Types of Breast Cancer

The three major biologic subtypes of breast cancer are defined by estrogen (ER) and progesterone receptors (PR) and HER2 expression (HER2): (1) ER- or PR-positive, (2) HER2positive, and (3) triple negative (negative for ER, PR, and HER2). These tissue biomarkers are used because they are validated to describe differences in prognosis and, above all, are predictive of the benefit of specific treatments. These biomarkers thereby guide and direct therapeutic choices.

Biomarkers

Biomarkers can serve multiple purposes, depending on the context of disease being evaluated. Prognostic markers can distinguish between good and poor risk, aggressive and indolent disease. Usually, this is in the context of patients receiving no therapy (i.e., the natural history of the tumor) or patients receiving a relatively homogeneous standard of care. These markers can be measured directly in tumor tissue as well as systemically in serum or urine. When measured in serum or urine rather than tumor tissue, they may reflect overall tumor burden. Predictive markers can identify host or tumor characteristics that differentiate a group that is likely to benefit or not from a specific treatment. These markers, which can be single or multiple, are integrated into a composite measure. Biomarkers can be both prognostic and predictive. Putative biomarkers may be causally related to the biologic behavior and/or the mechanism of action of the therapy given or can be “innocent bystanders,” which just track reasonably closely with these biologic behaviors, but are not directly involved with driving that phenotype or mechanism of action.

Biomarkers are verified when their presence has been confirmed by more than one methodology. Validation of an assay, however, requires more stringency. A test is said to be validated when it is reproducible in the initial laboratory developing the assay, when it has been demonstrated to correlate with outcomes in an independently derived dataset with no overall relation to the original discovery set, and when it has been tested for its correlation to outcomes (either prognostic or predictive) in the population of patients, tumors, and treatments for the question being addressed. For example, the OncotypeDx assay has been extensively validated for a relatively narrow and homogeneous subset of breast cancers (ER-positive, nodenegative, and largely HER2-negative), providing an assessment of residual risk of distant relapse after assuming the patient had taken 5 years of tamoxifen. This test can therefore be used to guide treatment choices in this subset of patients, but not in those with a different subtype of breast cancer.

On the other hand, the other commercially available multigene assay, Mammaprint, was initially developed as a prognosticator in a heterogeneous population of patients—with different tumor types, and variable treatments, including no treatment. Thus, this assay is now undergoing validation in the MINDACT (Microarray in Node-Negative Disease May Avoid Chemotherapy) trial to ask whether chemotherapy should be given to early-stage patients. In my opinion, there is no level 1 evidence to use these multiplex biomarkers to guide treatment choice yet; however, these tests are in widespread clinical use outside of clinical trials.

It is outside the scope of this review to provide a comprehensive and detailed explanation and critique of each technique used to detect biomarkers. Rather, a short summary is provided. Biomarkers include DNA (native, methylated, mutated, amplified, and deleted), various forms of RNA (mRNA, miRNA), proteins (native, modified [phosphorylated, glycosylated, acetylated, mutated, fixed]). Compared with RNA or protein, DNA tends to be the most stable and resistant to degradation during routine tissue handling and storage. Moreover, genetic changes tend to be characteristic of the tumor and drive its biologic behavior. On the other hand, the presence of DNA does not directly correlate with protein expression and therefore protein activity. mRNA is hardy to tissue handling and tends to correlate better with the level of protein. Assays of DNA and mRNA appear to be quantitative over a large dynamic range. On the other hand, proteins are the effectors of cell function. Levels of mRNA do not necessarily correlate strongly with protein concentration, nor with the vast array of post-translational modifications that proteins undergo during regulation of cellular processes. Thus, most biomarkers are still protein markers. Unfortunately, proteins are much less resistant to degradation during tissue handling and to proteases that remain active until the tissue is in deep freeze. Significant but variable loss of phosphorylation occurs within minutes of loss of blood supply. Another limitation in protein biomarkers is that most assays are linear over a narrow dynamic range and become nonlinear in clinically important concentrations.

Tumor tissue is most often queried with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Traditional markers by IHC include ER, PR, HER2, Ki67 or MIB-1 (markers of proliferation), and p63 (to determine invasiveness if uncertain) (Fig. 21-1). HER2 is the only marker currently ascertained routinely by FISH, although topoisomerase II amplification in HER2-positive breast cancer may become useful if validated (Fig. 21-2). IHC and FISH have the advantages of being cheap, reproducible in multiple laboratories, and able to demonstrate tumor heterogeneity. They also have the most long-term clinical usage and are well validated. IHC typically measures proteins (presumably the business end of gene expression), and FISH measures gene copy number (a more remote endpoint relative to protein function). Disadvantages of these techniques are that quality assurance among laboratories is poor or fair, they can interrogate only a few molecular pathways, and the intensity of IHC at least is linear only over a small range. Newer techniques use RT-PCR (reverse transcriptase polymerase chain reaction) or cDNA arrays to measure mRNA levels of various genes.

Figure 21-1 Immunohistochemistry for estrogen receptor.

(From www.nibib.nih.gov/nibib/Image/Eadvances/scannerERbiopsy2.jpg.)

Figure 21-2 Fluorescence in situ hybridization for HER2/neu overamplification.

(From Digital Atlas of Breast Pathology, Meenakshi Singh, MD, www.hsc.stonybrook.edu/gyn-atlas/breast-atlas/atlasrcp.htm)

By giving more sophisticated individualized information, tumor profiling can supplement the conventional prognostic methods in use (e.g., Adjuvant Online, AJCC staging, and Nottingham Prognostic Index) and provide potential predictive information to minimize overtreatment and reduce undertreatment in the adjuvant setting.

The Intrinsic Subtype Assay

As discussed in Chapter 19, Perou and colleagues^1–3 first described molecular profiles of subtypes of breast cancer, generally concordant with clinical thinking. The intrinsic subtype assay requires fresh frozen tissue, although newer assays depending on RT-PCR or antibody panels in fixed tissues are being developed (Table 21-1). Luminal A assays are ER-positive, generally low grade with low proliferative thrust, and HER2-negative. Luminal B assays are ERpositive, high grade with high proliferative thrust, and occasionally HER2-positive. Frequently, they may be PR-low. HER2-positive breast cancers are driven by the characteristic HER2 gene amplification and are biologically aggressive with high grade and proliferation indices. These may be ER-negative or -positive. Basaloid or triple-negative breast cancers are characterized by the lack of HER2, ER, and PR, but are heterogeneous in other respects. They have high grade and proliferation. Sub-subtypes can be identified, although to date, none of these patterns is validated nor carries clear therapeutic implications. This represents a fertile area for target and drug discovery. It is possible that a gene signature associated with BRCA1 dysfunction may become an important subtype given the provocative data generated in the PARP inhibitor trials.

Table 21-1 Breast Cancer Intrinsic Subtypes Assay

The OncotypeDx Recurrence Score

The OncotypeDx assay (Genomic Health, Inc, Redwood City, California) is a multiplex RT-PCR measurement of mRNA levels for 21 genes: 16 breast cancer–related and five housekeeping genes to normalize the data (Fig. 21-3). Fixed tissue is used. The expression levels of these 16 cancer genes were placed into an algorithm correlated with distant disease-free survival at 10 years in 447 patients. This assay was subsequently validated in ER-positive node-negative breast cancer tissues obtained from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 and B-20 trials.^4,5 The recurrence score (RS) as a continuous variable correlates with the distant disease-free survival (DDFS) at 10 years, assuming that the patient has taken tamoxifen for 5 years (Fig. 21-4). More recent work suggests that the RS also will correlate with outcome in node-positive breast cancers.^6,7 As highlighted in Chapter 19, this assay is being prospectively validated in the TAILORx trial (Trial Assigning IndividuaLized Options for Treatment) and is used extensively in clinical decision making.⁸

Figure 21-3 Gene panel used in OncotypeDX.

(From Paik S, Shak S, Tang G, et al: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351:2817–2826, 2004, Fig. 1.)

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