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 69% (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.

Microtubule inhibitor-based antibody-drug conjugates for cancer therapy.

The specificity of monoclonal antibodies represents a potential therapeutic advantage, but their use as single agents in oncology has proven limited to date. The development of antibody-drug conjugates (ADCs) takes advantage of the specificity of the monoclonal antibody and potent cytotoxic effect of chemotherapy, leading to enhanced cytotoxicity in target cells and limiting toxicity to normal tissue. Microtubules represent a validated oncologic target in a range of tumor types, with a number of anti-microtubule targeting cytotoxic drugs approved for cancer use. The systemic use of potent microtubule-binding agents is limited by their effects in normal cells, which leads to toxicity including myelosuppression and peripheral neuropathy. Linking these agents to monoclonal antibodies may limit toxicity to normal tissues and increase drug concentration in target tissues, also allowing the use of more potent agents which would be too toxic to administer in their unbound form. Two such ADCs have been approved for clinical use and many others are in development. Here we review the characteristics of each of the ADC components that have led to efficacious therapies and discuss some of the tubulin inhibitor-based ADCs in development for cancer therapy.

Resistin levels in lupus and associations with disease-specific measures, insulin resistance, and coronary calcification.

OBJECTIVE: To evaluate levels of resistin in female subjects with systemic lupus erythematosus (SLE) compared to age and race-matched controls and to determine the relationship between resistin and systemic inflammation, disease measures, and coronary artery calcification (CAC). METHODS: Resistin levels were measured on stored samples from 159 women with SLE and 70 controls as an extension of a previous cross-sectional study. Spearman correlations and multivariable regressions were used to examine whether resistin levels were associated with SLE, disease-specific and inflammatory markers, insulin resistance, and CAC. RESULTS: In a multivariable linear regression model, a diagnosis of SLE was significantly associated with higher resistin levels independent of age, race, renal function, body mass index (BMI), high-sensitivity CRP (hsCRP), hypertension, diabetes, and steroid use. In SLE, resistin levels correlated positively with Systemic Lupus International Collaborating Clinics Damage Index, glomerular filtration rate (GFR), hsCRP, erythrocyte sedimentation rate, homocysteine, and disease duration (all p < 0.03). Resistin level did not correlate with markers of insulin resistance or body adiposity, including homeostatic model assessment or BMI. Resistin levels were significantly elevated in SLE cases with CAC compared to cases without CAC (16.58 vs 13.10 ng/ml, respectively; p = 0.04). In multivariate logistic regression, the association was not present after adjustment for age, race, and GFR. CONCLUSION: SLE was independently associated with higher resistin levels. Among subjects with SLE, higher resistin level correlated positively with renal dysfunction, inflammatory markers, and disease damage but not with insulin resistance or BMI. SLE cases with CAC had higher resistin levels than cases without CAC; however, this relationship was dependent on other established risk factors.

Functional polymorphisms of folate-metabolizing enzymes in relation to homocysteine concentrations in systemic lupus erythematosus.

OBJECTIVE: To determine if functional polymorphisms of folate/homocysteine pathway enzymes are associated with homocysteine concentrations and/or coronary artery calcification (CAC) scores in patients with systemic lupus erythematosus (SLE) and controls. METHODS: We investigated 163 SLE patients and 160 controls. Functional polymorphisms in 6 genes in the folate/homocysteine pathway were genotyped: 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T, MTHFR 1298A>C, cystathionine ss-synthase (CBS) 844ins68, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, thymidylate synthase (TYMS) 1494del6, and dihydrofolate reductase (DHFR) c.86+60_78. RESULTS: Homocysteine levels were higher in African American SLE patients than Caucasian patients and African American controls. Genotype distributions were significantly different in African American and Caucasian controls for 6 of the 7 polymorphisms. Genotype distributions for each polymorphism did not differ significantly between SLE patients and controls even after stratification by race. Glomerular filtration rate was strongly negatively correlated to homocysteine levels, and was therefore adjusted for as a covariate in the models of the effects of the polymorphisms on homocysteine levels. In SLE patients none of the 7 polymorphisms was associated with homocysteine concentrations. In Caucasian controls only MTHFR 677C>T and 1298A>C showed effects on homocysteine similar to what would be expected from the literature. There were no genotypic associations with median CAC scores in SLE patients or controls with and without stratification by race. CONCLUSION: Polymorphisms in folate/homocysteine metabolizing enzymes do not predict higher homocysteine levels or CAC scores in patients with SLE.

Monocyte chemoattractant protein-1: plasma concentrations and A(-2518)G promoter polymorphism of its gene in systemic lupus erythematosus.

OBJECTIVE: To determine (1) whether the A(-2518)G polymorphism of CCL-2, the gene encoding monocyte chemoattractant protein-1 (MCP-1), is associated with disease, MCP-1 concentration, nephritis, or coronary artery calcification (CAC) in systemic lupus erythematosus (SLE); and (2) whether MCP-1 and homocysteine (Hcy) concentrations are correlated. METHODS: Statistical tests were applied to determine the relationships between CCL-2 A(-2518)G genotypes, plasma MCP-1 concentrations, and clinical variables in Caucasian and African American patients with SLE and controls. RESULTS: The CCL-2 (-2518)G allele was not significantly associated with SLE in the whole study sample (p = 0.07). Among Caucasians, but not African Americans, G allele carriers had significantly increased risk of SLE (OR 4.2, 95% CI 1.8-9.6, p < 0.0001). Genotype was not associated with nephritis, CAC, or MCP-1 concentrations when all patients or all controls were considered; however, among recently diagnosed patients, G allele carriers had significantly higher MCP-1 concentrations than AA homozygotes (p = 0.02). SLE patients had higher MCP-1 concentrations than controls (p < 0.0001), African American patients had higher concentrations than Caucasian patients (p = 0.006), and patients with nephritis had higher concentrations than those without nephritis (p = 0.02). Although not associated with CAC, MCP-1 concentrations were significantly positively correlated with Hcy. CONCLUSION. CCL-2 A(-2518)G genotype is a significant risk factor for SLE among Caucasians but not African Americans, suggesting that genetically mandated differences in MCP-1 expression contribute to SLE etiology in the former. The positive correlation between MCP-1 and Hcy concentrations is consistent with the hypothesis that active inflammation and hyperhomocysteinemia are etiologically linked.

Homocysteine levels and disease duration independently correlate with coronary artery calcification in patients with systemic lupus erythematosus.

OBJECTIVE: To compare the incidence and extent of coronary artery calcification (CAC) as measured by electron beam computed tomography (EBCT) in patients with systemic lupus erythematosus (SLE) and controls, and to identify variables associated with CAC in patients with SLE. METHODS: Female patients with SLE and matched controls were recruited; EBCT of the coronary arteries was performed, and laboratory values (including the homocysteine concentration, the lipid level, the high-sensitivity C-reactive protein [hsCRP] concentration, the glomerular filtration rate [GFR], and the level of soluble CD154 [sCD154]) were determined. For patients, the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index and the SLE Disease Activity Index scores were recorded. Tests of association between the CAC score and the above-mentioned variables were performed. RESULTS: The incidence of CAC was higher in patients with SLE than in controls (P = 0.009), and patients had a higher mean raw CAC (rCAC) score (87.9 versus 9.6 in controls; P = 0.02). In particular, more CAC-positive patients than CAC-positive controls had rCAC scores above the 75th percentile (P = 0.003). Among both patients and controls, those with CAC were approximately 10 years older than those without CAC. In addition to age, a significant determinant of positive CAC status in both groups was the number of cardiovascular risk factors. In patients with SLE, CAC was associated with a higher homocysteine concentration, a lower GFR, and longer disease duration. In controls, the total cholesterol level correlated positively with CAC. When multivariate logistic regression methods were applied to candidate explanatory variables, homocysteine concentration, age, and disease duration (but not the levels of sCD154 or hsCRP) contributed significantly to CAC status. The methylenetetrahydrofolate reductase C677T genotype was not a predictor of hyperhomocysteinemia or CAC status. CONCLUSION: Among patients with SLE, the homocysteine concentration, the GFR, age, and disease duration were associated with CAC. CAC occurred more frequently and was more extensive in patients with SLE than in controls, suggesting that EBCT could be used to detect premature atherosclerosis in the former group. An elevated homocysteine concentration might identify patients with SLE who are likely to have premature atherosclerosis and who would benefit from evaluation of CAC by EBCT.