- 2 Million New Cases of Breast Cancer in 2018—Earlier Detection Needed
- MicroRNA or Cell-Free DNA Biomarkers for Breast Cancer Are Emerging
- These Biomarkers, Found in Blood or Urine, Enable “Liquid Biopsies”
Every October, Breast Cancer Awareness Month (BCAM) is an annual international health campaign organized by major breast cancer charities in an effort to increase awareness of the disease, and raise funds for research into its cause, prevention, diagnosis, treatment, and cure. The campaign also offers information and support to those affected by breast cancer.
BCAM was launched in October of 1985 as a partnership between the American Cancer Society and the pharmaceutical division of Imperial Chemical Industries, which is now part of AstraZeneca, producer of several anti-breast cancer drugs. The Breast Cancer Research Foundation, established in 1993, adopted the now iconic pink ribbon as its symbol, which had been introduced two years earlier when the Susan G. Komen Foundation had handed out pink ribbons to participants in its New York City race for breast cancer survivors.
A variety of BCAM events around the world are organized in October, including walks and runs near you, which can be easily found using Google. Additionally, landmark buildings all over the world are illuminated in pink, including the White House, Buckingham Palace, and Tokyo Tower. This blog will provide introductory statistics about breast cancer, as well as information regarding conventional detection and standard treatment. Then, the focus will shift towards the importance of early detection using recently reported microRNA (miRNA) and cell-free DNA (cfDNA) tests. Readers interested in more comprehensive information about all aspects of breast cancer can consult these links to the US National Cancer Institute and Food & Drug Administration.
“Making Strides Against Breast Cancer of Tampa” on October 28, 2017, Tampa, FL.
Selected Statistics About Breast Cancer
Breast cancer core biopsy. Ductal in situ carcinoma detected by screening mammography. Tumor cells are confined to the mammary ducts. There is branching and budding with no invasion present.
Breast cancer is reported as the most commonly occurring cancer in women, and the second most common cancer overall. There were over 2 million new cases in 2018. The top 25 countries with the highest rates of breast cancer in 2018 are listed elsewhere. According to facts and figures for the US provided by the American Cancer Society, in 2017, an estimated 252,710 new cases of invasive breast cancer were diagnosed among women, and 2,470 cases were diagnosed in men. In addition, 63,410 cases of in situ carcinoma (i.e. abnormal cells that have not invaded nearby tissues, shown here) were diagnosed among women. Approximately 40,610 women and 460 men are expected to die from breast cancer in 2017.
A woman living in the US has a 12.4% or a 1-in-8 lifetime risk of being diagnosed with breast cancer. The age-specific 10-year probabilities of US women developing invasive breast cancer are listed here, whereas incidence and death rates for female breast cancer by race/ethnicity and geographical location in the US can be found elsewhere.
Current age 10-Year probability
Conventional Detection and Treatment
Based on the results of eight randomized trials, namely the NIH-ACS Breast Cancer Detection Demonstration Projects published in 1988, and other research studies that collectively showed that mammographic screening (pictured below) can reduce the mortality from breast cancer, routine mammographic screening is an accepted standard for the early detection. However, as discussed in the following section, there is considerable recent interest in improving early detection through the utlization of nucleic acid-based testing.
Ribbon diagram of the Fab fragment of trastuzumab bound to the extracellular domain of HER2. Taken from commons.wikipedia.org and free to use.
According to an NIH summary, breast-conserving surgery (lumpectomy) followed by local radiation therapy has replaced mastectomy as the preferred surgical approach for treating early-stage breast cancer. Combination chemotherapy is a standard of care in the adjuvant treatment of operable breast cancer. The goal of this systemic therapy is to eradicate cancer cells that may have spread beyond the breast.
The monoclonal antibody trastuzumab is an accepted treatment for breast cancers that overproduce a protein called human epidermal growth factor receptor 2, or HER2. This protein is produced in abnormally high amounts by about 20% of breast tumors, which tend to be more aggressive and more likely to recur. Trastuzumab targets the HER2 protein specifically, as depicted here, and this antibody, in conjunction with adjuvant chemotherapy, can lower the risk of recurrence of HER2-overproducing breast cancers by about 50%, when compared to chemotherapy alone.
Several breast cancer susceptibility genes have now been identified, including BRCA1, BRCA2, TP53, and PTEN/MMAC1. Approximately 60% of women with an inherited mutation in BRCA1 or BRCA2 will develop breast cancer sometime during their lives, compared to about 12% of women in the general population. Women with inherited BRCA1 or BRCA2 gene mutations also have an increased risk of developing ovarian cancer. These genetic links to breast cancer scientifically segue to the next sections on early detection using miRNA and cfDNA tests.
miRNA Biomarkers for Breast Cancer
Overview of miRNA processing, from transcription to the formation of the effector complex. Taken from commons.wikimedia.org and free to use.
An miRNA is a small non-coding RNA molecule (containing about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression by following their processing, as depicted here. miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced by one or more of the following processes: (1) cleavage of the mRNA strand into two fragments, (2) destabilization of the mRNA through shortening of its poly(A) tail, or (3) less efficient translation of the mRNA into proteins by ribosomes.
As described elsewhere, miRNAs resemble the small-interfering RNAs (siRNAs) of the RNA interference (RNAi) pathway. However, miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA. An analysis published in 2015 indicates that the number of human miRNAs is approximately 600. According to a review by Bertoli et al., dysregulation of miRNAs is involved in the initiation and progression of several human cancers, including breast cancer, as strong evidence has been found that miRNAs can act as oncogenes or tumor suppressor genes. The researchers add that some miRNAs have emerged as biomarkers of breast cancer diagnosis, or as indicators for prediction of therapeutic outcomes. Other miRNAs are of interest as new, easily accessible, affordable, non-invasive biomarkers for the personalized management of patients with breast cancer because they are circulating in body fluids.
Since 2015, there are 16,100 articles in Google Scholar that contain both the words “microRNA” and “biomarkers” alongside the phrase “breast cancer” anywhere in the article, which includes patents but excludes citations. Google Scholar can list these articles by date or relevance. The latter is possible thanks to algorithmic methodology described elsewhere, and provides a very useful way to find relatively high-impact items, which are listed in the first few pages.
Below are the titles for the first ten articles found in this search, together with an abbreviated citation with date and the “cited by” number in parentheses.
- MicroRNAs: new biomarkers for diagnosis, prognosis, therapy prediction and therapeutic tools for breast cancer G Bertoli et al. Theranostics 2015 (cited by 431).
- miRpower: a web-tool to validate survival-associated miRNAs utilizing expression data from 2178 breast cancer patients A Lánczky et al. Breast Cancer Res. 2016 (cited by 226).
- Plasma exosome microRNAs are indicative of breast cancer Hannafon et al. Breast Cancer Res. 2016 (cited by 130).
Formation of exosomes, which are extracellular vesicles. Taken from commons.wikimedia.org and free to use.
- Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers Haman et al. Cell Death & Disease 2017 (cited by 68).
- Dysregulation of microRNAs in breast cancer and their potential role as prognostic and predictive biomarkers in patient management Schooneveld et al. Breast Cancer Res. 2015 (cited by 159).
- MicroRNAs as biomarkers for early breast cancer diagnosis, prognosis and therapy prediction Nassar et al. Pharmacology & Therapeutics 2017 (cited by 61).
- Exosome‐encapsulated microRNAs as circulating biomarkers for breast cancer Joyce et al. International Journal of Cancer 2016 (cited by 51).
- Circulating cell-free miRNAs as biomarker for triple-negative breast cancer Shin et al. British Journal of Cancer 2015 (cited by 73).
- A novel label-free electrochemical miRNA biosensor using methylene blue as redox indicator: application to breast cancer biomarker miRNA-21 Rafiee-Pour et al. Biosensors and Bioelectronics 2016 (cited by 65).
- Feasibility of urinary microRNA detection in breast cancer patients and its potential as an innovative non-invasive biomarker Erbes et al. BioMed Central 2015 (cited by 71).
I think it’s is worth emphasizing that several of the aforementioned highly relevant articles feature either exosome-encapsulated miRNAs, or cell-free miRNAs. These are physically distinct types of miRNA that can be studied independently, as reported by Endzeliņš et al. in 2017 in a publication titled Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients. In this particular study, it was found that only a small fraction of the total cell-free miRNA was recovered from plasma extracellular vesicles (EVs). However, the EV-incorporated and whole plasma cell-free miRNA profiles were clearly different.
Release of cell-free circulating tumor DNA originating from several different types of tumors. Taken from commons.wikimedia.org and free to use.
cfDNA Biomarkers for Breast Cancer
According to a 2019 review by Yu et al., cells can release cfDNA that circulates in the bloodstream, as depicted here. More specifically, the cfDNA released from tumor cells is sometimes referred to as circulating tumor DNA (ctDNA). Although cfDNA is also present in healthy individuals, it is significantly increased in cancer subjects. cfDNA is recognized as a novel biomarker in the diagnosis of several types of cancer, including breast cancer. There are several detection strategies for breast cancer by cfDNA, such as the concentration of cfDNA, cfDNA integrity, microsatellite alteration, gene mutations, DNA methylation, and more.
When the Google Search of microRNA biomarkers for breast cancer was repeated with substitution of “cell-free DNA” for “microRNA,” 14,900 articles were found since 2015. Below are the titles for the initial (i.e. most relevant) ten articles detected by this search, alongside an abbreviated citation with date and the “cited by” number in parentheses.
- Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen Jansen et al. Oncotarget 2016 (cited by 20).
- Prevalence of ESR1 mutations in cell-free DNA and outcomes in metastatic breast cancer: a secondary analysis of the BOLERO-2 clinical trial Chandarlapaty et al. J. Amer. Med. Assoc. 2016 (cited by 162).
- Diagnostic Value of Concentration of Circulating Cell-Free DNA in Breast Cancer: A Meta-Analysis Yu et al. Frontiers in Oncology 2019 (cited by 2).
- Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence Cheng et al. Oncotarget 2017 (cited by 12).
- Methylation of cell-free circulating DNA in the diagnosis of cancer Warton and Samimi Frontiers in Molecular Biosciences 2015 (cited by 116).
Taken from commons.wikimedia.org and free to use.
- Cell-free DNA measured by a simple fluorescent assay to predict relapse in triple negative breast cancer patients receiving neoadjuvant chemotherapy Park et al. Amer. Assoc. Cancer Res. 2017 (Abstract, no citations).
- Cell-free DNA detected by “liquid biopsy” as a potential prognostic biomarker in early breast cancer Maltoni et a. Oncotarget 2017 (cited by 13).
- Mutation analysis of cell-free DNA captures heterogeneity of individual circulating tumor cells in metastatic breast cancer Guttery et al. Amer. Assoc. Cancer Res. 2016 (Abstract, no citations).
- Sensitive detection of mono-and polyclonal ESR1 mutations in primary tumors, metastatic lesions, and cell-free DNA of breast cancer patients Wang et al. Clinical Cancer Res. 2016 (cited by 119).
- Circulating cell-free DNA-based epigenetic assay can detect early breast cancer Uehiro et al. Breast Cancer 2016 (cited by 33).
Two of the above articles deal with estrogen receptor 1 (ESR1) mutations, and the schematic drawing shown here indicates how these mutations have been used by Allouchery et al. In brief, at the end of adjuvant treatment by aromatase inhibitor (AI), circulating ESR1 mutations were not detected among the 42 patients in this cohort. At relapse, 2/38 patients (5.3%) had a detectable circulating ESR1 mutation. At progression after re-exposure to AI as first-line metastatic treatment, 7/21 patients (33%) had a detectable circulating ESR1 mutation.
Circulating ESR1 mutation frequency during HR+ breast cancer history. AI aromatase inhibitor, CT chemotherapy, RT radiation therapy. Taken from Allouchery et al. Breast Cancer Research Vol. 20, Article number 40 (2018). Open Access with permission.
The sheer amount of Google Scholar articles I was able to find on miRNA and cfDNA biomarkers for detection of breast cancer point towards promising future clinical practices that may enable improved methods for earlier detection. This will become especially true if safe, simple, and convenient collection methods, such as blood or urine samples, could be used instead of invasive biopsies.
As usual, your comments are welcomed.
After this blog was written, I was able to find an FDA website that provides a comprehensive list of nucleic acid-based tests that have been cleared or approved by the Center for Devices and Radiological Health. These tests analyze variations in the sequence, structure, or expression of DNA or RNA in order to diagnose disease, medical conditions, or an infection by an identifiable pathogen. The tests can also determine genetic carrier status. This list includes nucleic acid-based companion diagnostic tests. There are currently 16 tests listed for breast cancer, each with a link to detailed information, as exemplified by an in situ hybridization (ISH) product available from Ventana Medical Systems, part of the Roche Group.