By Andi K. Cani, PhD, University of Michigan
Recent efforts to study the cellular changes driving breast cancer types have led to a better understanding of invasive lobular carcinoma (ILC) and its driver molecular defects. In the case of lobular breast cancer, loss of function of E-cadherin, a protein which normally participates in cell-cell adhesion (what keeps cells bound to each other in tissues), is considered a key molecular driver in this breast cancer subtype.
The loss of E-cadherin can be caused by harmful DNA mutations in its gene (CDH1) or when a cancer cell shuts down gene protein production through epigenetic modifications. The cause of mutations in cancer in general is still an active area of debate. The following discussion of tissue and liquid biopsies is intended to provide information on what these tests are, how these tests are currently used in ILC treatment and what holds promise for improving and creating ILC-specific treatments.
Tissue biopsy samples are commonly used to determine whether the tumor cells express estrogen and/or progesterone receptor (ER/PR) proteins, or the human epidermal growth factor receptor 2 (HER2). The presence or absence of these three proteins help to classify breast cancer of lobular or ductal morphology into subtypes. Tissue biopsies can also be used to detect genomic alterations among patients with metastatic breast cancer that may serve as targets for so-called precision therapies such as alpelisib (PIQRAY) and the very recently approved elacestrant (ORSERDU).
Cancer biomarkers detected from a tumor tissue biopsy help doctors determine its aggressiveness and guide therapy decisions. However, tissue biopsies are usually limited to a one-time procedure which can be invasive, prone to complications, and high-cost. In metastatic tumors and patients being treated or diagnosed with metastatic disease, a tissue biopsy is usually performed once early on, thus failing to reveal the states into which the tumor later evolves. Likewise, not all tumor cells in a patient, or even in a tumor mass, are identical at any time. The tumor is often heterogeneous, i.e. it is composed of various clonal cell populations* with different characteristics. One clone or another may become dominant or recede based on the pressures of therapy, the immune system, metabolic constraints, etc. A tissue biopsy, which samples only a small region, may miss the full picture of that tumor clonal makeup.
Liquid biopsies, defined as the detection of tumor-derived material in bodily fluids, most commonly blood, have been introduced as less invasive methods compared to tissue biopsies, since they can be obtained by a simple blood draw. They most commonly refer to circulating cell-free tumor DNA (ctDNA), which is DNA released by dying (or live) tumor cells that finds its way into blood.
Liquid biopsies offer the opportunity to obtain cancer molecular information that represents the entire disease, much more easily, and repeatedly, compared to tissue biopsies.
Liquid biopsies can be analyzed to detect mutations and their levels in blood, which is an indication of tumor burden, or how much cancer is in the body, since a higher tumor burden releases more DNA. Given the ease of obtaining blood, ctDNA enables tracking or monitoring of the patient mutational landscape over time.
CtDNA alone, however, is unable to provide information on the protein-based ER/PR and HER2 status with which to determine the breast cancer subtype. In addition, ctDNA is highly diluted in the much more abundant cell-free DNA from dying normal cells like white blood cells. This means that there is often only a low amount of tumor derived DNA in the blood and it provides no information on the heterogeneous and clonal makeup of the tumor. This makes conclusions drawn about mutation detection often questionable. In fact, ctDNA tests that find no mutations are not generally considered accurate without a confirmatory tissue test.
Circulating Tumor Cells (CTCs) In contrast, another type of liquid biopsy, CTCs, are whole, intact cells released from the tumor and found in blood, likely on the way to spread to other parts of the body. Like ctDNA levels, high CTC counts portend a worse prognosis. At the same time, CTC analysis provides a lot of tumor molecular information, but without the invasiveness and cost of a tissue biopsy.
Single-cell CTC analysis, pioneered by our group and others, allows not only prognostication, detection of relevant protein markers, and detection of targetable mutations, but also it can shed light on tumor heterogeneity. This refers to the fact that no two tumor cells are identical. As we are finding in the lab, while the vast majority of CTC in a lobular patient will have the CDH1 mutation/deletion, only some will harbor mutations in the estrogen receptor for example, since that mutation usually arises later and becomes only one “branch” of the tumor cell family “tree.” Tissue and ctDNA analysis as a bulk sample instead will provide a composite picture of the mutational landscape. As in ctDNA, serial CTC sampling allows tracking of tumor evolution, and also allows detection of the interplay between various subclones that arise and fall over time. CTCs are quite rare in circulation, with only half of metastatic breast cancer patients having more than five such cells in 7.5 mL (1/2 tablespoon) of blood. Lobular patients, however, have considerably increased rates of CTC released into circulation compared to ductal cancer as we and others have shown. This is perhaps a reflection of their mutant CDH1, i.e. defective cell-cell adhesion. CTC abundance makes lobular breast cancer particularly amenable to CTC liquid biopsy, and this remains an area of active investigation.
In the Future
Liquid biopsies have the potential to drive better predictions of therapeutic response, tumor evolution monitoring, and to enable early detection of resistance to systemic therapies. Interestingly, lobular breast cancer tends to have a higher tumor mutation burden, or the number of DNA mutations in cancer cells, compared to ductal cancer. Since misshapen proteins made from mutated genes can be recognized as foreign by the immune system, having many mutations may be associated with better response to immunotherapy. Our group has pioneered detection of tumor mutation burden and microsatellite instability (another marker of immunotherapy response) in CTC at the single-cell level.
Currently, ctDNA is used for the identification of PIK3CA mutations and more recently ESR1 mutations for clinical decision making. The use of liquid biopsy in the form of ctDNA, CTCs, and other types will have to be rigorously tested in order to show additional utility in treatment decision making and improving patient care. Together with tissue, these tests are likely to provide a complete molecular picture of the evolving tumor over time and enable real-time treatment adjustment for patients with metastatic breast cancer. (It should be noted that there is not yet data to support using liquid biopsy tests to detect a recurrence of ILC). This supports the continued investigation of CTC and ctDNA for patients diagnosed with metastatic breast cancer and monitoring to extend survival and eventually eradicate deaths from lobular breast cancer.
Andi Cani, PhD, is a research investigator in the breast cancer group of Dr. Daniel F. Hayes at the University of Michigan Rogel Cancer Center.
* Cells divide by mitosis and generally produce two genetically identical, or clonal daughter cells, although occasional DNA mutations may happen. Cells in a person’s body are usually clonal (as they arose from a single cell). In cancer, an initial normal cell has transformed by gaining one or more DNA errors/mutations/alterations. This makes it grow, divide, and proliferate at an increased rate. As this cell gives rise to other cells by copying DNA and passing on those initial mutations in successive generations, one cell, in addition to the original (truncal) mutations, will gain a new mutation. That cell and its progeny, not the rest of the cells, will carry that additional mutation together with the initial ones (forming a branch). The different clones or branches may be in cooperation or competition as the tumor evolves. The presence of many such branches makes curing cancer difficult. Treatments we use may be effective on one or more of such branches, but one or other branches may be resistant. They grow out and become dominant, leading to regrowth of cancer. That said, identification of variants within a population of cancer cells, particularly over time as the cells evolve, can inform the treatment of the disease.
