Hongchao Du¹, Wenfeng Liu², Yunfei Li³, Lijuan Zhang⁴, Fangfang Jiang⁴, Dandan Zhu⁴, Jingshuai Li⁴, Pan Hu³, Ningning Yan⁴, Mao Mao^3,5*, Shiyong Li^3*

1.New Ruipeng Pet Healthcare Group Co, Ltd, Beijing, China.

2.Shanghai Companion Animal Hospital, Shanghai, China.

3.Research & Development, TwixBio, Shenzhen, China.

4.Clinical Laboratories, Shenyou Bio, Zhengzhou, China.

5.Yonsei Song-Dang Institute for Cancer Research, Yonsei University, Seoul, Korea.

Hongchao Du, Wenfeng Liu, and Yunfei Li contributed equally to this work.

*Corresponding author.

Address correspondence to: Mao Mao, MD, PhD, TwixBio. Address: 11F, Building 2, DBH Life Sciences & Health Industrial Park, 2028 Shenyan Road, Yantian, Shenzhen, Guangdong 518000, China. E-mail: maomao@yuhs.ac. Shiyong Li, TwixBio Inc. E-mail: lishiyonglee@gmail.com.

Abstract

Objective: Cancer is currently the most common cause of death in adult dogs. Like humans, dogs also have a one-third chance of developing cancer in their lifetime. We used shallow whole-genome sequencing (sWGS) to analyze blood cell-free DNA (cfDNA) from four tumor-bearing dogs (one with benign and three with malignant tumors) and 38 healthy dogs.

Results: Similar to the results observed in healthy dogs, no copy number aberration (CNA) was detected in the dog with benign lipomas, and the distribution of cfDNA fragment size (FS) closely resembled that of healthy dogs. However, in the three dogs diagnosed with malignant tumors, each dog exhibited varying degrees and quantities of CNAs. Compared to the distribution of FS in healthy dogs, cancer dogs exhibited a noticeable shift towards shorter lengths. These findings indicated that CNA and FS values derived from sWGS genomic profiling can be used for non-invasive cancer detection in dogs.

Keywords: cfDNA, CNA, dog, FS, liquid biopsy, sWGS.

Introduction

Cancer is the leading cause of death in dogs, primarily due to most cases are diagnosed at an advanced stage, resulting in a poor prognosis [1]. Growing evidence of cancer as a genomic disease has led to the emergence of liquid biopsy. Liquid biopsy is widely used for various applications, including screening, auxiliary diagnosis, targeted therapy selection, treatment response monitoring, minimal residual disease detection, and recurrence monitoring [2]. It broadly refers to the collection and analysis of various body fluid samples (mainly blood, occasionally urine, cerebrospinal fluid, or other secretions) using minimally invasive or non-invasive methods. Blood-based liquid biopsy tests involve analyzing circulating tumor cells (CTCs), proteins, and circulating tumor DNA (ctDNA) from cancer patients. Among these, ctDNA is the most commonly employed method for liquid biopsy and is highly effective at identifying cancer patients in asymptomatic populations. This approach facilitates early diagnosis and advanced intervention treatment [3,4]. It is widely recognized that CNAs are prevalent in various types of cancer [5]. ctDNA, similar to tissue samples, can be used to detect genomic alterations (including CNA) caused by tumors, which has been demonstrated in both humans and dogs and used for cancer detection [6,7]. Moreover it is well established that the FS of ctDNA in human cancer patients is typically shorter than that of cfDNA in healthy individuals, which can increase the sensitivity of cancer detection [8,9]. Here, we performed sWGS analysis on 42 dogs, one with benign masses, three with malignant masses, and 38 healthy dogs, to evaluate differences in CNA and FS data and demonstrated that cfDNA-based sWGS analysis could be used for dog cancer detection.

Main Text

Methods

Materials

Four dogs were newly enrolled in this study, and informed consent was obtained from the owners. Additionally, a total of 38 healthy dogs from our previous study with no history of cancer or related symptoms were used as controls [9]. These sWGS data were generated by the same experiments and had the same sequencing depth.

sWGS

Peripheral blood was collected using 5 ml Cell-Free DNA Blood Collection Tubes (Ardent BioMed, Guangzhou, China), and cfDNA was subsequently extracted from the blood using the QIAamp Circulating Nucleic Acid Kit (QIAGEN, Dusseldorf, Germany) following the manufacturer's instructions. Subsequently, the cfDNA was utilized for library construction with the Kapa Hyper Prep Kit (Kapa Biosystems, Wilmington, USA) as per the manufacturer's instructions. The resulting libraries were sequenced on a NovaSeq system (Illumina, San Diego, USA) to generate paired-end 150 bp reads, resulting in approximately 3 Gb (~1x coverage) of raw data. After removing adapters and eliminating low-quality reads, the clean reads were aligned to the dog genome (https://hgdownload.soe.ucsc.edu/goldenPath/icanFam3/bigZips/).

CNA analysis

For CNA detection, a well-established methodology from our previous study was employed [5]. In brief, the dog genome was divided into non-overlapping 1 Mb bins, and read counts were tabulated, followed by GC correction. Read counts were then normalized using a cohort of 38 healthy dog samples from our previous study [9]. The normalization process yielded Z-scores, calculated by subtracting the mean value of the healthy sample dataset and dividing by the standard deviation. These Z-scores were then input into a circular segmentation algorithm, as provided by the R package “DNAcopy”, to make segmentation calls. Whole genome copy number changes were used to compute the chromosomal instability (CIN) score, determined by summing the Z-scores of all segments, each multiplied by its respective length.

CIN_Score = ∑V_{se gment} x L _{se gment} , where V represents the Z-score of a segment and L signifies its length.

FS analysis

FS analysis was performed based on the sWGS data [5]. Specifically, the size of cfDNA fragments was calculated based on the mapping position of the remaining paired-end reads. The number of cfDNA fragments within each size range was aggregated, and the distribution of cfDNA FSs was determined. The short fragment ratio, denoted as P150, was defined as the proportion of cfDNA fragments falling within the 50~150 bp range.

Results

One beagle was randomly selected from the 38 healthy dogs as a demonstration case (case 1). The CNA analysis revealed no amplifications or deletions across any of the 38 chromosomes. The CIN score of case 1 was 205.6 (Fig. 1a). The cfDNA fragmentation profiles displayed peaks at 50, 60, 70, 81, 92, 102, 112, 122, 133, 143, 153, and 164 bp, consistent with the peaks observed in all healthy dogs (Fig. 1b). The P150 value for case 1 was 35.9. The gray region represents the first and third quartiles of each FS proportion in all 38 healthy dogs. Both CNA and FS results fell within the comparison range of normal dogs, indicating the absence of a cancer signal in the blood of case 1.

Case 2, a 5-year-old male Corgi, had a sagging mass on the left abdomen. The CNA analysis revealed no amplifications or deletions across any of the 38 chromosomes, and the CIN score was 203.5 (Fig. 1c). Like those of healthy dogs, the cfDNA fragmentation profiles displayed peaks at positions 50, 60, 70, 81, 92, 102, 112, 122, 133, 143, 153, and 164 bp. Notably, the dominant peak of case 2 was at 164 bp, which was slightly longer than that observed in healthy dogs (Fig. 1d). The P150 value for case 2 was 29.7, which falls within the range of healthy dogs (range: 25.2-44.5; Fig. 2B). Both the CNA and FS results indicated that the absence of a cancer signal in the blood from case 2. Biopsy was conducted and the lesionswere assessed histologically. Based on the examination results, case 2 was diagnosed with a lipoma, a benign tumor, consistent with the sWGS analysis. Lipomas are benign neoplasms comprising focal fatty nodules derived from adipocytes in the subcutaneous tissue [10]. In general, the prognosis after surgical resection is good [11]. Follow-up results also showed that the dog remained alive and well 15 months after diagnosis, with no complementary treatment other than surgery.

Case 3, a 14-year-old male Schnauzer, had a mass in the oral cavity. While no apparent CNAs were detected, the CIN score was notably elevated at 615.6, surpassing that of healthy dogs (Fig. 1e; Fig. 2a). Furthermore, the cfDNA fragmentation profile indicated a shift towards shorter FS compared to healthy dogs and case 2, with a dominant peak at 143 bp and a P150 of 49.9 (Fig. 1f). These findings suggested the detection of cancer signals in case 3. It is a well-known fact that in humans, ctDNAs exhibit shorter fragment sizes than cfDNAs, and the proportion of short ctDNA fragments in plasma is positively correlated with the tumor DNA fraction [12]. In addition, biopsy was conducted and the lesions were assessed cytologically. Based on the examination results, case 3 was diagnosed with a oral melanoma, a common malignant tumor in dogs [13]. The dog owner did not choose to pursue further examination or treatment for the dog, and euthanasia was performed on the dog 10 days later.

Case 4, a 6-year-old female Labrador, had a size of 10.0 x 8.3 cm space-occupying mass in the abdomen. The CNA analysis revealed varying degrees of amplification on chromosomes 13 and 31, along with a high CIN score of 1585.1, approximately 5 times the median of healthy dogs (median: 289.8; Fig. 1g), indicating the presence of cancer signals in the blood. Similar to case 3, the cfDNA fragmentation profile displayed a shift towards shorter fragment lengths, with a dominant peak at 143 bp and a P150 of 51.7 (Fig. 1h). Biopsy was conducted and the lesions were assessed histologically. Based on the examination results, case 4 was diagnosed with a hemangiosarcoma, a highly aggressive and malignant tumor that originates from the cells lining blood vessels, with a poor prognosis and a high rate of metastasis [14]. Despite undergoing surgery and doxorubicin chemotherapy, splenic metastasis occurred four months post-surgery.

Case 5, a 12-year-old male Teddy, was presented with the stifle joint osteolysis of the left hind limb. According to the sWGS analysis, the genome of case 5 was quite different from that of the other samples. Approximately 40% (15/38) of the chromosomes had different degrees of amplification or deletion. Among them, chromosomes 5, 8, 13, 15, 17, 34, 35 and 36 were amplified, and chromosomes 3, 4, 8, 10, 18, 22, 24 and 29 were deleted. Its CIN score, 3735.6, was extremely high, about 13 times the median of healthy dogs (median: 289.8; Fig. 1i). Additionally, the cfDNA fragmentation profile demonstrated a higher proportion of shorter fragments, with a dominant peak at 143 bp and a P150 of 55.1(Fig. 1j), indicating the clear detection of cancer signals. Biopsy was conducted and lesion was assessed cytologically. Based on the examination results, osteosarcoma, the most common primary bone tumor found in dogs, accounting for 80% to 90% of all malignancies originating in the skeleton, was diagnosed in case 5 [15,16]. This dog's condition deteriorated further four months after the initial visit, with osteolysis spreading to the femur of the left hind limb, and he was euthanized one month later.

Comparative analysis of CIN scores and P150 values between the healthy controls and the tumor-bearing dogs. The median CIN score of the controls was 289.8 (range: 204.5-594.3). The CIN score of case 2 (203.5) was slightly below the minimum of healthy dogs, while the CIN scores of cases 3-5 (615.6, 1585.1 and 3735.6, respectively) were above them. In particular, cases 4 and 5 were outliers in the boxplot, 5-13 times greater than the median CIN in control dogs (Fig. 2a). For the P150 value, the controls had a median of 35.8, with a range of 25.2 to 44.5. Similarly, the P150 of case 2 was within the distribution of healthy dogs. Additionally, the P150 values of all 3 cancer dogs were 49.9, 51.7 and 55.1, respectively, which were outside the range of those of healthy dogs (Fig. 2b).

Discussion

With the advancement of sequencing technology, liquid biopsy has been widely used in cancer diagnosis and monitoring [17]. In our study, we used sWGS to detect cancer-related signals in 42 dogs. Higher CIN levels and obvious CNAs could be detected in the blood of two dogs with malignant tumors. Moreover, all three cancer cases had shorter cfDNA FS (P150) than did the healthy dogs. The degrees of genomic variation in the three malignant tumor cases were different, which was consistent with the conclusion that the tumor DNA fraction and the degree of genomic variation are related to tumor type and stage [18,19]. The CNA and FS results from the dog with a benign tumor were similar to those in the healthy control group. Our study demonstrated the potential of sWGS-based liquid biopsy for detecting cancer signals in dogs. These findings emphasiz the feasibility of utilizing cfDNA analysis for non-invasive cancer screening and diagnosis in canine patients. Early detection through liquid biopsy can facilitate timely intervention and expand treatment options. Moreover, this approach has the potential to become a more affordable testing option for pet owners, thereby enhancing access to advanced cancer diagnostic tools.

Limitations

1. The number of cases was too small. Further validation with additional cases is warranted to confirm the efficacy of sWGS-based cfDNA analysis in detecting cancer in dogs.

2. Due to the lack of consent from the dog owners for a postmortem examination, only the pathological results of the patients’ tumor tissue biopsies were currently available. Future observed cases will require additional clinical diagnostic information, such as postmortem results and tumor typing results.

List of abbreviations

sWGS: shallow whole-genome sequencing

cfDNA: cell-free DNA

CNA: copy number aberration

FS: fragment size

CTC: circulating tumor cell

ctDNA: circulating tumor DNA

CIN: chromosomal instability

Declarations

Data availability

The datasets used and/or analyzed during the current study could be requested from the corresponding author upon reasonable request.

Acknowledgments

We would like to express our sincere gratitude to the dogs and their owners that participated in this study.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Author Information

Authors and Affiliations

Hongchao Du: New Ruipeng Pet Healthcare Group Co, Ltd, Beijing, China. E-mail: oncovetdu@gmail.com.

Wenfeng Liu: Shanghai Companion Animal Hospital, Shanghai, China. E-mail: l10240822@126.com.

Yunfei Li: Research & Development, TwixBio, Shenzhen, China. E-mail: liyunfei@seekin.onaliyun.com.

Lijuan Zhang: Clinical Laboratories, Shenyou Bio, Zhengzhou, China. E-mail: ljuanzhang@foxmail.com.

Fangfang Jiang: Clinical Laboratories, Shenyou Bio, Zhengzhou, China. E-mail: jff@biosy.net.

Dandan Zhu: Clinical Laboratories, Shenyou Bio, Zhengzhou, China. E-mail: zdd@biosy.net.

Jingshuai Li: Clinical Laboratories, Shenyou Bio, Zhengzhou, China. E-mail: ljs@biosy.net.

Pan Hu: Research & Development, TwixBio, Shenzhen, China. E-mail: hupan@seekin.onaliyun.com.

Ningning Yan: Clinical Laboratories, Shenyou Bio, Zhengzhou, China. E-mail: yn@biosy.net.

Mao Mao: Research & Development, TwixBio, Shenzhen, China; Yonsei Song-Dang Institute for Cancer Research, Yonsei University, Seoul, Korea. E-mail: maomao@yuhs.ac.

Shiyong Li: Research & Development, TwixBio, Shenzhen, China. E-mail: lishiyonglee@gmail.com.

Contributions

M.M. and S.L. designed the study; H.D., W.L., and P.H. collected participants’ samples and clinical information; D.Z., J.L., and N.Y. performed experiments; S.L. and Y.L. designed bioinformatics pipelines and analyzed results; L.Z. and F.J. wrote the manuscript; S.L. and M.M. revised the manuscript. All authors read and approved the final manuscript.

Declaration of Conflicting Interests

P.H. is a full-time employee of and holds stock options in TwixBio. Y.L., D.Z., M.M., and S.L. hold stock options in TwixBio. All other authors declare no competing interest.

Consent for publication

Not applicable.

Ethics approval and consent to participate

No experimental studies were conducted on the dogs, and dogs were not experimental. According to Chapter 1, Article 2 of document No. 167 (2023) issued by the National Science and Technology Administration (on September 7, 2023), "Regulations on Science and Technology Ethical Review," this research did not require official or institutional ethics approval. The dogs in this study were examined with the written consent of their owner. Blood samples from dogs were collected by veterinarians from reputable veterinary hospitals.

ORCID iDs

Mao Mao: https://orcid.org/0000-0002-8570-8571

Shiyong Li: https://orcid.org/0000-0003-0416-526X

Lijuan Zhang: https://orcid.org/0009-0001-7156-1759

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Figure Legends

Figure 1. sWGS analysis results of cfDNA from 5 dog cases. a, c, e, g and i displayed the CNA profiles of cases 1-5, respectively. Amplifications were represented by orange, deletions were represented by blue, and neutral segments were represented by gray. The X-axis represented the ID of chromosomes, and the Y-axis represented the Z-score standardization based on the log R ratio compared to the healthy controls. b, d, f, h and j displayed the FS distributions of cases 1-5. The gray region represented the first and third quartile of the FS distribution among 38 healthy dog blood samples.

CNA: copy number aberration; CIN: chromosomal instability; FS: fragment size.

Figure 2. Comparative analysis of cases (n = 4) and controls (n = 38). Three dogs with malignant tumor were represented in red dots, benign tumor dog was represented in a blue dot, and healthy controls were represented in green dots. a. Displayed the CIN scores of 4 dog cases and 38 controls. b. Displayed the P150 for 4 cases and 38 controls.

CIN: chromosomal instability; P150: the proportion of cfDNA fragments falling within the 50~150 bp range.