The Potential Use of Urinary CtDNA Profiling in the Treatment of Breast Cancer
Abstract
Cell-free circulating tumor DNA (ctDNA), shed into the blood stream by apoptotic or necrotic tumor cells of either primary or metastatic sites, became an extensively investigated and very promising analyte in oncology research. If passing through the kidney barrier, ctDNA is likely to occur in urine. Literature research revealed a lack of studies aiming at diagnostic use of urinary ctDNA. Most studies investigating urinary ctDNA were performed in the field of urological cancers emphasizing, however, that urinary liquid biopsies were suitable to draw conclusive real time pictures of ctDNA alterations coming from circulation and hence strengthen the hypothesis that genetic profiling of urinary ctDNA could be valuable to gain tumor-related information also in other solid tumors such as breast cancer. Usually, clinical treatment decisions are based on mutation profiles that were received from initial tissue biopsies. Though, during therapy the genetic tumor profile might change e.g. gain and loss of genetic alterations that might be relevant for targeted therapy options or treatment resistance. Particularly patients with advanced breast cancer may acquire mutations during treatment cycles and might benefit from serial ctDNA sequencing to find new targetable mutations and gain access to tailored therapy. Here, the use of urinary ctDNA might offer an opportunity for non-invasive longitudinal genotyping and testing for actionable mutations. In contrast to plasma-derived ctDNA, only a few studies were performed using urinary ctDNA from patients with breast cancer and revealed that targeted NGS appeared to be a sensitive method to detect tumor-specific genetic features. In this mini review we sought to illuminate the potential use of urinary ctDNA for longitudinal disease monitoring at frequent intervals and low effort for patients with breast cancer.
Keywords: Breast cancer; Mutation profiling; Urinary ctDNA; NGS; Targeted therapy; Disease monitoring; Recurrence; Resistance
Abbreviations: cfDNA: cell free circulating DNA; ctDNA: cell free circulating tumor DNA; CNV: Copy Number Variation; SNV: Single Nucleotide Variant; ER: Estrogen Receptor; PR: Progesterone Receptor; HER2: Human Epidermal growth factor Receptor-2; ddPCR: digital droplet PCR; NGS: Next Generation Sequencing; PIK3CA: Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha; TP53: Tumor protein p53; CDH1: Cadherin-1; MLL3: Myeloid/lymphoid or mixed-lineage leukemia protein 3; ESR1: Estrogen Receptor 1; BRCA 1/2: Breast Cancer ½; DTC: Disseminated Tumor Cell
Case Report
Cell-free circulating DNA (cfDNA) became an extensively investigated and very promising analyte in oncology research. Particularly, DNA fragments originating from the tumor cells, socalled circulating tumor DNA (ctDNA), appear to be surrogates of the primary tumor or metastatic sites thereof. Usually, clinical treatment decisions are based on mutation profiles that were received from initial tissue biopsies. Therapy, however, might alter the genetic tumor profile and cause the occurrence of genetic changes that could be relevant for targeted therapy options or treatment resistance. In breast cancer, genetic profiling of ctDNA from blood plasma was shown to have good potential for clinical use and might help to find individual treatment options and to monitor metastatic relapse. Cancer recurrence is of high relevance for both, patients diagnosed with early breast cancer and pre treated women with advanced breast cancer. Due to limited therapy options, particularly, women diagnosed with the aggressive triple negative breast cancer subtype might benefit from mutation profiling using non-invasive tools allowing disease monitoring at frequent intervals. Here, the utility of urinary ctDNA might offer promising opportunities to obtain non-invasive liquid biopsies and make access to tailored individual treatment choices possible.
About Cell Free Circulating Tumor DNA (ctDNA)
Cell free circulating tumor (ctDNA) is shed into the blood stream by apoptotic or necrotic tumor cells. Characteristic features of apoptosis are DNA fragmentation and formation of apoptotic bodies. Consequently, the release of short nuclear DNA fragments of about 167 bp was shown to have a lower molecular weight compared to DNA fragments released by necrotic cells, thus, making it possible to determine tumor-derived DNA based on size distribution [1,2]. More precise approaches to identify ctDNA originating from tumor cells are based on the molecular alterations of tumor-specific genetic features like DNA mutations, methylation and copy number variations (CNVs) [3]. If circulating cell free DNA in the blood is passing through the kidney barrier it is likely to be found in urine and also called trans renal DNA or ucfDNA [4- 6]. Urinary cfDNA might also originate from apoptotic or necrotic cells coming in direct contact with urine such as cells from the genitourinary tract. Since glomerular filtration, which takes place in the pores of the glomerular barrier, works like separation by size, only small DNA fragments with a size of about 100 bp are able to pass and might appear in urine. Molecular analysis of urinary ctDNA could be useful to gain tumor-related information not only in patients with urological cancers but also with other solid tumors such as breast cancer [4,7]. The concentration of ctDNA extracted from body fluids like blood plasma or urine might vary hugely depending on numerous patient-individual parameters such as disease stage, treatment response and further physiological and patho-logical conditions as well as the body fluid itself. Circulating ctDNA in blood can be detected in serum and plasma, with the latter one containing up to 20-fold higher concentrations [8]. Hence, a variety of ctDNA extraction and detection methods were developed [9]. At the time being there is no standard protocol for isolation and detection of urinary cell free DNA, but plenty of techniques for isolation of low-molecular weight DNA fragments are available and appear to be sensitive and reproducible [10,11]. Literature research revealed a lack of studies aiming at diagnostic use of ucfDNA. Most publications described preliminary results based on small patient cohorts. Novel molecular technologies such as targeted next generation sequencing (NGS) or digital droplet PCR (ddPCR) are offering promising opportunities for the translation of ucfDNA based tumor profiling into the clinic though [2]. Most studies investigating urinary cfDNA were performed in the field of urological cancers including renal, bladder and prostate cancer emphasizing that urinary liquid biopsies were suitable to draw conclusive real time pictures of DNA alterations coming from circulation. At this point it appears noteworthy that a close similarity was described between bladder cancer and breast cancer [12] . Based on genomic expression and mutation analyses it was shown that bladder cancer was, similar to breast cancer, distinguishable into luminal and basal tumors and that those molecular subtypes appeared to be of prognostic relevance [13].
Molecular Features of Breast Cancer Tissue
Breast cancer is a very heterogeneous disease and can be classified into distinct molecular subtypes. Luminal A type tumors are hormone-receptor positive (estrogen-receptor (ER) and/or progesterone-receptor (PR) positive), human-epidermal-growthfactor- receptor-2 (HER2) negative and show low levels of the protein Ki-67, meaning low proliferation. Usually, they are of low are lowgrade, tend to grow slowly and patients have the best prognosis. The majority of patients is diagnosed with ER positive tumors and might receive endocrine therapy. Luminal B type tumors are also hormone-receptor positive (ER and/or PR positive), and either HER2 positive or HER2 negative, with high levels of Ki-67 meaning that they grow faster. The patient’s prognosis is slightly worse compared to luminal A. Patients suffering from HER2 (also called ERBB2) amplified tumors are prone to treatment with targeted therapy such as trastuzumab or pertuzumab. Triple negative breast cancer (TNBC) is hormone-receptor negative (neither ER nor PR) and HER2 negative with poor prognosis. The majority of TNBCs are of high grade and show an aggressive phenotype. Patients usually receive chemotherapy but have an increased risk of recurrence [14]. Molecular analysis of the breast cancer subtypes revealed that luminal A tumors, although being associated with good prognosis and therapy response, were presenting the majority of driver mutations such as PIK3CA ~ 45 %, GATA3 ~ 14 %, MAP3K1 ~ 13 %, TP53 ~ 12 %, CDH1 ~ 9 % [15]. In contrast, triple negative tumors appeared to lack driver mutations (PIK3CA ~ 9 %, MLL3 ~ 5 %), but showed mutations of the tumor suppressor gene TP53 in 80 % of the cases.
Somatic and Targetable Mutations in Breast Cancer
As mentioned above mutation profiling of breast cancer tissue revealed distinct molecular subtypes presenting characteristic somatic mutations, including single nucleotide variants (SNVs) and copy number variations (CNVs). Next to a huge number of low frequency variants, the usual suspects among mutated genes in breast cancer are TP53 and PIK3CA. In addition, to broaden the understanding of tumor initiation, promotion and progression it is of high medical relevance to identify and investigate somatic mutations that drive the cancer phenotype in order to find possible therapies targeted against the products of these abnormal genomic alterations. Currently, the genes ESR1 and HER2 are not only characteristic features of the respective molecular subtype, in fact they serve as therapeutic targets for endocrine and antibody-based therapeutics such as tamoxifen and trastuzumab or pertuzumab. Further, ESR1 mutations were found to be associated with tamoxifen resistance [16]. Among TNBC a variety of subgroups has been identified including high cellular proliferation, increased immunological infiltrate, basal-like and mesenchymal phenotype as well as deficiency in homologous recombination which was partly associated with loss of BRCA1 or BRCA2 function [17]. Although challenging, the molecular analysis of TNBC gave rise to potential options for tailored therapy strategies such as modified chemotherapy approaches targeting the DNA damage response, angiogenesis inhibitors, immune checkpoint inhibitors, or even anti-androgens, all of which are currently being evaluated in phase I to III clinical studies [17].
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