Homologous recombination deficiency and host anti-tumor immunity in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is associated with worse outcomes relative to other breast cancer subtypes. Chemotherapy remains the standard-of-care systemic therapy for patients with localized or metastatic disease, with few biomarkers to guide benefit. METHODS: We will discuss recent advances in our understanding of two key biological processes in TNBC, homologous recombination (HR) DNA repair deficiency and host anti-tumor immunity, and their intersection. RESULTS: Recent advances in our understanding of homologous recombination (HR) deficiency, including FDA approval of PARP inhibitor olaparib for BRCA1 or BRCA2 mutation carriers, and host anti-tumor immunity in TNBC offer potential for new and biomarker-driven approaches to treat TNBC. Assays interrogating HR DNA repair capacity may guide treatment with agents inducing or targeting DNA damage repair. Tumor infiltrating lymphocytes (TILs) are associated with improved prognosis in TNBC and recent efforts to characterize infiltrating immune cell subsets and activate host anti-tumor immunity offer promise, yet challenges remain particularly in tumors lacking pre-existing immune infiltrates. Advances in these fields provide potential biomarkers to stratify patients with TNBC and guide therapy: induction of DNA damage in HR-deficient tumors and activation of existing or recruitment of host anti-tumor immune cells. Importantly, these advances provide an opportunity to guide use of existing therapies and development of novel therapies for TNBC. Efforts to combine therapies that exploit HR deficiency to enhance the activity of immune-directed therapies offer promise. CONCLUSIONS: HR deficiency remains an important biomarker target and potentially effective adjunct to enhance immunogenicity of 'immune cold' TNBCs.

Research needs in breast cancer.

New research questions emerge as medical needs continue to evolve and as we improve our understanding of cancer biology and treatment of malignancies. Although significant advances have been made in some areas of breast cancer research resulting in improvements in therapies and outcomes over the last few decades, other areas have not benefited to the same degree and we continue to have many gaps in our knowledge. This article summarizes the 12 short and medium-term clinical research needs in breast cancer deemed as priorities in 2016 by a panel of experts, in an attempt to focus and accelerate future research in the most needed areas: (i) de-escalate breast cancer therapies in early breast cancer without sacrificing outcomes; (ii) explore optimal adjuvant treatment durations; (iii) develop better tools and strategies to identify patients with genetic predisposition; (iv) improve care in young patients with breast cancer; (v) develop tools to speed up drug development in biomarker-defined populations; (vi) identify and validate targets that mediate resistance to chemotherapy, endocrine therapy and anti-HER2 therapies; (vii) evaluate the efficacy of local-regional treatments for metastatic disease; (viii) better define the optimal sequence of treatments in the metastatic setting; (ix) evaluate the clinical impact of intra-patient heterogeneity (intra-tumor, inter-tumor and inter-lesion heterogeneity); (x) better understand the biology and identify new targets in triple-negative breast cancer; (xi) better understand immune surveillance in breast cancer and further develop immunotherapies; and (xii) increase survivorship research efforts including supportive care and quality of life.

Genetic variant predicts bevacizumab-induced hypertension in ECOG-5103 and ECOG-2100.

BACKGROUND: Bevacizumab has broad anti-tumour activity, but substantial risk of hypertension. No reliable markers are available for predicting bevacizumab-induced hypertension. METHODS: A genome-wide association study (GWAS) was performed in the phase III bevacizumab-based adjuvant breast cancer trial, ECOG-5103, to evaluate for an association between genotypes and hypertension. GWAS was conducted in those who had experienced systolic blood pressure (SBP) >160 mm Hg during therapy using binary analysis and a cumulative dose model for the total exposure of bevacizumab. Common toxicity criteria (CTC) grade 3-5 hypertension was also assessed. Candidate SNP validation was performed in the randomised phase III trial, ECOG-2100. RESULTS: When using the phenotype of SBP>160 mm Hg, the most significant association in SV2C (rs6453204) approached and met genome-wide significance in the binary model (P=6.0 × 10(-8); OR=3.3) and in the cumulative dose model (P=4.7 × 10(-8); HR=2.2), respectively. Similar associations with rs6453204 were seen for CTC grade 3-5 hypertension but did not meet genome-wide significance. Validation study from ECOG-2100 demonstrated a statistically significant association between this SNP and grade 3/4 hypertension using the binary model (P-value=0.037; OR=2.4). CONCLUSIONS: A genetic variant in SV2C predicted clinically relevant bevacizumab-induced hypertension in two independent, randomised phase III trials.

A phase II pilot trial incorporating bevacizumab into dose-dense doxorubicin and cyclophosphamide followed by paclitaxel in patients with lymph node positive breast cancer: a trial coordinated by the Eastern Cooperative Oncology Group.

BACKGROUND: E2104 was designed to evaluate the safety of two different strategies incorporating bevacizumab into anthracycline-containing adjuvant therapy as a precursor to a definitive randomized phase III trial. PATIENTS AND METHODS: Patients were sequentially assigned to one of two treatment arms. In addition to dose-dense doxorubicin and cyclophosphamide followed by paclitaxel (Taxol) (ddAC→T), all patients received bevacizumab (10 mg/kg every 2 weeks × 26) initiated either concurrently with AC (Arm A: ddBAC→BT→B) or with paclitaxel (Arm B: ddAC→BT→B). The primary end point was incidence of clinically apparent cardiac dysfunction (CHF). RESULTS: Patients enrolled were 226 in number (Arm A 104, Arm B 122). Grade 3 hypertension, thrombosis, proteinuria and hemorrhage were reported for 12, 2, 2 and <1% of patients, respectively. Two patients developed grade 3 or more cerebrovascular ischemia. Three patients in each arm developed CHF. There was no significant difference between arms in the proportion of patients with an absolute decrease in left ventricular ejection fraction of >15% or >10% to below the lower limit of normal post AC or post bevacizumab. CONCLUSIONS: Incorporation of bevacizumab into anthracycline-containing adjuvant therapy does not result in prohibitive cardiac toxicity. The definitive phase III trial (E5103) was activated with systematic and extensive cardiac monitoring to define the true impact of bevacizumab on cardiac function.

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.

Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.

Effect of matrix metalloproteinase inhibitor batimastat on breast cancer regrowth and metastasis in athymic mice.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in the invasion and metastasis of human cancers by mediating the degradation of extracellular matrix components. Therefore, these enzymes constitute promising targets in the development of anticancer therapies. Batimastat ([(4-N-hydroxyamino)-2R-isobutyl-3S-(thienyl-thiomethyl)succinyl]-L- phenyl-alanine-N-methylamide) is one of a new class of agents designed to inhibit MMP activity. PURPOSE: We asked whether batimastat, given as adjuvant therapy after primary tumor resection, could inhibit local-regional tumor regrowth and the formation of lung metastases in a human breast cancer xenograft model. We also explored possible effects of batimastat on breast cancer cell viability and on the accumulation of specific messenger RNAs (mRNAs). METHODS: Human MDA-MB-435 breast cancer cells were treated in vitro for 6 days with batimastat at concentrations ranging from 0.1 to 10.0 microM, and then viable cell counts were performed. The activity of collagenases, directly associated with cultured MDA-MB-435 cells or released into their culture fluids, was assessed by gelatin zymography after 1 and 3 days of batimastat treatment (drug range, 0.2-2.0 microM). Athymic nude mice were given daily intraperitoneal injections of batimastat (30 mg/kg body weight) after resection of MDA-MB-435 primary tumors grown in their mammary fat pads; the volumes of tumor regrowths and the numbers and volumes of lung metastases were calculated; neovascularization in the regrowths was assessed by immunohistochemical analysis with an antibody directed against CD31, an endothelial cell antigen. The effect of batimastat treatment on the accumulation of mRNAs encoding specific MMPs and the tissue inhibitor of metalloproteinases-2 (TIMP-2) in cultured cells, primary tumors, and tumor regrowths was measured by RNA dot blotting and hybridization with complementary probes. Linear regression analysis, Student's t tests, and chi-squared analysis were used to evaluate the data. RESULTS: The viability of cultured MDA-MB-435 cells was not affected by treatment with batimastat; however, measured activities for the 72-kd and 92-kd collagenases released by these cells were reduced after batimastat treatment. Intraperitoneal injection of batimastat significantly inhibited the local-regional regrowth of resected MDA-MB-435 tumors in athymic nude mice (in comparison with control mice, P = .035), and it reduced the incidence (P < .05), number (P = .0001), and total volume (P = .0001) of lung metastases. Batimastat treatment did not affect cellular levels of MMP or TIMP-2 mRNAs. CONCLUSION: Batimastat inhibits human breast cancer regrowth and metastasis in a nude mouse xenograft model. Potential mechanisms for batimastat's inhibitory activity do not include direct cell toxicity or alteration of MMP or TIMP mRNA levels.

Establishment in long term culture of megakaryocytic leukemia cells (EST-IU) from the marrow of a patient with leukemia and a mediastinal germ cell neoplasm.

Megakaryocytes are the bone marrow cells that generate platelets. They are relatively rare cells, comprising between 0.03 and 0.06% of all nucleated marrow cells (R. L. Berkow et al., J. Lab. Clin. Med., 103: 811-818, 1984). The study of human megakaryocyte differentiation and function has been hampered by the small number of these cells available for study. Recently we have established a human cell line (EST-IU) from the marrow of a patient with an acute nonlymphocytic leukemia and a mediastinal germ cell tumor. While this cell line seems to express many of the phenotypic characteristics of human megakaryocytes, it does not appear to express any phenotypic properties associated with cells of the erythroid, lymphoid, granulocytic, or monocytic lineages. Transmission electron microscopy demonstrates frequent multinucleated cells. Staining for platelet peroxidase reactivity revealed darkening of the perinuclear envelope and the endoplasmic reticulum, a characteristic of cells of the megakaryocytic lineage. Indirect immunofluorescence assays reveal that EST-IU expresses reactivity with anti-platelet glycoprotein antisera, anti-Factor VIII-related antigen antisera, anti-Factor V antisera, anti-thrombocyte antisera, Tab (monoclonal anti-platelet glycoprotein IIb-IIIa), and anti-fibronectin antisera. Flow cytometry-derived DNA histograms demonstrate populations with multiple ploidies, ranging from approximately 4N to 32N. Based upon morphological and histochemical characteristics, antigenic expression, and nuclear characteristics, EST-IU cells appear to have a phenotype that closely resembles human megakaryocytes. This cell line should be useful in the further cell study of the molecular and cell biology of human megakaryocytopoiesis.

Relation of proliferative activity to survival in patients with advanced germ cell cancer.

Patients with advanced disseminated germ cell tumors of the testis, retroperitoneum, and mediastinum have impaired survival compared to other patients with disseminated germ cell tumors having less bulky metastatic disease. Among patients with advanced disseminated germ cell tumors, we currently lack adequate predictors of long-term survival. Flow cytometric analysis of the paraffin-embedded, formalin-fixed tumor blocks of 50 of these patients suggests that proliferative activity is significantly correlated with survival (p less than 0.001) in multivariate analysis. Log (beta-human chorionic gonadotropin) is the only other useful predictor of long-term survival in multivariate analysis of prognostic factors in this group of patients. Flow cytometric DNA analysis may be useful in predicting survival in patients with advanced disseminated germ cell tumors.

Neovascularization in clinical stage A testicular germ cell tumor: prediction of metastatic disease.

Increased numbers of blood vessels (angiogenesis or neovascularization) in certain primary tumors correlates with an increased risk for metastatic disease. We therefore conducted a blinded review of the resected testicular germ cell tumors of 65 clinical stage A patients to evaluate the usefulness of angiogenesis in identifying those patients with clinically occult nodal metastases (pathological stage B). Angiogenesis was assessed in the primary tumors using an immunohistochemical stain for factor VIII-related antigen assay for quantitation of microvessel counts. Of 65 clinical stage A patients, 43 had pathological stage B disease at retroperitoneal lymph node dissection. Eleven patients had microvessel counts > 30 microvessels/x 400 high powered field, and all of these patients had pathological stage B disease (P = 0.02 in univariate analysis). Multiple regression analysis using microvessel count and other histological findings found to be prognostic (venous invasion, lymphatic invasion, presence of embryonal carcinoma, and absence of yolk sac tumor) showed that only the absence of a yolk sac tumor component was significantly predictive of occult metastases. This study shows that angiogenesis, as measured by quantitation of microvessel counts in the primary tumor of germ cell neoplasms, is significantly predictive of occult nodal metastatic disease by univariate analysis in clinical stage A patients. The prospective use of angiogenesis quantitation needs to be defined.

Aberrant expression of the myeloid zinc finger gene, MZF-1, is oncogenic.

The zinc finger gene MZF-1 is preferentially expressed in primitive hematopoietic cells and plays an important role in regulating myelopoiesis. Regulators of development are potential targets for neoplastic transformation. This study investigated whether unregulated expression of MZF-1 could function as an oncogene. Retroviral transduction and subsequent overexpression of MZF-1 resulted in loss of contact inhibition, loss of substrate dependence, and more rapid cell cycling in NIH 3T3 cells. The MZF-1-transformed 3T3 cells formed aggressive tumors in athymic mice. Disruption of the tight lineage- and stage-specific regulation of MZF-1 can result in neoplastic transformation of embryonic fibroblasts. Therefore, MZF-1 represents a novel oncogene.

Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer.

A monoclonal antibody (323/A3) with a high degree of selectivity for binding to breast cancer cells was produced by immunization of mice with MCF-7 human breast cancer cells. The antigen recognized by 323/A3 on MCF-7 appears to be surface localized, and by enzyme-linked immunosorbent assay, the antibody was found to bind strongly with four of six breast cancer cell lines examined while no binding was detectable with nonbreast cancer cell lines. In vivo distribution of the 323/A3 antigen was screened by immunoperoxidase staining of formalin-fixed paraffin sections of normal human tissues and tumors. Among breast tissues, positive staining was detected with 75% (6 of 8) of metastatic lymph nodes, 59% (76 of 128) of primary breast tumors, 20% (13 of 63) of benign breast lesions, and 0% (0 of 10) of normal breast. No immunostaining was detected with a large variety and number of other normal human tissues with the exception of staining observed with epithelium of normal colon. Antigen distribution appears not to be disease specific, since positive staining was also observed with adenocarcinomas other than breast. The antigen recognized by the 323/A3 antibody was identified by Western blot analysis as a Mr 43,000 protein. The glycoprotein nature of the antigen was demonstrated by its binding to concanavalin A, specific elution with sugar, and immunoprecipitation of a Mr 43,000 radiolabeled protein from extracts of MCF-7 cells after pulse labeling with [3H]glucosamine. The 323/A3 antigen appears to be the same Mr 43,000 protein in cell lines as in breast tumors in vivo. Based on a comparison with the molecular weights of other known tumor-associated antigens and with their immunocytochemical tissue distribution, the Mr 43,000 glycoprotein described here represents a tumor-associated antigen previously undescribed in breast cancer or in other tumors. Since the Mr 43,000 glycoprotein is present on the surface of most breast cancer cells and is either absent or expressed at very low levels in most normal tissues including normal breast, the monoclonal antibody described here may have potential applications in diagnosis and management of breast cancer.

Binding of insulin-like growth factors to Tera-2 human embryonal carcinoma cells during differentiation.

Differentiation of Tera-2 human embryonal carcinoma cells by exposure to 2.1 mM alpha-difluoromethylornithine resulted in changes in morphology, a decrease in growth rate, and changes in the expression of SSEA-1 differentiation antigen. While the binding of 125I-insulin-like growth factor I (IGF-I) remained relatively constant during differentiation, binding of 125I-IGF-II increased 2-3-fold. Further, the binding of IGF-II was 87 times greater than IGF-I in both undifferentiated and differentiated cells. Undifferentiated Tera-2 cells exhibited a single class of binding sites for both IGF-I (KD = 1.2 nM, 7.0 x 10(3) sites/cell) and IGF-II (KD = 8.3 nM, 3.4 x 10(5) sites/cell). Following differentiation, IGF-I continued to bind to a single class of binding sites (KD 1.0 nM, 6.7 x 10(3) sites/cell) whereas IGF-II bound to both high-affinity sites (KDH 0.3 nM, 2.2 x 10(5) sites/cell) and low-affinity sites (KDL 15.1 nM, 1.6 x 10(7) sites/cell). The binding of iodinated IGF-II was blocked by unlabeled IGF-II but not IGF-I. In contrast, 125I-IGF-I binding was prevented by either IGF-I or IGF-II. Affinity cross-linking experiments demonstrated the presence of both type I and type II IGF receptors along with a number of IGF binding proteins. IGF-I failed to stimulate the incorporation of [3H]thymidine in both undifferentiated and differentiated cells. Although IGF-II caused a significant increase in [3H]thymidine incorporation in both undifferentiated and differentiated Tera-2 cells, the magnitude of the response and the sensitivity of the cells to IGF-II stimulation was diminished following differentiation. The observed changes in IGF-II binding, which occur in conjunction with cellular differentiation, may be an important feature of the expression of the differentiated phenotype by human germ cell tumors.

The antiangiogenic property of docetaxel is synergistic with a recombinant humanized monoclonal antibody against vascular endothelial growth factor or 2-methoxyestradiol but antagonized by endothelial growth factors.

Numerous chemotherapeutic agents have been shown to have an inhibitory effect on endothelial cell proliferation and migration, and tubule formation. In this study, we examined the antiangiogenic activity of docetaxel. Docetaxel inhibited endothelial cell proliferation and tubule formation in vitro in a dose-dependent fashion. Docetaxel treatment also inhibited angiogenesis in an in vivo Matrigel plug assay. The endothelial stimulating factors, vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor are able to protect endothelial cells from the antiangiogenic properties of docetaxel. This protective effect can be overcome by a recombinant humanized monoclonal antibody directed against VEGF in both in vitro and in vivo models. Similarly, combination of docetaxel with the antiangiogenic agent 2-methoxyestradiol also overcomes the protective effect of VEGF in both in vitro and in vivo models. These data suggest that microenvironmental factors (e.g., local release of VEGF and basic fibroblast growth factor) could play a role in decreasing the antiangiogenic effects of docetaxel, whereas agents such as 2- methoxyestradiol and recombinant humanized monoclonal antibody directed against VEGF may reverse this protective effect.

Antitumor activity and immunotherapeutic properties of Flt3-ligand in a murine breast cancer model.

Flt3-Ligand (Flt3-L) is a stimulatory cytokine for a variety of hematopoietic lineages, including dendritic cells and B cells. The antitumor properties of Flt3-L were evaluated in C3H/HeN mice challenged with the syngeneic C3L5 murine breast cancer cell line. Eighty % of animals receiving 500 microg/kg/day of Chinese hamster ovary-derived human Flt3-L for 10 days were protected from tumor growth, whether the tumor challenge was administered on the first or fourth days of Flt3-L administration. The protection provided by soluble Flt3-L was transient. All tumor-free animals rechallenged 4 weeks after the primary challenge developed tumor. Transduction of C3L5 with retroviral vectors expressing human or murine Flt3-L did not influence in vitro growth or MHC expression but decreased in vivo tumor development to 0 and 10% of mice, respectively. This compares with tumor growth of 52% with interleukin-2 transduced C3L5 and over 85% with untransduced and control vector-transduced C3L5. Unlike animals treated with soluble Flt3-L, administration of Flt3-L as a tumor vaccine protected mice from a subsequent challenge with untransduced C3L5 in 60-78% of mice, compared to 0% of controls. Our initial work used the most common Flt3-L isoform, which is membrane bound but can undergo proteolytic cleavage to generate a soluble form. To evaluate the role of the various Flt3-L isoforms in preventing tumor formation, retroviral vectors encoding only the membrane-bound form or only the soluble isoform were evaluated in the C3L5 model. Tumor formation was similar with either isoform, preventing tumor formation in 80-90% of mice after the primary challenge and 88-89% after the secondary challenge. Splenocytes obtained 4 weeks after the secondary challenge conferred adoptive immunity to naive mice in 60% of animals. This initial report of antitumor activity by Flt3-L is consistent with its known stimulatory effect on antigen-presenting cells and suggests it may enhance the development of tumor vaccines.