For decades, tissue-conserving surgery coupled with chemotherapy (either preceding or following surgery, depending on the size of the tumor(s)) followed by radiation has been the standard of care for the majority of cancers. Over the past fifteen years, chemoradiotherapy (concurrently dosing a patient with chemotherapy and radiation) has improved the overall survival rates especially for head, neck and colorectal cancer patients as some chemotherapy agents sensitize the body to radiation which leads to an enhanced systemic effect (1). More recently, next generation sequencing (NGS) has emerged as a leading approach to clinical care given the growing number of identified tumor biomarkers for oncologists to test and hence, to help make better informed treatment decisions.
Cancer Biomarker Discoveries
“Tumor markers” is a broad term and generally refers to a protein; gene mutation, duplication, deletion or rearrangement; or other molecules which are known to play a role in cancer cell growth, multiplication, death and response to the body’s own immunity system. Also known as “biomarkers”, changes in the average or normalized expression levels of these molecules can be used as a metric to detect the presence of cancer by analyzing blood, urine or body tissues. In contrast, predictive genetic testing, now widely popular and readily available to consumers as DIY-mouth swabbing kits and offered by a variety of vendors, analyzes inherited gene mutations which can be scored as a prognostic indicator for the likelihood (i.e. lifetime risk) that a person will develop the disease being tested for. Note: the American Cancer Society has prepared a good overview of genetic testing for cancer (2).
Perhaps the best known cancer biomarkers, infamous or famous depending on your perspective, are BRCA1 and BRCA2 which are associated with hereditary breast and ovarian cancers. These two genes are also known to increase the risk of other cancers such as melanoma as well as both prostate and breast cancer in men. Following diagnosis of breast cancer, blood is routinely drawn from patients to test for the following three serum tumor antigens:
- CA 27.29 with levels >40 U/ml;
- CA 15-3 with levels >30 U/ml;
- CEA (carcinoembryonic antigen) with levels >3 ng/ml
Similarly, there are numerous other serum tumor markers, such as CA 19.9, which is the primary tumor marker used to detect pancreatic cancers if levels are >37 U/ml. And CA 125, which may be indicative of pancreatic, ovarian, endometrial, fallopian or peritoneal cancer if levels are >35 U/ml. Note: the Mayo Clinic has prepared a good overview of tumor marker testing, results and clinical significance (3).
Women, and to a lesser degree men also, with mutations in the gene, PALB2 (aka FANCN), are at an increased risk for breast and pancreatic cancers due to an impaired ability to repair damaged DNA and halt tumor growth as a result of suppressed bone marrow function (and hence, a weakened immune system)(4). Mutations in the genes such as CHEK2 and ATM, which code for serine-threonine kinases (these act as tumor suppressors halting cell growth) result in a significantly higher risk of pancreatic cancer (5).
The Variant Problem
After countless billions of dollars and hours have been poured into cancer research, we have collectively advanced treatment but only recently come to the agreement that cancer is a spectrum disease, where we have learned that individual tumors do not even share the same genetics from one side to the other. Pleiotropy and elucidating the function of oncology genes – in combination – with respect to their collective impact on the progression of cancer is already enormously complex, and made even more challenging by gene variants, particularly those low frequency ones (6). Next Generation Sequencing (NGS) has changed how we study complex diseases and test the impact of genetic mutations at both the somatic and germline level. To this end, digital DNA sequencing is one of the best tools currently available to address the low frequency variants problem.
Specifically, targeted DNA panels have been designed to utilize molecular barcodes as a means of tagging variants of interest. This approach can be used to distinguish a unique, low frequency variant from a library construction error such as PCR duplication or false positives and these panels can help overcome gene library bias. Digital DNA panels work effectively even with low amounts (>10 ng DNA) and low-quality DNA typically provided as FFPE and circulating free DNA (cfDNA) samples and can be systematically cycled through various buffers and conditions to achieve high coverage across all sequences including GC-rich regions. Some clinically relevant panels, such as those offered by QIAGEN® can accommodate up to nearly 400 samples per sequencing run and enable testing against a multiple cancers including breast, colorectal, lung and solid tumors.
Oncologists are increasingly relying on the genetics (plus cytology) of tumors to inform treatment decisions and can screen for multiple mutations (i.e. biomarkers) in parallel using a single DNA panel and patient sample. NGS has made massive scale sequencing initiatives accessible to both large and small labs, as well as hospitals and clinics to accelerate on-site sequencing efforts and improve patient care. Through NGS, our collective understanding of disease is expanding and bringing us closer to understanding disease at the level of n=1 patient, 1 disease, which is the currently elusive “dangling carrot” targeted in the pursuit of precision medicine.
The Role of Sequencing in Precision Medicine
Via NGS, researchers can characterize polymorphic markers such as single-nucleotide polymorphisms (SNPs) at a genome-wide level to identify – with great precision – which genomic loci are contributing to the cancer phenotype (7). Teetering on the edge of moving from the bench to the hospital bedside is the liquid biopsy based on the concept that circulating nuclear and mitochondrial DNA, as well as micro-RNA, originating from but not encapsulated by cancer cells could serve as biomarkers for tumors (8)(9). Telomerase mRNA, harvested from both peripheral blood and circulating tumor cells, was recently sequenced and used to diagnose and inform specific immuno-therapy treatment options for nasopharyngeal carcinoma patients (10).
Perhaps the best illustration of sequencing oncology genes to inform treatment hails from the recent TailorX study (11). Over 10,000 women were tested for 21 genes known to be associated with breast cancer. The study definitively demonstrated that women with risk scores in the mid-range did not benefit from chemotherapy more than women with risk scores in the low-range. As a result, only women with high-range scores will be treated with chemotherapy which can help impede cancer growth but can also impose life-altering negative effects on the patient’s physical, emotional and social well-being.
Indeed, the future looks brighter than ever before with NGS providing a beacon of hope and understanding to aid oncologists and patients with optimized, personalized treatment plans.
- Vale, Claire et al.. “Reducing uncertainties about the effects of chemoradiotherapy for cervical cancer: individual patient data meta-analysis”. John Wiley & Sons. Cochrane Database Syst Rev. 2010 Jan 20;(1): CD008285.
- “Understanding Genetic Testing for Cancer”. Published by the American Cancer Society. (2018). Retrieved from: https://www.cancer.org/cancer/cancer-causes/genetics/understanding-genetic-testing-for-cancer.html
- “CA-125 Test”. Published by The Mayo Clinic. (2018). Retrieved from: https://www.mayoclinic.org/tests-procedures/ca-125-test/about/pac-20393295
- Antonis, C. et al.. “Breast Cancer Risk in Families with Mutations in PALB2”. N Engl J Med 2014; 371: 497-506.
- Qu, M. et al.. “Current early diagnostic biomarkers of prostate cancer”. Asian J Androl 2014 Jul-Aug; 16(4): 549–554.
- Bush, W.S. et al.. “Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network”. Clin Pharmacol Ther 2016 Aug; 100(2): 160–169.
- Mardis, E.R. and Wilson, R.K.. “Cancer genome sequencing: a review”. Hum Mol Genet 2009 Oct 15; 18(R2): R163-8.
- Kohler, C. et al.. “Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors”. Mol Cancer 2009 Nov 17; 8:105.
- Zanutto, S. et al.. “Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer”. Br J Cancer 2014 Feb 18; 110(4): 1001–1007.
- Fu, X. et al.. “Joint quantitative measurement of hTERT mRNA in both peripheral blood and circulating tumor cells of patients with nasopharyngeal carcinoma and its clinical significance”. BMC Cancer 2017; 17: 479.
- Sparano, J.A. et al.. “Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer”. N Engl J Med 2018; 379: 111-121.