Rapid Changes in Circulating Tumor DNA in Serially Sampled Plasma During Treatment of Breast Cancer: A Case Report

Background

Breast cancer is the most frequent malignancy in females, with 1.7 million new cases developing annually worldwide [1] and 60 000 new cases in Japan [2]. Although most metastatic breast cancer is incurable, progress in developing anti-cancer drugs has improved the survival of breast cancer patients [3]. Recently, the utility of liquid biopsy, such as circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), has emerged [4]. These modalities are important in the progress of personalized therapies.

The analysis of ctDNA is expected to be a non-invasive method to determine the real-time status of patients with cancer, such as tumor burden, existence or potency of cancer metastases, and response to anti-cancer therapies [5]. However, a variety of mutated genes and their dynamic changes observed in cell-free DNA make this difficult.

We found a dynamic change in ctDNA (TP53, p.Arg158fs with 7 nucleotides deletion) as a “marker” in plasma of a patient treated with bevacizumab and paclitaxel. This mutation was easily confirmed as a tumor-derived mutated gene and we detected very small amount until 2–5×10−5 of allelic fraction (AF). This case indicated the utility of and issues regarding the measurement of ctDNA in cancer patients.

Case Report

GENOME ANALYSIS:

The patient provided written informed consent for the genetic research studies, which were performed in accordance with protocols approved by the Institutional Review Board at our hospital [6]. The tumor sample was obtained from formalin-fixed paraffin-embedded tissue by laser-capture microdis-section from the axillary lymph node removed after primary therapy. The primary breast tumor was not suitable for subsequent genomic analysis because of severe DNA fragmentation and scant DNA. Peripheral blood samples were collected at pretreatment for metastases and at 30, 60, 120, 150, 180, and 210 days after the first administration of the treatment serially. Tumor and buffy coat samples were subjected to next-generation sequencing analysis using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA), which targets the hotspot regions of 50 oncogenes and tumor-suppressor genes (Table 1). A matched buffy coat sample was used as a control to detect somatic mutations.

As a result, TP53 frameshift mutation (TP53 p.Arg158fs) was identified in the tumor, and its variant allelic fraction was 79% (coverage: 1522/1931) (Figure 3). Then, we serially analyzed the 7 obtained plasma samples (at 0 as pretreatment and 30, 60, 120, 150, 180, 210 days) during the treatment by targeted amplicon sequencing using primers flanking the TP53 p.Arg158fs mutations site. We obtained high-depth sequencing data (average coverage depth: 7322820, (range: 94432–1201464) and detected TP53 p.Arg158fs mutation at the bottom of 2–5×10−5 of AF (Table 2). We performed Sanger sequencing and validated the TP53 p.Arg158fs mutations in the tumor and plasma taken at 120 days after treatment (Figure 4). The other plasma samples (pretreatment and days 30 and 60) with AF values less than 1.4% were not detected by Sanger sequencing due to the detection limit [7].

The change in CEA values and AF of TP53 mutation during the treatment with bevacizumab and paclitaxel is shown in Figure 5. Approximately 60 days after the start of chemotherapy, AF of TP53 mutation dropped from 1.4% to 0% (despite CEA lingering around 15.7 ng/ml), but CEA gradually fell afterwards to a low of 4.0–4.3 ng/ml. But at the low of CEA (4.3 ng/ml), AF jumped to 21% (255,656 reads of 1201,464 coverage), detectable even by Sanger method (Figure 4). CEA slowly started to climb and reached 14.1 ng/ml at 90 days from the AF 21% time. Clinical worsening was now recognized after 3-month delay. However, the TP53 mutation was decreased to 4–5×10−4 of AF at 150, 180, and 210 days during the treatment.

Discussion

Since the existence of fragment of DNA in plasma was initially reported by Mandel et al. in 1948 [8], the utility of circulating cell-free DNA (cfDNA) has been studied in various areas of medicine. Progress in next-generation sequencing made it possible to utilize cfDNA in clinical practice, such as in prenatal assessment in high-risk infants [9], renal failure [10], myocardial infarction [11], and trauma [12]. In oncology, detection of cfDNA derived from tumors has been an exciting target to pursue as a tool to assess cancer patients in real time, such as tumor burden, existence or potency of cancer metastases, and response to anti-cancer therapies [13–16].

The definition of ctDNA is presented as a portion of cell-free DNA (cfDNA) derived from cancer cells [4]. It is thought to originate from lytic, apoptotic, or necrotic tumor cells, by active secretion from macrophages that have phagocytized necrotic cells, or from circulating tumor cells themselves.

There are several issues in measuring ctDNA in cancer management: how to discriminate ctDNA from normal cfDNA, what level of concentration to measure, and when to take samples to see the change in ctDNA. The presented case may indicate some suggestions for these issues.

Most tests for the detection of tumor-derived cfDNA target characteristic genetic or epigenetic modifications, such as mutations in tumor-suppressor genes, activated oncogenes, hypermethylation, or chromosomal disorders [7]. The 7-nucleotide deletion of TP53 mutation detected in axillary metastases could be an adequate target to analyze the ctDNA in this case. Instead of using a single-nucleotide change, which often suffers from sequencing errors, we could measure a very small amount of ctDNA by targeting a related large deletion (7 nucleotides of TP53) [17].

Regarding the second issue – the level of concentration to be measured – the concentration of ctDNA in cfDNA is usually considered very small if it is less than 0.1%. However, there is still no standard method to detect and measure the ctDNA. There are several methods used to analyze ctDNA, such as Sanger sequencing, digital PCR, targeted amplicon sequencing and multi-gene parallel sequencing. The sensitivities of these methods vary: more than 10% of ctDNA in total DNA can be detected in Sanger sequence, while less than 0.01% can be detected in digital PCR or target amplicon sequencing [16,18].

The value of AF decreases according to the effect of treatment before the change in CEA. Plasma sample taken at 30 days after the chemotherapy already showed almost zero value (0.005% of AF), indicating ctDNA had very rapid turnover. This amount of ctDNA is usually not detected and might be defined as a major molecular response (MMR). It was reported that ctDNA has a comparatively short half-life time (approximately 2 h), allowing us to observe the change in hours rather than weeks or months [18]. Therefore, the plasma sample taken after several days provided important information on the therapeutic effect.

Then, we observed that the ctDNA level suddenly increased 2 months earlier than the CEA. This surge indicates that a large amount of ctDNA was released at that time due to some dynamic change in the metastatic lesion. Subsequently, the value was stable at 2–4×10−4, regardless of the progression of disease and elevation of CEA value. Compared with the protein markers CEA or CA15-3, ctDNA can more sensitively detect rapid changes in tumor burden [19].

Why was the high value of AF not consistent during the progression of cancer metastases? The metastatic site (meningeal metastases) progressed at terminal stage might have tumor heterogeneity bearing another mutational profile without 7-nucleotide deletion of TP53. Otherwise, the total cfDNA changed in a substantial proportion might affect the decreased value of AF for ctDNA. It was difficult to determine the mechanism of dynamic change in ctDNA during the progression of cancer metastases in this case. Quantitative analysis might be needed determine the changes in ctDNA. Further investigations are needed to reveal the significance of these high-sensitivity changes.

Conclusions

Although this is a single case experience, it strongly suggests that “liquid biopsy” or ctDNA in plasma could be a modality to determine cancer status in real time. However, several factors affect the value of ctDNA, and further investigations are needed to reveal the significance of these high-sensitivity changes.