Domestic cat hepadnavirus detection in blood and tissue samples of cats with lymphoma

Figures & data

Figure 1. Histopathological features of DCH infection in lymphoma tissue of cats. (A, E-J) Sections from case no. 3. (A) The lymphoid follicles are diffusely expanded by large neoplastic lymphocytes separating coarse fibrovascular stroma. Neoplastic lymphocytes have moderate amounts of cytoplasm with distinct cellular borders. The nuclei are 2 times larger than red blood cells, round, and finely stippled, containing a prominent nucleolus (square box is indicated for the inset). (B-D) DCH qPCR-positive lymphoma section of case no.6. (B) Diffuse, generalized cytoplasmic IHC staining for CD79. (C) Prominent nuclear IHC staining for pax-5 in multiple neoplastic cells. (D) Cytoplasmic IHC staining for CD3 in some single cells in lymphoma tissue. (E) In situ hybridization (ISH) for DCH DNA staining (red precipitates) in lymphoma tissue revealed intensely intranuclear staining in diffuse round cells (square box is indicated for the inset). (F) Prominent nuclear staining for DCH hybridization in large, pleomorphic round cells. No DCH labeling was observed within the small round cells. (G) Dual DCH ISH (red precipitates) and CD79 IHC (green color) revealed marked, diffuse staining in the nucleus and cytoplasm of neoplastic cells, respectively. (H) Higher magnification reveals intense DCH labeling (red precipitates) in the nucleus of the cells that were cytoplasmically stained with CD79 IHC (green color). (I) Negative control using unrelated probe. No hybridization signals were present in the DCH qPCR-positive lymphoma section that was incubated with the FBoV-3 ISH probe. (J) Negative control using DNase-treated section. No hybridization was present in the DCH qPCR-positive lymphoma section treated with DNase prior to incubation with the DCH probe. (K) Negative control for dual ISH/IHC labeling. Cytoplasmic IHC CD79 staining of the DCH qPCR-positive lymphoma section incubated with the FBoV-3 probe. Bars indicate 180 µm for A, B, D and E; 80 µm for F; 120 µm for C, G, I, and J; and 20 µm for H and K.

Table 1. Detection of DCH DNA in blood and lymph node tissues of investigated cats.

Samples	Sample characteristics		Lymphoma		Non-lymphoma		P- value (Odd ratio, 95% CI)
			No. of cases (A)^a %		No. of cases	%
EDTA Blood	Total case	N = 594					0.002 (5.1496, 2.33–11.36)
	DCH qPCR	No. of cases	68 (42)	100	526	100
		Positive	11 (5)	16.18	19	3.61
		Negative	57 (37)	83.82	507	96.39
FFPE Tissue	Total case	N = 123					0.028 (13.678, 0.75–248.36)
	DCH qPCR	No. of cases	63 (35)	100	60	100
		Positive	6 (3)	9.52	0	0
		Negative	57 (32)	90.48	60	100

A = number of cases that were co-detected with feline retroviruses. ^aThere was no statistical significance between DCH detection and retrovirus status in lymphoma cases (p = 0.3).

Table 2. Morphological characteristics of lymphoma cats and DCH DNA detection in paraffin-embedded lymphoma tissues.

Lymphoma classification	Number of tested cases (n)	DCH qPCR		P-value (Odd ratio, 95% CI)
		Positive	Negative
Subtypes	B-cell (56)	6	50	P >0.9 (1.93, 0.09–37.89)
	T-cell (7)	0	7
Anatomical form	Multicentric (31)	6	24	P = 0.008 (1.327, 0.06–31.18)
	Others (32)	0	32

Table 3. Histological characteristics of DCH-positive feline lymphoma cases.

Case no.	Lymphoma subtype/anatomical form	Histological Grading	WHO classification	DCH loads (copies/ml)	ISH score^a	ISH staining pattern	Feline retroviral status^b
1	B-cell/Multicentric	Low grade	Peripheral B cell	2.35 × 10^4	+	Focal, dot-like pattern	Negative
2	B-cell/Multicentric	High grade	Diffuse large B-cell	2.82 × 10^4	+	Focal, dot-like pattern	FeLV
3	B-cell/Multicentric	Low grade	B-cell small lymphocytic	3.84 × 10^6	+++	Multifocal, multiple dot-like pattern	Negative
4	B-cell/Multicentric	Low grade	Follicle center	1.80 × 10^7	+++	Diffuse, intensity	FeLV
5	B-cell/Multicentric	High grade	Diffuse large B-cell	1.28 × 10^6	++	Patchy, multiple dot-like pattern	Negative
6	B-cell/Multicentric	Low grade	Follicle center	1.11 × 10^6	++	Focally extensive, multiple dot-like pattern	FeLV

^aThere is no significant correlation between DCH loads and DCH staining pattern (p = 0.067).

^bFeLV: feline leukemia virus.

Data availability statement

All data generated or analysed during this study are included in this published article.