Tetrathiomolybdate-Associated Copper Depletion Decreases Circulating Endothelial Progenitor Cells in Women With Breast Cancer at High Risk of Relapse

S. Jain; J. Cohen; M. M. Ward; N. Kornhauser; E. Chuang; T. Cigler; A. Moore; D. Donovan; C. Lam; M. V. Cobham; S. Schneider; S. M. Hurtado Rúa; S. Benkert; C. Mathijsen Greenwood; R. Zelkowitz; J. D. Warren; M. E. Lane; V. Mittal; S. Rafii; L. T. Vahdat


Ann Oncol. 2013;24(6):1491-1498. 

In This Article

Abstract and Introduction


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.


Despite improvements in adjuvant therapy of breast cancer over the past two decades, there is significant risk of relapse in a high-risk subset of patients. Although the definition of high risk has been evolving, it still includes patients with stage 3 breast cancer and those with stage 4 with no evidence of disease (NED). The risk of relapse in stage 3 patients is 50%–75% over 5 years, and patients with stage 4 breast cancer will inevitably recur even when temporarily rendered disease-free by surgery, radiation, or chemotherapy.[1] Reflecting the increasing importance of biology over stage, now included in the definition is the triple-negative subset [lack of expression of estrogen receptor (ER)/progesterone receptor (PR), nor overexpression of human epidermal growth factor 2 (HER2)]. These patients have a poor prognosis in earlier stages and represent a disproportionately increased percentage with metastatic disease.[2–4]

The tumor microenvironment and its components, including stromal fibroblasts and endothelial and inflammatory cells, play a major role in the establishment, progression, and metastatic dissemination of cancer.[5–11] Using preclinical models of breast cancer that metastasize to the lungs, the premetastatic niche is comprised of recruited bone marrow-derived cells, including endothelial progenitor cells (EPCs; CD45dim, CD133+, VEGFR2+), that modulate the angiogenic switch for the progression of avascularized micrometastases to vascularized macrometastases. VEGFR1+ hematopoietic progenitor cells (HPCs) and CD11b+ myeloid progenitor cells establish the premetastatic niche and recruit EPCs, among other cells, to activate this angiogenic switch.[12,13] EPC deficiency results in impaired macrometastatic formation as a result of severe angiogenesis inhibition.[9] We extended these analyses to breast cancer patients, in which significant increases in EPCs and HPCs were observed immediately before overt relapse, suggesting that these cells comprise a critical component for the propagation of macrometastases.[14]

While there are many important components of angiogenesis, copper is emerging as essential through experiments that demonstrate decreased endothelial cell proliferation, blood vessel formation, and tumor growth with copper depletion.[15–18] Copper is a required cofactor in the expression, activation, and secretion of key activators of angiogenesis through multiple mechanisms including NF-κB and HIF-1 alpha. Copper is a key component of enzymes, including superoxide dismutase-1, vascular adhesion protein-1, and lysyl oxidase, implicated in the priming of the tumor microenvironment.[19–22] Copper may also play a role in migration and invasion as perinuclear copper is translocated to the leading edge of endothelial cell projections during angiogenesis and is transported across the cell membrane.[23]

Tetrathiomolybdate (TM), an oral copper chelator developed for the treatment of Wilson's disease, blocks angiogenesis through the inactivation of copper chaperones and decreased incorporation of copper into copper-containing enzymes.[24] Copper levels needed for physiologic functions are lower than those favored by tumor angiogenesis; therefore, copper must be depleted to a therapeutic window. This is achieved by measuring serum ceruloplasmin (Cp), a major extracellular copper transporter, used as a surrogate marker of copper availability.[25] In non-human primates, copper depletion has been shown to decrease peripheral circulation of EPCs.[26] In an HER2/neu breast cancer mouse model, TM was studied as a chemo-preventive agent and it delayed tumor development by >200 days, suggesting that TM maintained these micrometastatic tumors in a dormant-like state.[20,27] Phase I/II studies of TM in advanced malignancies demonstrated safety and promising efficacy, particularly in patients with minimal residual disease.[28–30]

Encouraged by these data, we investigated TM as a drug to promote tumor dormancy in breast cancer patients with NED but at high risk of relapse. We hypothesized that targeting the tumor microenvironment via copper depletion prevents relapse by disrupting the EPC-mediated angiogenic switch required for the progression of micro- to macrometastasis. We further hypothesized that TM might promote tumor dormancy, as reflected by a decrease in circulating EPCs. We report here the results for the first 12 months of copper depletion.