Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15INK4B tumor suppressor gene reactivation, hypomethylation of the p15INK4B promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4–11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin – whether used as a single agent or as an adjuvant – for AML treatment.
A phase I study to determine the maximum tolerated dose (MTD) of bortezomib (B) when combined with weekly paclitaxel in patients with advanced solid tumors.
Patients and methods
Eligible patients received escalating doses of intravenous (IV) bortezomib (0.6–2 mg/m2) on days 2 and 9 and IV paclitaxel at 100 mg/m2 on days 1 and 8 of a 21-day cycle. Dose escalation was based on two end-points: not exceeding 80% 20S-proteasome inhibition (20-S PI) and the development of dose-limiting toxicity defined as grade 3 or greater non-hematologic or grade 4 hematologic toxicities.
Forty-five patients with advanced solid tumors and a median of 3 prior chemotherapy regimens (range 0– 9), received 318 doses (median 5, range 1–34) of bortezomib and paclitaxel. Dose-related inhibition of 20-S PI was observed with a maximum inhibition of 70–80% at the MTD of 1.8 mg/m2 of bortezomib. At the MTD (N = 9) the following toxicities were observed: grade 4 neutropenia without fever (n = 2) and cerebrovascular ischemia (n = 1); grade 3 neutropenia (n = 3), diarrhea (n = 2), nausea (n = 1), and fatigue (n = 1); grade 2 fatigue (n = 5), diarrhea (n = 4), and dyspnea (n = 2). There was one partial response in a patient with an eccrine porocarcinoma. Stabilization of disease was observed in 7 (16%) patients, 3 of whom had advanced pancreatic cancer.
Sequential paclitaxel and bortezomib in previously treated patients with advanced solid tumors resulted in acceptable toxicity and no evidence of interaction. The recommended phase II dose of bortezomib in combination with weekly paclitaxel was 1.8 mg/m2.
Bortezomib; Phase I; Solid tumors; Paclitaxel
Multiple myeloma (MM) is an incurable plasma-cell neoplasm for which most treatments involve a therapeutic agent combined with dexamethasone. The preclinical combination of lenalidomide with the mTOR inhibitor CCI-779 has displayed synergy in vitro and represents a novel combination in MM.
Patients and Methods
A phase I clinical trial was initiated for patients with relapsed myeloma with administration of oral lenalidomide on days 1 to 21 and CCI-779 intravenously once per week during a 28-day cycle. Pharmacokinetic data for both agents were obtained, and in vitro transport and uptake studies were conducted to evaluate potential drug-drug interactions.
Twenty-one patients were treated with 15 to 25 mg lenalidomide and 15 to 20 mg CCI-779. The maximum-tolerated dose (MTD) was determined to be 25 mg lenalidomide with 15 mg CCI-779. Pharmacokinetic analysis indicated increased doses of CCI-779 resulted in statistically significant changes in clearance, maximum concentrations, and areas under the concentration-time curves, with constant doses of lenalidomide. Similar and significant changes for CCI-779 pharmacokinetics were also observed with increased lenalidomide doses. Detailed mechanistic interrogation of this pharmacokinetic interaction demonstrated that lenalidomide was an ABCB1 (P-glycoprotein [P-gp]) substrate.
The MTD of this combination regimen was 25 mg lenalidomide with 15 mg CCI-779, with toxicities of fatigue, neutropenia, and electrolyte wasting. Pharmacokinetic and clinical interactions between lenalidomide and CCI-779 seemed to occur, with in vitro data indicating lenalidomide was an ABCB1 (P-gp) substrate. To our knowledge, this is the first report of a clinically significant P-gp–based drug-drug interaction with lenalidomide.
Biliary cancers (BCs) carry a poor prognosis, but targeting the RAS/RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is of significance. Selumetinib is an inhibitor of MEK1/2, so this trial was designed to determine the safety and efficacy of selumetinib in BC.
Patients and Methods
This was a multi-institutional phase II study of selumetinib at 100 mg given orally twice per day to patients with advanced BC. The primary end point was response rate. All patients were required to provide tissue before enrolling. The levels of phosphorylated ERK (pERK) and AKT (pAKT) were assessed by immunohistochemistry. Tumors were genotyped for the presence of BRAF- and/or RAS-activating mutations.
Twenty-eight eligible patients with a median age of 55.6 years were enrolled. Thirty-nine percent of patients had received one prior systemic therapy. Three patients (12%) had a confirmed objective response. Another 17 patients (68%) experienced stable disease (SD), 14 of whom (56%) experienced prolonged SD (> 16 weeks). Patients gained an average nonfluid weight of 8.6 pounds. Median progression-free survival was 3.7 months (95% CI, 3.5 to 4.9) and median overall survival was 9.8 months (95% CI, 5.97 to not available). Toxicities were mild, with rash (90%) and xerostomia (54%) being most frequent. Only one patient experienced grade 4 toxicity (fatigue). All patients had tissue available for analysis. No BRAF V600E mutations were found. Two patients with short-lived SD had KRAS mutations. Absence of pERK staining was associated with lack of response.
Selumetinib displays interesting activity and acceptable tolerability in patients with metastatic BC. Our results warrant further evaluation of selumetinib in patients with metastatic BC.
Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.
ABCB1; leukemia; multi-drug resistance; P-glycoprotein; silvestrol
A sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of the plant natural product silvestrol in mice, using ansamitocin P-3 as the internal standard. The method was validated in plasma with a lower limit of quantification of 1 ng/mL, accuracy ranging from 87 to 114%, and precision (coefficient of variation) below 15%. The validated method was used to characterize pharmacokinetics in C57BL/6 mice and metabolism in mouse, human and rat plasma, and liver microsomes. Mice were dosed with silvestrol formulated in hydroxypropyl-β-cyclodextrin via intravenous, intraperitoneal, and oral routes followed by blood sampling up to 24 h. Intraperitoneal systemic availability was 100%, but oral administration resulted in only 1.7% bioavailability. Gradual degradation of silvestrol was observed in mouse and human plasma, with approximately 60% of the parent drug remaining after 6 h. In rat plasma, however, silvestrol was completely converted to silvestric acid (SA) within 10 min. Evaluation in microsomes provided further evidence that the main metabolite formed was SA, which subsequently showed no cytotoxic or cytostatic activity in a silvestrol-sensitive lymphoblastic cell line. The ability of the analytical assay to measure tissue levels of silvestrol was evaluated in liver, brain, kidney, and spleen. Results indicated the method was capable of accurately measuring tissue levels of silvestrol and suggested it has a relatively low distribution to brain. Together, these data suggest an overall favorable pharmacokinetic profile of silvestrol in mice and provide crucial information for its continued development toward potential clinical testing.
liquid chromatography–tandem mass spectrometry; mouse plasma; pharmacokinetics; silvestrol
Tumor lysis syndrome (TLS) has been described in over 40% of patients with chronic lymphocytic leukemia (CLL) treated with the cyclin dependent kinase inhibitor, flavopiridol. We conducted a retrospective analysis to determine predictive factors for TLS. In 116 patients, the incidence of TLS was 46% (95% CI: 36%-55%). In univariable analysis, female gender, greater number of prior therapies, Rai stages III-IV, adenopathy ≥ 10 cm, splenomegaly, del(11q), decreased albumin, and increased absolute lymphocyte count, white blood cell count (WBC), β2-microglobulin, and lactate dehydrogenase (LDH) were associated (p<0.05) with TLS. In multivariable analysis, female gender, adenopathy ≥ 10 cm, elevated WBC, increased β2-microglobulin, and decreased albumin were associated with TLS (p<0.05). With respect to patient outcomes, 49% and 44% of patients with and without TLS, respectively, responded to flavopiridol (p=0.71). In a multivariable analysis controlling for number of prior therapies, cytogenetics, Rai stage, age, and gender, progression-free survival (PFS) was inferior in patients with TLS (p=0.01). Female patients and patients with elevated β2-microglobulin, increased WBC, adenopathy ≥ 10 cm, and decreased albumin were at highest risk and should be monitored for TLS with flavopiridol. TLS does not appear to be predictive of response or improved PFS in patients receiving flavopiridol.
chronic lymphocytic leukemia; flavopiridol; tumor lysis syndrome
Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this report we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 Acute Myeloid Leukemia (AML) cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. Liquid chromatography mass spectrometry profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin dependent kinase inhibitor, flavopiridol, and the Hsp90 inhibitor, 17AAG (17-(Allylamino)-17-demethoxygeldanamycin). In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.
Histone; Acute Myeloid Leukemia; Chemotherapy; Phosphorylation
Lenalidomide is effective in myeloma and low-risk myelodysplastic syndromes with deletion 5q. We report results of a phase I dose-escalation trial of lenalidomide in relapsed or refractory acute leukemia.
Patients and Methods
Thirty-one adults with acute myeloid leukemia (AML) and four adults with acute lymphoblastic leukemia (ALL) were enrolled. Lenalidomide was given orally at escalating doses of 25 to 75 mg daily on days 1 through 21 of 28-day cycles to determine the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD), as well as to provide pharmacokinetic and preliminary efficacy data.
Patients had a median age of 63 years (range, 22 to 79 years) and a median of two prior therapies (range, one to four therapies). The DLT was fatigue; 50 mg/d was the MTD. Infectious complications were frequent. Plasma lenalidomide concentration increased proportionally with dose. In AML, five (16%) of 31 patients achieved complete remission (CR); three of three patients with cytogenetic abnormalities achieved cytogenetic CR (none with deletion 5q). Response duration ranged from 5.6 to 14 months. All responses occurred in AML with low presenting WBC count. No patient with ALL responded. Two of four patients who received lenalidomide as initial therapy for AML relapse after allogeneic transplantation achieved durable CR after development of cutaneous graft-versus-host disease, without donor leukocyte infusion.
Lenalidomide was safely escalated to 50 mg daily for 21 days, every 4 weeks, and was active with relatively low toxicity in patients with relapsed/refractory AML. Remissions achieved after transplantation suggest a possible immunomodulatory effect of lenalidomide, and results provide enthusiasm for further studies in AML, either alone or in combination with conventional agents or other immunotherapies.
In preclinical models, non-cytotoxic suramin (concentrations <50 μM) potentiates the activity of multiple chemotherapeutic agents. The present study evaluated the safety and tolerability of suramin in combination with docetaxel or gemcitabine in previously chemotherapy-treated patients with advanced non-small cell lung cancer.
Patients received suramin intravenously in combination with either docetaxel on day 1 or gemcitabine on days 1 and 8, of each 21-day treatment cycle. After 3 cycles, patients with partial response (PR) or better continued on the same combination, whereas patients with stable disease (SD) or worse crossed-over to the other combination. Pharmacokinetic analyses were performed before and after each treatment.
Eighteen patients received a total of 79 courses (37 suramin plus docetaxel, 42 suramin plus gemcitabine). The dose-limiting toxicity (DLT) was febrile neutropenia, observed in three of six patients treated with suramin and docetaxel 75 mg/m2. No DLTs were observed with suramin plus docetaxel 56 mg/m2 or suramin plus gemcitabine 1,250 mg/m2. Common adverse events included neutropenia, thrombocytopenia, anemia, fatigue, nausea, vomiting, skin rash, hyperglycemia, and electrolyte abnormalities. The target plasma suramin concentration range of 10–50 μM was achieved in 90% of treatments. Discernable antitumor activity was noted in 11 patients (2 PR, 9 SD).
Non-cytotoxic suramin, in combination with docetaxel 56 mg/m2 or gemcitabine 1,250 mg/m2, was reasonably well-tolerated with a manageable toxicity profile. Target plasma concentrations were correctly predicted by our previously described dosing nomogram. The observed preliminary evidence of antitumor activity encourages evaluation of this strategy in efficacy trials.
Suramin; Docetaxel; Gemcitabine; Chemosensitizer; Modulator; Non-small cell lung cancer
Hematologic malignancies account for a substantial percentage of cancers worldwide, and the heterogeneity and biological characteristics of leukemias and lymphomas present unique therapeutic challenges. Although treatment options exist for most of these diseases, many types remain incurable and the emergence of drug resistance is pervasive. Thus, novel treatment approaches are essential to improve outcome. Nearly half of the agents used in cancer therapy today are either natural products or derivatives of natural products. The enormous chemical diversity in nature, coupled with millennia of biological selection, has generated a vast and underexplored reservoir of unique chemical structures with biologic activity. This review will describe the investigation and application of natural products derived from higher plants in the treatment of leukemia and lymphoma and the rationale behind these efforts. In addition to the approved vinca alkaloids and the epipodophyllotoxin derivatives, a number of other plant compounds have shown promise in clinical trials and in preclinical investigations. In particular, we will focus on the discovery and biological evaluation of the plant-derived agent silvestrol, which shows potential for additional development as a new therapeutic agent for B-cell malignancies including chronic lymphocytic leukemia.
Epipodophylloxin derivatives; flavopiridol; hematological malignancies; leukemia; lymphoma; natural products; silvestrol; vinca alkaloids
Targeting aberrant DNA hypermethylation in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) with decitabine may reverse epigenetic silencing in B-cell malignancies.
Twenty patients were enrolled in two phase I trials to determine the minimum effective pharmacologic dose (MEPD) of decitabine in patients with relapsed/refractory CLL (n=16) and NHL (n=4).
Patients received 1–3 cycles of decitabine. Dose limiting toxicity (DLT) was observed in 2 of 4 CLL and 2 of 2 NHL patients receiving decitabine at 15 mg/m2/d days 1–10, consisting of grade 3–4 thrombocytopenia and hyperbilirubinemia. Six patients with CLL received decitabine at 10 mg/m2/d days 1–10 without DLT; however, re-expression of methylated genes or changes in global DNA methylation were not observed. Therefore, a 5-day decitabine schedule was examined. With 15 mg/m2/d decitabine days 1–5, DLT occurred in 2 of 6 CLL and 2 of 2 NHL patients, consisting of grade 3–4 neutropenia, thrombocytopenia, and febrile neutropenia. Eight patients had stable disease. In 17 patients, there were no significant changes in genome-wide methylation or in target gene re-expression.
Dose-limiting myelosuppression and infectious complications prevents dose escalation of decitabine to levels associated with changes in global methylation or gene re-expression in CLL and NHL.
decitabine; methylation; chronic lymphocytic leukemia; non-Hodgkin’s lymphoma
The in vitro evaluation of histones and their post-translational modifications has drawn substantial interest in the development of epigenetic therapies. The differential expression of histone isoforms may serve as a potential marker in the classification of diseases affected by chromatin abnormalities. In this study, protein profiling by liquid chromatography and mass spectrometry was used to explore differences in histone composition in primary CLL cells. Extensive method validations were performed to determine the experimental variances that would impact histone relative abundance. The resulting data demonstrated that the proposed methodology was suitable for the analysis of histone profiles. In 4 normal individuals and 40 CLL patients, a significant decrease in the relative abundance of histone H2A variants (H2AFL and H2AFA/M*) was observed in primary CLL cells as compared to normal B cells. Protein identities were determined using high mass accuracy mass spectrometry and shotgun proteomics.
Chronic lymphocytic leukemia; histone; variant; RPLC-MS
We have shown that the combination of pentostatin (P), cyclophosphamide (C) and rituximab (R) achieves an overall response (OR) rate >90% with more than 40% complete responses (CR) in patients with untreated CLL. To evaluate if the tolerability of this regimen could be enhanced without sacrificing efficacy, we conducted a phase II trial of P and R without cyclophosphamide, using a higher P dose (4 mg/m2). Among the 33 patients enrolled, 82% were male, median age was 65 (9 patients ≥70 years) and 64% were Rai stage III-IV. The OR rate was 76% with 9 CR (27%), 5 nPR, and 11 PRs. At the time of this analysis, 29/33 patients are still alive and the median follow up for patients still alive is 14 months (range: 1-34.8 months). Four (12%) patients experienced grade 3 or higher hematologic toxicity and 5 (15%) experienced grade 3 or higher non-hematologic toxicity. Comparison of this trial to our previous PCR trial showed that patients treated with PCR had a higher OR rate (91% vs. 76%) and CR rate (41% vs. 27%) compared to patients treated with PR. Median treatment-free survival for all accrued patients was notably longer in PCR treated patients compared to PR (30 vs. 16 months). These findings suggest that increasing the dose of the purine nucleoside analogue does not eliminate the need for cyclophosphamide in chemoimmunotherapy for treatment of CLL.
pentostatin; rituximab; cyclophosphamide; chemoimmunotherapy; response rates; B-CLL
Previous studies in our laboratory have shown the progressive methylation and suppression of the gene encoding protein tyrosine phosphatase, PTPRO, in the livers of rats fed a methyl-deficient diet that induces hepatocarcinogenesis. Subsequently, we observed the methylation of PTPRO in primary human lung tumors and also showed its potential tumor suppressor characteristics. The present study was undertaken to investigate whether the truncated form of PTPRO (PTPROt), specifically expressed in naïve B lymphocytes, was also methylated and suppressed in chronic lymphocytic leukemia (CLL), a disease generally affecting B lymphocytes.
Experimental Design and Results
Initial screening showed that 60% of the 52 CLL samples analyzed using methylation-specific PCR assay were methylated compared with B lymphocytes from normal individuals, which were not methylated. The expression of PTPROt, as measured by semiquantitative reverse transcription-PCR, inversely correlated with methylation in the few samples tested. Analysis of additional samples (n = 50) by combined bisulfite restriction analysis showed that the PTPRO CpG island was methylated in 82% of patients with CLL compared with B lymphocytes from normal individuals. Furthermore, overall expression of PTPRO was reduced in CLL relative to normal lymphocytes. The PTPRO gene was also suppressed by methylation in the CLL cell lineWaC3CD5, where it could be reactivated upon treatment with the DNA hypome-thylating agent 5-AzaC. Ectopic expression of PTPROt in a nonexpressing cell line increased growth inhibition with fludarabine treatment, a therapy commonly used for CLL.
This study reveals the potential role of PTPRO methylation and silencing in CLL tumorigenesis and also provides a novel molecular target in the epigenetic therapy.
To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells.
A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools.
The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 μMin cell lysate. The within-day coefficients of variation (CVs, n=5) were found to be 12.0–18.0% at the LLOQ and 3.0–9.0% between 500 and 5,000 nM for dNTPs and 8.0–15.0% and 2.0–6.0% for NTPs. The between-day CVs (n=5) were 9.0–13.0% and 3.0–11.0% for dNTPs and 9.0–13.0% and 3.0–6.0% for NTPs. The within-day accuracy values were 93.0–119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected.
This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.
dNTP/NTP levels; GTI-2040; LC-MS/MS
Flavopiridol has shown promising activities in hematologic and solid tumor models, as well as in clinical trials in chronic lymphocytic leukemia patients. Flavopiridol has relatively low solubility and high plasma protein-binding. To address these issues and to provide an alternative strategy to achieve clinical efficacy, we encapsulated flavopiridol into a liposomal carrier and characterized its physicochemical and pharmacokinetic properties. The liposomes, comprising hydrogenated soy phosphatidylcholine (HSPC), cholesterol and poly (ethylene glycol) 2000-distearoyl phosphatidylethanolamine (PEG-DSPE), were prepared by polycarbonate membrane extrusion and then loaded with flavopiridol by a pH-gradient driven remote loading procedure. The liposomes had a mean diameter of 120.7 nm and a flavopiridol entrapment efficiency of 70.4%. Pharmacokinetic study in mice after i.v. bolus injection showed that the liposomal flavopiridol had an increased elimination phase half-life (T1/2β, 339.7 min vs. 57.0 min), decreased clearance (CL, 0.012 L/min vs. 0.036 L/min), and increased area under the plasma concentration–time curve (AUC, 10.8 min μmol/L vs. 3.4 min μmol/L) compared to the free drug. This indicates a significant and potentially beneficial change in flavopiridol pharmacokinetics for the liposomal formulation. Further preclinical studies are warranted to define the toxicity and therapeutic efficacy of this novel formulation.
Liposomes; Flavopiridol; Physicochemical properties; Pharmacokinetics; Nanotechnology
Flavopiridol downmodulates antiapoptotic proteins associated with resistance to fludarabine and rituximab and is effective against p53-mutated chronic lymphocytic leukemia (CLL). We conducted a phase I study of flavopiridol, fludarabine, and rituximab (FFR) in patients with mantle-cell lymphoma (MCL), indolent B-cell non-Hodgkin's lymphomas (B-NHL), and CLL to determine the activity of FFR.
Patients and Methods
Therapy included fludarabine 25 mg/m2 intravenously (IV) days 1 to 5 and rituximab 375 mg/m2 day 1 every 28 days for 6 cycles. We administered flavopiridol 50 mg/m2 by 1-hour IV bolus (IVB) day 1 (n = 15); day 1 to 2 (n = 6); 20 mg/m2 30-minute IVB + 20 mg/m2 4-hour IV infusion (n = 3); or 30 mg/m2 + 30 mg/m2 (n = 14).
Thirty-eight patients (median age, 62 years) with MCL (n = 10); indolent B-NHL including follicular (n = 9), marginal zone (n = 4), lymphoplasmacytic (n = 1), or small lymphocytic lymphoma (n = 3); and CLL (n = 11), were enrolled. Twenty-two patients were previously untreated; 16 had received one to two prior therapies. Two patients in cohort 2 developed grade 3 dose-limiting toxicity (seizures, renal insufficiency). The median number of treatment cycles was 4, with cytopenias (n = 10) and fatigue (n = 3) the most common reasons for early discontinuation. Overall response rate was 82% (complete response, 50%; unconfirmed complete response, 5%; partial response, 26%), including 80% of patients with MCL (median age, 68; seven complete responses, one partial response). Median progression-free survival (PFS) was 25.6 months. Median PFS of patients with nonblastoid variant MCL (n = 8) was 35.9 months.
FFR was active in MCL, indolent B-NHL, and CLL and should be studied for older patients with MCL who are not candidates for aggressive chemotherapy.
Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated.
Patients and Methods
Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy.
Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P ≤ .01), resulted in improved tolerability and treatment delivery.
Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.
Our preclinical work showed a dramatic synergy between interleukin-12 (IL-12) and trastuzumab for stimulation of natural killer cell cytokine secretion. We aimed to determine the safety profile of IL-12 when given in combination with trastuzumab and paclitaxel to patients with meta-static HER2-overexpressing cancers. Paclitaxel was given i.v. at 175 mg/m2 every 3 weeks. Trastuzumab was given on day 1 each week (4 mg/kg initially and 2 mg/kg thereafter) in combination with injections of IL-12 on days 2 and 5 starting in cycle 2. This trial accrued 21 patients with metastatic HER2-positive tumors (breast, 7; colon, 6; esophagus, 4; stomach, 2; pancreas, 1; thyroid, 1). The IL-12 component was dose-escalated in cohorts of three patients. The dose-limiting toxicity was grade 3 fatigue at the 300 ng/kg dose level in two patients. The recommended phase II dose was 200 ng/kg administered s.c. There was one complete response in a patient with breast cancer, partial responses in 4 patients (breast, 2; esophageal, 2), and stabilization of disease lasting 3 months or greater (SD) in 6 other patients. All but one response occurred in patients with HER2 3+ disease. Two SD patients completed 1 year of therapy. Ten patients had progressive disease. There was increased activation of extracellular signal–regulated kinase in peripheral blood mononuclear cells and increased levels of IFN-γ and several chemokines in patients with clinical benefit (complete response, partial response, or SD), but not in patients with progressive disease. IL-12 in combination with trastuzumab and paclitaxel therefore exhibits an acceptable toxicity profile and has activity in patients with HER2-overexpressing cancers.
Inhibition of ribonucleotide reductase reduces the availability of the endogenous pool of deoxycytidine and may increase cytarabine (AraC) cytotoxicity. We performed a phase I dose escalation trial of AraC combined with GTI-2040, a 20-mer antisense oligonucleotide shown in preclinical studies to decrease levels of the R2 subunit of ribonucleotide reductase, to determine the maximum tolerated dose in adults with relapsed/refractory acute myeloid leukemia.
Twenty-three adults (ages 18–59 years) were enrolled in this dose escalation phase I trial, receiving high-dose AraC twice daily combined with infusional GTI-2040. An ELISA-based assay measured plasma and intracellular concentrations of GTI-2040. R2 protein changes were evaluated by immunoblotting in pretreatment and post-treatment bone marrow samples.
The maximum tolerated dose was 5 mg/kg/d GTI-2040 (days 1–6) and 3 g/m2/dose AraC every 12 hours for 8 doses. Neurotoxicity was dose limiting. Eight patients (35%) achieved complete remission. Mean bone marrow intracellular concentration of GTI-2040 were higher at 120 hours than at 24 hours from the start of GTI-2040 (P = 0.002), suggesting intracellular drug accumulation over time. Reductions in bone marrow levels of R2 protein (>50%) were observed at 24 and 120 hours. Higher baseline R2 protein expression (P = 0.03) and reductions after 24 hours of GTI-2040 (P = 0.04) were associated with complete remission.
GTI-2040 and high-dose AraC were coadministered safely with successful reduction of the intended R2 target and encouraging clinical results. The clinical efficacy of this combination will be tested in an upcoming phase II study.
To evaluate the maximum-tolerated dose (MTD), safety profile, and immunogenicity of two chimeric, B-cell epitopes derived from the human epidermal growth factor receptor (HER2) extracellular domain in a combination vaccine with a promiscuous T-cell epitope (ie, MVF) and nor-muramyl-dipeptide as adjuvant emulsified in SEPPIC ISA 720.
Patients and Methods
Eligible patients with metastatic and/or recurrent solid tumors received three inoculations on days 1, 22, and 43 at doses of total peptide that ranged from 0.5 to 3.0 mg. Immunogenicity was evaluated by enzyme-linked immunosorbent assay, flow cytometry, and HER2 signaling assays.
Twenty-four patients received three inoculations at the intended dose levels, which elicited antibodies able to recognize native HER2 receptor and inhibited both the proliferation of HER2-expressing cell lines and phosphorylation of the HER2 protein. The MTD was determined to be the highest dose level of 3.0 mg of the combination vaccine. There was a significant increase from dose level 1 (0.5 mg) to dose level 4 (3.0 mg) in HER2-specific antibodies. Four patients (one each with adrenal, colon, ovarian, and squamous cell carcinoma of unknown primary) were judged to have stable disease; two patients (one each with endometrial and ovarian cancer) had partial responses; and 11 patients had progressive disease. Patients with stable disease received 6-month boosts, and one patient received a 20-month boost.
The combination vaccines were safe and effective in eliciting antibody responses in a subset of patients (62.5%) and were associated with no serious adverse events, autoimmune disease, or cardiotoxicity. There was preliminary evidence of clinical activity in several patients.
Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.
Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.
Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.
Hepatocellular carcinoma (HCC) is on the rise worldwide. HCC responds poorly to chemotherapy. Lapatinib is an inhibitor of EGFR and HER2/NEU both implicated in hepatocarcinogenesis. This trial was designed to determine the safety and efficacy of lapatinib in HCC.
A Fleming phase II design with a single stage of 25 patients with a 90% power to exclude a true response rate of < 10% and detect a true response rate of ≥30% was utilized. The dose of lapatinib was 1,500 mg/d administered orally in 28-day cycles. Tumor and blood specimens were analyzed for expression of HER2/NEU/CEP17 and status of downstream signal pathway proteins.
Twenty-six patients with HCC enrolled on this study. 19% had one prior therapy. Most common toxicities were diarrhea (73%), nausea (54%) and rash (42%). No objective responses were observed. Ten (40%) patients had stable disease (SD) as their best response including 6 (23%) with SD lasting > 120 days. Median progression-free-survival was 1.9 months and median overall survival 12.6 months. Patients who developed a rash had a borderline statistically significant longer survival. Tissue and blood specimens were available on >90% of patients. No somatic mutations in EGFR (exons 18–21) were found. In contrast to our previous findings, we did not find evidence of HER2/NEU somatic mutations. PTEN, P-AKT and P70S6K expression did not correlate with survival.
Lapatinib is well-tolerated but appears to benefit only a subgroup of patients for whom predictive molecular or clinical characteristics are not yet fully defined.
Serum levels of pro-(vascular endothelial growth factor [VEGF]) and anti-(thrombospondin-1 [TSP]) angiogenic cytokines were prospectively measured in a phase II trial of chemoimmunotherapy (CIT) for chronic lymphocytic leukemia (CLL) patients(n=56). Pretreatment VEGF levels were lower among patients who achieved complete remission (CR) or nodular partial remission (nPR) relative to those with partial remission (PR) or stable/progressive disease (median 122.0 pg/ml vs. 246.8 pg/ml; p=0.03). VEGF:TSP ratio was lower (anti-angiogenic phenotype) among patients who achieved CR/nPR. The pretreatment VEGF:TSP ratio also correlated with overall survival (p=0.008). A pro-angiogenic profile appears associated with diminished response and inferior survival in CLL patients receiving CIT.
Chronic lymphocytic leukaemia; angiogenesis; therapy; VEGF, prognostic factors