Detection and differential diagnosis of cancer relapse using circulating tumor DNA profiling in a patient with synchronous breast and rectal cancer

ABSTRACT

Background: In Chinese women, breast and colorectal cancers are highly prevalent. In the early stage, the primary treatment for these cancers is surgical resection. However, many patients develop a metastatic recurrence. Thus, tools that help estimate the risk of recurrence are critical. Although synchronous breast and rectal cancer is uncommon, estimating recurrence risk is even more challenging in patients with two histologically distinct malignancies. Methods: Next generation sequencing (NGS) allows the comprehensive detection of simultaneous genome abnormalities. NGS-based circulating tumor DNA (ctDNA) profiling is a new molecular technique that has demonstrated great potential in the detection and differential diagnosis of cancer relapse. Results: We present a 43-year-old female patient with synchronous breast and rectal cancer that was surgically removed 2 years prior. During regular follow-up, elevated carcinoembryonic antigen (CEA) levels were detected. ctDNA profiling revealed multiple somatic mutations that were identical to those found in rectal cancer samples. Thus, we suspected relapse of rectal cancer. Positron emission tomography-computed tomography (PET-CT) and pathogenic analysis confirmed lung metastasis of rectal cancer. Conclusions: This case demonstrated the utility of ctDNA profiling in the detection and differential diagnosis of cancer relapse in a patient with synchronous breast and rectal cancer.

Introduction

In China, breast and colorectal cancers are the first and third most common cancers in women, respectively. For both early stage breast and colorectal cancers, the primary treatment is surgical excision. However, approximately 25% of post-surgery patients experience metastatic recurrence, with the greatest risk for relapse occurring during the first 5 years post-diagnosis. Currently, estimating recurrence risk in these patients originates from the work of C. E. Dukes in the 1930s. This estimation process is complicated, particularly in patients with two histologically distinct malignancies. Thus, reliable diagnostic analyses and markers are critical, especially in patients with synchronous cancers.

In early stage breast cancer, one potential tool for predicting disease relapse and detecting micrometastatic disease is profiling of post-surgical levels of circulating tumor DNA (ctDNA)¹. Analysis of ctDNA has also shown great sensitivity and specificity in monitoring disease burden following colorectal cancer surgery, and has demonstrated the potential to provide clinically relevant lead times compared to conventional detection methods².

Molecular genetic analysis is also increasingly used for late stage cancers. Despite intra-tumor mutational heterogeneity, there is high concordance between primary tumor and metastasis in colorectal and lung cancer when observing common driver mutations such as EGFR, KRAS, NRAS, and BRAF^3,4. One commonly used tool that is highly useful for evaluating genetic profiles is next generation sequencing (NGS). In the current study, we hypothesized that NGS-based ctDNA analysis of liquid biopsies could not only predict relapse of previous cancers, but also indicate the origin of metastasis for patients with two or more cancers.

Here, we present a case in which NGS-based liquid biopsy facilitated the diagnosis of a pulmonary metastasis of rectal cancer in a patient with synchronous breast and rectal cancer. This study highlights the potential of using ctDNA profiling to compare molecular gene profiles between primary tumors as a diagnostic tool in a clinical setting.

Case presentation

A 43-year-old Chinese woman was admitted to our hospital in October 2014 for a mass in her left breast. Magnetic resonance imaging (MRI) revealed that this mass was approximately 1.2 × 0.7 cm and was located in the left upper quadrant of her left breast. Ultrasound-guided puncture of the mass indicated grade II invasive ductal breast cancer. Breast conserving surgery with a sentinel lymph node biopsy was performed. Pathological analysis of the surgical samples confirmed grade II focal invasive ductal carcinoma of the breast. Immunohistochemical staining showed: ER (60%++), PR (80%++), Her-2 (-), and Ki-67 (5%). Moreover, we observed a large number of intravascular tumor emboli and micro-metastasis in the left axillary sentinel node (2/3) (Figure 1A). As the patient was at the pT1N1micM0 stage, she received chemotherapy (Pirarubicin + Docetaxel + Cycloph-osphamide). After five cycles of chemotherapy, the patient presented to our hospital again for bloody stool. A colonoscopic biopsy confirmed rectal adenocarcinoma for which a laparoscopic radical resection of rectal cancer was performed. The pathological diagnosis was moderately differentiated adenocarcinoma of the rectum. The cancer had invaded all layers of the intestinal wall, with para-intestinal lymph node metastasis (3/7) (Figure 1B). As the patient was at the pT4bN1M0 stage, four cycles of postoperative chemotherapy (Oxaliplatin + Capecitabine) and concurrent adjuvant radiotherapy were administrated. Subsequently, the patient continued with the sixth breast cancer chemotherapy and whole breast and axillary radiotherapy.

Figure 1.

Hematoxylin and eosin (H&E) staining of breast and rectal cancer samples. (a) Grade II focal invasive ductal carcinoma of the breast, and (b) moderately differentiated adenocarcinoma of the rectum. Original magnification 100 × .

Following the completion of chemo- and radiotherapy, the patient began regular follow-up. She was uneventful for two years until July 2017, when her carcinoembryonic antigen (CEA) levels were elevated from 4.79 ng/mL in December 2016 to 13.99 ng/mL in April 2017 and finally 32.33 ng/mL in July 2017 (CEA reference: 0.00–5.00 ng/mL). Because cell-free DNA (cfDNA) tests can detect residual/recurrent disease earlier than standard-of-care radiologic imaging in post-surgery lung cancer patients⁵, we performed genetic profiling of ctDNA and samples from breast cancer and rectal cancer simultaneously. In the breast and rectal cancer samples, 10 and 15 somatic mutations were identified, respectively. In ctDNA, nine of the ten somatic mutations identified were identical to those found in the rectal cancer sample, while the remaining AKT1 p.E17K mutation was similar to the mutation with the highest allele frequency in breast cancer (Table 1).

Table 1.

Tissue and liquid mutation profiling results.

Tissue	Gene	DNA change	Amino acid change	Mutation frequency (tissue)	Mutation frequency (plasma)
Rectal cancer	TP53	c.377A> G	p.Y126C	42.90%	1.30%
	TRPC5	c.212G> A	p.R71Q	33.60%	2.50%
	APC	c.1213C> T	p.R405*	29.00%	1.40%
	APC	c.2240C> G	p.S747*	28.40%	1.40%
	IMPG1	c.175C> T	p.R59*	28.40%	ND
	MYCN	c.1375G> A	p.E459K	27.10%	ND
	KRAS	c.34G> A	p.G12S	26.50%	1.60%
	APC	c.4669_4670delAT	p.I1557*fs*1	23.00%	1.10%
	LRRFIP2	c.1382G> A	p.R461H	16.20%	1.30%
	NCF4	c.941_942insG	p.F314Lfs*11	13.20%	ND
	NOTCH4	c.4223T> C	p.L1408P	2.90%	0.09%
	SMARCA4	c.1764G> T	p.K588N	1.20%	ND
	FAT2	c.733C> T	p.H245Y	1.10%	ND
	CDH23	c.4664G> A	p.R1555H	1.10%	ND
Breast cancer	AKT1	c.49G> A	p.E17K	11.80%	0.10%
	XBP1	c.315AGA[3 > 2]	p.E106[3 > 2]	6.80%	ND
	NOTCH3	c.1180G> A	p.E394K	6.00%	ND
	LRP1B	c.8880G> C	p.K2960N	4.90%	ND
	TP53	c.307_310delTACC	p.Y103Rfs*19	4.80%	ND
	IFITM3	c.160G> A	p.V54M	2.20%	ND
	PCLO	c.8458A> G	p.M2820V	1.40%	ND
	SRD5A2	c.560C> T	p.T187M	1.20%	ND
	MAP2K1	c.885G> T	p.R295S	1.10%	ND
	CDKN1A	c.47A> G	p.K16R	1.00%	ND

Abbreviations: ND, not detected.

Suspecting a rectal cancer relapse, we next performed a positron emission tomography-computed tomography (PET-CT) scan. This revealed a mass in the anterior segment of the upper lobe of the left lung measuring approximately 4.6 cm in transverse maximal dimension with a maximum standard uptake value (SUVmax) of 8.85. A round nodule was found in the dorsal segment of the left lower lobe of the left lung, with a high metabolism, an SUVmax measuring up to 10.06, and a transverse maximal dimension of 2.4 cm (Figure 2A). Biopsy of both nodules confirmed metastatic adenocarcinoma originating from the gastrointestinal tract (Figure 2B). Subsequently, chemotherapy was continued for four cycles (Oxaliplatin + Capecitabine).

Figure 2.

Positron emission tomography-computed tomography (PET-CT) imaging and H&E staining of lung lesions.

(a) PET-CT imaging identified two hypermetabolic lesions in the anterior segment of the upper lobe and the dorsal segment of the left lower lobe of the left lung, respectively. (b) H&E staining of the lung nodule, original magnification: (top) 100× and (bottom) 200 × .

Discussion

The molecular information provided by ctDNA analysis has numerous applications in advanced cancer patients, including companion diagnostics for actionable mutations and patient stratification, monitoring therapy response, and identification of resistance mechanisms. Currently, ctDNA analysis is being extended to applications early stage cancer patients, including detection of minimal residual disease and prediction of relapse.

Early detection or relapse prediction assume that there is a curative window of minimal disease burden, and that earlier treatment of subclinical metastatic disease can lead to meaningfully longer disease control in a relatively early stage cancer patient. Using ctDNA profiling is appealing, as to date, conventional imaging or serum protein markers have not shown any benefit of monitoring for metastatic disease after primary treatment. One explanation for this lack of benefits is that imaging and protein biomarkers lack sufficient sensitivity and/or specificity for relapse monitoring. Alternatively, ctDNA analysis is reported to have considerably superior sensitivity and specificity in detecting minimally invasive disease compared to conventional radiographic or laboratory analyses^1,6.

In the current study, the patient was quickly diagnosed with metastatic rectal cancer in the lung after detecting a slight elevation in CEA. Considering the lag time between elevation of ctDNA and serum markers, we cannot exclude the possibility that ctDNA could have detected recurrence before the elevation of CEA. Currently, there are many clinical trials recruiting postoperative cancer patients to evaluate the efficacy of ctDNA in early detection of recurrence, including trials for ovarian cancer (NCT03614689, NCT03302884), colon cancer (NCT03614689), and lung cancer (NCT03172156). In this case, it was not possible to evaluate the potential benefit of early treatment in ctDNA-detected recurrent disease. Importantly, the c-TRAK-TN trial (A Trial Using ctDNA Blood Tests to Detect Cancer Cells After Standard Treatment to Trigger Additional Treatment in Early Stage Triple Negative Breast Cancer Patients, NCT03145961) will assess the efficacy of a PD-1 inhibitor, pembrolizumab, in ctDNA-positive patients versus the observation arm.

Ultimately, this case demonstrates the utility of ctDNA profiling in the detection and diagnosis of cancer recurrence in a patient with synchronous cancer. Future studies should prospectively evaluate the efficacy ctDNA analysis in early recurrence detection.

Materials and methods

Patient

This study was approved by the Ethics Committee of Daping Hospital. After receiving informed consent from the patient, we ordered NGS of the patient’s tumor and cfDNA (Geneplus-Beijing, China).

Histological analyses

Tissues were fixed for histology in 10% neutral-buffered formalin for 24 h. Hematoxylin and eosin (H&E) staining was performed on 4-µm sections according to common procedures. Results were analyzed by two experienced pathologists.

NGS testing of tumor DNA and cfdna

Circulating DNA was isolated from 4–5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Tumor DNA was extracted using the DNeasy Blood Kit. Sequencing libraries were prepared for cfDNA using the KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA), and gDNA sequencing libraries were prepared with the Illumina TruSeq DNA Library Preparation Kit (Illumina, San Diego, CA). Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Roche NimbleGen, Madison, WI, USA) covering ~ 1.1 Mbp of sequence. DNA sequencing was performed with the HiSeq CN 500 Sequencing System (Illumina, San Diego, CA). After removing terminal adaptor sequences and low-quality data, the reads were mapped to the reference human genome. GATK (https://www.broadinstitute.org/gatk/, The Genome Analysis Toolkit) and MuTect were used to call small insertions and deletions (indels) and single nucleotide variants (SNVs) in the somatic DNA by filtering peripheral blood (PBL) sequencing data. Contra was used to detect copy number variants, and BreakDancer was used to detect cancer-associated structural variants.

Biography

Yan Xu performed the experiments and analyzed the data; C.H., J.F., and B.Z contributed materials; Y.X. and W.W performed surgeries; X.M., R.C., and X.X. analyzed the genetic data and contributed to paper writing; Y.X. wrote the paper and provided financial support for this project.

Funding Statement

This work was supported by the Key Laboratory of the Ministry of Education of China [2013jszl13]; National Natural Science Foundation of China (NSFC) [81472482].

Acknowledgments

We are thankful to all referring surgeons, pathologists, and specialists for their contributions to this study. This work was supported by the National Natural Science Foundation of China under Grant 81472482; and the Key Laboratory of the Ministry of Education of China under Grant No. 2013jszl13.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.