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.

Clinical and Translational Results of a Phase II, Randomized Trial of an Anti-IGF-1R (Cixutumumab) in Women with Breast Cancer That Progressed on Endocrine Therapy.

PURPOSE: This phase II trial evaluated the efficacy and safety of cixutumumab, a human anti-insulin-like growth factor receptor 1 (IGF-1R) monoclonal IgG1 antibody, and explored potential biomarkers in postmenopausal women with hormone receptor-positive breast cancer. EXPERIMENTAL DESIGN: Patients with hormone receptor-positive breast cancer that progressed on antiestrogen therapy received (2:1 randomization) cixutumumab 10 mg/kg and the same antiestrogen (arm A) or cixutumumab alone (arm B) every 2 weeks (q2w). Primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS) and safety. Correlative analyses of IGF-1R, total insulin receptor (IR), and IR isoforms A (IR-A) and B (IR-B) expression in tumor tissue were explored. RESULTS: Ninety-three patients were randomized (arm A, n = 62; arm B, n = 31). Median PFS was 2.0 and 3.1 months for arm A and arm B, respectively. Secondary efficacy measures were similar between the arms. Overall, cixutumumab was well tolerated. IGF-1R expression was not associated with clinical outcomes. Regardless of the treatment, lower IR-A, IR-B, and total IR mRNA expression in tumor tissue was significantly associated with longer PFS [IR-A: HR, 2.62 (P = 0.0062); IR-B: HR, 2.21 (P = 0.0202); and total IR: HR, 2.18 (P = 0.0230)] and OS [IR-A: HR, 2.94 (P = 0.0156); IR-B: HR, 2.69 (P = 0.0245); and total IR: HR, 2.72 (P = 0.0231)]. CONCLUSIONS: Cixutumumab (10 mg/kg) with or without antiestrogen q2w had an acceptable safety profile, but no significant clinical efficacy. Patients with low total IR, IR-A, and IR-B mRNA expression levels had significantly longer PFS and OS, independent of the treatment. The prognostic or predictive value of IR as a biomarker for IGF-1R-targeted therapies requires further validation.

Pro1170 Ala polymorphism in HER2-neu is associated with risk of trastuzumab cardiotoxicity.

BACKGROUND: Variations in single nucleotide polymorphisms (SNPs) have been associated with enhanced drug efficacy and toxicity in cancer therapy. SNP variations in the ErbB2 gene have been identified that alter the protein sequence of the HER2-neu protein, but how these polymorphisms affect prognosis and response to HER2 targeted therapy is unknown. We examined eleven ErbB2 SNPs that alter the HER2-neu amino acid sequence to determine whether any of these particular polymorphisms were associated with increased trastuzumab cardiotoxicity in a case-control study. METHODS: 140 subjects were enrolled from a single institution under Weill Cornell Medical College IRB protocol #0804009734. Patients were eligible if they had histologically or cytologically proven HER2-neu positive breast cancer and more than 3 months of trastuzumab therapy. Cases had either symptomatic CHF or a decline in LVEF of 15% (or if the LVEF <55%, a decline in LVEF of 10%) that resulted in at least temporary discontinuation of trastuzumab, whereas controls had no decline in their LVEF. Eleven ErbB2 single gene SNPs that resulted in an alteration in the HER2-neu protein amino acid sequence were studied. Single gene SNP analysis was carried out using SNP genotyping assays from genomic DNA obtained from peripheral blood or buccal swab. RESULTS: Only two of the ErbB2 SNPs (Ile 655 Val and Pro 1170 Ala) were found to have variation. There was no association between codon 665 and cardiotoxicity; however the proline variant of amino acid 1170 was more likely than the alanine variant to be found in cases with trastuzumab cardiotoxicity (35% of case patients as compared to 17% of controls, p = 0.04). This association remained significant in multivariable analysis taking into account age, race, and history of hypertension (adjusted OR = 2.60, 95% CI = 1.02, 6.62, p = 0.046). CONCLUSIONS: The Her2/neu Pro 1170 Ala polymorphism can be used to identify a subset of patients who are at increased risk of cardiotoxicity from trastuzumab therapy. Her2/neu single nucleotide polymorphisms may be useful in conjunction with other biomarkers to risk stratify patients in order to optimize clinical management.

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 Dean's 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. Dean's 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.

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

BACKGROUND: 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. METHODS: Ten breast cancer patients receiving Ix underwent total neuropathy score clinical (TNSc) assessment, distal leg skin biopsies at cycle (Cy) 3 (80-90 mg/m(2)), Cy5 (160-190 mg/m(2)), and Cy7 (>200 mg/m(2)) and were compared to 5 controls. Skin blocks were processed for EM and ultrastructural morphometry of Remak axons done. RESULTS: At baseline, Ix-treated subjects had higher TNSc values (4.5 ± 0.8 vs. 0.0 ± 0.0), greater percentage of empty (denervated) Schwann cells (29% vs. 12%), altered axonal diameter (422.9 ± 17 vs. 354.9 ± 14.8 nm, P = 0.01), and axon profiles without mitochondria tended to increase compared to control subjects (71% vs. 70%). With increasing cumulative Ix exposure, an increase in TNSc values (Cy3: 5.4 ± 1.2, Cy7: 10 ± 4, P < 0.001), empty Schwann cells (39% by Cy7), and dilated axons (in nm, Cy3: 506.3 ± 22.1, Cy5: 534.8 ± 33, Cy7: 527.8 ± 24.4; P < 0.001) was observed. In addition, axon profiles without mitochondria (Cy3:74%, Cy7:78%) and mitochondria with abnormal morphology (grade 3 or 4) increased from 24% to 79%. Schwann cells with atypical mitochondria and perineuronal macrophage infiltration in dermis were noted. INTERPRETATION: This study provides functional and structural evidence that Ix exposure induces a dose-dependent toxicity on small sensory fibers with an increase in TNSc scores and progressive axonal loss. Mitochondria appear to bear the cumulative toxic effect and chemotherapy-induced toxicity can be monitored through serial skin biopsy-based analysis.

Induction of antigen-specific immunity with a vaccine targeting NY-ESO-1 to the dendritic cell receptor DEC-205.

Immune-based therapies for cancer are generating substantial interest because of the success of immune checkpoint inhibitors. This study aimed to enhance anticancer immunity by exploiting the capacity of dendritic cells (DCs) to initiate T cell immunity by efficient uptake and presentation of endocytosed material. Delivery of tumor-associated antigens to DCs using receptor-specific monoclonal antibodies (mAbs) in the presence of DC-activating agents elicits robust antigen-specific immune responses in preclinical models. DEC-205 (CD205), a molecule expressed on DCs, has been extensively studied for its role in antigen processing and presentation. CDX-1401 is a vaccine composed of a human mAb specific for DEC-205 fused to the full-length tumor antigen NY-ESO-1. This phase 1 trial assessed the safety, immunogenicity, and clinical activity of escalating doses of CDX-1401 with the Toll-like receptor (TLR) agonists resiquimod (TLR7/8) and Hiltonol (poly-ICLC, TLR3) in 45 patients with advanced malignancies refractory to available therapies. Treatment induced humoral and cellular immunity to NY-ESO-1 in patients with confirmed NY-ESO-1-expressing tumors across various dose levels and adjuvant combinations. No dose-limiting or grade 3 toxicities were reported. Thirteen patients experienced stabilization of disease, with a median duration of 6.7 months (range, 2.4+ to 13.4 months). Two patients had tumor regression (~20% shrinkage in target lesions). Six of eight patients who received immune-checkpoint inhibitors within 3 months after CDX-1401 administration had objective tumor regression. This first-in-human study of a protein vaccine targeting DCs demonstrates its feasibility, safety, and biological activity and provides rationale for combination immunotherapy strategies including immune checkpoint blockade.

Breast cancer subtypes in Asian-Americans differ according to Asian ethnic group.

Breast cancer prognosis and breast cancer molecular subtype vary by race/ethnicity. We determined whether the distribution of breast cancer subtypes varies among different Asian ethnic groups. Using immunohistochemical surrogates for the four molecularly defined breast cancer subtypes, we characterized breast cancer subtype for 346 Asian subjects treated at two New York City institutions. We found that Chinese and Japanese had a higher proportion of good-prognosis luminal A cancers (66.7 and 80.0%, respectively) compared to Filipinos and Koreans (48.5 and 47.1%) (P = 0.001). Filipinos had a higher proportion of HER-2/neu positive cancers (45.6%) compared to other ethnic groups (23.6%) (P = 0.002).Koreans had a higher proportion of triple negative cancers(23.5%) compared to other ethnic groups (7.5%) (P =0.06). The results suggest that differences exist in breast cancer tumor biology among distinct Asian ethnic groups and have implications for cancer care and research. Future studies of breast cancer in Asian-Americans should distinguish among the different ethnic groups.

Incremental increase in VEGFR1⁺ hematopoietic progenitor cells and VEGFR2⁺ endothelial progenitor cells predicts relapse and lack of tumor response in breast cancer patients.

Animal models have demonstrated the critical role of bone marrow-derived VEGFR1(+) hematopoietic progenitor cells (HPCs) and VEGFR2(+) endothelial progenitor cells (EPCs) in metastatic progression. We explored whether these cells could predict relapse and response in breast cancer (BC) patients. One hundred and thirty-two patients with stages 1-4 BC were enrolled on 2 studies. Circulating CD45(+)/CD34(+)/VEGFR1(+) HPCs and CD45(dim)/CD133(+)/VEGFR2(+) EPCs were assessed from peripheral blood mononuclear cells using flow cytometry. Changes in HPCs and EPCs were analyzed in (1) patients without overt disease that relapsed and (2) metastatic patients according to response by RECIST. At study entry, 102 patients were without evidence of disease and 30 patients had metastatic BC. Seven patients without evidence of BC by exam, labs, and imaging developed recurrence while on study. Median HPC/ml (range) increased from 645.8 (23.5-1,914) to 2,899 (1,176-37,336), P = 0.016, followed by an increase in median EPC/ml from 21.3 (4.7-42.5) to 94.7 (28.2-201.3), P = 0.016, prior to clinical relapse. In metastatic patients with progressive disease, median HPC/ml increased from 1,696 (10-16,470) to 5,124 (374-77,605), P = 0.0009, and median EPC/ml increased from 26 (0-560) to 71 (0-615) prior to progression, P = 0.10. In patients with responding disease, median HPC/ml decreased from 6,147 (912-85,070) to 633 (47-18,065), P = 0.05, and EPC/ml decreased from 46 (0-197) to 23 (0-105), P = 0.41, at response. There were no significant changes in these cells over time in patients with stable disease. Circulating bone marrow-derived HPCs and EPCs predict relapse and disease progression in BC patients.

Phase II study of eribulin mesylate, a halichondrin B analog, in patients with metastatic breast cancer previously treated with an anthracycline and a taxane.

PURPOSE: Eribulin mesylate (E7389), a nontaxane microtubule dynamics inhibitor, is a structurally simplified, synthetic analog of the marine natural product halichondrin B. This open-label, single-arm, phase II study evaluated efficacy and tolerability of eribulin in heavily pretreated patients with metastatic breast cancer (MBC). METHODS: MBC patients who were previously treated with an anthracycline and a taxane received eribulin mesylate (1.4 mg/m(2)) as a 2- to 5-minute intravenous (IV) infusion on days 1, 8, and 15 of a 28-day cycle. Because of neutropenia (at day 15), an alternative regimen of eribulin on days 1 and 8 of a 21-day cycle was administered. The primary end point was overall response rate. RESULTS: Of the 103 patients treated, the median number of prior chemotherapy regimens was four (range, one to 11 regimens). In the per-protocol population (n = 87), eribulin achieved an independently reviewed objective response rate (all partial responses [PRs]) of 11.5% (95% CI, 5.7 to 20.1) and a clinical benefit rate (PR plus stable disease > or = 6 months) of 17.2% (95% CI, 10.0 to 26.8). The median duration of response was 171 days (5.6 months; range, 44 to 363 days), the median progression-free survival was 79 days (2.6 months; range, 1 to 453 days), and the median overall survival was 275 days (9.0 months; range, 15 to 826 days). The most common drug-related grades 3 to 4 toxicities were as follows: neutropenia, 64%; leukopenia, 18%; fatigue, 5%; peripheral neuropathy, 5%; and febrile neutropenia, 4%. CONCLUSION: Eribulin demonstrated activity with manageable tolerability (including infrequent grade 3 and no grade 4 neuropathy) in heavily pretreated patients with MBC when dosed as a short IV infusion on days 1 and 8 of a 21-day cycle.

Circulating endothelial progenitor cells correlate to stage in patients with invasive breast cancer.

Tumor growth and metastasis is dependent on the formation and assembly of new blood vessels, a process known as neo-angiogenesis. Both pre-existing and circulating vascular cells have been shown to contribute to the assembly of tumor neo-vessels in specific tumors. Mobilization of endothelial progenitor cells (EPCs) from the bone marrow constitutes a crucial step in the formation of de novo blood vessels, and levels of peripheral blood EPCs have been shown to be increased in certain malignant states. However, the role of circulating EPCs in breast cancer is largely unknown. We recruited twenty-five patients with biopsy-proven invasive breast cancer at Weill Cornell Breast Center to participate in a pilot study investigating the correlation of circulating EPCs to extent of disease and initiation of chemotherapy. For each patient, a baseline sample was drawn before systemic treatment, and for seventeen of those patients, a second sample was taken after the first round of chemotherapy. Levels of peripheral blood EPCs, as defined by co-expression of CD133 and VEGFR2, were quantified by flow cytometry. Breast cancer patients with stage III & IV disease had statistically higher levels of circulating EPCs than did patients with stage I & II disease (median = 165,000 EPCs/5 x 10(6)MNCs vs. median = 6,920 EPCs/5 x 10(6)MNCs, respectively, P < 0.0001). In addition, in late-stage patients, levels of EPCs demonstrated a statistically significant drop after initiation of chemotherapy (median = 162,500 EPCs/5 x 10(6)MNCs [pre] vs. median = 117,500 EPCs/5 x 10(6)MNCs [post], P = 0.01). These results suggest that circulating EPCs may serve as a potential tumor biomarker in breast cancer and that EPCs may represent a plausible target for future therapeutic intervention.

Concurrent invasive ductal carcinoma and chronic lymphocytic leukemia manifesting as a collision tumor in breast.

Collision tumors are rare clinical entities in which two histologically distinct tumor types show involvement in the same site. The occurrence of these tumors in the breast is extremely rare. Here, we present a case of a patient with both invasive ductal carcinoma and chronic lymphocytic leukemia in the breast. Wide excision with sentinel lymph node biopsy revealed palpably abnormal lymph nodes negative for breast carcinoma on frozen section. Histopathological examination of these lymph nodes showed extensive involvement by lymphoma and review of the breast specimen demonstrated the same lymphoma at the periphery of the ductal carcinoma. We review the literature and discuss possible etiologies for the dual presentation of both cancers.

Transgenic expression of CTLA-4 controls lymphoproliferation in IL-2-deficient mice.

IL-2-deficient mice develop a lymphoproliferative and autoimmune disease characterized by autoimmune hemolytic anemia (AHA) and inflammatory bowel disease. We have previously reported that IL-2 is necessary for optimal up-regulation of CTLA-4, an inducible negative regulator of T cell activation. In this study, we have tested the hypothesis that reduced expression of CTLA-4 in IL-2-deficient T cells contributes to the pathogenesis of disease in IL-2-deficient mice. Expression of CTLA-4 as a transgene completely prevented lymphoaccumulation and AHA in IL-2-deficient mice. The normalization of T cell numbers was due to inhibition of expansion of conventional CD4+CD25- T cells rather than to rescue of the numbers or function of CD4+CD25+ regulatory T cells, suggesting that CTLA-4 expression on conventional T cells plays a role in maintaining normal T cell homeostasis. In addition, the inhibitory effect of the CTLA-4 transgene on T cell expansion was at least in part independent of CD28 expression. Our results suggest that deficient CTLA-4 expression on conventional T cells contributes to the pathophysiology of the lymphoproliferative disease and AHA in IL-2-deficient mice. Thus, restoring CTLA-4 expression in T cells may be an attractive strategy to control clinical autoimmune diseases in which CTLA-4 expression is reduced.

Tyrosine-mediated inhibitory signals contribute to CTLA-4 function in vivo.

The ability of CTLA-4 to inhibit T cell activation may be either negatively or positively regulated by a critical tyrosine at position 201 (Y201) within the CTLA-4 cytoplasmic domain. By binding to the clathrin-associated adaptor complex AP-2 and inducing endocytosis, Y201 reduces the amount of CTLA-4 on the cell surface, thereby down-regulating CTLA-4 inhibitory function. Alternatively, Y201 may function to transmit CTLA-4 inhibitory signals, perhaps through binding to intracellular proteins that oppose TCR- and/or CD28-induced signal transduction. Results from studies performed in vitro have cast doubt on whether this second mechanism contributes significantly to CTLA-4 function. In order to determine if a role existed for Y201 in mediating CTLA-4 inhibitory signaling in vivo, we studied lymphocyte activation and homeostasis in CTLA-4(-/-) mice that were reconstituted with a transgenic CTLA-4 receptor in which Y201 was mutated to valine (Y201V/CTLA-4(-/-)). We found that despite augmented levels of CTLA-4 on the cell surface of T cells, Y201V/CTLA-4(-/-) mice developed a lymphoproliferative syndrome characterized by lymphadenopathy and the accumulation of T cells that secreted IL-4. Mutant T cells exhibited increased cell division when treated with suboptimal doses of mitogenic stimuli in vitro. These results demonstrate that in addition to down-modulating CTLA-4 expression on the cell surface of T cells, the Y201 residue also functions to transmit CTLA-4 inhibitory signals in vivo. Elucidating the biochemical pathways downstream of Y201 will be important for a full understanding of the molecular basis for CTLA-4 function.