ALBERT

Description: The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., 〉90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Description: Purpose: Mantle cell lymphoma (MCL) is characterized by a t(11;14) resulting in overexpression of cyclin D1, a member of the phosphatidylinosital 3 kinase (PI3K) pathway. This study tested whether CCI-779, which inhibits the PI3K pathway at the level of the mammalian target of rapamycin (mTOR) could produce tumor responses in patients (pts) with MCL. Patients and Methods: Eligible pts had biopsy-proven, cyclin D1 positive MCL and had relapsed or were refractory to therapy. Pts received CCI-779 250 mg IV every week as a single agent. Pts were re-staged after 1 cycle (4 doses) and every 3 cycles thereafter. Pts with a tumor response after 6 cycles were eligible to continue drug for a total of 12 cycles or 2 cycles after complete remission (CR) and then were observed. Results: Thirty-five pts were enrolled and evaluable for toxicity; 1 patient had MCL by histology but was cyclin D1 negative and ineligible for efficacy evaluation. The median age was 70 years (range, 38–89), 91% were stage 4, and 69% had ≥ 2 extranodal sites. Pts had received a median of 3 prior therapies (range, 1–11) and 54% were refractory to their last treatment. The overall response rate was 38% (13/34) with 1 CR (3%) and 12 PRs (35%), surpassing the pre-defined criteria for a promising agent. Responses tended to occur rapidly with median time to response of 1 month (range, 1–8). To date, 26 patients have progressed, with a median time-to-progression of 6.8 months (95% CI: 3.8 – 9.7). Median duration of response for the 13 responders was 5.7 months (95% CI: 5.2 – 13.2). Overall, 32 out of 35 patients who received treatment had grade 3 or 4 toxicity. The most common toxicities were hematologic with grade 3 (n=24) or grade 4 (n=4). Thrombocytopenia was the most frequent grade 3/4 toxicity (n=25) and the largest cause of dose-reductions, although counts typically recovered within one week. Only 4 patients could tolerate sustained 250 mg per week throughout their treatment (including one who went on to alternate treatment after 1 cycle) and the median dose/month was 175 mg. Conclusions: Single-agent CCI-779 has substantial anti-tumor activity in relapsed MCL. This study demonstrates that agents, which selectively target cellular pathways dysregulated in MCL cells can produce therapeutic benefit. The high response rate warrants further studies of this agent in MCL, but the high incidence of hematologic toxicity suggests that a lower dose should be explored. CCI-779 at 25mg is currently being evaluated in MCL through an NCCTG trial

Description: Introduction Antisense oligonucleotides are used to inhibit messenger RNA function and inhibit protein translation. Bcl-2 expression proteins have been implicated in mediating resistance to apoptotic cell death in Waldenstrom Macroglobulinemia (WM). Oblimersen sodium is a specific inhibitor of Bcl-2 expression and has shown activity in multiple myeloma and in Waldenstrom cell lines. The in vitro data led to the development of a phase I study for WM patients with relapsed or refractory disease. Materials and Methods Eligible patients had symptomatic WM who had failed prior cytotoxic chemotherapy. All patients had to have one of the following: a hemoglobin

Description: Background: In B-CLL, the observation of interphase cells with hemizygous D13S319 deletion at 13q14 (13q-x1) as a sole anomaly in blood is widely considered a favorable prognosis. The observation of cells with 11q-, +12 or 17p- has been associated with a relatively poor prognosis. Over the past 1.5 yrs, 16.2% (174/1,076) of patients (pts) referred for fluorescence in situ hybridization (FISH) testing for B-CLL in our clinical practice had a clone with homozygous D13S319 deletion (13q-x2), but the prognostic significance of this observation is poorly understood. Moreover, 39.3% (142/361) of pts with unfavorable FISH anomalies have 13q-x1 and/or 13q-x2 and the clinical significance of this observation is also unknown. Thus, we investigated pts with 13q- (with or without other chromosome anomalies) to establish the relative clinical significance of 13q- in B-CLL. Methods: We studied 333 pts with B-CLL sampled between 9/1999 and 6/2004 who had FISH performed on interphase nuclei from blood. The FISH probe set was designed to detect 6q-, 11q-, +12, 13q-, 17p-, and translocations involving IgH at 14q32. We classified pts into four groups: 13q-x1 only (group 1), 13q-x1 and 13q-x2 only (group 2), 13q-x2 only (group 3) and 13q-x1 and/or 13q-x2 plus other FISH anomalies (group 4). FISH groups were compared with gender, age, Rai stage, treatment status, time to treatment, CD38 and IgVH mutation. Results: Of the 333 pts, 171 (51.3%) had a 13q-: 71 were in group 1, 25 in group 2, 26 in group 3 and 49 in group 4. %CD38+ differed significantly across FISH groups; in pairwise analyses, the proportion of pts with 〉30% CD38+ was significantly greater for pts in group 4 vs. group 3 (p=0.0015) although no significant differences were observed for group 3 vs. group 1 or vs. group 2. Pts in group 3 were not significantly different from other FISH groups for Rai stage, IgVH mutation or gender. The median percentage of abnormal nuclei for pts with group 1 was 54.5% vs. 79.5% for pts in group 4 (p

Description: Gene promoter methylation is a major mechanism of transcriptional silencing in cancer cells that is mediated by CpG methylation and histone deacetylase activity, with methylation being dominant. Importantly, pharmacological treatment of cancer cells with inhibitors of DNA methylation and histone deacetylation have been shown to synergistically reactivate transcription of previously silenced genes. In an effort to identify novel genes silenced by DNA methylation in human acute myeloid leukemia (AML), we used cDNA microarray technology to screen for genes upregulated in the human cell line AML193 following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, Trichostatin A (TSA). Analysis of 1,700 gene microarrays in triplicate revealed seven genes consistently upregulated by combined 5-Aza-dC and TSA treatment, all of which were confirmed by semi-quantitative RT-PCR. Interestingly, four of these genes (MT1H, MT1E, MT2A, MT1G) are members of the metallothionein family of cysteine-rich small molecular weight proteins. These proteins are considered to be important mediators of cellular detoxification and are induced by toxic heavy metals, UV irradiation and reactive oxygen species. As well, MT1G promoter methylation has been implicated in the pathogenesis of sporadic papillary thyroid carcinoma. The methylation status of candidate metallothionein genes reactivated by 5-Aza-dC and TSA treatment in AML193 cells was assessed by methylation specific PCR (MSP) in seven human AML cell lines. Methylated promoter alleles were detected in MT1H, MT1E and MT1G in 71%, 29% and 29% of cell lines, respectively, and there was no methylation of MT2A in any of the cell lines tested. To investigate for the occurrence of MT1H, MT1E and MT1G methylation in human AML in vivo, we analyzed AML patient blasts (n=39) and non-leukemic controls (n=13) by MSP. MT1H, MT1E and MT1G methylation was detected in 51%, 0% and 3% of AML patient samples, respectively, and there was no methylation of any of these genes in control samples. Our findings implicate MT1H promoter methylation in the pathogenesis of human AML and suggest that the use of cDNA microarray technology following pharmacological manipulation is a useful approach for identifying novel epigenetically silenced genes in this disease.

Description: RIZ1 (PRDM2) is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 functions as a tumor suppressor gene in a number of human cancers and is down regulated in some human acute myeloid leukemias. We previously found RIZ-1 to be silenced in K562 erythroleukemia cells by promoter hypermethylation. Furthermore, expression of RIZ1 in K562 promotes erythroid differentiation and also potentiates TGF-β1 mediated differentiation. To investigate similarities between genes altered by RIZ1 expression and the TGF-β1 pathway, we used SELDI to compare the protein profiles of K562 against K562 + RIZ1 and K562 + TGF-β1. Protein extracts for SELDI profiling were separated into six fractions according to their isoelectric points. Proteins from each fraction were then bound to two different protein chip surfaces (H50-hydrophoboic and CM10-cation exhange) and their mass/charge determined using SELDI. We analyzed four replicates from each sample and classified proteins as differentially expressed if their P-values were below 0.05. In total, we observed 104 differentially expressed proteins (60 upregulated and 44 down regulated) between K562 and K562 + RIZ1 and 176 proteins (96 upregulated and 80 down regulated) between K562 and K562 + TGF-β1. We used 2D-PAGE to identify differentially expressed proteins identified by SELDI analysis and located 48 proteins that were over expressed in K562 + RIZ1 and K562 + TGF-β1 relative to K562. To establish whether these proteins were the same proteins observed using SELDI, we determined if the proteins had the same pI and molecular weight and if the gel-eluted proteins bound to the same protein chip surface with the same mass/charge. 15 of 48 proteins passed the above criteria and we determined their identities using Trypsin-based peptide mapping strategies with molecular weight and pI restrictions. We identified two candidate proteins (14-3-3ε and S100/A13) that are similarly over expressed in K562 + RIZ1 and K562 + TGF-β1. These proteins have been shown to be associated with TGF-β1 signaling. Schistosomal 14-3-3ε interacts with SmRK1, a divergent type I transforming growth factor β1 receptor (TR-I) present on the surface of adult parasites and also binds to and activates human TR-I. S100/A13 belongs to a family of low molecular weight proteins characterized by the presence of two calcium-binding EF-hand motifs that includes S100C/A11, a member recently shown to play a key role in a PKCα mediated pathway essential for the growth inhibition of normal human keratinocytes by TGF-β1. In summary, we demonstrate the potential for using SELDI to identify novel proteins involved in regulating and connecting cellular growth and differentiation pathways.

Description: Background: Myelofibrosis with myeloid metaplasia (MMM) is a progressive and fatal myeloproliferative disorder with limited therapeutic options. R115777 is a non-peptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) which we recently reported has in vitro activity against aberrant myeloid progenitors from MMM patients (Leukemia2003;17:849). This observation, coupled with the clinical activity demonstrated in high risk acute myeloid leukemia (Blood2001;97:3361) and chronic myeloid disorders (Blood2003;101:1692) led us to perform a Phase II trial in patients (pts) with MMM. Methods: Eligible pts had histologically confirmed MMM and were symptomatic, defined by anemia (hemoglobin

Description: Light chain–associated amyloidosis (AL) is a plasma cell dyscrasia in which the secreted monoclonal immunoglobulin (Ig) light chains form amyloid fibrils. There is considerable heterogeneity in clinical presentation, and prognosis of the disease relates to the severity of organ dysfunction induced by amyloid deposits. The mechanisms by which the amyloid fibrils are deposited as well as the predilection for specific organ sites have not been clearly elucidated. This study characterizes the repertoire of immunoglobulin light chain variable genes used by the clonal B cell in AL amyloid patients, and the association of light chain variable region (VL) genes with clinical presentation and outcome is assessed in 58 (32 λ and 26 κ) patients. A preferential use of VL germ-line genes was noted for both AL κ and λ patients. There was a significant correlation between the use of the Vλ VI germ-line donor, 6a, and renal involvement as well as the Vλ III gene, 3r, with soft-tissue AL. The use of a biased VL gene repertoire also correlated with clinical outcome, revealing important trends for predicting prognosis. The use of Vλ II germ-line genes was associated with cardiac amyloidosis and affected survival adversely. The presence of multiple myeloma also correlated with a poor prognosis. The presence of renal disease, on the other hand, was associated with improved survival. Therefore, identification of the clonal VL gene in AL has important implications in determining clinical outcome.

Description: Background: Increasingly thalidomide (Thal) plus dexamethasone (Dex) is being used as initial therapy for multiple myeloma (MM), but there is a need to minimize non-hematologic toxicity with this regimen. CC-5013 (lenalidomide; Revlimid™) is a more potent analog of thalidomide with significantly fewer non-hematologic toxicities that has shown promising results in relapsed refractory myeloma. We report the initial results of the first phase II trial using the combination of CC-5013 plus Dex (Rev/Dex) as initial therapy for newly diagnosed MM. Methods: The trial is designed to accrue 31 eligible patients; 13 patients (pts) (11 male and 2 female) were analyzed in this interim report. Patients were enrolled between February 2004 and July 2004. CC-5013 was given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dex was given orally at a dose of 40 mg daily on days 1–4, 9–12, 17–20 of each cycle. Patients also received an aspirin once daily as thrombosis prophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours. Responses were assessed on an intent to treat basis. Results: The median age was 61 years (range, 32–78). 8 patients (62%) had Stage III myeloma. 11 of the 13 patients achieved an objective response yielding a response rate of 85% within 1–2 months of therapy. So far 6 patients have experienced grade 3 adverse events. These include one episode each of CD4-count 〈 200/mm3, anemia, neutropenia, increased liver enzymes, muscle weakness, agitation, hyperglycemia, cardiac arrhythmia, pneumonitis, and colonic perforation (underlying diverticulitis and dexamethasone suspected). No deep vein thrombosis or grade 4 or higher adverse events have been observed so far. Conclusions: Rev/Dex appears active and well tolerated in the treatment of newly diagnosed MM and is a potential alternative to Thal/Dex. However, these results are preliminary and responses are still being evaluated and need to be confirmed in the final analysis of this trial. A large randomized trial using Rev/Dex as initial therapy for MM is expected to be activated by the Eastern Cooperative Oncology Group later this year.

Description: Background: Though STI-571 (Gleevec™) was designed to specifically inhibit the bcr-abl gene product, it inhibits other receptor tyrosine kinases at physiologically attainable concentrations, including c-kit (steel factor receptor, stem cell factor receptor, SCF-R or CD 117). Studies have shown that about one-third of patients with multiple myeloma or monoclonal gammopathy of undetermined significance have plasma cells that display reactivity for c-kit. Others have shown that several myeloma cell lines and fresh myeloma bone marrow cells proliferate in response to stem cell factor. mRNA transcripts for c-kit ligand and, more commonly, its receptor have been detected in myeloma cell lines RPMI 8226, JJN3, U266 B1, NCI-H929, ARH77 and HS-Sultan by RT-PCR. Methods: Patients were eligible for study if they presented with relapsed or refractory myeloma, an ECOG performance score 〈 3, ability to sign informed consent, serum creatinine