Carboxyamido-triazole (CAI)--a novel "static" signal transduction inhibitor induces apoptosis in human B-cell chronic lymphocytic leukemia cells.

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 microM) 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 microM (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 microM 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 microM) 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 II studies of CAI in patients with B-CLL.

Gemcitabine demonstrates in vitro activity against human B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is the most common type of leukemia diagnosed in the Western Hemisphere and remains incurable with currently available therapy. In an attempt to identify new potential therapy for CLL, we explored the pre-clinical activity of gemcitabine in human B-CLL cells (n =11). Mononuclear cell isolates were exposed to varying concentrations of gemcitabine (0.01-100 microM) for 4, 24, and 96 h. Viability, as determined by the tetrazolium salt (MTT) assay, after a 4 h incubation with gemcitabine declined in 6 of 8 (75%) evaluable patients at a concentration < 30 microM (a physiologically attainable level), and 3 of 8 of the B-CLL cells had an LC50 (concentration where 50% loss of viability is observed) < 30 microM. At 4 days of drug exposure, 82% (9/11) of patients had an LC50 < 30 microM. Annexin-V/propidium iodine staining demonstrated apoptosis in CLL cells exposed to 30 microM of gemcitabine. Examination of changes in apoptosis related proteins demonstrated no significant change in bcl-2, bax or p53 protein expression with gemcitabine (23 microM) at 4, 24, or 48 h. These data demonstrate that gemcitabine has pre-clinical activity in B-CLL and supports its exploration as a single agent and in combination with other active agents in this disease.

Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity.

PURPOSE: Rituximab has been reported to have little activity in small lymphocytic lymphoma (SLL)/chronic lymphocytic leukemia (CLL) and to be associated with significant infusion-related toxicity. This study sought to decrease the initial toxicity and optimize the pharmacokinetics with an alternative treatment schedule. PATIENTS AND METHODS: Thirty three patients with SLL/CLL received dose 1 of rituximab (100 mg) over 4 hours. In cohort I (n = 3; 250 mg/m(2)) and cohort II (n = 7; 375 mg/m(2)) rituximab was administered on day 3 and thereafter three times weekly for 4 weeks using a standard administration schedule. Cohort III (n = 23; 375 mg/m(2)) administered rituximab similar to cohort II for the first two treatments and then over 1 hour thereafter. RESULTS: A total of 33 CLL/SLL patients were enrolled; only one patient discontinued therapy because of infusion-related toxicity. Thirteen patients developed transient hypoxemia, hypotension, or dyspnea that were associated with significant changes in baseline interleukin-6, interleukin-8, tumor necrosis factor alpha, and interferon gamma compared with those not experiencing such reactions. Infusion-related toxicity occurred more commonly in older (median age 73 v 62 years; P =.02) patients with no other pretreatment clinical or laboratory features predicting occurrence of these events. The overall response rate was 45% (3% CR, 42% PR; 95% CI 28% to 64%). Median response duration for these 15 patients was 10 months (95% CI, 6.8-13.2; range, 3 to 17+). CONCLUSION: Rituximab administered thrice weekly for 4 weeks demonstrates clinical efficacy and acceptable toxicity. Initial infusion-related events seem to be cytokine mediated and resolve by the third infusion making rapid administration possible. Future combination studies of rituximab with other therapies in CLL seem warranted.

Novel therapies for chronic lymphocytic leukemia in the 21st century.

Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia diagnosed in the Western Hemisphere. Introduction of the purine analogs has improved outcome for patients with CLL as measured by improvements in disease-free survival and has made it possible to attain a complete remission in a minority of patients with this disease. The therapeutic success with the purine analogs in CLL has led to preclinical and early clinical investigation of a variety of other therapeutic agents. This, combined with advances in the understanding the genetic and immunobiology of CLL, leaves hope that CLL may become a curable disease in this century.

Theophylline, pentostatin (Nipent), and chlorambucil: a dose-escalation study targeting intrinsic biologic resistance mechanisms in patients with relapsed lymphoproliferative disorders.

In spite of the chemosensitivity seen with the initial treatment of malignant lymphoid disorders, relapse is common and death most often occurs as a result of disease progression. This is related to a multitude of resistance mechanisms associated with the various lymphoproliferative disorders. As a result, therapies targeting intrinsic drug-resistance mechanisms are evolving and have become an active area of research. In vitro studies of human chronic lymphocytic leukemia cells incubated with theophylline, a phosphodiesterase inhibitor, resulted in downregulation of bcl-2 concomitant with induction of apoptosis. We describe the preclinical basis for a novel combination therapy involving pentostatin (Nipent; SuperGen, San Ramon, CA), chlorambucil, and theophylline in the treatment of patients with relapsed chronic lymphoproliferative disorders. An ongoing study based on such justification, which is currently accruing patients, is also described. Results from this trial appear promising, and a phase II study is now being planned.

Pentostatin (Nipent) and chlorambucil with granulocyte-macrophage colony-stimulating factor support for patients with previously untreated, treated, and fludarabine-refractory B-cell chronic lymphocytic leukemia.

Renewed interest in chronic lymphocytic leukemia (CLL) has led to an unprecedented number of investigators contributing to all aspects of research in this disease. In fact, the evolution of research in the area of molecular aberrations in CLL and their impact on treatment resistance alone is striking. These data, along with the advent of the purine analogs, have been central to this paradigm shift. The inferior response rate, the abbreviated response duration, and the inability to prolong survival with alkylating agents such as chlorambucil have resulted in purine analogs being used as first- and second-line therapy for patients with CLL. In fact, patients treated with fludarabine have a higher overall and complete response rate as well as a disease-free survival advantage compared with patients treated with alkylator-based therapy. Pentostatin, the first purine analog to enter clinical trials, was never subjected to extensive schedule optimization despite its demonstrated efficacy and its paucity of significant myelosuppression compared with the other purine analogs. However, pentostatin induced a 25% to 30% response rate in heavily pretreated CLL patients, including some who had received prior fludarabine, suggesting possible non-cross-resistance. Based on preclinical data demonstrating synergistic activity when a DNA damaging agent (eg, an alkylating agent) is followed by an inhibitor of DNA repair (a purine analog), a number of purine analog/alkylator combinations have been and are presently being examined in a variety of lymphoid neoplasms. While the clinical data conflict, at least two phase II studies examining a combination of a purine analog and an alkylator in untreated patients with CLL have generated promising data. This report describes the scientific justification and the design of a new phase II study examining the combination of pentostatin and chlorambucil with granulocyte-macrophage colony-stimulating factor support for patients with untreated, treated, and fludarabine-refractory B-cell CLL.

Depsipeptide (FR901228): a novel therapeutic agent with selective, in vitro activity against human B-cell chronic lymphocytic leukemia cells.

Therapy of B-cell chronic lymphocytic leukemia (CLL) has been limited by both the nonselectivity of therapeutic agents toward normal residual immune cells and inherent drug resistance. Identification of agents that spare normal immune effector cells, thus facilitating addition of immune-based therapies, and that modulate factors associated with drug resistance in CLL might represent a major therapeutic advance. Depsipeptide (FR901228) is a novel agent entering clinical trials that has selective in vitro activity against resistant leukemia cell lines. To assess its in vitro activity in CLL, we exposed peripheral mononuclear cells from CLL patients (n = 10) to varying concentrations of this agent. Viability of the CLL cells was reduced by 50% (LC(50)) at 4 hours, 24 hours, and 4 days at depsipeptide concentrations of 0.038, 0.024, and 0.015 micromol/L, respectively. Depsipeptide had marked selective cytotoxicity when compared with normal blood mononuclear cells, in which the LC(50) was 3.44 micromol/L at 4 hours (P =.03), 0.965 micromol/L at 24 hours (P =.01), and 0.0318 micromol/L at 96 hours (P =.04). Inhibition of bone marrow progenitor cell growth was also minimal after incubation with 0.015 micromol/L (19% inhibition of colony forming unit-granulocyte-macrophage [CFU-GM]; 17% inhibition burst forming unit-erythroid [BFU-E]) and 3.44 micromol/L (24% inhibition of CFU-GM; 57% inhibition BFU-E) of depsipeptide for 4 hours, followed by a 14-day incubation period. Expression of apoptotic proteins after depsipeptide exposure (0.015 micromol/L) included no change in bcl-2, elevation of bax, and decreased expression of p27. These data demonstrate that depsipeptide has significant selective in vitro activity against human CLL cells concurrent with favorable alterations of the bcl-2:bax protein ratio and decrease in p27 expression. Such findings strongly support the early introduction of depsipeptide into clinical trials for patients with CLL.

Stem-cell transplantation in chronic lymphocytic leukemia: the time for designing randomized studies has arrived.

Stem-cell transplantation in conjunction with myeloablative therapy has evolved as a standard treatment option for patients with several hematologic malignancies, including chemosensitive, relapsed non-Hodgkin's lymphoma and untreated multiple myeloma. The pharmacologic basis for this treatment includes a favorable tumor dose-response curve that abrogates intrinsic drug resistance associated with these diseases and facilitates cure or prolongation of survival even in the absence of a cure. The belief that chronic lymphocytic leukemia (CLL) is a palliative disease of the elderly has been perpetuated, limiting the application of more aggressive therapies. The introduction of fludarabine and its use in combination with other agents has increased the morphologic complete response rate observed in the initial treatment of CLL, providing the rationale to explore further disease-consolidative therapies. Concomitant with this, several phase II studies have demonstrated the feasibility of performing both allogeneic and autologous stem-cell transplantation in patients with CLL. In this regard, allogeneic transplantation has produced prolonged remissions in young patients with relapsed and refractory CLL, but at the cost of high treatment-related morbidity and mortality. Application of "minitransplantation" regimens may temper the frequency of these complications and warrants further study. Autologous stem-cell transplantation has also been explored with promising disease-free survival outcomes in less heavily pretreated patients. However, relapses continue to be the most frequent source of late mortality, as has been observed previously with multiple myeloma. With scientific justification established in similar diseases and demonstrated feasibility with low morbidity, we believe the time for a randomized comparison of standard chemotherapy versus autologous stem-cell transplantation in CLL has arrived. Despite promising results observed with allogeneic transplantation, further refinements that broaden the patient eligibility and lower treatment mortality will be required before similar investigations can occur with this modality.

Rituximab therapy in hematologic malignancy patients with circulating blood tumor cells: association with increased infusion-related side effects and rapid blood tumor clearance.

PURPOSE: Rituximab was recently approved for use in relapsed, low-grade non-Hodgkin's lymphoma; however, few data exist regarding the safety of this agent in patients with a high number of tumor cells in the blood. METHODS AND RESULTS: After the observation at our institution of a rapid reduction of peripheral-blood tumor cells with associated severe pulmonary infusion-related toxicity in two patients with refractory hematologic malignancies, data on three additional cases were collected from physician-submitted reports of adverse events related to rituximab treatment. Five patients with hematologic malignancies possessing a high number of blood tumor cells were treated with rituximab and developed rapid tumor clearance. The median age was 68 years (range, 26 to 78 years). Patients were diagnosed with B-cell prolymphocytic leukemia (n = 2), chronic lymphocytic leukemia (n = 2), or transformed non-Hodgkin's lymphoma (n = 1). All of these patients had bulky adenopathy or organomegaly. All five patients developed a unique syndrome of severe infusion-related reactions, thrombocytopenia, rapid decrement in circulating tumor cell load, and mild electrolyte evidence of tumor lysis, and all required hospitalization. In addition, one patient developed ascites. These events resolved, and four patients were subsequently treated with rituximab without significant complications. CONCLUSION: Rituximab administration in patients who have a high number of tumor cells in the blood may have an increased likelihood of severe initial infusion-related reactions. These data also suggest that rituximab may have activity in a variety of other lymphoid neoplasms, such as chronic lymphocytic leukemia and B-cell prolymphocytic leukemia.

Women with thrombophilia: assessing the risks for thrombosis with oral contraceptives or hormone replacement therapy.

Women with inherited or acquired thrombophilia are at increased risk for venous thromboembolism (VTE) when they use oral contraceptives (OCs) of either the second or third generation. For women who are heterozygous for Factor V Leiden, the risk is probably 28 to 50 of 10,000 women-years compared to 2 to 5 of 10,000 years for those not known to have thrombophilia. The thrombotic risk is highest during the first year that OCs are used. Whether women with thrombophilia are at increased risk for VTE when they use hormone replacement therapy (HRT) has not been assessed in any study. For women without thrombophilia, the risk for VTE associated with HRT is probably 2 to 3 of 10,000 years. The benefits of HRT include reduced risk for myocardial infarction and Alzheimer's disease, and increased bone density. The physiological changes induced by HRT are not the same as those induced by OCs. Small studies have suggested that for women who have additional risks of thrombosis (i.e., perioperative setting, underlying systemic lupus erythematosus), HRT does not confer the same increased risk of thrombosis, as does the use of OCs. Until data are available to address the magnitude of any increase in thrombotic risk induced by HRT for women with thrombophilia, physicians probably serve their patients best by providing information about the benefits of HRT, emphasizing that the risk of VTE is unknown, and encouraging patients to take an active role in decisions about their healthcare.

Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53.

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 micromol/L (95% confidence interval [CI] +/-0.31), 0.18 micromol/L (95% CI +/-0.04), and 0.16 micromol/L (95% CI +/-0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 micromol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol's dependence on intact p53 by exposing splenocytes from wild-type (p53(+/+)) and p53 null (p53(-/-)) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.

Preservation of nucleic acids for polymerase chain reaction after prolonged storage at room temperature.

A guanidinium isothiocyanate (GITC) lysis solution was evaluated for its efficacy in preserving nucleic acids for subsequent analysis after prolonged storage at room temperature. Aliquots of thyroid crude cell lysates were stored at 22 degrees C for 8 weeks in the GITC solution. Crude cell lysates stored for 2 weeks in the GITC solution consistently provided adequate RNA and DNA for reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, respectively, of beta 2-microglobulin, thyroid stimulating hormone receptor (TSHR), and thyroglobulin. Interestingly, the thyroglobulin and TSHR messages from thyroid fine needle aspirate samples were detected by RT-PCR only when stored at room temperature for less than 1 week, whereas the RT-PCR product for beta 2-microglobulin was detectable in 95% of the samples at 3 months. In addition, thyroglobulin DNA was amplified by PCR in nearly all samples stored at 22 degrees C for 2 months. These data suggest that GITC solutions can be used to preserve nucleic acids in most circumstance until transported to laboratories equipped and staffed with appropriate resources.

Management of progressive metastatic prostate cancer.

Metastatic prostate cancer is a growing health problem and is the second leading cause of cancer death in men. While the response of patients with metastatic prostate cancer to initial hormonal manipulation is excellent, the majority of patients eventually progress. As a result, a growing number of patients and their physicians need-to-find acceptable therapeutic alternatives. Fortunately, the number of therapies in the management armamentarium is growing and includes: alternative hormonal therapies, chemotherapy, radioisotopes, and investigational agents. The major focus of treatment has shifted to palliation and quality of life. The decline of prostate-specific antigen (PSA) has become another important end point as evidence supporting a correlation with prolonged survival mounts. Enrolling eligible patients in clinical trials is critical to the development of new treatment strategies for this difficult disease.