Sarah Schneider

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.

Efficacy of Scalp Cooling in Preventing Chemotherapy-Induced Alopecia in Breast Cancer Patients Receiving Adjuvant Docetaxel and Cyclophosphamide Chemotherapy

BACKGROUND Chemotherapy-induced alopecia (CIA) is a distressing adverse effect of many chemotherapy agents. The TC (docetaxel [Taxotere] and cyclophosphamide) chemotherapy regimen is typically associated with complete alopecia. Scalp cooling with cold caps has been reported to minimize or prevent CIA. We conducted a prospective study to assess efficacy of scalp cooling in preventing CIA among women receiving adjuvant TC chemotherapy for breast cancer. METHODS Women at the Weill Cornell Breast Center who independently elected to use scalp cooling with cold caps during adjuvant TC chemotherapy were asked to participate. Degree of hair loss was assessed by a single practitioner using Deans alopecia scale (grade 1/excellent [< 25% hair loss], grade 2/good [25%-50% hair loss], grade 3/moderate [50%-75% hair loss], grade 4/poor [> 75% hair loss]), by digital photographs, and by patient self-report of hair thinning or the need to wear a wig/head covering, or both. Assessments were made before each chemotherapy treatment and at follow-up visits between 3 weeks and 3 months after completion of chemotherapy. RESULTS Of 20 evaluable patients, 10% reported a need to wear a wig/head covering at the follow-up visit. Deans alopecia score was excellent for 65% of patients, good for 25% of patients, and moderate or poor for 10% of patients. The majority of patients reported hair thinning after every chemotherapy cycle. No patient discontinued therapy because of an intolerance to cold caps. CONCLUSION Scalp cooling with cold caps appears to be effective in preventing CIA among the majority of women undergoing treatment with TC chemotherapy.

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.

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 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.

Abstract 2699: Phase II study evaluating the effect of tetrathiomolybdate on circulating endothelial progenitor cells in patients with high risk for breast cancer recurrence

Background: Bone marrow-derived endothelial progenitor cells (EPCs) are critical for metastatic progression. Tetrathiomolybdate (TM), a copper-depleting compound inhibits angiogenesis and maintains tumor dormancy. This study explores the effect of TM on EPCs in patients (pts) at high risk for breast cancer (BC) recurrence. Methods: This phase II study enrolled Stage 3, 4 without evidence of disease (NED), and any node-positive triple negative (TN) BC pts. Only concomitant hormonal therapy was allowed. Pts received induction TM 180 mg daily at baseline (with exception of one pt) followed by an equal or lower daily dose (median 100mg, range 0-140) to maintain ceruloplasmin (Cp) level Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2699. doi:1538-7445.AM2012-2699

Abstract 4720: Incremental increase in VEGFR1+ and VEGFR2+ hemangiogenic progenitor cells predict relapse and tumor response in breast cancer (BC) patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: BC can relapse years after initial diagnosis. In animal models, bone marrow (BM)-derived VEGFR1+ cells define the premetatastic niche and VEGFR2+ cells are critical for the transition from micro- to macrometastatic disease. We sought to define the temporal relationship of these circulating hemangiogenic progenitor cells in a cohort of BC patients (pts) that developed recurrent disease and whether quantitative changes in these cells could predict response to therapy in pts with established metastases. Methods: 125 pts with Stages I to IV BC enrolled in 2 studies. In Study #1 circulating VEGFR1+ (CD45+/CD34+/VEGFR1+) hematopoietic progenitor cells (HPCs) and VEGFR2+ (CD45dim/CD133+/ VEGFR2+) endothelial progenitor cells (EPCs) of adjuvant patients were assessed at baseline (BL), every 3 months (mo) for the first year, then every 6 mo. Stage IV pts had levels measured at BL (defined as when initiating new treatment (tx)), with subsequent 1 mo interval blood draws and accompanied by a clinical evaluation. Tx could include chemo-, hormone or biologic therapy. In Study #2, these cells were examined monthly in pts without overt evidence of BC. HPCs/EPCs were quantified from peripheral blood mononuclear cells using flow cytometry with commercially available antibodies. Statistical analysis is by Wilcoxon signed-rank test. Results: Data from both studies were combined to analyze 1) pts without overt BC who relapse and 2) stage IV pts according to response. Seven pts without evidence of BC based on physical exam, labs, and imaging developed recurrence while on study. In all 7 pts, there was a median (med) increase of 1,111% in HPCs preceding overt relapse (range 283% to 5800%). Med HPCs at BL 0.65/ul (range 0.02 to 1.22/ul) increased to 2.90/ul prior to relapse (range 1.18 to 34.94/ul), p=0.016. In 5 of 7 relapsed pts a 433% increase in EPCs occurred as HPCs decreased: med EPCs at BL 0.03/ul (range 0.015 to 0.21/ul) and at relapse 0.16/ul (range 0.03 to 0.27/ul). This pattern was not seen in non-relapsed pts. In 22 stage IV pts, HPCs and EPCs were evaluated over the course of 40 tx. For the 20 tx (16 pts) in which progression of disease (POD) was the outcome, HPCs increased prior to POD (median 7.45/ul, range 0.37 to 77.6/ul) from BL (med 1.70/ul, range 0.01 to 16.47/ul), p=0.001. Similarly, EPCs increased at relapse (med 0.07/ul, range 0 to 0.62/ul) from BL (med 0.03/ul, range 0 to 0.21/ul), p=0.04. For the 12 tx (11 pts) with disease responding to systemic tx, there was a reduction in HPCs (BL med 6.15/ul, range 0.91 to 85.1/ul) to a 3 mo time point (med 0.63/ul, range 0.05 to 18.1/ul), p=0.05. A trend was noted in med EPCs (0.05/ul at BL to 0.02/ul at 3 mo), p=0.37. There was no change in HPCs/EPCs in 8 tx (6 pts) with stable disease. Conclusion: BM-derived progenitor cells are important in the metastatic cascade and may represent a novel biomarker in following disease status and a new target for therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4720. doi:10.1158/1538-7445.AM2011-4720

Abstract P2-16-14: The Effect of Tetrathiomolybdate (TM) on Circulating Endothelial Progenitor Cells in Women at Moderate to High Risk of BC Recurrence

Background: Bone marrow (BM) derived endothelial progenitor cells (EPCs) are critical to tumor angiogenesis, are increased in BC patients (pts) and are probably an early marker for paclitaxel response. Copper is required for angiogenesis, and pre-clinical data suggest that TM, a copperdepleting compound, inhibits angiogenesis and maintains tumor dormancy through unknown mechanisms. We sought to measure the effect of TM on BM derived EPCs in pts at high risk of BC recurrence and to evaluate the effect of copper depletion on their absolute number. Methods: This analysis is part of an ongoing phase II study of TM in BC pts at high risk of recurrence defined as any node positive triple negative (TN) BC, Stage III or IV with no evidence of disease (NED). 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 EPCs (CD45dim, CD133+, VEGFR2+), CEA, CA15-3, and Cp at baseline and monthly. Imaging studies are done every 6 months (mos). Initial study duration is 24 mos. Extension study for an additional 24 mos in selected pts. Results: 40 pts are enrolled and 566 cycles of TM have been administered. Adjuvant: 28 pts, Stage 4 NED: 12 pts, Triple negative: 11 pts (4 stage 4 NED, 7 Adjuvant). Median age is 51 yrs (range: 29-64). Median number of positive lymph nodes among Stage 2/3 pts is 7 (4-42). Median baseline Cp level is 29 mg/dL (21-47). Among 36 pts who have reached target Cp, the median time to target is 4 wks (2-20 wks). Four pts discontinued treatment before reaching target. The median baseline EPCs is 0.01 cells/ml (0.0-0.286), and the majority of pts’ EPCs were maintained below baseline when Cp levels remained below target (i.e. copper depleted). Toxicity: Grade 3/4 neutropenia occurred in 15 cycles (2.6%) with 1 pt with febrile neutropenia. One cycle was complicated by Grade 3 anemia. All resolved 5-13 days later with TM held and resumed at a lower dose. No other grade 3/4 toxicity was observed. Six pts were diagnosed with recurrent breast cancer at 1, 2, 2, 9, 10 and 10 mos. An EPC rise preceded an abnormal marker or overt relapse by 3-5 months in 4 of 6 pts (2 pts too early to tell). Conclusions: TM is well tolerated in breast cancer patients. TM might contribute to maintaining EPCs below baseline in pts who are copper depleted. We postulate that the increased EPCs noted in 4 pts with recurrent disease 2-4 months prior to overt relapse could represent the turning on of an angiogenic switch, resulting in an outpouring of BM derived EPCs to the new site of metastasis. Other studies geared toward understanding the mechanism for metastases are underway. The trial continues to accrue. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-16-14.