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Table 1. AUC of cfDNA and the DNA integrity index for

screening distant metastasis.

AUC (95% CI)

Cut off value

cfDNA

DNA integrity index

0.833 (0.714–0.952)

0.752 (0.611–0.893)

852 ng/mL

0.62

Accuracy (%)

84.1

71.4

Sensitivity (%)

73.7

63.2

Specificity (%)

88.6

81.8

PPV (%)

73.7

52.0

NPV (%)

88.6

84.2

AUC = area under the receiver operating characteristics curve;

cfDNA = cell-free DNA; CI = confidence interval; NPV =

negative predictive value; PPV = positive predictive value.

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Table 2. Clinical characteristics of 6 patients assessed during follow-up study.

Age

Sex/

(y)

status

10

Castrated

Mix

10

Castrated

FB

13

Spayed

Patient

Breed

Tumor site

Urinary

Tumor

Stagea)

type

1st-line

2nd-line

Metastasis

Recurrence,

treatment

treatment

site, timing

timing

Lung,

TCC

T2N0M0

Surgery

COX2i

Subcutaneous

STS

T4N0M0

Surgery

LDCPA

Mix

Spleen

HS

Surgery

DOX

Male

Mix

MC

LSA

Stage Vb

Chemotherapy -

Castrated

FB

MC

LSA

Stage Va

Chemotherapy -

Castrated

CHH

Leukemia

Chemotherapy -

bladder

Spleen, liver,

PB, LN

T2N0M0,

stage II

day102

Peritoneum,

day60

Local site,

day48

Mandibular

LN, day220

Spleen, Liver,

PB, day205

a) According to the WHO staging system.23

CHH = Chihuahua; COX2i = cyclooxygenase-2 inhibitor; DOX = doxorubicin; FB = French Bulldog; HS = hemangiosarcoma; LDCPA =

low-dose cyclophosphamide; LN = lymph node; LSA = lymphoma; MC = multicentric; PB = peripheral blood; STS = soft tissue sarcoma;

TCC = transitional cell carcinoma.

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Figure 1. A. Box-plot of plasma cfDNA concentrations in dogs with malignant tumors or

335

benign diseases and healthy controls. B. cfDNA-integrity index. Each box indicates the 25th

336

and 75th percentiles. The horizontal line inside the box indicates the median, and the whiskers

337

indicate the extreme measured values.

338

cfDNA = cell-free DNA.

339

Figure 2. A. Box-plot of plasma cfDNA concentrationsin dogs with LSA, HS, and other

340

malignant tumors. B. cfDNA-integrity index. Each box indicates the 25th and 75th

341

percentiles. The horizontal line inside the box indicates the median, and the whiskers indicate

342

the extreme measured values.

343

cfDNA, cell-free DNA; HS, hemangiosarcoma; LSA, lymphoma/leukemia.

344

Figure 3. A. Box-plot of plasma cfDNA concentrations in malignant-tumor dogs with DM or

345

without DM. B. cfDNA-integrity index. Each box indicates the 25th and 75th percentiles. The

346

horizontal line inside the box indicates the median, and the whiskers indicate the extreme

347

measured values. cfDNA, cell-free DNA; DM, distant metastasis.

348

Figure 4. Receiver operating characteristic and AUC of plasma cfDNA concentrations (A)

349

and cfDNA-integrity index (B) to distinguish patients with distant metastasis from other

350

patients and controls. The AUC for cfDNA concentrations and the cfDNA-integrity index was

351

0.833 (95% CI: 0.714–0.952; p = 0.002) and 0.752 (95% CI: 0.611–0.893; p < 0.001),

352

respectively.

353

AUC, area under the curve; cfDNA, cell-free DNA; CI, confidence interval.

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Figure 5. Comparison of cfDNA and cfDNA-integrity index according to treatment response.

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cfDNA levels (solid line) and the cfDNA-integrity index (dotted line) at various time points in

356

6 patients. The vertical axis on the left represents cfDNA concentration, and the vertical axis

357

on the right represents the cfDNA-integrity index. The horizontal axis labels represent the

358

time points of plasma-sampling day and tumor status. A. Patient 1, transitional carcinoma of

359

the bladder treated with surgery and cyclooxygenase-2 inhibitor; B. Patient 2, undifferentiated

360

sarcoma treated with surgery; C. Patient 3, splenic hemangiosarcoma treated with surgery and

361

chemotherapy; D. Patient 4, lymphoma treated with chemotherapy; E. Patient 5, lymphoma

362

treated with chemotherapy; F. Patient 6, leukemia treated with chemotherapy. CR, complete

363

response; PD, progressive disease; PR, partial response.

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