Discordance in Oncotype DX Breast Recurrence Score® Results for Bilateral Breast Cancer.

BACKGROUND: The Oncotype DX Breast Recurrence Score® assay is a clinically useful tool to determine the benefit of chemotherapy in the treatment of early-stage, hormone-receptor-positive breast cancer. Bilateral breast cancer (BBC) is found in ~ 5% of patients with breast cancer, and data regarding discordance of Oncotype DX results between BBC defined by current TAILORx subgroups are limited. Our goals are to study the rate of Oncotype DX discordance between BBC and investigate whether such differences can affect chemotherapy treatment discussions. METHODS: Patients with BBC were identified in US samples submitted to Genomic Health for 21-gene testing between January 2019 and July 2020. The risk categories were defined as 0-25 and 26-100 as well as 0-17, 18-30, and 31-100 for all patients. Subgroup analysis was also performed for node-negative women age ≤ 50 years with Recurrence Score results of 0-15, 16-20, 21-25, and 26-100. RESULTS: 944 BBC patients with known nodal status (702 node negative, 242 node positive) were identified and included. Among node-negative patients aged > 50 years, the rate of discordance in Recurrence Score by group (0-25 and 26-100) was 4.2% (n = 598). For node-negative patients aged ≤ 50 years, the risk group was discordant in < 3% when considering the risk grouping of 0-25 and 26-100. However, upon subgroup analysis based on TAILORx analysis, the rate of discordance was 48.1% in these younger patients (n = 104). CONCLUSIONS: This study shows that a clinically relevant rate of discordance in Oncotype DX results in patients with BBC may impact medical decision-making regarding chemotherapy.

Malignant and High-Risk Lesions in the Contralateral Breast Symmetry Mastopexy and Reduction Specimens When Performing Large-Volume Displacement Oncoplastic Surgery.

BACKGROUND: Large-volume displacement oncoplastic surgery using mastopexy/reduction mammaplasty designs is becoming increasingly popular in breast cancer surgery. A contralateral symmetry operation using similar mastopexy or breast reduction designs is also commonly performed by the plastic surgeon. Our goal was to analyze contralateral symmetry specimens to review the prevalence of high-risk or malignant lesions. METHODS: We conducted a retrospective study of the first consecutive 100 large-volume displacement oncoplastic surgeries at our institution between August 2015 and June 2018. Eighty-five patients had an immediate symmetry operation performed on the contralateral breast. Information on malignant lesions and high-risk lesions was obtained from the patient's pathology report. RESULTS: Seven different surgical techniques were used for both the cancerous and contralateral breasts. The WISE pattern skin incision pattern was most frequently used, along with the superomedial pedicle design. Fourteen cases (16.5%) had malignant and/or high-risk lesions incidentally detected. Specifically, there was a breast cancer prevalence of 4.8% in the contralateral symmetry breast specimen. CONCLUSION: Plastic surgeons should be aware that there is a substantial minority of contralateral specimens that have high-risk or malignant lesions, which underscores the importance of specimen orientation and communication with the associated pathologist.

Anatomic and terminological description and processing of breast pathologic specimens from oncoplastic large volume displacement surgeries.

Oncoplastic surgery provides breast cancer patients with greater aesthetic satisfaction without compromising disease-free survival or overall survival rate. Large volume displacement oncoplastic surgical techniques have become increasingly popular as a strategy for improving aesthetic outcomes and extending the option of breast conservation therapy. They often involve breast reduction or mastopexy reconstructive techniques to facilitate resection of large breast volumes on the side of the breast cancer and accompanied with symmetry contralateral breast reductions or mastopexies. However, dissection of large volume displacement oncoplastic surgical specimens presents unique challenges. Compared with traditional mastectomy specimens, they are relatively complicated, which requires the pathologist to understand the surgical procedure and the anatomy of the specimens. Given this, we introduce the standard anatomical and terminological description for the breast pathologic specimens of five large volume displacement oncoplastic surgical techniques commonly performed in our institution for breast cancer management. The individual surgical specimen is composed of one or several components, which include lateral wall, superior keyhole, medial wall, lateral wing, inferior pole, and medial wing. We also present specimen documentation and sectioning procedures used in our institution. The advantages for the patient provided by large volume reduction oncoplastic surgery must be supported by proper evaluation of the surgical pathology specimen. Therefore, we recommend that each section taken from the oncoplastic specimen be labeled as to its specific location in the specimen components. Standardized nomenclature and technique will assist pathologists in accurately evaluating the surgical margins.

American Joint Committee on Cancer's Staging System for Breast Cancer, Eighth Edition: What the Radiologist Needs to Know.

The TNM staging system for cancer was developed by Pierre Denoix in France in the 1940s and 1950s. The North American effort to standardize the TNM system for cancer staging was first organized in 1959 as the American Joint Committee for Cancer Staging and End-Results Reporting, which is now the American Joint Committee on Cancer (AJCC). The most recent edition of the AJCC Cancer Staging Manual, the eighth edition, was globally adopted on January 1, 2018. Previous editions of the manual have relied on anatomic methods of staging alone, which used population-based survival data to predict clinical outcomes. In the era of precision medicine, the major change in the eighth edition is the incorporation of prognostic biomarkers to more accurately predict clinical outcomes and treatment response on an individual basis, without relying solely on the anatomic extent of disease. Factors such as tumor grade, hormone receptor and oncogene expression, and multigene panel recurrence scores are now integrated with anatomic information to yield a final prognostic stage group, which will provide better stratification of patient prognosis. The purpose of this article is to review the major changes in the AJCC eighth edition for breast cancer staging, review anatomic TNM staging, familiarize the radiologist with prognostic biomarkers and prognostic staging, and identify key sites of disease that may alter clinical management. ©RSNA, 2018.

The fibroblast Tiam1-osteopontin pathway modulates breast cancer invasion and metastasis.

BACKGROUND: The tumor microenvironment has complex effects in cancer pathophysiology that are not fully understood. Most cancer therapies are directed against malignant cells specifically, leaving pro-malignant signals from the microenvironment unaddressed. Defining specific mechanisms by which the tumor microenvironment contributes to breast cancer metastasis may lead to new therapeutic approaches against advanced breast cancer. METHODS: We use a novel method for manipulating three-dimensional mixed cell co-cultures, along with studies in mouse xenograft models of human breast cancer and a histologic study of human breast cancer samples, to investigate how breast cancer-associated fibroblasts affect the malignant behaviors of breast cancer cells. RESULTS: Altering fibroblast Tiam1 expression induces changes in invasion, migration, epithelial-mesenchymal transition, and cancer stem cell characteristics in associated breast cancer cells. These changes are both dependent on fibroblast secretion of osteopontin and also long-lasting even after cancer cell dissociation from the fibroblasts, indicating a novel Tiam1-osteopontin pathway in breast cancer-associated fibroblasts. Notably, inhibition of fibroblast osteopontin with low doses of a novel small molecule prevents lung metastasis in a mouse model of human breast cancer metastasis. Moreover, fibroblast expression patterns of Tiam1 and osteopontin in human breast cancers show converse changes correlating with invasion, supporting the hypothesis that this pathway in tumor-associated fibroblasts regulates breast cancer invasiveness in human disease and is thus clinically relevant. CONCLUSIONS: These findings suggest a new therapeutic paradigm for preventing breast cancer metastasis. Pro-malignant signals from the tumor microenvironment with long-lasting effects on associated cancer cells may perpetuate the metastatic potential of developing cancers. Inhibition of these microenvironment signals represents a new therapeutic strategy against cancer metastasis that enables targeting of stromal cells with less genetic plasticity than associated cancer cells and opens new avenues for investigation of novel therapeutic targets and agents.

Defining the cellular precursors to human breast cancer.

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM(+)) and basal/myoepithelial (CD10(+)). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM(+) epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10(+) cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10(+) breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues.

Anatomical localization of progenitor cells in human breast tissue reveals enrichment of uncommitted cells within immature lobules.

INTRODUCTION: Lineage tracing studies in mice have revealed the localization and existence of lineage-restricted mammary epithelial progenitor cells that functionally contribute to expansive growth during puberty and differentiation during pregnancy. However, extensive anatomical differences between mouse and human mammary tissues preclude the direct translation of rodent findings to the human breast. Therefore, here we characterize the mammary progenitor cell hierarchy and identify the anatomic location of progenitor cells within human breast tissues. METHODS: Mammary epithelial cells (MECs) were isolated from disease-free reduction mammoplasty tissues and assayed for stem/progenitor activity in vitro and in vivo. MECs were sorted and evaluated for growth on collagen and expression of lineages markers. Breast lobules were microdissected and individually characterized based on lineage markers and steroid receptor expression to identify the anatomic location of progenitor cells. Spanning-tree progression analysis of density-normalized events (SPADE) was used to identify the cellular hierarchy of MECs within lobules from high-dimensional cytometry data. RESULTS: Integrating multiple assays for progenitor activity, we identified the presence of luminal alveolar and basal ductal progenitors. Further, we show that Type I lobules of the human breast were the least mature, demonstrating an unrestricted pattern of expression of luminal and basal lineage markers. Consistent with this, SPADE analysis revealed that immature lobules were enriched for basal progenitor cells, while mature lobules consisted of increased hierarchal complexity of cells within the luminal lineages. CONCLUSIONS: These results reveal underlying differences in the human breast epithelial hierarchy and suggest that with increasing glandular maturity, the epithelial hierarchy also becomes more complex.

Pregnancy-associated breast cancers are driven by differences in adipose stromal cells present during lactation.

INTRODUCTION: The prognosis of breast cancer is strongly influenced by the developmental stage of the breast when the tumor is diagnosed. Pregnancy-associated breast cancers (PABCs), cancers diagnosed during pregnancy, lactation, or in the first postpartum year, are typically found at an advanced stage, are more aggressive and have a poorer prognosis. Although the systemic and microenvironmental changes that occur during post-partum involution have been best recognized for their role in the pathogenesis of PABCs, epidemiological data indicate that PABCs diagnosed during lactation have an overall poorer prognosis than those diagnosed during involution. Thus, the physiologic and/or biological events during lactation may have a significant and unrecognized role in the pathobiology of PABCs. METHODS: Syngeneic in vivo mouse models of PABC were used to examine the effects of system and stromal factors during pregnancy, lactation and involution on mammary tumorigenesis. Mammary adipose stromal cell (ASC) populations were isolated from mammary glands and examined by using a combination of in vitro and in vivo functional assays, gene expression analysis, and molecular and cellular assays. Specific findings were further investigated by immunohistochemistry in mammary glands of mice as well as in functional studies using ASCs from lactating mammary glands. Additional findings were further investigated using human clinical samples, human stromal cells and using in vivo xenograft assays. RESULTS: ASCs present during lactation (ASC-Ls), but not during other mammary developmental stages, promote the growth of carcinoma cells and angiogenesis. ASCs-Ls are distinguished by their elevated expression of cellular retinoic acid binding protein-1 (crabp1), which regulates their ability to retain lipid. Human breast carcinoma-associated fibroblasts (CAFs) exhibit traits of ASC-Ls and express crabp1. Inhibition of crabp1in CAFs or in ASC-Ls abolished their tumor-promoting activity and also restored their ability to accumulate lipid. CONCLUSIONS: These findings imply that (1) PABC is a complex disease, which likely has different etiologies when diagnosed during different stages of pregnancy; (2) both systemic and local factors are important for the pathobiology of PABCs; and (3) the stromal changes during lactation play a distinct and important role in the etiology and pathogenesis of PABCs that differ from those during post-lactational involution.

The melanocyte differentiation program predisposes to metastasis after neoplastic transformation.

The aggressive clinical behavior of melanoma suggests that the developmental origins of melanocytes in the neural crest might be relevant to their metastatic propensity. Here we show that primary human melanocytes, transformed using a specific set of introduced genes, form melanomas that frequently metastasize to multiple secondary sites, whereas human fibroblasts and epithelial cells transformed using an identical set of genes generate primary tumors that rarely do so. Notably, these melanomas have a metastasis spectrum similar to that observed in humans with melanoma. These observations indicate that part of the metastatic proclivity of melanoma is attributable to lineage-specific factors expressed in melanocytes and not in other cell types analyzed. Analysis of microarray data from human nevi shows that the expression pattern of Slug, a master regulator of neural crest cell specification and migration, correlates with those of other genes that are important for neural crest cell migrations during development. Moreover, Slug is required for the metastasis of the transformed melanoma cells. These findings indicate that melanocyte-specific factors present before neoplastic transformation can have a pivotal role in governing melanoma progression.

Analytical Validation of NavDx, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Cancers.

The NavDx® blood test analyzes tumor tissue modified viral (TTMV)-HPV DNA to provide a reliable means of detecting and monitoring HPV-driven cancers. The test has been clinically validated in a large number of independent studies and has been integrated into clinical practice by over 1000 healthcare providers at over 400 medical sites in the US. This Clinical Laboratory Improvement Amendments (CLIA), high complexity laboratory developed test, has also been accredited by the College of American Pathologists (CAP) and the New York State Department of Health. Here, we report a detailed analytical validation of the NavDx assay, including sample stability, specificity as measured by limits of blank (LOBs), and sensitivity illustrated via limits of detection and quantitation (LODs and LOQs). LOBs were 0-0.32 copies/µL, LODs were 0-1.10 copies/µL, and LOQs were <1.20-4.11 copies/µL, demonstrating the high sensitivity and specificity of data provided by NavDx. In-depth evaluations including accuracy and intra- and inter-assay precision studies were shown to be well within acceptable ranges. Regression analysis revealed a high degree of correlation between expected and effective concentrations, demonstrating excellent linearity (R2 = 1) across a broad range of analyte concentrations. These results demonstrate that NavDx accurately and reproducibly detects circulating TTMV-HPV DNA, which has been shown to aid in the diagnosis and surveillance of HPV-driven cancers.

Dissecting genetic requirements of human breast tumorigenesis in a tissue transgenic model of human breast cancer in mice.

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

Chemotherapy-Induced Collagen IV Drives Cancer Cell Motility through Activation of Src and Focal Adhesion Kinase.

Triple-negative breast cancer (TNBC) is the most aggressive and deadly subtype of breast cancer, accounting for 30,000 cases annually in the United States. While there are several clinical trials ongoing to identify new agents to treat TNBC, the majority of patients with TNBC are treated with anthracycline- or taxane-based chemotherapies in the neoadjuvant setting, followed by surgical resection and adjuvant chemotherapy. While many patients respond well to this approach, as many as 25% will suffer local or metastatic recurrence within 5 years. Understanding the mechanisms that drive recurrence after chemotherapy treatment is critical to improving survival for patients with TNBC. It is well established that the extracellular matrix (ECM), which provides structure and support to tissues, is a major driver of tumor growth, local invasion, and dissemination of cancer cells to distant metastatic sites. In the present study, we show that decellularized ECM (dECM) obtained from chemotherapy-treated mice increases motility of treatment-naïve breast cancer cells compared with vehicle-treated dECM. Tandem-mass-tag proteomics revealed that anthracycline- and taxane-based chemotherapies induce drug-specific changes in tumor ECM composition. The basement membrane protein collagen IV was significantly upregulated in the ECM of chemotherapy-treated mice and patients treated with neoadjuvant chemotherapy. Collagen IV drove invasion via activation of Src and focal adhesion kinase signaling downstream of integrin α1 and α2, and inhibition of collagen IV-driven signaling decreased motility in chemotherapy-treated dECM. These studies provide a novel mechanism by which chemotherapy may induce metastasis via its effects on ECM composition. SIGNIFICANCE: Cytotoxic chemotherapy induces significant changes in the composition of tumor ECM, inducing a more invasive and aggressive phenotype in residual tumor cells following chemotherapy.

Detection of Occult Recurrence Using Circulating Tumor Tissue Modified Viral HPV DNA among Patients Treated for HPV-Driven Oropharyngeal Carcinoma.

PURPOSE: Despite generally favorable outcomes, 15% to 25% of patients with human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) will have recurrence. Current posttreatment surveillance practices rely on physical examinations and imaging and are inconsistently applied. We assessed circulating tumor tissue modified viral (TTMV)-HPV DNA obtained during routine posttreatment surveillance among a large population of real-world patients. EXPERIMENTAL DESIGN: This retrospective clinical case series included 1,076 consecutive patients across 108 U.S. sites who were ≥ 3 months posttreatment for HPV-driven OPSCC and who had one or more TTMV-HPV DNA tests (NavDx, Naveris Laboratories) obtained during surveillance between February 6, 2020, and June 29, 2021. Test results were compared with subsequent clinical evaluations. RESULTS: Circulating TTMV-HPV DNA was positive in 80 of 1,076 (7.4%) patients, with follow-up available on all. At first positive surveillance testing, 21 of 80 (26%) patients had known recurrence while 59 of 80 (74%) patients were not known to have recurrent disease. Among these 59 patients, 55 (93%) subsequently had a confirmed recurrence, 2 patients had clinically suspicious lesions, and 2 had clinically "no evidence of disease" (NED) at last follow-up. To date, the overall positive predictive value of TTMV-HPV DNA testing for recurrent disease is 95% (N = 76/80). In addition, the point-in-time negative predictive value is 95% (N = 1,198/1,256). CONCLUSIONS: These findings highlight the clinical potential for circulating TTMV-HPV DNA testing in routine practice. As a surveillance tool, TTMV-HPV DNA positivity was the first indication of recurrence in the majority of cases, pre-dating identification by routine clinical and imaging exams. These data may inform future clinical and guideline-endorsed strategies for HPV-driven malignancy surveillance. See related commentary by Colevas, p. 4171.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines.

INTRODUCTION: Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells involving various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear whether they retain a similar cellular heterogeneity as to that found within breast tissues. METHODS: We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. RESULTS: Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines were enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Although normal human mammary epithelial cell lines exhibited features of bi-potent progenitor cells they were unable to differentiate into mature luminal breast epithelial cells. CONCLUSIONS: As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues.

Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis.

The extracellular matrix (ECM), a major component of the tumor microenvironment, promotes local invasion to drive metastasis. Here, we describe a method to study whole-tissue ECM effects from disease states associated with metastasis on tumor cell phenotypes and identify the individual ECM proteins and signaling pathways that are driving these effects. We show that decellularized ECM from tumor-bearing and obese mammary glands drives TNBC cell invasion. Proteomics of the ECM from the obese mammary gland led us to identify full-length collagen VI as a novel driver of TNBC cell invasion whose abundance in tumor stroma increases with body mass index in human TNBC patients. Last, we describe the mechanism by which collagen VI contributes to TNBC cell invasion via NG2-EGFR cross-talk and MAPK signaling. Overall, these studies demonstrate the value of decellularized ECM scaffolds obtained from tissues to identify novel functions of the ECM.

Akt1 ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice.

Ample evidence to date links the phosphatidylinositol 3-kinase-regulated protein kinase Akt with the induction and progression of human cancer, including breast cancer. However, there are three Akt isoforms with limited information about their specificity during oncogenesis. This study addresses the role of the three isoforms in polyoma middle T (PyMT) and ErbB2/Neu-driven mammary adenocarcinomas in mice. The effects of ablation of Akt1, Akt2, and Akt3 on the induction and the biology of these tumors were dramatically different, with ablation of Akt1 inhibiting, ablation of Akt2 accelerating, and ablation of Akt3 having a small, not statistically significant, inhibitory effect on tumor induction by both transgenes. Whereas PyMT-induced tumors are all invasive, Akt1(-/-)Neu-induced tumors are more invasive than Akt2(-/-)Neu-induced tumors. Invasiveness, however, does not always correlate with metastasis. Ablation of individual Akt isoforms does not affect the development of the mammary gland during puberty or the expression of the transgenes. Akt ablation, therefore, influences tumor induction by modulating transgene-induced oncogenic signaling. Immunostaining for Ki-67 and cyclin D1 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays on tissue sections revealed that the delay of tumor induction in Akt1 knockout mice is due to the inhibitory effects of Akt1 ablation on cell proliferation and survival. Given that these animal models exhibit significant similarities to human breast cancer, the results of the present study may have significant translational implications because they may influence how Akt inhibitors will be used in the treatment of human cancer.

Systemic stromal effects of estrogen promote the growth of estrogen receptor-negative cancers.

Numerous hormonal factors contribute to the lifetime risk of breast cancer development. These include inherited genetic mutations, age of menarche, age of menopause, and parity. Inexplicably, there is evidence indicating that ovariectomy prevents the formation of both estrogen receptor (ER)-positive and ER-negative breast cancers, suggesting that ER-negative breast cancers are dependent on ovarian hormones for their formation. To examine the mechanism(s) by which this may be occurring, we investigated the hypothesis that steroid hormones promote the outgrowth of ER-negative cancers by influencing host cell types distinct from the mammary epithelial cells. We used a novel xenograft mouse model of parturition-induced breast carcinoma formation, in which the tumors that arise following pregnancy lack the expression of nuclear hormone receptors, thereby recapitulating many clinical cases of this disease. Despite lacking ER expression, the tumors arising following pregnancy in this model require circulating estrogens for their formation. Moreover, increasing the levels of circulating estrogens sufficed to promote the formation and progression of ER-negative cancers, which was accompanied by a systemic increase in host angiogenesis and was attendant with the recruitment of bone marrow-derived stromal cells. Furthermore, bone marrow cells from estrogen-treated mice were sufficient to promote tumor growth. These results reveal a novel mechanism by which estrogens promote the growth of ER-negative cancers.

Distinct roles of the three Akt isoforms in lactogenic differentiation and involution.

The three Akt isoforms differ in their ability to transduce oncogenic signals initiated by the Neu and PyMT oncogenes in mammary epithelia. As a result, ablation of Akt1 inhibits and ablation of Akt2 accelerates mammary tumor development by both oncogenes, while ablation of Akt3 is phenotypically almost neutral. Since the risk of breast cancer development in humans correlates with multiple late pregnancies, we embarked on a study to determine whether individual Akt isoforms also differ in their ability to transduce hormonal and growth factor signals during pregnancy, lactation and post-lactation involution. The results showed that the ablation of Akt1 delays the differentiation of the mammary epithelia during pregnancy and lactation, and that the ablation of Akt2 has the opposite effect. Finally, ablation of Akt3 results in minor defects, but its phenotype is closer to that of the wild type mice. Whereas the phenotype of the Akt1 ablation is cell autonomous, that of Akt2 is not. The ablation of Akt1 promotes apoptosis and accelerates involution, whereas the ablation of Akt2 inhibits apoptosis and delays involution. Mammary gland differentiation during pregnancy depends on the phosphorylation of Stat5a, which is induced by prolactin, a hormone that generates signals transduced via Akt. Here we show that the ablation of Akt1, but not the ablation of Akt2 or Akt3 interferes with the phosphorylation of Stat5a during late pregnancy and lactation. We conclude that the three Akt isoforms have different roles in mammary gland differentiation during pregnancy and this may reflect differences in hormonal signaling.

A mouse model of human breast cancer metastasis to human bone.

Currently, an in vivo model of human breast cancer metastasizing from the orthotopic site to bone does not exist, making it difficult to study the many steps of skeletal metastasis. Moreover, models used to identify the mechanisms by which breast cancer metastasizes to bone are limited to intracardiac injection, which seeds the cancer cells directly into the circulation, thus bypassing the early steps in the metastatic process. Such models do not reflect the full process of metastasis occurring in patients. We have developed an animal model of breast cancer metastasis in which the breast cancer cells and the bone target of osteotropic metastasis are both of human origin. The engrafted human bone is functional, based on finding human IgG in the mouse bloodstream, human B cells in the mouse spleen, and normal bone histology. Furthermore, orthotopic injection of a specific human breast cancer cell line, SUM1315 (derived from a metastatic nodule in a patient), later resulted in both bone and lung metastases. In the case of bone, metastasis was to the human implant and not the mouse skeleton, indicating a species-specific osteotropism. This model replicates the events observed in patients with breast cancer skeletal metastases and serves as a useful and relevant model for studying the disease.

BALB/c alleles for Prkdc and Cdkn2a interact to modify tumor susceptibility in Trp53+/- mice.

In mice heterozygous for p53 (Trp53(+/-)), the incidence of mammary tumors varies among strains, with C57BL/6 being resistant and BALB/c being susceptible. Mammary tumor phenotypes were examined in female Trp53(+/-) F1 mice (C57BL/6 x BALB/c;n = 19) and N2 backcross mice [(C57BL/6 x BALB/c) x BALB/c] (n = 224). Susceptibility to mammary tumors segregated as a dominant phenotype in F1 females, but a higher frequency and shorter latency in N2 mice indicated a contribution from recessive-acting modifiers. Segregation of the hypomorphic BALB/c alleles for DNA-dependent protein kinase catalytic subunit (Prkdc) and p16(INK4A) (Cdkn2a) was analyzed in the N2 mice. The time to first tumor (considering all tumor types) was significantly different among the four genotype combinations (P = 0.01). Cdkn2a had a strong effect (P = 0.008) but was restricted to Prkdc(B/B) mice (P = 0.001), indicating a strong interaction between the loci. Differences in mammary tumor occurrence among genotypes for Prkdc and Cdkn2a in N2 mice were not statistically significant. This study indicates that BALB/c Prkdc and Cdkn2a alleles do modify tumor incidence in Trp53(+/-) mice and highlights the complexity of gene interaction effects in determining cancer phenotypes but discounts these alleles as major recessive loci contributing to spontaneous mammary tumor susceptibility.