Familial adenomatous polyposis in dogs: hereditary gastrointestinal polyposis in Jack Russell Terriers with germline APC mutations

Abstract

Many hereditary disorders in dogs have equivalents in humans and thus attract attention as natural animal models. Breed predisposition to certain diseases often provides promising clues to explore novel hereditary disorders in dogs. Recently, cases of gastrointestinal (GI) polyps in Jack Russell Terriers (JRTs) have increased in Japan. In 21 affected JRTs, polyps were found in either or both the stomach and colorectum, with a predilection for the gastric antrum and rectum. Multiple polyps were found in 13 of 21 examined dogs, including 5 dogs with both gastric and colorectal polyps. Some dogs were found to have GI polyps at an early age, with the youngest case being 2.3 years old. Histopathologically, 43 of 46 GI polyps (93.5%) were diagnosed as adenomas or adenocarcinomas. Immunohistochemical analysis revealed cytoplasmic and nuclear accumulation of β-catenin in the tumor cells. As in the case of human patients with familial adenomatous polyposis, all examined JRTs with GI polyps (n = 21) harbored the identical heterozygous germline APC mutations, represented by a 2-bp substitution (c.[462A>T; 463A>T]). The latter substitution was a non-sense mutation (p.K155X) resulting in a truncated APC protein, thus suggesting a strong association with this cancer-prone disorder. Somatic mutation and loss of the wild-type APC allele were detected in the GI tumors of JRTs, suggesting that biallelic APC inactivation was involved in tumor development. This study demonstrated that despite differences in the disease conditions between human and dog diseases, germline APC mutation confers a predisposition to GI neoplastic polyps in both dogs and humans.

Introduction

Many hereditary neoplastic syndromes have been identified in humans (1). In contrast, there are no reports on hereditary tumors in dogs, except for renal cystadenocarcinoma in German Shepherd dogs (2). In addition, a high prevalence of a germline mutation in the MET proto-oncogene has been reported in Rottweiler dogs, a breed predisposed to several types of cancers (3). In dogs, hereditary disorders usually occur with greater frequency in a particular dog breed (4,5). Considering that some dog breeds are known to be at increased risk for certain types of tumors (5,6), it is highly likely that some hereditary tumor syndromes remain undiscovered in dogs.

Unlike in humans, gastrointestinal (GI) epithelial cancers are rare in dogs (7–10); the reported incidence of gastric or intestinal adenocarcinoma is less than 1% (9,10). Recently, cases of GI neoplastic polyps in Jack Russell Terriers (JRTs) have increased in Japan. Several dog breeds have been reported to be predisposed to GI epithelial cancers (5,9–14), and recent studies have revealed possible risk factors of gastric polyps in dogs (15,16). However, the cause for the recent increase in the number of JRTs with GI polyps remains unknown; thus, it is suspected to be a novel hereditary disorder.

Familial adenomatous polyposis (FAP) is a highly penetrant autosomal dominant disorder caused by germline mutations in the Adenomatous polyposis coli (APC) tumor-suppressor gene (17). FAP is characterized by the development of hundreds to thousands of adenomatous polyps in the colon and rectum (17). Colorectal adenomatous polyps become detectable in the second or third decade of life and increase in number and size with age. If untreated, virtually all FAP patients inevitably develop colorectal carcinomas by 50 years of age, with a mean age of about 40 years (17). FAP patients frequently develop extracolonic manifestations including upper GI lesions. Antral adenoma also occurs in 5–20% of FAP patients (17–19). The risk of gastric cancer among FAP patients is higher than that in the general population, especially in countries such as Japan and Korea where gastric cancer is prevalent (20,21).

In this study, we investigated the clinical and pathological features of GI polyposis in JRTs, its association with germline APC mutations and the somatic alterations involved in their development.

Materials and methods

Case information

In total, 21 JRTs with GI neoplastic polyps were investigated (Table 1). Eleven cases were prospectively recruited between 2016 and 2019 at Gifu University Teaching Animal Hospital or at private animal hospitals. Furthermore, we retrospectively collected 10 cases that were histopathologically examined between 2008 and 2019 at the Laboratory of Veterinary Pathology, Gifu University.

Table 1.

Summary of clinical information from Jack Russell Terriers with gastrointestinal polyps

Case No.	Agea		Sex	Clinical symptoms	Number and location of gastrointestinal polypsb
					Stomach					Colorectum
	Initial occurrence	Recurrence
					Cardia	Corpus	Antrum	Unrecorded	Total	Colon	Rectum	Total
1	4y 0m		F	(−)	ND	ND	ND	ND	ND	ND	1	1
		5y 4m		(−)	ND	ND	ND	ND	ND	ND	1	1
		7y 8m		Vomiting, bloody stool	0	0	1	0	1	ND	1	1
		8y 6m	F (Spayed)	No symptoms	0	0	1	0	1	ND	ND	ND
		9y 2m		No symptoms	0	0	7	0	7	ND	ND	ND
		11y 4m		Bloody stool	0	0	0	0	0	0	1	1
2	7y 10m		M	Bloody stool	1	0	1	0	2	1	0	1
		8y 8m		Bloody stool	0	0	1	0	1	1	0	1
3	6y 2m		F	Vomiting, anorexia	0	2	3	0	5	0	0	0
4	8y 0m		M	Melena, soft stool, anorexia	0	1	0	0	1	ND	ND	ND
5	12y 1m		F (Spayed)	Vomiting, hematemesis, melena, emaciation	0	0	2	0	2	0	0	0
6	6y 6m		F (Spayed)	Vomiting	0	0	1	0	1	0	0	0
		(8y 6m)c		Vomiting, hematemesis, melena	0	2	1d	0	3	0	0	0
7	10y 3m		F (Spayed)	Bloody stool, soft stool	0	0	0	3	3	0	1	1
8	5y 2m		F	Vomiting, anorexia	0	0	0	1	1	ND	ND	ND
		8y 9m		Bloody stool, tenesmus	1	0	0	0	1	1	0	1
9	3y		F	Vomiting	0	0	1	0	1	ND	ND	ND
10	13y		F	Tenesmus	ND	ND	ND	ND	ND	ND	1	1
11	5y		M(Neutered)	Vomting, bloody stool, tenesmus, anorexia	0	0	1	0	1	0	1	1
12	7y 8m		F (Spayed)	Vomiting, melena	0	0	1	0	1	ND	ND	ND
		13y 10m		Vomiting	1	0	1	0	2	ND	ND	ND
13	5y 3m		M	Bloody stool	ND	ND	ND	ND	ND	0	1	1
		7y 3m		Bloody stool, vomiting	ND	ND	ND	ND	ND	0	2	2
14	6y		M(Neutered)	Diarrhea, prolapse of polyp	ND	ND	ND	ND	ND	ND	1	1
15	10y 5m		M(Neutered)	Vomiting, emaciation	0	1	1	0	2	ND	ND	ND
16	8y 10m		F	(−)	ND	ND	ND	ND	ND	ND	1	1
17	7y 3m		M(Neutered)	(−)	0	0	1	0	1	ND	1	1
		8y 4m		(−)	0	0	1	0	1	ND	1	1
		8y 11m		Bloody stool	0	0	1	0	1	ND	3	3
18	12y 4m		M(Neutered)	Bloody stool	ND	ND	ND	ND	ND	ND	1	1
19	2y 3m		M	Vomiting	0	0	4	0	4	ND	ND	ND
20	11y 8m		F (Spayed)	Bloody stool, anorexia, rectal prolapse	ND	ND	ND	ND	ND	ND	1	1
21	9y 9m		M(Neutered)	(−)	ND	ND	ND	ND	ND	0	1	1

(−), No information was available; ND, Endoscopic examination was not performed.

^aAge at which gastrointestinal polyps were resected surgically or endoscopically.

^bNumber and location of gastrointestinal polyps based on endoscopic findings and microscopic observation at the time of surgery.

^cSecond endoscopic examination was performed due to the exacerbation of digestive symptoms. Newly developed lesions were detected in addition to enlargement of the primary lesion.

^dAntral lesion.

Histopathological analysis

In total, 46 GI polyps from 21 JRTs, which included 26 gastric and 20 colorectal polyps, were examined (Supplementary Tables 1 and 2, available at Carcinogenesis Online). All samples were fixed in formalin and routinely processed. Sections were stained with hematoxylin and eosin and were immunostained for β-catenin. See Supplementary Methods, available at Carcinogenesis Online, for details.

Molecular analysis of germline mutation in APC

Peripheral blood was collected from 11 JRTs with GI polyps (Cases 1–11) and 6 control dogs. JRTs treated for other diseases served as the control subjects. Genomic DNA was extracted from ethylenediaminetetraacetic acid-anticoagulated blood samples and PCR-direct sequencing was performed to examine the entire coding region of the canine APC gene. In the retrospective analysis of 10 cases (Cases 12–21), genomic DNA was extracted from normal tissues on formalin-fixed paraffin-embedded (FFPE) sections. A selected region of APC-containing codons 154 and 155 was examined by PCR-direct sequencing. See Supplementary Methods, available at Carcinogenesis Online, for details.

Molecular analysis of somatic gene alterations of APC in GI tumors

Genomic DNA was extracted from fresh tumor samples obtained from six polyps of five dogs, including four gastric and two rectal polyps. In three dogs, samples were also collected from normal-appearing mucosa surrounding the lesions as controls. In addition, genomic DNA was extracted from gastric and colorectal tumors (n = 13 and 8, respectively) and the surrounding normal tissues on FFPE sections. When fresh tumor tissues were available, the entire APC-coding region was examined by PCR-direct sequencing to detect somatic mutations. In addition, to detect the loss of wild-type APC allele, PCR–restriction fragment length polymorphism and TaqMan real-time PCR assays were performed using both fresh and FFPE tumor tissues. The protocol is described in detail in Supplementary Methods, available at Carcinogenesis Online.

Results

Clinical features of JRTs with GI polyps

Age and sex distribution

The mean age at which GI polyps were surgically or endoscopically resected in 21 affected dogs was 7.7 years (median: 7.7 years; range: 2.3–12.6 years). Some cases presented digestive symptoms for months or years before presentation to animal hospitals and therefore, the age of disease onset could be younger. Notably, six dogs developed GI polyps at 5 years or younger, with the youngest case being 2 years and 3 months old. There was no sex predisposition.

Clinical symptoms

With the exception of two dogs in the retrospective analysis, we could obtain the clinical information of JRTs with GI polyps (Table 1). In dogs with gastric polyps, vomiting was the most frequent symptom and was recorded as a chief complaint in 10 of 14 dogs. Hematemesis and/or melena were found in four dogs with gastric polyps. In dogs with colorectal polyps, a bloody stool was the most common presenting symptom and was found in 9 out of 11 dogs. At presentation, no dogs showed detectable metastasis.

Distribution and the number of GI polyps

Fourteen dogs had polyps in the stomach and 13 dogs had colorectal polyps (Table 1). Notably, six dogs developed both gastric and colorectal polyps. When the stomach and colorectum were endoscopically examined at the same time, more than half of the dogs had polyps in both the organs (five of eight dogs, 62.5%). In the stomach, a small number of lesions developed in the fundus or corpus (n = 3 and 6, respectively), whereas most polyps were located in the antrum (n = 29). In the colorectum, polyps were concentrated in the rectum (21 out of 23 colorectal polyps, 91.3%).

Importantly, more than half of the affected dogs (13 out of 21, 57.1%) developed multiple polyps (Table 1). The mean cumulative number of GI polyps was 2.95 ± 2.68 (mean ± standard deviation), with 13 polyps in Case 1 being the highest. Multiple polyps were recorded more frequently in the stomach (10 of 14 dogs with gastric polyps, 71.4%) than in the colorectum (3 of 13 dogs with colorectal polyps, 23.1%).

Recurrence and prognosis

Five dogs showed recurrence of GI polyps after surgical resection (Table 1). Case 1 had a history of repeated recurrence of GI polyps (Supplementary Figure 1, available at Carcinogenesis Online). Disease recurrence occurred at 1–6 years after complete surgical resection of GI polyps (Table 1), implying that it could be attributed to newly developed lesions, rather than regrowth of residual tumor tissues. In fact, the recurrent polyps were detected in different regions from which the primary lesions developed in most cases.

Prognostic information was collected through interviews with clinical veterinarians and dog owners, enabling follow-up of more than 1 year in 12 cases and of more than 2 years in 10 cases. Among these cases, none of the dogs died within the first or second year after initial surgical or endoscopic treatment: the 1-year and 2-year survival rates for the examined cases were thus 100% (n = 12 and 10, respectively). Furthermore, seven of eight cases (87.5%) survived for more than 3 years after initial treatment. The maximum survival period after diagnosis was found to be 9 years (Case 1). The present study included four deceased cases. The deceased cases died 2–4 years after the initial diagnosis of GI polyps with a mean survival time of 3.5 years (see also Supplementary information, available at Carcinogenesis Online).

Pathological findings

Endoscopic and gross appearance

The gastric and colorectal lesions in JRTs had a similar gross and endoscopic appearance. Most lesions were sessile polyps with glossy, smooth, or slightly lobulated surfaces, sometimes with reddish areas (Figure 1A and B). In some cases, consistent with the main complaint of vomiting, multiple polyps protruded into the lumen and caused severe narrowing of the gastric antrum lumen (Figure 1A).

Figure 1.

Endoscopic appearance of gastrointestinal polyps in Jack Russell Terriers. (A) Multiple polyps in the pyloric antrum. The protruded lesions cause severe narrowing of the pylorus. (B) Polyps in the rectum.

Histopathological findings

Stomach

According to the WHO classification of tumors in domestic animals (7), gastric polyps were diagnosed as hyperplastic polyps, adenoma and adenocarcinoma (n = 3, 16 and 7, respectively; Supplementary Table 1, available at Carcinogenesis Online). In adenomas and adenocarcinomas, tumor cells formed glandular and papillary structures with branching and budding (Figure 2A–D). All the adenocarcinomas were low-grade tumors that showed slightly increased signs of dysplasia than adenomas (7). Notably, most adenocarcinomas contained benign adenomatous areas, suggesting that they arose within adenomas (Figure 2E and F). The tumors developed mainly in the upper epithelial layer of the gastric mucosa overlying normal or dilated glands (Figure 2E). Tumor invasion into the lamina propria with lymphovascular infiltration was observed in an adenocarcinoma (Figure 2G and H), but invasive proliferation through the muscularis mucosa was not observed in any case (Supplementary Table 1, available at Carcinogenesis Online).

Figure 2.

Histopathology of the gastrointestinal lesions in Jack Russell Terriers. (A–D) Representative photomicrographs of adenoma (A, B) and adenocarcinoma (C, D) in the stomach. (B) and (D) Higher magnification of (A) and (C), respectively. The lesions were diagnosed as tubulopapillary adenoma and tubulopapillary adenocarcinoma, respectively. (E) Carcinoma in adenoma. The arrowheads indicate a carcinomatous component growing within an adenoma. Note also that the lesion developed mainly in the upper epithelial layer overlaying the normal or dilated gastric glands. (F) Higher magnification of the boxed area in (E). The boundary between adenoma (AD) and adenocarcinoma (ADC) components. (G) Adenocarcinoma with invasion into the lamina propria. (H) Higher magnification of the boxed area in (G). Lymphovascular invasions are also observed (arrows). (I–L) Representative photomicrographs of adenoma (I, J) and adenocarcinoma (K, L) in the rectum. (J) and (L) Higher magnification of (I) and (K), respectively. (M) Multiple tumors developed in the small area of rectal mucosa. In addition to the tumors (black arrowheads), there is also a single crypt adenoma (white). (N–P) Single crypt adenomas in the rectal mucosa. Hematoxylin and eosin staining (N, O) and immunostaining for β-catenin on serial sections (P). (O) Higher magnification of (N). Multiple lesions are scattered in the rectal mucosa (arrowheads). Note the nuclear and cytoplasmic accumulation of β-catenin in the aberrant crypt. Bars = 500 µm (A, C, I and K), 50 µm (B, D, J, L, O and P), 5 mm (E and M), 100 µm (F), 1 mm (G), 200 µm (H and N).

Colorectum

Except for a rectal polyp diagnosed as adenoma, all other colorectal polyps (n = 19) were adenocarcinomas (Figure 2I–L; Supplementary Table 2, available at Carcinogenesis Online) (7). The majority of gastric tumors developed in the upper epithelial layer of colorectal mucosa. When evaluating the depth of cancer invasion in surgical specimens (n = 15), although lamina propria invasion was observed in an adenocarcinoma, tumor invasion through the muscularis mucosa was not found in any lesions (Supplementary Table 2, available at Carcinogenesis Online). Notably, we detected multiple lesions in a small area of the rectal mucosa on a single section (Figure 2M).

Single crypt adenoma in the colorectum

A single crypt adenoma, also termed as an aberrant crypt focus, is the earliest microscopically recognizable lesion in FAP patients (17). In this study, we identified strikingly similar lesions comprising a single dysplastic crypt in normal-appearing colorectal mucosa surrounding the tumors in two JRTs (Figure 2N and O). While multiple single crypt adenomas in the colorectum are considered pathognomonic for FAP (17), we found two or three lesions scattered in the colorectal mucosa of dogs with colorectal polyps (Figure 2N).

Immunohistochemical findings

In the normal GI mucosa surrounding the polyps, β-catenin was localized in the cell membrane (Figure 3A–D). β-catenin was still localized in the cell membrane in hyperplastic polyps of the stomach (data not shown), whereas cytoplasmic and nuclear accumulation of β-catenin was observed in adenomas and adenocarcinomas of both the stomach and colorectum (Figure 3A–E; see also Supplementary information, available at Carcinogenesis Online). Moreover, cytoplasmic and nuclear β-catenin accumulation was observed in single crypt adenomas (Fig. 2P).

Figure 3.

β-Catenin expression in the gastrointestinal neoplastic polyps of affected dogs. (A) The boundary between adenoma and surrounding normal tissues in the gastric antrum. (B) Higher magnification of the normal and neoplastic tissues in (A). (C) The boundary between the rectal adenocarcinoma and surrounding normal tissues. (D) Higher magnification of the normal and neoplastic tissues in (C). Although the normal gastric and rectal epithelial cells show membranous β-catenin expression with no intracellular expression (B, D), cytoplasmic and nuclear accumulation of β-catenin is observed in tumor cells (B, D). Bars = 500 µm (A and C), 100 µm (B and D). (E) Percentage of cells with nuclear β-catenin accumulation in gastrointestinal lesions.

Germline APC mutations of JRTs with GI polyps

Based on the similarities in clinical and pathological features, GI polyposis in JRTs was suspected to be a canine counterpart of FAP. Therefore, we examined the entire coding region of APC using DNA extracted from peripheral blood and identified heterozygous germline mutations in JRTs with GI polyps. Importantly, these mutations were not detected in any of the control dogs (n = 6), whereas all of the examined dogs with GI polyps (n = 11) harbored identical mutations, a 2-bp substitution (c.[462A>T;463A>T]) in exon 4 of APC (Figure 4A). The former substitution is a missense mutation at codon 154 (p.Q154N), whereas the latter substitution is a non-sense mutation at codon 155 (p.K155X), which results in a truncated APC protein. Subsequently, the germline APC mutation status was evaluated in 10 additional dogs with GI polyps using DNA obtained from retrospectively collected FFPE samples. As expected, the same mutations at the codons 154 and 155 were also detected in all the examined dogs (Figure 4A). In human FAP, disease severity is correlated with the location of germline APC mutations (Figure 4B) (18). Germline mutation of JRTs with GI polyps was detected in the 5′ region of APC, corresponding to the region where mutations of patients with an attenuated form of FAP are located (Figure 4B) (17,18).

Figure 4.

Germline APC mutations identified in Jack Russell Terriers with gastrointestinal polyps. (A) A sequence of codons 153–156 in APC of the affected and control dogs. DNA samples were isolated from peripheral blood (left and middle panels) or paraffin-embedded tissue (right panel). Arrowheads indicate germline mutations. Heterozygous 2-bp substitutions at codons 154 and 155 were identified in dogs with gastrointestinal polyps (middle and left panels), but not in control dogs (left panels). (B) Location of the germline APC mutation detected in Jack Russell Terriers and comparison with those in human FAP. The mutations of the dogs (arrow) are positioned in the 5′ region of APC where the mutations of patients with attenuated FAP are located (blue-colored area). Colored areas represent regions of mutations associated with each FAP phenotype in humans. (C) Family tree conducted according to pedigree certificates of Cases 1 and 6. There was a blood relationship between these cases: mother dog of Case 1 and grandmother dog of Case 6 were littermates. Square and circle show male and female, respectively, and the stripe pattern indicates a potential carrier of the germline APC mutation.

Single nucleotide polymorphisms were also detected in the APC gene of JRTs (see Supplementary information, available at Carcinogenesis Online).

Pedigree analysis of the affected dogs

Pedigree certificates of five affected JRTs (Cases 1, 3, 6, 10 and 21) were available with the cooperation of the dog owners. The certificates contained the information on three generations in the ancestry of the affected JRTs. We found that Cases 1 and 6 were descended from a common breeding pair (Figure 4C), implying the hereditary nature of this disease.

Somatic gene alterations of APC in GI tumors

In accordance with the two-hit theory of carcinogenesis, the wild-type APC allele is lost or mutated in FAP-associated tumors including colorectal and gastroduodenal tumors (17,22–25). Therefore, we investigated whether there were somatically acquired APC alterations in the GI tumors of JRTs.

Somatic mutations

Analysis of the entire coding region of APC using freshly frozen samples (n = 6) revealed a frameshift mutation at codon 548 (c.1643dup), which generates a premature termination codon at codon 559 (p.Leu548PhefsX12) in gastric adenoma (Figure 5A, right panel). This mutation was not detected in the normal-appearing mucosa surrounding this lesion (Figure 5A, left panel).

Figure 5.

Somatic APC alterations in gastrointestinal tumors of Jack Russell Terriers. (A) A frameshift mutation identified in APC of the gastric adenocarcinoma. Right panel, the gastric tumor (Lesion No. 05S01). Left panel, the surrounding normal mucosa. Arrowhead indicates a single base insertion in codon 548, forming a premature termination codon at codon 559. (B–D) Loss of heterozygosity (LOH) at the APC locus determined by PCR-restriction fragment length polymorphism assay using the Agilent 2100 bioanalyzer. (B) Electropherograms of MseI-digested PCR products amplified from normal (left panel) and tumor (right panel) samples. The reduction of the wild-type allele-derived fragment (109 bp) is evident in the tumor sample (arrow); Lesion No. 06S01. (C) Virtual gel image of MseI-digested PCR products on the bioanalyzer. Representative results of tumors with or without reduction of the wild-type allele-derived fragment (lanes 2 and 4) and the corresponding normal tissues (lanes 1 and 3). The asterisk shows a reduction in the wild-type allele-derived fragment. (D) Wild-type to mutant allelic ratio in gastrointestinal tumors (black bars) relative to the corresponding normal tissues (white bars). Relative reduction of the wild-type fragment is shown in some GI lesions. (E and F) LOH at the APC locus determined by TaqMan real-time PCR assay. (E) Amplification plots of wild-type (blue curve) and mutant (red curve) APC copies in the gastric tumor; Lesion No. 06S01. Amplification of the wild-type allele copies requires approximately four cycles more than that of the mutant allele to reach the threshold level in the gastric tumor (right panel), whereas both amplifications reach the threshold level after nearly the same cycles in the surrounding normal tissues (left panel). (F) Wild-type to mutant allelic ratio in gastrointestinal tumors (black bars) relative to the corresponding normal tissues (white bars). Data are mean ± SD of duplicate PCR reactions. There are marked reductions in the allele ratio in some tumors including gastric adenocarcinomas (Cases 1, 6 and 12) and a rectal adenocarcinoma (Case 17). (G) DNA sequences of APC encompassing the germline mutation sites as determined by PCR-direct sequencing. There are double peaks of A and T at the mutation sites in the normal mucosa (left panel), whereas the chromatogram appears as single peaks of A in the gastric tumor (right panel, Lesion No. 06S01) indicating loss of the wild-type allele. Arrowheads indicate the sites of germline APC mutations.

Loss of heterozygosity at the APC locus

We first examined whether the wild-type APC allele was lost in GI tumors using PCR–restriction fragment length polymorphism analysis wherein the mutant allele-derived fragments were cleaved into smaller pieces (51 and 58 bp) than the wild-type allele-derived fragments (109 bp) by the restriction enzyme MseI (Figure 5B and C; Supplementary Figure 3, available at Carcinogenesis Online). Quantitative comparison of the signal intensity between wild-type and mutant allele-derived fragments showed relative reductions in the wild-type fragments in some of the examined GI lesions (Figure 5B–D; Supplementary Figure 3, available at Carcinogenesis Online).

To quantitatively assess the loss of wild-type alleles in GI lesions, the relative amount of wild-type and mutant alleles was examined by TaqMan real-time PCR assay. Notably, the ratios of the wild-type to mutant alleles in some of the examined GI lesions were markedly reduced as compared with the values in corresponding normal tissues (Figure 5E and F). Consistently, nucleotide sequences at the sites of germline APC mutations determined by PCR-direct sequencing showed that the sequences of the wild-type allele were lost in the gastric tumors (06S01) (Figure 5G, right panel), whereas heterozygosity remained unchanged in corresponding normal tissues (Figure 5G, left panel).

Discussion

In dogs, discovery of a hereditary disease often begins with the identification of breed predisposition to a certain disease. In the middle of the 2010s, we noticed an increase in the number of cases of JRTs with GI neoplastic polyps through daily clinical practice and pathological examination (Supplementary Figure 4, available at Carcinogenesis Online). According to the reports from the Japan Kennel Club, the recent increase of the affected JRTs cannot be attributed to a rapid increase in their population in Japan.

The majority of germline APC mutations in FAP patients are non-sense or frameshift mutations producing a truncated protein with abnormal function (17,26). Consistently the mutation identified in the affected JRTs was a non-sense mutation (p.K155X). In humans, more than 700 different germline mutations have been identified throughout APC in FAP-affected families (17,18) and correlations between the location of APC germline mutations and disease severity have been well documented (Figure 5B) (18). The germline mutations of affected JRTs were located in the 5′ region of APC, where mutations of patients with attenuated FAP are located in humans (Figure 4B) (17,18,27–30). Although FAP patients typically develop hundreds to thousands of colorectal polyps, attenuated FAP is characterized by the development of 100 or less polyps, with an average number of approximately 30 polyps. Consistently, the affected JRTs developed a small number of GI polyps, with an average of 2.3 polyps. The much smaller number of polyps in JRTs might reflect a lower prevalence of sporadic GI cancers in dogs (7–10). Considering the genomic location of APC mutations and the disease severity, GI polyposis in JRTs could be a canine counterpart of attenuated FAP in humans.

In the present study, the hereditary nature of this disease was implied by the analysis of pedigree certificates of the affected JRTs. However, because the dogs examined in the present study were household dogs, it was impossible to examine the germline APC mutation status of their close relatives, including their parents and littermates, which potentially harbored these mutations. The adult onset of this disease made it more difficult to trace their relatives. Further studies are thus needed to provide direct evidence of familial transmission of APC mutations in JRTs.

The present study demonstrated that GI lesions of JRTs developed through processes that were morphologically and molecularly similar to those of colorectal adenomas in FAP patients. Interestingly, adenomas in FAP begin as dysplastic aberrant crypt foci also called single crypt adenomas (17), and this microscopic lesion was also detected in JRTs with the germline APC mutation. The presence of single crypt adenomas in grossly normal colorectal mucosa indicates that dogs with germline APC mutations are still at increased risk of disease recurrence even after complete surgical resection. In fact, five dogs experienced recurrences after surgical resection in the present study. Moreover, molecular analysis revealed somatic APC alterations in the GI tumors of JRTs, suggesting that inactivation of the residual APC allele would be a critical step in the molecular pathogenesis of GI tumors in dogs with the germline APC mutation, as in the colorectal adenomas of FAP patients (17,22–25). Consistently, as previously shown in colorectal lesions of FAP patients (31), loss of functional APC protein was also suggested by immunohistochemical analysis showing intracellular accumulation of β-catenin in the GI tumors of JRTs.

There was an obvious difference in the most common site of tumor development between human and canine disease. Unlike human patients with FAP, the lesions in dogs with germline APC mutations most commonly developed in the stomach, rather than in the colorectum. Gastric adenomas are recognized as an extracolonic manifestation of FAP and occur at 5–20% frequency in FAP patients (18). However, two-thirds of the JRTs with APC mutations had gastric adenomas and/or adenocarcinomas, with multiple lesions frequently observed in the stomach rather than in the colorectum. This dissimilarity in the common site has also been noted in murine FAP models. It is well known that Apc^Min/+ mice, the best-known mouse model for FAP, develop a greater number of tumors in the small intestine than in the large intestine (32,33). Similarly, the predominance of small-bowel adenomas was reported in another mouse model for FAP (34). These findings suggest that, although germline APC mutations invariably confer a predisposition to GI tumors, the common site of tumor development could vary depending on the animal species.

Another difference between the human and canine disease was that, unlike in FAP, almost all the colorectal lesions of JRTs were diagnosed as adenocarcinomas, rather than adenomas. However, this difference would arise mainly from a difference in classification criteria between human and animal tumors. Although we made the diagnosis of adenocarcinoma mainly based on cellular and structural atypia irrespective of the tumor invasion depth (7), colorectal carcinomas are defined as tumors with submucosal invasion in the WHO classification of human tumors (35). In fact, canine colorectal lesions were diagnosed as adenoma with high-grade dysplasia using the human classification (Supplementary Table 2, available at Carcinogenesis Online).

Histopathological analysis clarified that the hereditary GI cancers of JRTs had features different from those of sporadic canine cases. Classification of the gastric tumors of JRTs based on histological growth patterns showed no signet-ring cell carcinoma despite it being the most common type of sporadic gastric cancer in dogs (7,9). More importantly, in striking contrast with sporadic GI cancers, tumor cell invasion through the muscularis mucosae was not observed in the GI tumors of JRTs indicating a poorly invasive phenotype. Consistently, the sporadic GI tumors of dogs are typically advanced with nodal and distant metastasis at presentation (11,36,37), whereas JRTs with GI tumors did not have any detectable metastatic lesions at presentation and showed much better survival rates after treatment compared to dogs with sporadic GI tumors (7,9,11,36,37).

To date, there have been no reports suggesting the existence of the hereditary disease in JRTs in countries other than Japan, even in European countries, where JRTs are a popular breed. Due to the lower prevalence of the germline APC mutation and a consequently infrequent occurrence of GI polyps in JRTs outside Japan, the disease might not have attracted much attention. It is highly likely that this cancer-prone disorder is prevalent only among JRTs in Japan. Considering that all the affected JRTs examined in the present study were born during the first decade of the 2000s, the causative germline APC mutations could be propagated widely among JRTs in Japan during this decade, possibly due to extensive use of popular sires such as dog-show champions, which harbored these mutations (4,5). It is known that small closed populations of purebred dogs are subject to popular sire effects (5). Furthermore, the pedigree analysis revealed that, although there was a blood relationship between two of the affected dogs, the remaining three affected dogs did not have any relationships in their past three generations. This finding implies that the causative APC mutations might not be confined to closely related bloodlines and could be widely spread among JRTs in Japan. Further studies are thus needed to clarify the prevalence rate of germline APC mutations in JRTs in Japan and in other countries.

In summary, we showed that GI polyposis in JRTs is a novel hereditary disease in dogs caused by identical APC germline mutations. This is the first report of a naturally occurring hereditary GI cancer in animals. Our data demonstrate that despite differences in the disease conditions between humans and dogs, a germline APC mutation confers a predisposition to GI neoplastic polyps in both dogs and humans.

Abbreviations

Abbreviations
 
• APC

  adenomatous polyposis coli

 
• FAP

  familial adenomatous polyposis

 
• FFPE

  formalin-fixed paraffin-embedded

 
• GI

  gastrointestinal

 
• JRT

  Jack Russell Terrier

Funding

This work was supported by Grant-in-Aid for Scientific Research (KAKENHI) (C) grant number 18K06969 from Japan Society for the Promotion of Science (JSPS).

Acknowledgements

We thank Ms Chikako Iriyama, Mr Gaku Ieda, Mr Hiroyuki Matsushita, Drs Kayoko Yonemaru, Mami Murakami, Ryota Iwasaki and Kou Hamada and Prof. Tokuma Yanai at Gifu University and clinical veterinarians for their assistance.

Conflict of Interest Statement: None declared.