Since its inception, Focused Research has been a main pillar of the CBCP. Using the high-quality biospecimens maintained by the Biobank, we are able to study a range of disease stages from normal/benign breast conditions to invasive breast carcinomas.
Areas of research have included racial disparities in breast cancer, genetic and molecular factors controlling metastasis, the role of the tumor microenvironment in breast tumorigenesis, cancer predisposition genes, specific breast cancer subtypes including HER2+ and triple negative breast cancers, the effect of chemical exposures on breast tumor development, and breast cancer in young women.
Previously, the bulk of these studies utilized genomic technologies such as DNA sequencing, microsatellite markers, SNP and copy number arrays and assays, gene expression microarrays, and qRT-PCR. Several years ago, we began conducting next-generation sequencing studies both in-house and in collaboration with researchers from other institutions that have included WES, WGS, and total RNA-Seq platforms. We also participated in a limited number of studies that included proteomic analyses such as global and phosphoproteomics. Currently, with the advent of the APOLLO program, we are performing even more in-depth genomic and proteomic studies that will allow us to gain a more comprehensive molecular understanding of breast cancer through our various research projects, including the identification of proteogenomic differences across molecular subtypes, races, age groups, etc.
Below are some of the major research areas we are currently investigating.
Breast cancer incidence and mortality vary among different racial/ethnic groups in the United States. While incidence is higher among white women, mortality is highest in black women. Socioeconomic factors, such as access to healthcare and treatment differences, have been proposed to play a role in the disparate breast cancer outcomes among races. However, biological differences between breast tumors in whites and blacks may also play a role. For example, breast tumors in blacks tend to be larger, more aggressive, and of higher grade than those diagnosed in white women. Moreover, black women are more likely to be diagnosed with triple negative breast cancer, the most aggressive subtype, which unlike other subtypes, cannot be treated with targeted therapies.
Earlier work from our Institute investigated whether there were molecular differences between breast tumors from black and white women. Microarray analysis of invasive breast tumors from 26 pairs of black and white women matched by age, tumor grade, and ER status, identified 23 differentially expressed genes between blacks and whites. This same study also analyzed gene expression differences in normal breast tissue from disease-free black and white women and found 13 genes to be differentially expressed (Field, Love et al. 2012). A subsequent study comparing breast tumors of the triple negative subtype from black and white women identified differential expression of a single gene, CRYBB2P1, a pseudogene (Sturtz, Melley et al. 2014).
More recently, as part of the TCGA-Breast Cancer Analysis Working Group (TCGA-BCAWG), we conducted a comprehensive molecular study comparing breast tumors from blacks and whites using data from TCGA. After adjusting for subtype, 16 methylation probes, 4 copy number segments, 1 protein, and 142 genes were found to be differentially expressed, and the gene-based signature performed excellently in distinguishing breast tumors from black and white patients (Huo, Hu et al. 2017). This same paper also examined breast cancer-free interval (BCFI) within the TCGA-BC dataset. Using genomically-determined race, it was found that BCFI was worse among black patients compared to whites (P=0.043).
The modest number of molecular differences that have been observed in these studies suggests that breast tumors from blacks and whites are more similar than different, and it is possible that the observed differences are, in fact, due to differences in normal breast biology between blacks and whites. As part of the APOLLO project, we will further investigate racial disparities in breast cancer outcomes between black and white women. Our overall goal is to identify and characterize the molecular features of breast tumors from black and white patients and determine if and how they affect breast cancer outcomes.
All patients included in this study are members or dependents of the military and receive standardized treatment within an equal-access healthcare system. This minimizes any confounding effects that differences in healthcare may have on our analyses. Using specimens from patients enrolled in the CBCP, we are designing new studies using the standard APOLLO molecular platforms of WGS, total RNA-Seq, and global- and phosphoproteomics, as well as other molecular platforms, to obtain a more comprehensive, proteogenomic characterization of tumors and other tissues from black and white patients to better understand the reported clinical outcome and molecular differences between these two races.
Field, L. A., B. Love, et al. (2012). “Identification of differentially expressed genes in breast tumors from African American compared with Caucasian women.” Cancer 118(5): 1334-1344.
Huo, D., H. Hu, et al. (2017). “Comparison of Breast Cancer Molecular Features and Survival by African and European Ancestry in The Cancer Genome Atlas.” JAMA Oncol 3(12): 1654-1662.
Sturtz, L. A., J. Melley, et al. (2014). “Outcome disparities in African American women with triple negative breast cancer: a comparison of epidemiological and molecular factors between African American and Caucasian women with triple negative breast cancer.” BMC Cancer 14: 62.
Breast cancer accounts for more than 40% of all cancers diagnosed in women under the age of 40. Although only about 7% of all breast cancer cases occur in women under age 40, studies have shown that they have worse prognoses and poorer outcomes than women diagnosed at older ages. Breast tumors in young women are often aggressive, high grade, ER-negative, and more often observed in black women; however, multivariate analysis has demonstrated that young age itself is an independent predictor of poor outcome.
Several reasons have been proposed to explain the worse outcomes among young women with breast cancer. Among these is that tumors from young women have more unfavorable molecular features than those from older women. For example, young women may be more likely to develop more aggressive subtypes of breast cancer, and in fact, both the triple-negative and luminal B subtypes of breast cancer have been shown to contribute to the poorer outcomes in young women. On the other hand, genomic analyses of primary breast tumors in young women have been largely unsuccessful in identifying mutational events or differentially expressed genes that are specific to breast tumors in young women and/or associated with the worse outcomes observed in young women. A second possibility is that the ability to metastasize is greater in young women. In order to grow successfully at a distant site, the “seed” or tumor cell must be able to disseminate from the primary tumor to a site with “congenial soil” or a favorable host environment. The physiology of the host environment in young women may be more amenable to the development of metastases, leading to the development of more aggressive breast cancers with worse outcomes compared to older women. Finally, it has been proposed that disparities in treatment patterns may also contribute to the worse outcomes observed in young women with breast cancer.
As part of the APOLLO 4 project, a bioinformatics analysis of breast cancer in young women was conducted using the Military Health System Repository and the DoD Central Registration databases (Zimmer, Zhu et al. 2018). The military patient population is unique in that a disproportionate number of patients are young women, and all members have equal-access to the same standard of care. This analysis included 10,066 women diagnosed with breast cancer from 1998-2007 broken down into three age groups: (1) <40 years old, (2) 40-49 years old, and (3) ≥50 years old. Women under age 40 were more likely than the two older age groups to be diagnosed at a higher stage of disease and with high-grade and ER negative breast tumors. Young patients <40 years old were also more likely than their older counterparts to undergo unilateral and bilateral mastectomies. They were also most likely to receive chemotherapy as part of their treatment, regardless of disease stage or ER status. Finally, recurrence was highest among the <40 years old group (15%) compared to the 40-49 (11.8%) and ≥50 (10.8%) years old groups. Overall survival was highest among the 40-49 year old group, with survival of young women <40 being similar to that of women 50 and older despite receiving more aggressive treatment.
We are also performing molecular studies using retrospectively collected invasive breast tumors from CBCP participants to further characterize breast tumors from younger and older women. Using the standard APOLLO molecular platforms of WGS, total RNA-Seq, global- and phosphoproteomics, as well as other molecular platforms, we will obtain additional insight into the spectrum of proteogenomic alterations present in breast tumors from young women compared to older women and determine if and how these changes impact patient outcomes.
Zimmer, A. S., K. Zhu, et al. (2018). “Analysis of breast cancer in young women in the Department of Defense (DOD) database.” Breast Cancer Res Treat 168(2): 501-511.
Tumors do not grow in isolation but rather require a complex interplay between tumor epithelial cells and cells of the surrounding microenvironment. The breast microenvironment, composed of extracellular matrix and stromal cells such as fibroblasts, immune cells, adipocytes, and endothelial cells, plays a pivotal role in all aspects of breast tumorigenesis, including tumor development, progression, and response to therapy. In addition, the metastatic microenvironment allows for breast tumor cells that have disseminated from the primary tumor to successfully colonize and grow at foreign sites including lymph nodes and distant organs.
Our previous work investigating the role of the tumor microenvironment in breast tumorigenesis focused primarily on genomic approaches. Using a panel of microsatellite markers to study allelic imbalance (AI) and SNP arrays for copy number analysis, very few to no AI events or copy number alterations were detected in breast stroma, leading to the conclusion that the breast microenvironment is genetically stable (Rummel, Valente et al. 2012). Additional studies using microarrays found that gene expression patterns in adipose tissue vary with proximity to the tumor, with tumor-adjacent adipose demonstrating an immunotolerant phenotype, which likely promotes tumor development (Sturtz, Deyarmin et al. 2014). Finally, in studies of the metastatic microenvironment, colonized lymph nodes demonstrated an altered immune response, increased mesenchymal-epithelial transition, and increased cellular proliferation compared to lymph nodes free of metastatic tumors, suggesting that metastatic sites are not simply overrun by tumor cells but play an active role in metastatic colonization (Valente, Kane et al. 2014).
Currently, as part of the APOLLO project, we continue to investigate the role of the tumor microenvironment in breast tumorigenesis. For all specimens in which tumor-adjacent stromal tissue is available, samples will undergo total RNA-Seq, global- and phosphoproteomics, and reverse-phase protein array (RPPA), allowing us to measure transcript and protein levels in the stroma that may influence breast tumorigenesis. Moreover, these studies will allow us to determine if differential gene and protein expression in the tumor microenvironment impacts outcome disparities between (1) black and white women and (2) younger and older women with breast cancer.
Rummel, S., A. L. Valente, et al. (2012). “Genomic (in)stability of the breast tumor microenvironment.” Mol Cancer Res 10(12): 1526-1531.
Sturtz, L. A., B. Deyarmin, et al. (2014). “Gene expression differences in adipose tissue associated with breast tumorigenesis.” Adipocyte 3(2): 107-114.
Valente, A. L., J. L. Kane, et al. (2014). “Molecular response of the axillary lymph node microenvironment to metastatic colonization.” Clin Exp Metastasis 31(5): 565-572.