Gwen Nichols

Restoration of p53 function for selective Fas-mediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide

We have shown that a COOH-terminal peptide of p53 (amino acids 361–382, p53p), linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction, induced rapid apoptosis in human premalignant and malignant cell lines. Here, we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas, which express sufficient quantities of endogenous mutant or WT p53, may be restored or activated, respectively, by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system, which circumvents the blood-brain barrier. [Mol Cancer Ther 2006;5(1):20–8]

Treating Anemia of Cancer with Every-4-Week Darbepoetin Alfa: Final Efficacy and Safety Results from a Phase II, Randomized, Double-Blind, Placebo-Controlled Study

Darbepoetin alfa is an erythropoiesis-stimulating agent (ESA) approved for treating chemotherapy-induced anemia. This phase II, double-blind, placebo-controlled study examined the efficacy of darbepoetin alfa for treating anemia of cancer (AoC) in patients not receiving chemotherapy or radiotherapy. Patients were randomized 3:1 to receive darbepoetin alfa (6.75 microg/kg) or placebo every 4 weeks; the end of the study was at week 17. The primary endpoint was the percentage of patients with a hematopoietic response. Secondary endpoints included transfusion incidence and safety parameters. Efficacy analyses were performed on 162 patients in the darbepoetin alfa group and 56 patients in the placebo group. The Kaplan-Meier percentages of patients who achieved a hematopoietic response (darbepoetin alfa, 69%; placebo, 24%) or achieved the target hemoglobin (darbepoetin alfa, 85%; placebo, 50%) differed significantly between treatment groups. The transfusion incidence did not differ between treatment groups probably because of the low baseline transfusion rates in AoC patients. The incidence of adverse events (including on-study deaths) was similar in both groups. In conclusion, darbepoetin alfa appeared to be well tolerated and significantly increased hemoglobin levels in these AoC study patients.

Therapy-related myelodysplastic syndrome after autologous stem cell transplantation for breast cancer

Therapy-related myelodysplastic syndrome and acute myelogenous leukemia (t-MDS/AML) are serious complications of chemotherapy and radiotherapy for cancer. High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) may be associated with an increased incidence of these complications. The frequency of t-MDS/AML after ASCT for breast cancer is uncertain. We reviewed our database of 379 consecutive breast cancer ASCT patients treated with alkylator-based chemotherapy, followed for a median of 1.52 years (range 0–8.97), with a median survival of 6.16 years. Three patients have developed tMDS/AML. The probability of developing this complication at 5 years is 0.032 in our series. We have used pathologic, cytogenetic and molecular methods to evaluate which portions of therapy may have predisposed to the development of this complication. Cytogenetic abnormalities were not found in the stem cell harvests of these patients by metaphase analysis or by fluorescence in situ hybridization (FISH). One patient demonstrated a clonal X chromosome inactivation pattern in her stem cell harvest, indicating pre-transplant chemotherapy may have been responsible for the development of her leukemia. As two of our patients developed this complication at greater than 4 years post-transplant, the number of cases may increase with longer follow-up. While the incidence appears to be low, further prospective and retrospective analysis will be necessary to determine which portions of therapy predispose to the development of t-MDS/AML in patients undergoing ASCT for treatment of breast cancer.

Coexistence of independent myelodysplastic and Philadelphia chromosome positive clones in a patient treated with hydroxyurea

We report a patient with Philadelphia chromosome positive (Ph +ve) chronic myelogenous leukemia (CML), treated with hydroxyurea alone, who upon disease progression developed an additional Ph - ve clone containing chromosomal abnormalities typical of myelodysplastic syndrome (MDS). Retrospective analysis of a cryopreserved stem cell specimen from diagnosis confirmed that this second clone developed during the course of treatment. The development of a clone with additional cytogenetic abnormalities in CML has only been reported after leukemogenic treatment, stem cell transplantation or interferon. We report a case of secondary Ph - ve MDS/AML during blast crisis in a patient treated with hydroxyurea for CML.

Redirecting Apoptosis to Aponecrosis Induces Selective Cytotoxicity to Pancreatic Cancer Cells through Increased ROS, Decline in ATP Levels, and VDAC

Pancreatic cancer cell lines with mutated ras underwent an alternative form of cell death (aponecrosis) when treated concomitantly with clinically achievable concentrations of arsenic trioxide, ascorbic acid, and disulfiram (Antabuse; AAA). AAAs major effects are mediated through generation of intracellular reactive oxygen species (ROS) and more than 50% decline in intracellular ATP. N-acetyl cysteine and a superoxide dismutase mimetic prevented aponecrosis and restored intracellular ATP levels. DIDS (4,4′-diisothiocyanatostilbene-2, 2′ disulfonic acid), the pan- Voltage-Dependent Anion Channel (VDAC), -1, 2, 3 inhibitor and short hairpin RNA (shRNA) to VDAC-1 blocked cell death and ROS accumulation. In vivo exposure of AAA led to a 62% reduction in mean tumor size and eliminated tumors in 30% of nude mice with PANC-1 xenografts. We concluded that early caspase-independent apoptosis was shifted to VDAC-mediated “targeted” aponecrosis by the addition of disulfiram to arsenic trioxide and ascorbic acid. Conceptually, this work represents a paradigm shift where switching from apoptosis to aponecrosis death pathways, also known as targeted aponecrosis, could be utilized to selectively kill pancreatic cancer cells resistant to apoptosis. Mol Cancer Ther; 12(12); 2792–803. ©2013 AACR.

Phase II trial of sequential high-dose chemotherapy with paclitaxel, melphalan and cyclophosphamide, thiotepa and carboplatin with peripheral blood progenitor support in women with responding metastatic breast cancer

A single high-dose cycle of chemotherapy can produce response rates in excess of 50%. However, disease-free survival (DFS) is 15–20% at 5 years. The single most important predictor of prolonged DFS is achieving a complete response (CR). Increasing the proportion of patients who achieve a complete response may improve disease-free survival. Women with metastatic breast cancer and at least a partial response (PR) to induction chemotherapy received three separate high-dose cycles of chemotherapy with peripheral blood progenitor support and G-CSF. The first intensification was paclitaxel (825 mg/m2), the second melphalan (180 mg/m2) and the third consisted of cyclophosphamide 6000 mg/m2 (1500 mg/m2/day × 4), thiotepa 500 mg/m2 (125 mg/m2/day × 4) and carboplatin 800 mg/m2 (200 mg/m2/day × 4) (CTCb). Sixty-one women were enrolled and 60 completed all three cycles. Following the paclitaxel infusion most patients developed a reversible, predominantly sensory polyneuropathy. Of the 30 patients with measurable disease, 12 converted to CR, nine converted to a PR*, and five had a further PR, giving an overall response rate of 87%. The toxic death rate was 5%. No patient progressed on study. Thirty percent are progression-free with a median follow-up of 31 months (range 1–43 months) and overall survival is 61%. Three sequential high-dose cycles of chemotherapy are feasible and resulted in a high response rate. The challenge continues to be maintenance of response and provides the opportunity to evaluate strategies for eliminating minimal residual disease.

Pilot study of tandem high-dose chemotherapy and autologous stem cell transplantation with a novel combination of regimens in patients with poor risk lymphoma.

Summary:In an effort to improve the outcome of poor-risk lymphoma patients, we evaluated a novel regimen of tandem high-dose chemotherapy (THDC) with autologous stem cell transplantation. A total of 41 patients (median age 40 years, range 15–68 years) with poor-risk non-Hodgkins lymphoma and Hodgkins disease were enrolled. THDC consisted of melphalan (180u2009mg/m2) and escalating dose mitoxantrone (30–50u2009mg/m2) (MMt) for the first conditioning regimen, and thiotepa (500u2009mg/m2), carboplatin (800u2009mg/m2), and escalating dose etoposide phosphate (400–850u2009mg/m2), (ETCb) as the second regimen. In all, 31 patients (76%) completed both transplants, with a median time between transplants of 55 days (range 26–120). The maximum tolerated dose was determined as 40u2009mg/m2 for mitoxantrone and 550u2009mg/m2 for etoposide phosphate. The overall toxic death rate was 12%. Following high-dose chemotherapy, 10 of 24 evaluable patients (42%) were in CR. The two-year overall survival and event-free survival is 67% (95% CI, 52–81%) and 45%, (95% CI, 29–61%) for the 41 patients enrolled; and 69% (95% CI, 525–586%) and 48% (95% CI, 30–67%) for the 31 patients completing both transplants. This THDC regimen is feasible but with notable toxicity in heavily pretreated patients; its role in the current treatment of high-risk lymphoma remains to be determined.

Tyrosine kinase inhibitors as cancer therapy.

Since the 1980s, research in molecular biology has expanded our understanding of the complex signal transduction networks that regulate normal and malignant cell growth. Tyrosine kinases (TKs), enzymes responsible for phosphorylation of protein tyrosine residues, play a pivotal role in these networks. Adenosine triphosphate (ATP)-mediated transfer of energy by phosphorylation of key signal transduction intermediates allows signals from outside the cell to direct changes both in protein conformation and in protein–protein associations. These changes activate and inactivate further enzymes and second messengers, creating networks of interacting proteins. Regulation of these cascades occurs through receptor specificity, feedback loops, and substrate localization. Over 175 protein kinases have been described in mammals, and with mapping of the human genome, it is anticipated that the actual number may be even greater. Given the large variety of signal transduction intermediates, highly complex and specific networks of proteins direct cellular processes. These signaling pathways appear to be highly conserved throughout eukaryotic development. The connection between TK signal transduction and malignant growth became apparent with the discovery of oncogenic TKs, such as v-src and v-abl. It was recognized that the normal cellular counterparts of these oncogenes were TKs and that virally mediated overexpression of TKs could lead to malignant transformation. Although viral oncogenesis does not play a large part in human cancer, other mechanisms of aberrant activation of TKs play an important role in human malignancies.

Randomized trial of low-dose interleukin-2 vs cyclosporine a and interferon-γ after high-dose chemotherapy with peripheral blood progenitor support in women with high-risk primary breast cancer

High-risk primary breast cancer patients treated with high-dose chemotherapy (HDC) and stem cell support (SCS) have shown prolonged disease-free survival (DFS) in many studies; however, only one trial has demonstrated an overall survival benefit (OS). We hypothesize that the period following myeloablative therapy is ideal for immunologic manipulation and studied the effects of two different methods of immunotherapy following HDC with SCS aimed at the window of immune reconstitution. Seventy-two women with high-risk stage II or III breast cancer were randomized following HDC to receive either interleukin 2 (IL-2) at 1u2009million units/m2 SQ daily for 28 days or combined cyclosporine A (CsA) at 1.25u2009mg/kg intravenously daily from day 0 to +28 and interferon gamma (IFN-γ) 0.025u2009mg/m2 SQ every 2 days from day +7 to +28. At a median follow-up of 67 months, no significant difference was observed in DFS or OS between the two treatment groups. The IL-2 arm had a 59% DFS (95% CI (0.45, 0.78)) and a 72% OS (95% CI (0.58, 0.88)) at 5 years. The CsA/INF-γ arm had a similar outcome with a 55% DFS (95% CI (0.40, 0.76)) and a 78% OS (95% CI (0.65, 0.94)) at 5 years. Treatment was well tolerated, without increased toxicity.

57 – Gender Differences in Hematologic Malignancies

There are consistent gender differences in the incidence of all hematologic malignancies in adults including acute and chronic leukemias, Hodgkins disease (HD) and non-Hodgkins lymphoma (NHL), and multiple myeloma (MM). Such gender differences, although small, are consistent. A number of environmental and occupational exposures have been associated with the development of MM. These include a wide range of occupations that are not always consistent from study to study including painters, teachers (both male and female), metalworkers, farmers, and chemical workers. Gender differences in both the underlying illness and the types of treatment needed may play a role in the development of hematologic malignancies. Pregnancy, as a sex-specific condition, may affect both incidence and treatment of hematologic malignancies. Gender-related differences in response to therapy have long been a part of prognostic categorization in the hematologic malignancies. Gender differences appear to play a role in the incidence of graft-versus-host disease (GVHD), and therefore the morbidity of allogeneic stem cell transplantation. In HD, female sex has been shown to be a positive prognostic factor generally attributed to a more favorable subtype, which is more frequent in women and diagnosis at an earlier stage of disease among females. Gender itself is not an independent prognostic factor, but it is associated with favorable differences in presentation.