Maureen Ward

Tetrathiomolybdate-associated copper depletion decreases circulating endothelial progenitor cells in women with breast cancer at high risk of relapse

BACKGROUND Bone marrow-derived endothelial progenitor cells (EPCs) are critical for metastatic progression. This study explores the effect of tetrathiomolybdate (TM), an anti-angiogenic copper chelator, on EPCs in patients at high risk for breast cancer recurrence. PATIENTS AND METHODS This phase 2 study enrolled breast cancer patients with stage 3 and stage 4 without evidence of disease (NED), and stage 2 if triple-negative. TM 100 mg orally was administered to maintain ceruloplasmin <17 mg/dl for 2 years or until relapse. The primary end point was change in EPCs. RESULTS Forty patients (28 stage 2/3, 12 stage 4 NED) were enrolled. Seventy-five percent patients achieved the copper depletion target by 1 month. Ninety-one percent of triple-negative patients copper-depleted compared with 41% luminal subtypes. In copper-depleted patients only, there was a significant reduction in EPCs/ml by 27 (P = 0.04). Six patients relapsed while on study, of which only one patient had EPCs maintained below baseline. The 10-month relapse-free survival was 85.0% (95% CI 74.6%-96.8%). Only grade 3/4 toxicity was hematologic: neutropenia (3.1% of cycles), febrile neutropenia (0.2%), and anemia (0.2%). CONCLUSIONS TM is safe and appears to maintain EPCs below baseline in copper-depleted patients. TM may promote tumor dormancy and ultimately prevent relapse.

Competitive repopulation of retrovirally transduced haemopoietic stem cells.

Gene transfer into haemopoietic stem cells (HSC) may be useful in gene therapy for a variety of inherited and acquired human diseases. Cell division is required for retroviral transduction, and cytokine stimulation is often used to increase mitosis of quiescent HSC. Exposure to cytokines has been shown to have an unfavourable effect on the engraftment of these cells when competed with unmanipulated HSC. We now show that a similar engraftment defect is present when HSC are manipulated and transduced with the human multiple drug resistance (MDR) gene. The extent of the unfavourable competition depended on the relative numbers of cytokine‐treated and fresh cells when the two populations of cells were administered simultaneously into marrow‐ablated isogenic mice. When the manipulated transduced cells were given 2 or 4 d before the unmanipulated cells there was a much greater engraftment of the manipulated cells. The data suggested that the manipulated cells were at a relative disadvantage for marrow engraftment as compared to fresh cells, presumably due to the more efficient homing and engraftment properties of these latter unmanipulated cells. However, the manipulated cells had no intrinsic inability to engraft when they were the predominant donor cell population. In all cases the percent of MDR transduced cells in the engrafting manipulated cells remained relatively constant at about 25–30%. These results have implications for the use of manipulated transduced stem cells in gene therapy, suggesting that administering them before adding fresh cells can overcome their engraftment defect.

Ixabepilone-induced mitochondria and sensory axon loss in breast cancer patients.

We sought to define the clinical and ultrastructure effects of ixabepilone (Ix), a microtubule‐stabilizing chemotherapy agent on cutaneous sensory nerves and to investigate a potential mitochondrial toxicity mechanism.

Overcoming the Response Plateau in Multiple Myeloma: A Novel Bortezomib-Based Strategy for Secondary Induction and High-Yield CD34+ Stem Cell Mobilization

Purpose: This phase II study evaluated bortezomib-based secondary induction and stem cell mobilization in 38 transplant-eligible patients with myeloma who had an incomplete and stalled response to, or had relapsed after, previous immunomodulatory drug-based induction. Experimental Design: Patients received up to six 21-day cycles of bortezomib plus dexamethasone, with added liposomal doxorubicin for patients not achieving partial response or better by cycle 2 or very good partial response or better (≥VGPR) by cycle 4 (DoVeD), followed by bortezomib, high-dose cyclophosphamide, and filgrastim mobilization. Gene expression/signaling pathway analyses were conducted in purified CD34+ cells after bortezomib-based mobilization and compared against patients who received only filgrastim ± cyclophosphamide. Plasma samples were similarly analyzed for quantification of associated protein markers. Results: The response rate to DoVeD relative to the pre-DoVeD baseline was 61%, including 39% ≥VGPR. Deeper responses were achieved in 10 of 27 patients who received bortezomib-based mobilization; postmobilization response rate was 96%, including 48% ≥VGPR, relative to the pre-DoVeD baseline. Median CD34+ cell yield was 23.2 × 106 cells/kg (median of 1 apheresis session). After a median follow-up of 46.6 months, median progression-free survival was 47.1 months from DoVeD initiation; 5-year overall survival rate was 76.4%. Grade ≥3 adverse events included thrombocytopenia (13%), hand–foot syndrome (11%), peripheral neuropathy (8%), and neutropenia (5%). Bortezomib-based mobilization was associated with modulated expression of genes involved in stem cell migration. Conclusion: Bortezomib-based secondary induction and mobilization could represent an alternative strategy for elimination of tumor burden in immunomodulatory drug-resistant patients that does not impact stem cell yield. Clin Cancer Res; 19(6); 1534–46. ©2013 AACR.

Influencing the Tumor Microenvironment: A Phase II Study of Copper Depletion Using Tetrathiomolybdate in Patients with Breast Cancer at High Risk for Recurrence and in Preclinical Models of Lung Metastases

Purpose: Bone marrow–derived progenitor cells, including VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent pathways, model the tumor microenvironment. We hypothesized that copper depletion using tetrathiomolybdate would reduce EPCs in high risk for patients with breast cancer who have relapsed. We investigated the effect of tetrathiomolybdate on the tumor microenvironment in preclinical models. Experimental Design: Patients with stage II triple-negative breast cancer (TNBC), stage III and stage IV without any evidence of disease (NED), received oral tetrathiomolybdate to maintain ceruloplasmin (Cp) between 8 and 17 mg/dL for 2 years or until relapse. Endpoints were effect on EPCs and other biomarkers, safety, event-free (EFS), and overall survival (OS). For laboratory studies, MDA-LM2-luciferase cells were implanted into CB17-SCID mice and treated with tetrathiomolybdate or water. Tumor progression was quantified by bioluminescence imaging (BLI), copper depletion status by Cp oxidase levels, lysyl oxidase (LOX) activity by ELISA, and collagen deposition. Results: Seventy-five patients enrolled; 51 patients completed 2 years (1,396 cycles). Most common grade 3/4 toxicity was neutropenia (3.7%). Lower Cp levels correlated with reduced EPCs (P = 0.002) and LOXL-2 (P < 0.001). Two-year EFS for patients with stage II–III and stage IV NED was 91% and 67%, respectively. For patients with TNBC, EFS was 90% (adjuvant patients) and 50% (stage IV NED patients) at a median follow-up of 6.3 years, respectively. In preclinical models, tetrathiomolybdate decreased metastases to lungs (P = 0.04), LOX activity (P = 0.03), and collagen crosslinking (P = 0.012). Conclusions: Tetrathiomolybdate is safe, well tolerated, and affects copper-dependent components of the tumor microenvironment. Biomarker-driven clinical trials in high risk for patients with recurrent breast cancer are warranted. Clin Cancer Res; 23(3); 666–76. ©2016 AACR.

The effect of tetrathiomolybdate on circulating endothelial progenitor cells in patients with breast cancer at high risk of recurrence.

Abstract #1036 Background: Endothelial progenitor cells are critical to tumor angiogenesis and are increased in breast cancer patients. Copper is required for angiogenesis, and pre-clinical data suggest that tetrathiomolybdate (TM), a copper-depleting compound, inhibits angiogenesis and maintains tumor dormancy. We sought to measure circulating endothelial progenitor cells (CEPCs) in patients at high risk of breast cancer recurrence and to evaluate the effect of copper depletion on CEPCs.
 Methods: This analysis is part of an ongoing phase II study of TM in breast cancer patients at high risk of recurrence defined as Stage III or IV with no evidence of disease. All therapy other than hormonal was completed at least 6 weeks prior to study. Treatment: TM 180 mg daily to achieve a target ceruloplasmin (Cp) level of 5-15 mg/dL (copper depletion), and then 100 mg daily. We monitored levels of CEPCs (CD45dim, CD133+, VEGFR2+), CEA, CA15-3, and Cp at baseline and monthly. CEPCs were also measured in 6 healthy controls.
 Results: To date we have enrolled 16 patients with a median age of 51 years (range: 29-64). 14 had a history of Stage III disease, while 2 were considered to be Stage IV with no evidence of disease. The median number of positive lymph nodes among Stage III patients was 7 (1-42), with 2 patients having received neoadjuvant therapy. The median baseline Cp level was 28 mg/dL (21-41). Among 12 patients who have reached target Cp, the median time to target was 1 month (1-3 months). The median follow-up of the 4 patients who have not yet achieved target is 2.5 months. 1 of these discontinued treatment before reaching target. The median baseline CEPCs was lower in patients than healthy controls: 0.022 cells/μL (0.000-0.286) vs. 0.123 cells/μL (0.058-0.418); p=0.03. There was no statistically significant change in CEPCs from baseline over time.
 One patient was diagnosed with recurrent breast cancer at month 10. A rise in her CEPCs preceded a rise in a CEA and overt relapse by 1 and 5 months, respectively.
 Toxicity: Grade 3/4 neutropenia occurred in 3 patients. TM was held, and this resolved 5-13 days later, after which TM was resumed. No other grade 3/4 toxicity was observed. One patient discontinued TM due to diarrhea attributed to the lactose used in the compounding of TM.
 Conclusions: TM is well tolerated in breast cancer patients. We postulate that the increased CEPCs noted in one patient at month 4, 6 months prior to overt relapse, could represent the “turning on” of an angiogenic switch, resulting in an outpouring of CEPCs to the new site of metastasis. The trial is ongoing, and with additional follow-up other trends might emerge.
 Supported by Komen for the Cure Foundation, Anbinder Foundation, NY Community Trust and Breast Cancer Alliance of Greenwich. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1036.

Abstract CT309: Influencing the tumor microenvironment: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence

Background: Bone marrow derived endothelial progenitor cells (EPCs) and copper-dependent angiogenic pathways are critical to the metastatic process. Copper depletion (CD) therapy inhibits tumor metastases in preclinical models. We hypothesized that TM-associated CD would reduce EPCs in pts at high risk for BC recurrence, and we explored the relationship between CD and its effects on the tumor microenvironment in pre-clinical models. Methods: In this single arm, phase II study, BC pts at high risk for recurrence, defined as node positive triple negative (TN), stage 3 and 4 with no evidence of disease (NED) were enrolled on a trial of CD with TM. We CD’d to maintain ceruloplasmin (Cp) between 5-17 mg/dl for 2 years or until relapse. The primary endpoint was change in EPCs measured before and during treatment with TM. Secondary endpoints included tolerability, safety, and efficacy of CD. Laboratory studies: MDA-LM2-luciferase cells were implanted into CB17-SCID mice gavaged with water or TM. The tumors were quantified by bioluminescence images (BLI). We measured Cp oxidase to determine copper status. Western blots were used to assess LOX activity, and IHC was used to quantify collagen cross-linking and CD11b+ macrophage infiltration. Results: We enrolled 43 pts. Treatment duration was 24 cycles (each cycle is 28 days) for the primary study. A total of 752 cycles were completed in 2 years. The mean age was 49 (range 29-66). Mean Cp level decreased from 29 at baseline to 16 (p Citation Format: Nancy Chan, Naomi Kornhauser, Maureen Ward, Amy Willis, Tessa Cigler, Ellen Chuang, Anne Moore, Diana Donovan, Sarah E. Schneider, Christina Lam, David J. Warren, Anna Rubinchik, Sandra Hurtado Rua, Sharrell Lee, Maureen Lane, Vivek Mittal, Linda Vahdat. Influencing the tumor microenvironment: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT309. doi:10.1158/1538-7445.AM2014-CT309

Abstract P6-05-06: Association of HER2/neu single nucleotide polymorphism with trastuzumab-related cardiotoxicity

Background: Treatment with trastuzumab prolongs overall survival when given to patients (pts) with Her2/neu+ breast cancer (BC). The primary toxicity of trastuzumab is cardiotoxicity and the incidence is estimated at 2-4% in the adjuvant setting. The mechanism for trastuzumab-induced cardiotoxicity is not known. Although Her2neu expression is usually not seen on cardiac myocytes, its expression has been shown to be upregulated after chemotherapy. Trastuzumab is a monoclonal antibody that binds to the extracellular domain of Her2/neu. We hypothesized that single nucleotide polymorphisms (SNPs) in the Her2/neu receptor may play a role in trastuzumab associated cardiotoxicity. Methods: 140 pts with BC who were treated with chemotherapy and trastuzumab were enrolled into an IRB approved protocol at the Weill Cornell Medical College between July 2008 and March 2013. Cardiotoxicity was defined as either symptomatic CHF, or a decline in LVEF of 15% (or if LVEF Results: 140 subjects (29 with cardiotoxicity and 111 without) had 11 SNPs sequenced. Median age of subjects was 56 years (range: 32-85), mean baseline LVEF was 65% (±6%). 16.4% of subjects had hypertension (HTN). 80% of patients were Caucasian, 10% East Asian, 7.1% African American, 2.9% South Asian. There were two SNPs for which there was variation seen among subjects: rs 1136201 (corresponding to codon 655) and rs61552325 (codon 1170). The frequencies of the codon 655 polymorphisms were: AA (Ile/Ile) 67.9%, AG (Ile/Val) 29.3%, and GG (Val/Val) 2.9%. The frequencies of the codon 1170 polymorphisms were: CC (Pro/Pro) 20.7%, GC (Ala/Pro) 45.7%, and GG (Ala/Ala) 33.6%. There was no association observed between the codon 655 polymorphism and cardiotoxicity (p = 0.96). A significant association between cardiotoxicity and the codon 1170 polymorphism was observed, with subjects having cardiotoxicity being more likely to carry the CC allele compared with subjects without cardiotoxicity (34.5% vs 17.1%, p = 0.04). This association persisted after multivariable adjustment for age, race, and HTN status (adjusted OR = 2.60, 95% CI = 1.02-6.62, p = 0.046). Conclusion: In this study, the Her2/neu 1170 Pro/Pro polymorphism was associated with trastuzumab cardiotoxicity. If confirmed in a larger series, this polymorphism could be used to identify pts who may be at increased risk for cardiotoxicity and who may benefit from treatments associated with less cardiotoxicity. Furthermore, the Her2/neu 1170 SNP has previously been implicated as a minor histocompatibility antigen, and our findings raise the possibility that immune mediated mechanisms may play a role in trastuzumab related cardiotoxicity. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-06.

Abstract P1-15-07: Ixabepilone-associated peripheral neuropathy in metastatic breast cancer patients and its effects on the ultrastructure of neurons

Background: Peripheral neuropathy is a dose-limiting toxicity of most microtubule-stabilizing chemotherapeutic agents. Ixabepilone, a semisynthetic analog of the natural epothilone B, has activity in a wide range of tumors including taxane-resistant disease. In this study, we sought to understand the effect of ixabepilone on the development of peripheral neuropathy both clinically and its effect at the ultrastructural level of the peripheral nerves and circulating factors over time. Parallel studies in animal models of neuropathy were performed at the same time (Proc AACR 2010 Abstract 4184). Methods: This open-label, non-randomized phase II study enrolled 14 patients with metastatic breast cancer. Ixabepilone was administered by 2 schedules: the FDA approved dose of 40 mg/m 2 every 3 weeks (q3w) and 16 mg/m 2 on day 1, 8, and 15 of a 28-day cycle (weekly). Five controls, 2 with residual taxane-associated peripheral neuropathy and 3 with no prior chemotherapy or peripheral neuropathy, were also accrued. The primary objectives were to characterize the natural history of ixabepilone-associated peripheral neuropathy using the Total Neuropathy Score Clinical (TNSc) assessment tool prior to each cycle and to correlate changes in the ultrastructure of dermal myelinated nerve fibers via a 3 mm punch biopsy of an area 10 cm above the lateral malleolus every 2 cycles with electron microscopy (EM), as well as circulating factors (both inflammatory and neurotrophic) considered to be important in the pathogenesis of chemotherapy-induced peripheral neuropathy. Secondary objectives included progression-free survival (PFS) and non-neurologic toxicity. Results: 14 patients were enrolled and were equally divided between the 2 schedules of ixabepilone chemotherapy. There were no differences in baseline characteristics between the two groups. Mean age was 54 years (range 32–71). Mean number of previous chemotherapy regimens was 3.5 (range 0–8). 57% of patients had received a taxane in the adjuvant setting and 64% in the metastatic setting. The mean neuropathy score (TNSc) at baseline was 4.6 (range 1–11). At a mean cumulative dose of 185 mg/m 2 , the TNSc with ixabepilone q3w schedule was 3.7 points higher/worse (95% CI: 2.2–5.3, p = 0.03) than the mean score observed in patients on the weekly schedule. The sensory component was most significantly affected, predominantly numbness. In 3 patients, the chemotherapy schedule was changed from every 3 weeks to weekly due to > grade 2 toxicity at a mean cumulative dose of 107 mg/m 2 , and TNSc decreased/improved by 2.7 points. PFS in patients on q3w ixabepilone was 133 days (range 28–280) and in patients on weekly ixabepilone was 179 days (range 66–336), nonsignificant. Evaluation of EM and circulating factors is ongoing. Conclusions: Weekly ixabepilone appears to have a more favorable neurotoxicity profile compared to the standard q3w schedule. Integration of the EM data and the circulating factor data are underway and will be presented. Ixabepilone-associated peripheral neuropathy may improve in patients switched to weekly ixabepilone without compromising efficacy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-15-07.

Abstract P6-11-04: Targeting the tumor microenvironment: tetrathiomolybdate decreases circulating endothelial progenitor cells in women with breast cancer at high risk of relapse.

Background: Bone marrow-derived endothelial progenitor cells (EPCs) constitute an important part of the tumor microenvironment and are critical for metastatic progression in preclinical models and breast cancer patients (Jain et al, Breast Cancer Res Treat, 2012). Tetrathiomolybdate (TM), a copper-depleting compound inhibits angiogenesis, tumor growth, and metastasis. This study explores the effect of TM on EPCs in patients at high risk for breast cancer recurrence. Methods: This phase II study enrolled stage 3, 4 without evidence of disease (NED), and any node-positive triple negative breast cancer patient. Only concomitant hormone therapy was allowed. Patients received induction TM 180 mg daily at baseline followed by an equal or lower daily dose (median 100 mg, range 0–140) to maintain ceruloplasmin (Cp) level Results: 50 patients (33 adjuvant, 17 Stage 4 NED, and 22 triple negative) were enrolled. In the first 40 patients enrolled who had received at least 24 months of TM, EPC and Cp data were available for analysis. Of these 40 patients, 1 patient did not take TM due to patient preference, and 736 cycles of TM (average 18.9 per patient) were administered. Median age was 50 years (range 29–66). Median number of tumor size and positive lymph nodes among adjuvant patients were 3.5 cm (range 1.2–7) and 9 (range 0–42), respectively. Of the patients receiving hormone therapy, 11 patients were on tamoxifen and 16 patients were on an aromatase inhibitor. Median baseline Cp level was 30 mg/dL (range 20–47). 71% patients adequately copper depleted at month 1 to a mean Cp of 14.8 mg/dL. A larger proportion of triple negative patients copper depleted (82%) compared to hormone receptor positive subtypes (47%) and HER2/neu positive subtypes (67%). Median EPCs/ml decreased from baseline to last dose by 16 in patients that achieved the copper depletion target, p = 0.014. Conversely, in patients that did not copper deplete, median EPCs/ml increased by 136, p = 0.005. Of the 50 patients on study, 7 patients relapsed in which a significant increase in EPCs preceded an objective clinical relapse and a tumor marker rise by a median of 1 month. Only grade 3/4 toxicity was hematologic, occurred in 49 cycles (6.7%), and resolved in 5–13 days with TM held and resumed at a lower dose. Conclusions: TM is a well-tolerated oral copper chelator that may contribute to maintaining EPCs below baseline in copper-depleted patients. Molecular subtype may impact on the ability to copper deplete. EPCs may have potential as a surrogate marker for early relapse and as a therapeutic target for interrupting the metastatic progression. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-04.