A total of 662 dogs were included in this study (528 dogs with cancer and 134 healthy dogs). The most common cancers evaluated included lymphoma (LSA; n=126), Hemangiosarcoma (HSA; n=77), osteosarcoma (OSA; n=49), soft tissue sarcoma (STS; 51), malignant melanoma (n=49), mast cell tumors (MCT; n=126) and histiocytic sarcoma (n=26) (Figure 1). A variety of carcinomas (mammary, pulmonary, hepatocellular, squamous cell, urothelial cell and anal sac carcinomas) as well as a few miscellaneous tumors (multiple myeloma, acute leukemias, insulinoma, nasal chondrosarcoma, sertoli cell tumor and others) were also evaluated. These cases are described here however, due to the low case numbers of any specific histology, they are not included in the statistical analyses reported here.
Of the 528 dogs with cancer, 244 were spayed females, 15 were intact females, 237 were neutered males and 30 were intact males. Gender was unknown for 2 of the dogs. These dogs ranged in age from 1 – 19 years (median 9 years, mean 9.06 years) and ranged in weight from 5 - 74.5 kg (median 30.9 kg, mean 29.63 kg). The most commonly represented breeds included mixed breed dogs (n=126), Labrador retriever (n=58), golden retriever (n=53) and boxers (n=19).
A total of 134 healthy dogs were recruited for this study ranging in age from 10 months to 14 years (median 6 years). There were 61 spayed femles, 4 intact females, 66 neutered males and 3 intact males. The most common breeds represented were mixed breed dogs (n= 28), Labrador retreivers (n=15) and Australian cattle dogs (n=10). Dogs were determined to be healthy based on results from a client questionnaire as well as a physical exam by the attending veterinarian. The median nucleosome concentration for all healthy dogs was 31.1 ng/mL (mean 32.07 ng/mL, SEM 1.118). For a specificity of 100% the cut off for the healthy range was set at 67.5 ng/mL (nucleosome range for all healthy dogs was 6.33 - 67.42 ng/mL).
When all cancer cases were considered together in comparison to healthy dogs the sensitivity of this assay was 49.8% with a specificity of 97% (Figure 1). A receiver operator characteristic (ROC) curve was generated and the AUC was determined to be 68.74% (Figure 2). The top 3 malignancies detected by the test included lymphoma, hemangiosarcoma and histiocytic sarcoma. The malignancies least likely to be detected using this assay were soft tissue sarcomas and mast cell tumors.
H3.1 Nucleosome Levels Across Multiple Cancer Types
For the purposes of group comparisons, a single specificity of 97% was chosen and the sensitivity was then calculated using this parameter. The cancers with the highest sensitivities were lymphoma (76.98%), hemangiosarcoma (81.82%), visceral histiocytic sarcoma (61.9%) and malignant oral melanoma (50%). The cancers with the lowest sensitivity were cutaneous melanoma (0%), Mast cell tumors (19.05% all grades, grade 1: 33.33%, grade 2: 11.49% and grade 3: 34.62%) and primary bone histiocytic sarcomas (20%) (Table 1). Just under 35% (17/49) of the osteosarcoma cases demonstrated elevated plasma nucleosome concentrations at diagnosis. Interestingly, the dog with the highest plasma nucleosome concentration in the osteosarcoma group (446 ng/mL) was the one dog that presented with metastatic disease at diagnosis (Tables 1 and 2).
When considering the plasma nucleosome concentrations of dogs with a variety of carcinomas, eight of the sixteen cases had elevated levels at the time of diagnosis. Two of the three of the anal sac apocrine gland adenocarcinomas had elevated plasma nucleosome levels (101.2 ng/mL, 816.2 ng/mL, and 33.8 ng/mL). Two of the three urothelial cell carcinoma cases had elevated plasma nucleosome concentrations at the time of diagnosis (162.3 ng/mL, 129.6 ng/mL and 16.7 ng/mL). There were 5 dogs with sinonasal or oral carcinomas in this study. Two of the 5 had elevated plasma nucleosome concentrations at the time of diagnosis (101.8 ng/mL, 110.7 ng/mL, 53.5 ng/mL, 42.1 ng/mL, 8.2 ng/mL). The two dogs with elevated levels were both diagnosed with oral squamous cell carcinomas. There were 3 dogs in this group with primary lung carcinomas, all of which had elevated nucleosome concentrations (76.2 ng/mL, 94.4 ng/mL, 548.5 ng/mL). One dog with mammary carcinoma and one dog with hepatocellluar carcinoma were also included in this study. Neither dog had elevated nucleosome concentrations (25.0 ng/mL and 28.5 ng/mL respectively).
Several other tumors were included in this study as well. Both dogs with acute leukemia had elevated plasma nucleosome concentrations (lymphoblastic leukemia – 262.8 ng/mL, myeloblastic leukemia 423.5 ng/mL). We also saw elevated plasma nucleosome concentrations in a dog with a sertoli cell tumor (118 ng/mL), a dog with a pharyngeal sarcoma (135.4 ng/mL), a dog with nasal chondrosarcoma (109.25 ng/mL) and a dog with multiple myeloma (71.7 ng/mL). There were three cases that did not have elevated plasma nucleosome concentrations including a dog with an undifferentiated splenic sarcoma (16.4 ng/mL), a dog with insulinoma (8.5 ng/mL) and a dog with multilobular osteochondrosarcoma (20.3 ng/mL).
Table 1
Summary of the Sensitivity and specificity of plasma nucleosomes to detect a variety of canine cancers.
Cancer Histology
|
Number of cases
|
Elevated Nu.Q® level
|
Sensitivity
|
Specificity
|
AUC
|
All Cancers
|
504
|
251/504
|
49.8%
|
97%
|
68.74%
|
Lymphoma
|
126
|
97/126
|
76.98%
|
97%
|
87.83%
|
Hemangiosarcoma
|
77
|
63/77
|
81.82%
|
97%
|
91.74%
|
Histiocytic Sarcoma
|
26
|
14/26
|
53.85%
|
97%
|
83.01%
|
Hist Sarc - bone
|
5
|
1/5
|
20%
|
97%
|
81.04%
|
Hist Sarc Visceral
|
21
|
13/21
|
61.9%
|
97%
|
83.48%
|
Melanoma (all)
|
49
|
21/49
|
42.86%
|
97%
|
70.36%
|
Melanoma (20)
|
42
|
21/42
|
50%
|
97%
|
75.05%
|
Melanoma (cutaneous)
|
7
|
0/7
|
0%
|
97%
|
42.22%
|
Mast Cell Tumor
|
126
|
24/126
|
19.05%
|
97%
|
43.68%
|
Grade 1 MCT
|
9
|
3/9
|
33.33%
|
97%
|
44.1%
|
Grade 2 MCT
|
87
|
10/87
|
11.49%
|
97%
|
11.49%
|
Grade 3 MCT
|
26
|
9/26
|
34.62%
|
97%
|
60.7%
|
Osteosarcoma
|
49
|
17/49
|
34.69%
|
97%
|
60.17%
|
Soft tissue sarcoma
|
51
|
15/51
|
29.41%
|
97%
|
48.19%
|
Table 2
Nucleosome concentrations by histology
H3.1 (ng/mL)
|
n
|
Minimum
|
Maximum
|
Median
|
Mean
|
SEM
|
p
|
HSA
|
77
|
6.54
|
1956.90
|
198.30
|
414.62
|
53.14
|
6.33E-24
|
LSA
|
126
|
0.10
|
6544.00
|
211.05
|
570.87
|
90.85
|
5.62E-26
|
OSA
|
49
|
0.10
|
446.00
|
43.20
|
72.70
|
12.85
|
0.0354
|
STS
|
51
|
0.10
|
2500.00
|
25.09
|
200.07
|
82.24
|
0.7043
|
MCT
|
126
|
0.10
|
969.45
|
24.65
|
64.21
|
11.67
|
0.0784
|
MCT Grade unk
|
4
|
11.02
|
174.58
|
83.77
|
88.29
|
43.03
|
0.8341
|
MCT Grade 3
|
26
|
8.39
|
969.45
|
38.21
|
149.74
|
49.46
|
0.0849
|
MCT Grade 2
|
87
|
3.10
|
337.23
|
22.67
|
37.52
|
5.85
|
0.0029
|
MCT Grade 1
|
9
|
0.10
|
313.81
|
18.26
|
64.41
|
33.04
|
0.5579
|
Melanoma
|
49
|
7.28
|
1234.50
|
44.59
|
115.73
|
27.72
|
2.53E-05
|
Histiocytic sarcoma
|
26
|
21.81
|
1800.00
|
69.59
|
261.22
|
81.31
|
1.06E-07
|
Healthy
|
134
|
6.33
|
67.42
|
31.10
|
32.07
|
1.12
|
1
|
Detailed evaluations of plasma nucleosome concentrations in dogs with lymphoma and hemangiosarcoma by stage, location or phenotype have been described elsewhere (18, 19). For the cases with melanoma there were two main groups. The first represents cutaneous melanoma of haired skin which are often considered benign (n=7) and those in the oral cavity (n=42) which are often malignant. The median plasma nucleosome concentration for the dogs with cutaneous melanoma was 24.8 ng/mL (mean 27.6 ng/mL, range 7.3-43.9) and the median size of these tumors was 3 cm (mean 4.7 cm, range 1.8-12 cm) (Figure 3). The median plasma nucleosome concentration for dogs with oral melanoma was 60.0 ng/mL (mean 130.422, range 14.0-1234.5 ng/mL) and the median size of these tumors was 4 cm (mean 4.5 ng/mL, range 1.5-15 cm)(Figure 3). Mitotic index was not available for evaluation.
Mast cell tumors were divided by recorded grade. All tumors in the DCTD biobank were categorized according to the Patnaik 3 tier grading scheme. Grade 3 tumors had the highest mean plasma nucleosome concentrations (38.2 and 149.7 ng/mL respectively) followed by grade 1 tumors. Grade 2 tumors had the lowest nucleosome concentrations of the group (Figure 4,Table 2). Follow up information regarding overall survival was not available for the dogs with elevated plasma nucleosome concentrations.
Overall, visceral histiocytic sarcomas as a group had elevated plasma nucleosome concentrations similar to lymphoma and hemangiosarcoma. Those cases involving the spleen had the highest median plasma nucleosome concentrations. Histiocytic sarcomas involving the bone had the lowest median plasma nucleosome concentrations (Figure 5)(19).