N/A, Breast Cancer

Cancers are thought to arise from a collection of permissive factors which interact within and between different cells of a tissue or tumor to promote tumor growth and invasive characteristics (Sonnenschein and Soto 1999; Hanahan and Weinberg 2011; Floor, Dumont et al. 2012; Goodson, Lowe et al. 2015; Schwarzman, Ackerman et al. 2015; Smith, Guyton et al. 2016; Grashow, De La Rosa et al. 2018). Permissive factors or hallmarks include changes to the cell’s dependence on growth signals, proliferation, metabolism, apoptosis, senescence, angiogenesis, and invasion and metastasis. These hallmarks are modified by other factors including growth factors, inflammation, oxidative stress, changes to the microenvironment, DNA damage, and changes in gene expression.

The mammary gland is a hormone responsive organ with multiple phases of development from embryogenesis into adulthood. Consequently, certain hallmarks and contributing factors including proliferative response to growth signals, growth factors, changes to the microenvironment, and changes in gene expression play a larger role in this organ, and the importance of various factors shifts depending on developmental stage (Rudel, Fenton et al. 2011). Established risk factors of breast cancer extend beyond genetic contributors (principally alterations in DNA damage response genes) and DNA damaging environmental agents to include exposure to pharmaceutical hormones, timing of puberty and first birth, and lifetime exposure to estrogen and progesterone ((IOM) Institute of Medicine 2012). 

Hormonal and other environmental influences during proliferation and differentiation alter the pace and structure of cellular or mammary gland development to leave tissue in the adult gland more susceptible to cancer. In addition, the elevated hormone concentrations associated with the menstrual cycle and pregnancy provide a regular proliferative stimulus to any pre-cancerous cells present in the breast (Rudel, Fenton et al. 2011). A substantial majority of breast cancers express hormone receptors, and these cancers are particularly responsive to hormones (Badowska-Kozakiewicz, Patera et al. 2015).

Consistent with the importance of growth factors and DNA damage in the development of cancer, driver mutations (mutations that favor the success of the nascent cancer cells and are therefore selected) commonly appear in the growth factor related signaling pathways (BRAF, EGRF, RAS, PI3K, STK11) and in DNA damage response and cell cycle checkpoint signal pathways (ATM, TP53, CHEK2, CDKN2B (P15), CDK4) (Greenman, Stephens et al. 2007; Croce 2008; Kaufmann, Nevis et al. 2008; Stratton, Campbell et al. 2009; Vandin, Upfal et al. 2012). These and other mutations are acquired over the development of a cancer and contribute to the evolution of the cancer (Wang, Waters et al. 2014; Yates, Gerstung et al. 2015; Begg, Ostrovnaya et al. 2016).

In breast cancer, TP53, PI3K and GATA3 are each mutated in more than 10% of cancers, amplification or mutation of the RB1 pathway are common, and HER2 (an EGFR receptor) is amplified in HER2 type cancers (CGAN 2012). EGFR, HER2, BRAF, RAS, and PI3K participate in the EGFR (growth factor) signaling pathway. Activating mutations in PI3K generate growth factor independent proliferation of mammary epithelial cells, possibly via the RB1 pathway (Gustin, Karakas et al. 2009). GATA is a transcription factor that maintains luminal epithelial cell differentiation and suppresses proliferation, and mutation results in the proliferation of undifferentiated cells (Kouros-Mehr, Slorach et al. 2006; Shahi, Wang et al. 2017).

Environmental factors contribute significantly to the total number of breast cancers. Women exposed to the synthetic hormone DES or the pesticide DDT in utero are up to two to four times more likely to be diagnosed with breast cancer in their fifties (Palmer, Wise et al. 2006; Cohn, La Merrill et al. 2015). A study in 2002 found that recipients of hormone replacement therapy (HRT) around menopause are 26% more likely to be diagnosed with breast cancer (Narod 2011). When prescriptions of HRT began to fall in response to the study, so did cancer diagnoses. Over the next few years, approximately 5% fewer cancers were diagnosed in women over 45 (Glass, Lacey et al. 2007) with an estimated 126,000 fewer cases of breast cancer over the next ten years (Roth, Etzioni et al. 2014).

In rodent bioassays, tumors can be detected via visual observation or palpation of live animals, necropsy of dead animals, and via microscopic examination of tissue. Malignant tumors including carcinomas in situ are distinguishable from benign tumors on the basis of the thickness or shape of the epithelial cell layer, regularity of the lumen or the presence of cribiform luminae, inflammation or desmoplastic reaction of the stroma, dominance of a less differentiated cell type, and larger nuclei, while diagnosis of invasiveness depends on the identification of metastases or invasion of neoplastic cells into surrounding tissue (Russo and Russo 2000).

In humans, lumps are commonly detected by palpation or mammogram. Further imaging, biopsy, and/or surgical excision of the affected tissue are used to differentiate benign, cancerous, and invasive tumors (McDonald, Clark et al. 2016).

This can be applied to adult women and men and mice.

Because of the long latency of mammary tumors, the two-year rodent carcinogenicity bioassay is the primary assay for this adverse outcome. The assay is included in the OECD Test No. 451 and 453 for carcinogenicity and combined toxicity and carcinogenicity (OECD 2009; OECD 2009), and is also used by the US National Toxicology program (Chhabra, Huff et al. 1990), and the FDA (FDA (Food and Drug Administration) 2007), and referenced by the EPA (EPA (Environmental Protection Agency) 2005) in guidelines for risk assessments. Other assays from short term (2-4 weeks) and subchronic (90 day) to chronic (1 year) toxicity also call for the documentation of mammary tumors (FDA (Food and Drug Administration) 2007; OECD (Organisation for Economic Cooperation and Development) 2018), so these assays could capture the early onset of tumors, and could be modified to report earlier key events like proliferation and inflammation.

Several characteristics of classic cancer bioassays limit the sensitivity of these assays to mammary gland carcinogens. First, no assays require prenatal or early post-natal exposures for carcinogenicity testing. The US NIH’s National Toxicology Program assays start exposures at five to six weeks of age and OECD regulatory assay exposures suggest (but do not require) exposures beginning after weaning and before eight weeks of age. Assays initiating exposures at later ages have diminished sensitivity to agents that affect breast development and increase future susceptibility to cancer, such as estrogenic hormones, DDT and dioxins (EPA (Environmental Protection Agency) 2005; Rudel, Fenton et al. 2011). Agents with similar activity to ionizing radiation and DNA damaging chemicals may not be fully captured in some of these assays, since sensitivity appears to peak around or before week seven for these agents (around puberty) (Imaoka, Nishimura et al. 2013). Second, carcinogenicity assay guidelines do not require the best methods for detecting tumors in mammary gland: whole mount preparations of mammary gland coupled with longitudinal sections (dorsoventral sections parallel to the body) of mammary gland for histology (Tucker, Foley et al. 2017). Palpation and transverse sections of mammary gland can easily miss tumors or lesions of interest. Interestingly the NTP reproductive toxicity guidelines do specify these preferable methods for mammary gland analysis.

Two additional factors affect the sensitivity of standard carcinogenicity assays. First, benign tumors are not always considered to be an indicator of carcinogenicity, leading to a possible underestimation of risk.  NTP and EPA guidance suggest that benign tumors provide additional weight of evidence if malignant tumors are also present or if studies suggest benign tumors can progress to carcinogenicity. In a short-term study, benign tumors may indicate a need for a longer-term study. However, benign mammary tumors (fibroadenomas) almost always coincide with carcinogenic tumors in mammary gland or other organs, and carcinomas sometimes grow from fibroadnomas (Rudel, Attfield et al. 2007; Russo 2015) suggesting that benign tumors may be an underutilized indicator of carcinogenicity.

Finally, the dose selection guidance in carcinogenicity testing typically calls for a high dose that is sufficiently toxic to suppress body weight (OECD 2009). However, body weight interacts with risk of breast cancer (Haseman, Young et al. 1997; Rudel, Attfield et al. 2007), reducing the sensitivity of the upper end of the dose range and the likelihood of a positive dose-response.

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