therapy for progressive and/or symptomatic disease. Patients confirmed to have remained untreated were censored for this endpoint. Expression of CD38 was defined as the proportion of CD38-stained CLL cells that had fluorescence intensity above a defined threshold. Similarly, cases were defined as ZAP-70+ when 20% or more of the ZAP-70-stained CLL cells had fluorescence intensity above a defined threshold, as described (NEJM 351:893, 2004). CLL cells were classified as expressing mutated IgVH genes if they expressed IgVH with <98% homology with a known germline IgVH gene. The median percentage of cells expressing CD38 in our cohort was 11.5% (range 0.1% to 99.5%). In patients with multiple samples, the CD38 expression level appeared constant over time, with a median S.D. of 0.8%. We defined patient samples that had CD38 expression levels at or below the median as having low expression. There was a significant association between low expression of CD38 and lack of ZAP-70 or expression of a mutated IgVH (p < 0.0001 for each assessment by the Fisher exact test). Median level of CD38 among cases expressing mutated IgVH was 4.0%, compared to 27.4% among cases expressing unmutated IgVH (p < 0.0001 by the Wilcoxon rank sum test). Median level of CD38 among ZAP-70 negative cases was 5.3%, compared to 27.2% among ZAP-70+ cases (p < 0.0001 by the Wilcoxon rank sum test). Patients with low-level expression of CD38 had a median time to initiation of therapy of 8.2 years; whereas, patients with high-level expression of CD38 had a median time to initiation of therapy of 4.0 years (p<0.0001). Although high-level expression of CD38 was associated with a shorter time from diagnosis to initiation of therapy (p < 0.0001 in a Cox proportional hazards regression), the inclusion of IgVH mutation status (p < 0.0001) or expression of ZAP-70 (p < 0.0001) in these models reduced the contribution of CD38 status to a p-value of 0.051 (ZAP-70 model) or 0.052 (mutation status model). Although ZAP-70 expression and mutation status each contributed significantly to the hazard of initiating therapy in a Cox proportional hazards model, high-level expression of CD38 did not add significantly to the model that already addresses ZAP-70 and mutational status, p=0.425. As such, although knowledge of the CD38 expression level can be used as a predictor of the time from diagnosis to the initiation of therapy it provides only marginal value if either the IgVH mutational status or expression of ZAP-70 is known and no value if knowledge of both ZAP-70 expression and IgVH mutational status is available.
The combination of fludarabine and cyclophosphamide (FC) has been noted to produce a high complete response rates in previously untreated patients with CLL. However, it may be accompanied by ...increased toxicity. To further evaluate its efficacy and toxicity, a phase III randomized study of FC versus Fludarabine (F) was conducted in patients with previously untreated CLL. The study, which was open to accrual from December 23, 1999 to March 19, 2004, closed with 278 patients enrolled, 141 on the FC arm and 137 on the F arm. Four patients declined to receive protocol treatment, including one who was later found to be ineligible. Five additional patients were also deemed ineligible. All patients with data are included in this analysis (intent to treat). Patients on the FC arm received C 600 mg/m2 iv day 1 and F 20 mg/m2 iv days 1 through 5, followed by filgrastim 5 mg/kg SC starting approximately day 8. Patients randomized to the F arm received F 25 mg/m2 iv days 1 through 5. In April 2004, the ECOG Data Monitoring Committee conducted a planned review at 76% information, and determined that the null hypothesis of no difference in CR rates could be rejected. The Data Monitoring Committee gave permission for the submission of abstracts to ASH. The median age of patients was 62 years (34–86), and the median performance status was 1 (0 to 2). As is expected in CLL, 70% of patients were male (194) and 30% were female (83). At study entry, 56% of cases were Rai stages, 0,1 or 2, while 44% were in stage 3 or 4. 57% of patients received the maximum of 6 cycles of therapy. Toxicity data is available on 127 CF and 125 F patients. There were two deaths due to infection with grade 3 or 4 neutropenia (one in each arm). In the CF arm, 17% of patients suffered grade 4 or higher non-hematologic toxicities, while in the F alone arm, 13% had higher grade toxicities (p= 0.48). Additionally, 17% of patients in the FC arm suffered infections versus 11% in the F alone arm (p= 0.21). Response data was available on 246 of the 278 patients. In the FC arm (125 cases), 28 patients achieved CR (22.4%), 60 patients achieved PR (48.0%) for a total of 88 objective responses (70.0%). In contrast, on the F alone arm (121 cases), there were 7 CRs (5.8%), 53 PRs (43.8%) for a total of 60 objective responses (49.6%). The Fisher exact test for the difference in CR rates gives a p-value of 0.0002, while the test for difference in OR rate was 0.001. Currently, 235 patients are alive and 42 have died. Among the 229 patients with information on time to progression, 131 are alive without progression, 78 have progression, and 20 have died without progression. 58 of the 78 with progressive disease remain alive. The preliminary estimates of the median progression free survival time are 41.0 months for the FC arm, and 17.7 months for the F alone arm ( p <0.001). It is noteworthy that the CR rate of F alone is similar to that reported by the GCLLSG (Eichhorst #243 ASH 2003). In summary, FC is a highly effective and tolerable regimen that produces more durable remissions than F in patients with previously untreated CLL.
The CD23 antigen is expressed at high density on the cell surface of certain B-cell malignancies, including chronic lymphocytic leukemia (CLL). Lumiliximab (L-mab), a macaque-human chimeric anti-CD23 ...monoclonal antibody, has been reported to have antitumor activity against CLL in preclinical studies. In this Phase I multicenter study, the safety, efficacy, and pharmacokinetics of single-agent L-mab were evaluated in 46 patients with relapsed or refractory CLL. Therapy consisted of intravenous L-mab given as 6 regimens: (1) 125 mg/m2/wk for 4 weeks; (2) 250 mg/m2/wk for 4 weeks; (3) 375 mg/m2/wk for 4 weeks; (4) 500 mg/m2/wk for 4 weeks; (5) 500 mg/m2 for 3 doses during Week 1, then 500 mg/m2/wk during Weeks 2 to 4; and (6) 500 mg/m2 three times a week for 4 weeks. Patient characteristics were as follows: median age of 62 years (range 47 to 80 years), 93% Caucasian, 72% male, 54% fludarabine-refractory, 48% Rai stage III/IV, and 78% WHO Performance Status 1. At study entry, patients had progressive CLL after 1 to 13 prior treatment regimens (median = 4 prior regimens). Antibody infusions, administered over 2 hours in an outpatient setting, were well tolerated. Study-related adverse events (probable, possible, or unknown relationship to study treatment) were reported in 40 of 46 patients (87%). The majority of events were Grade 1 or 2; the most common were headache, constipation, nausea, and cough. Grade 3 or 4 study-related adverse events were reported in 7 of 46 patients (15%) and included neutropenia and dyspnea. Evidence of clinical activity consisted of reductions in absolute lymphocyte counts (ALC) and lymphadenopathy. Decreases in ALC were observed in 42 of 46 (91%) patients, and decreases ≥ 50% were observed in 11 of 40 (28%) patients enrolled at 375 mg/m2/week or higher. Of 37 patients evaluated for change in lymphadenopathy, reductions were observed in 22 (59%). Flow cytometry revealed that L-mab saturated CD23 sites on CLL cells at doses above 375 mg/m2/week without down regulating CD23 expression. These results suggest that single-agent L-mab can be administered safely with evidence of clinical activity in patients with heavily pretreated CLL. Ongoing clinical studies are assessing the potential of L-mab in combination with rituximab and fludarabine-based chemotherapy.
Deletions or mutations of the tumor suppressor p53 have been associated with more aggressive disease in many types of cancer including CLL, and are more commonly found in relapsed or refractory ...patients. We studied symptomatic patients (pts) enrolled on ECOG 2997, a randomized phase III trial of fludarabine monotherapy (F) versus fludarabine plus cyclophosphamide (CF) for previously untreated CLL. A total of 235 pretreatment samples were analyzed for p53 mutations in exons 5–9 using denaturing gradient gel electrophoresis (DGGE), followed by confirmation of mutations using automated sequencing. Cytogenetic analysis was also performed on 243 patient samples by Fluorescence In Situ Hybridization (FISH) using a probe to detect deletions in 17p13.1, the chromosomal location of the p53 gene. In the analysis by DGGE, a total of 25 pts (10.6%) were found to have p53 mutations. In 22 cases there was a single mutation, and in three cases there were two mutations. Mutations in exon 6 were the most common, and in each of the cases with two mutations, one of these was in exon 6. Results by exon are as follows: Exon 5 (1 pt, 0.4%), Exon 6 (15 pts, 6.4%), Exon 7 (5 pts, 2.1%), Exon 8 (3 pts, 1.3%), Exon 9 (4 pts, 1.7%). The interphase cytogenetic analysis included 24 of the 25 pts with a p53 mutation by DGGE. Of the 19 (7.8%) pts with del(17p13.1) as the leading cytogenetic anomaly, seven were cases that also had mutations in p53 by DGGE. The remaining 17 pts with mutations in p53 by DGGE had as leading cytogenetic anomaly: del(11q) (2 pts); trisomy 12 (3 pts), normal cytogenetics (2 pts), and del(13q) (10 pts). Results from both the DGGE and FISH analyses were combined to identify a group of 37 pts with either del(17p) or p53 mutation. This constituted 16% of the 230 pts in which either FISH or p53 mutational analysis was performed.
Immunocytochemistry staining for p53 was performed on all pts, and no correlation was found between over-expression of p53 protein with mutation and/or deletion of this gene. The pts were then assessed by treatment arm for response and progression-free survival (PFS), relative to patients without p53 abnormality either by DGGE or FISH analysis. In the FC group: 13 pts had either a p53 mutation or del(17p), nine (69%) of whom achieved a CR or a PR, versus 56/74 (76%) of those with normal p53 (p=0.73). In the F group: 15 pts had either a p53 mutation or del(17p), of whom four (27%) achieved a CR or a PR, versus 38/70 (54%) of those with normal p53 (p=0.09). Pts with a detectable abnormality in p53 had a significantly reduced PFS, regardless of whether they received fludarabine alone (p=0.02) or fludarabine plus cyclophosphamide (p=0.005). This study suggests that routine immunocytochemistry staining should not be substituted for p53 mutational studies. While some short responses were observed in patients with p53 mutation and/or deletion receiving fludarabine and/or fludarabine cyclophosphamide, the time to progression was significantly shorter, emphasizing the prognostic significance of p53 abnormalities for this disease.
To evaluate BMT outcomes in patients (pts) with newly diagnosed or relapsed mantle cell lymphoma, 58 consecutive pts were selected through the BMT registry at Johns Hopkins. Flow cytometric and ...immunohistochemical features were confirmed. Fifty-four pts (88%) had stage III or IV disease and 39 (67%) had documented bone marrow involvement at diagnosis. First-line therapy was CHOP +/− rituximab in 71% and was fludarabine-based in 17%. Thirty-seven pts (64%) were transplanted after first partial or complete remission, 14 pts (24%) after one or more relapses, and 12% after primary induction failure (PIF). Median age at BMT was 55 years. Thirty-eight pts (66%) received autologous (auto) BMT, 19 pts (33%) allogeneic (allo) BMT, and 1 pt syngeneic BMT. Preparative regimens in all but 1 case consisted of cyclophosphamide plus busulfan or total body irradiation. The estimated 3-year event-free survival (EFS) following BMT was 51% (95% CI, 35–65%), probability of relapse 31% (17–51%), and overall survival 59% (42–73%). Median follow-up is 16 months (range < 1 month to 79 months) for all pts and 23 months for surviving pts. Twelve relapses were documented, 8 after auto BMT. Four auto pts and 1 allo pt are alive with relapsed disease, and 3 auto pts developed MDS or AML. Actuarial EFS at 3 years was 57% (CI 35–74%) after auto BMT and 37% (17–57%) after allo BMT. Actuarial relapse rate at 3 years was 34% following auto BMT and 19% following allo BMT. On multiple regression analysis, BMT after one or more relapses (HR 3.0, CI 1.2–7.4, P = 0.02), PIF (HR 5.4, CI 1.9–15.4, P = 0.002), and allo BMT (HR 3.0, CI 1.3–6.8, P = 0.007) predicted an inferior EFS;
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whereas PIF and residual bone marrow involvement independently predicted relapse. Notably, however, EFS curves were not statistically different for auto and allo BMT performed in first remission, with the 3 year EFS approaching or equaling 70%.
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The benefit of BMT for mantle cell lymphoma is thus most apparent when performed in first remission. While the comparative efficacy of auto and allo BMT remains to be determined, allo BMT warrants continued study especially early in the disease course.
Bortezomib (formerly PS-341, VELCADE®) is a selective inhibitor of the ubiquitin-proteasome pathway, which plays an important role in degrading regulatory proteins that control cell cycle functions, ...neoplastic growth and tumor spread. It has been approved by the FDA for the treatment of relapsed and refractory myeloma patients (pts). Phase I and II trials have suggested activity in B-cell indolent and aggressive lymphomas including mantle cell lymphoma. Preclinical studies have also shown that B-CLL cells undergo apoptotic cell death following inhibition of the proteasome pathway. We therefore conducted a multicenter phase II study to assess the safety and efficacy of 3 doses of bortezomib in pts with B cell CLL who were refractory to or intolerant to fludarabine. Pts with an ANC ≥ 1 x 109/L and platelets ≥ 30 x 109/L were eligible. Bortezomib was given as i.v. push on days 1, 4, 8, and 11 of a 21 day course for a maximum of 9 courses. Pts were initially randomized to 1.0 (n=5) versus 1.3mg/m2/dose (n=9) and later, after review of preliminary data, to 1.3 versus 1.5mg/m2/dose (n=8). Twenty-two pts were enrolled. Median age 62 yrs (range 46-83). Median number of prior therapies 4 (2–11). Median number of yrs from diagnosis to therapy 5.5 (0.8–14.2). b2-microglobulin > 5.5 mg/L in 10 pts (45%). Fifteen pts (68%) had ≥ Rai stage 3. Of 19 evaluable pts no responses according to NCIWG criteria were demonstrated stable disease 8 pts (42%), progressive disease 11 pts (58%) based on investigator assessment. When analyzed by specific sites of disease, five pts (23%) experienced ≥ 50% decrease in ALC and 6 pts (27%) showed ≥ 50% decrease in lymphadenopathy. Bortezomib was well tolerated throughout all 3 dosing groups. Most common toxicities were ≤ grade 2 and included nausea (n=9;42%), headache (6;27%), diarrhea (5;23%), vomiting (5;23%), dyspnea (5;23%), anorexia (5;23%), and peripheral neuropathy (3;14%/ grade 3 in 1 pt). Pharmacodynamic evaluation of 20S proteasome inhibition at 1 hour postdose was similar to that reported for these doses in other studies, and was highest in the 1.5mg/m2 dosing group. We conclude that bortezomib as single agent is not effective in pts with fludarabine-refractory B-cell CLL at these doses. However, in view of the evidence of clinical activity, further exploration of bortezomib in combination with other agents might be the appropriate next step to define the optimal role of this new agent in the management of advanced CLL.
The critical importance of specific proteins including Bcl-2, Mcl-1, XIAP, Bax, Caspase-3, Traf-1, and ZAP70 in predicting disease progression, prognosis, and response to therapy in CLL have been ...extensively studied through in vitro experiments. We now report molecular data obtained from a large prospective clinical trial involving 278 previously untreated patients with CLL. This ECOG-led intergroup trial involved participation from CALGB and SWOG, and currently provides quantitative information regarding protein levels on 217 cases assessed immediately prior to treatment. NCI criteria for therapy in CLL were met before patients were randomized to receive either Fludarabine or Fludarabine plus Cytoxan. On-study analyses included: interphase cytogenetics by Fluorescence In Situ Hybridization (FISH), p53 mutational analysis, immunoglobulin variable heavy chain (IgVH) gene rearrangement, and quantitative analysis of the aforementioned specific proteins. Peripheral blood mononuclear cells were obtained from each patient at registration before starting therapy. Lysates were prepared from the patient cells and separated by SDS-PAGE. Proteins were detected by immunoblot analysis using the multiple antigen detection method previously developed by our group. Detection was performed by an enhanced chemiluminescence method and quantified by densitometry. Expression levels were calculated relative to expression in the Jurkat T cell line (for ZAP70) or the RS11846 lymphoblastoid cell line (for apoptosis-related proteins). Using a two-sided Wilcoxon rank sum test, molecular data were compared with FISH and IgVH mutational status. Patients with either p53 mutations or del(17p) had significantly lower levels of XIAP (p=0.022). Patients with del(11q) had significantly higher Bcl-2/Bax ratios (p=0.052). Patients with trisomy 12 had lower levels of Bcl-2 (p=0.066); higher Mcl-1 (p=0.026); higher Bax (p=0.09); and higher Caspase-3 (p=0.019) levels. Interestingly, these specimens also had lower ratios of Bcl-2/Bax (p=0.0016). Patients with del(13q) had significantly higher levels of Bcl-2 (p=0.0059) lower levels of Caspase-3 (p=0.047), and higher Bcl-2/Bax ratios (p=0.0569). IgVH mutated patients had significantly lower levels of Caspase-3 using either a 98% cut-off (p=0.0008) or the 97% cut-off (p<0.0001). Finally, patients with higher ZAP70 protein expression had significantly higher levels of Caspase-3 (p=0.0099) and Traf-1 (p=0.016). In 163 patients evaluable for both response and the molecular studies, either elevation of Bcl-2 protein (p= 0.01) or an abnormality of p53 (either del 17p or a p53 mutation) (p=0.08) correlated significantly with attainment of a complete remission. Potential opportunities to predict response to therapy in CLL will hopefully identify those patients likely to benefit from therapy. Based upon our previous work, these molecular parameters can be modulated both in vitro and in vivo. Therefore, understanding the relationship of these important proteins to therapeutic response can facilitate strategic plans to circumvent drug resistance to chemotherapy and immunotherapy in CLL.
Background: T cells can be activated and expanded ex vivo using the Xcellerate™ Process, in which peripheral blood mononuclear cells (PBMC) are incubated with anti-CD3 and anti-CD28 antibody-coated ...magnetic beads (Xcyte™-Dynabeads®). In an ongoing trial of Xcellerated T Cells in subjects with chronic lymphocytic leukemia, marked and sustained reductions in lymphadenopathy and splenomegaly were observed (Wierda et al., ASCO 2004). Increases in neutrophil, platelet and NK cell counts were also documented. This study is designed to determine if similar effects can be observed in subjects with indolent non-Hodgkin's lymphoma (NHL).
Methods: Subjects must have indolent NHL (follicular, small lymphocytic, marginal zone, or mantle cell lymphoma), have relapsed or refractory disease, and have received at least 1 but not more than 4 prior treatment regimens. PBMC are collected by leukapheresis for the Xcellerate Process, and subjects subsequently receive two infusions of 20–60 x 109 Xcellerated T Cells separated by 6–8 weeks. Approximately 40 subjects will be treated.
Results: Seven subjects have been enrolled and Xcellerated T Cells have been manufactured in 5 subjects to date. T cells expanded 181.8 ± 88.5 fold and the final product was >99.0 ± 0.0% T cells (mean + SD). One subject with small lymphocytic lymphoma has been treated with two infusions of 38.6 x 109 Xcellerated T Cells. There have been no serious adverse events to date. Following the first treatment, the lymphocyte count increased from 1.8 x 109/L to 2.9 x 109/L on Day 28. The neutrophil count also increased from 2.9 x 109/L to 5.5 x 109/L six weeks following infusion. The subject had a significant reduction in cervical lymphadenopathy and a slight decrease in bulky mesenteric lymphadenopathy six weeks following the first infusion.
Conclusions: Xcellerated T Cells can be manufactured in subjects with indolent NHL. Treatment leads to significant increases in T cell and neutrophil counts. Preliminary data suggest a reduction in peripheral lymphadenopathy. Data on additional subjects will be presented.
Signal transduction is a key mechanism by which both proliferative and apoptotic processes of B-cell chronic lymphocytic leukemia (CLL) cells are mediated. Carboxyamido-triazole (CAI) is a cytostatic ...signal transduction inhibitor currently being tested in phase II clinical trials. Based on this, we investigated the in vitro activity of CAI in mononuclear cell isolates from patients with B-CLL (n = 11). Viability, utilizing the MTT assay, was assessed at varying concentrations (0.01-100 μM) of CAI for 4 days. The CAI concentration required for 50% inhibition of cell viability (LC50), determined by the tetrazolium dye (MTT) assay, at 4 days was 53.5 μM (range 29-74.6; 95% CI × 14.8). Cells from 6 of 11 patients (3 of whom were clinically fludarabine refractory) had a 27 percent (range 11-43) mean decline in viability at 10 μM after a 4 day drug exposure, a concentration readily attainable in humans. To assess if loss of viability was due to apoptosis, we incubated cells from 4 additional CLL patients with media or CAI (10 μM) for 4 days. Annexin-V/propidium iodine labeling subsequently demonstrated CAI significantly (p = 0.049) induces apoptosis (40.1%; 95% CI × 18.1) as compared to media matched control cells (18.3%; 95% CI × 11.2). These data provide evidence that CAI can induce apoptosis in human CLL cells in vitro at drug concentrations attainable in vivo. These findings justify phase n studies of CAI in patients with B-CLL.
