Clinical validation of a ctDNA-Based Assay for Multi-Cancer Detection: An Interim Report from a Vietnamese Longitudinal Prospective Cohort Study of 2795 Participants

Figures & data

Figure 1. Overall flow chart of study subject selection and follow-up of K-DETEK. K-DETEK is a multi-center, prospective cohort study recruiting participants aged 40 or older who present at the Outpatient Clinic of one of the 13 hospitals and research institutes in five regions in Vietnam for periodic follow-up visits for their chronic conditions or for annual health check-ups beginning in April 2022. The estimated number of participants to be enrolled in the study is 3000. The inclusion criteria include: (i) 40 years of age or older, (ii) have neither clinical suspicion of cancer nor history of confirmed cancer, (iii) agree to be contacted at 6 months and 12 months following enrollment to collect information about general health status and progress of cancer, (iv) have no history of blood transfusion or bone marrow transplantation within the past 3 years, and (v) have no clinical manifestation of dementia. Eligible participants provided 10 ml of blood for analyzing cfDNA by SPOT-MAS assay. After 30 days, each participant received the results of either “ctDNA signal detected” or “ctDNA signal not detected.” The study participants were scheduled for follow-up visits at 6 and 12 months. This study reported interim results of 2795 participants 6 months from study initiation.

Table 1. Imaging tests for “signal-detected” cases.

Type of cancer	Imaging test^a
Liver cancer	Abdominal CT scan with contrast
Lung cancer	Chest CT scan with or without contrast
Breast cancer	Mammography or breast ultrasound/breast MRI for participants who have had mammography within the past three months
Colorectal cancer	Colonoscopy
Gastric cancer	Endoscopy
Other cancers	Suggest full-body C.T. scans at physicians’ discretion

^aTest recommended by The National Comprehensive Cancer Network (NCCN) guideline for cancer screening. CT: Computed tomography; MRI: Magnetic resonance imaging.

Table 2. Clinical characteristics of 2795 study participants.

Characteristics	Total
	N = 2795	%
Age (years, S.D.)
Median	51
Min	40
Max	88
Sex
Male	1180	42.2
Female	1615	57.8
High-risk factors^a
Yes	887	31.7
No	1908	68.3
Smoking status
Yes	385	13.8
No	2340	83.7
UNK	70	2.5
Drinking habit
Yes	539	19.3
No	2185	78.2
UNK	71	2.5
Hepatitis B
Yes	151	5.4
No	2369	84.8
UNK	275	9.8
Hepatitis C
Yes	17	0.6
No	2465	88.2
UNK	313	11.2
First-degree relatives diagnosed with two types of cancers
Yes	70	2.5
No	2622	93.8
UNK	103	3.7
First-degree relatives diagnosed with cancers and younger than 45 years old
Yes	56	2.0
No	2624	93.9
UNK	115	4.1
Carrying cancer inherited mutation
Yes	21	0.8
No	1197	42.8
UNK	1577	56.4

^aHigh-risk factors include smoking/drinking habits, hepatitis B/C infection, first-degree relatives diagnosed with cancer (<45-year-old or ≥2 cancers), cancer inherited mutation’s carriers; UNK, unknown/missing data.

Figure 2. Geographical location of participating centers and their contribution to the total participants in K-DETEK study. (A) K-DETEK is a prospective, multi-center clinical study that enrolled 2795 participants from 13 hospitals and 1 research institute in Vietnam between April 2022 and July 2022​. These research centers were grouped into five regions including two major cities (Hanoi and HCMC) and three remote regions (Central, Southeast and Mekong Delta). (B) Pie chart showing the distribution of participants across five regions.

Figure 3. The influences of sample processing time and plasma hemolysis on sequencing library yield of cfDNA. (A,B) Bar plots showing the differences in logistic time (A) and plasma hemolysis rate (B). (C,D) Violin plots showing the impact logistic time (C) and plasma hemolysis rate (D) on cfDNA amount per ml plasma. (E,F) Violin plots showing the impacts of sample processing time (E) and hemolysis rates (F) on the efficiency of library yields. Mann–Whitney–Wilcoxon test two-sided with Bonferroni correction was performed to compare the fold changes in DNA library yields across different patient groups. ns: not significant; *, p < 0.05; ****, p < 0.00001.

Figure 4. Association between participants’ clinical features and plasma cfDNA concentration. (A,B) Box plots showing the differences in cfDNA amount stratified by gender (A) and median age (B). (C) Box plots showing the differences in cfDNA stratified by both age and gender. (D) Box plots showing the differences in cfDNA concentration between medium risk and high-risk participants. Wilcoxon rank sum test performed to compare the median of cell free DNA amount across different patient groups. ***, p < 0.0001; ****, p < 0.00001.

Table 3. Associations between participants’ clinical features and amount of cfDNA per ml plasma.

Clinical features	N = 2795	cfDNA amount (ng) per ml plasma
		Median	IQR	Range	p-Value^a
Age
<51	1428	3.30	2.4–4.5	0.6–39	2.239E-20
> =51	1367	3.90	2.7–5.7	0.6–52.9
Gender
Female	1615	3.30	2.4–4.8	0.6–51.9	1.653E-10
Male	1180	3.90	2.7–5.4	1.2–39
Liver hepatitis
Yes	404	3.90	3–5.4	1.2–24.6	2.486E-07
No	2391	3.60	2.4–5.1	0.6–51.9
Alcohol consumptions
Yes	539	3.90	2.7–5.4	1.2–22.5	0.07807
No	2256	3.60	2.4–5.1	0.6–51.9
Smoking
Yes	385	3.90	2.7–5.4	1.2–22.5	0.1004
No	2410	3.60	2.4–5.1	0.6–51.9
High-risk factors
Yes	887	3.90	3–5.4	0.6–51.9	1.552E-12
No	1908	3.60	2.4–4.8	0.6–39

^ap-Values were calculated by a two-sided Mann–Whitney–Wilcoxon test with Bonferroni correction.

Table 4. Cases with signal-detected reports.

Signal-detected cases n = 13	Clinical features	ctDNA status	Tumor prediction	Diagnosis test	Confirmed diagnosis
With diagnostic test
1	Female, age 54	+	Other cancer	Endoscopy	Endometrial cancer
2	Female, age 65 Type 2 diabetes	+	Liver/ colorectal cancer	Colonoscopy	Colorectal carcinoma
3	Male, age 64 Epigastric pain	+	Liver cancer	Endoscopy	Stomach adenocarcinoma
4	Female, age 68 Gastritis	+	Breast cancer	Mammography, breast U.S.	Breast adenocarcinoma
5	Male, age 60 Hypertension, type 2 diabetes	+	Liver cancer	C.T. scan, abdominal ultrasound	Hepatocellular Carcinoma
6	Male, age 67 Echinococcus infection	+	CRC/Liver	CT scan	Cholangiocarcinoma – liver metastasis
7	Female, age 64	+	Breast/ colorectal cancer	Colonoscopy, MRI	Negative Colonoscopy: Hyperplastic sigmoid colon polyp (benign lesion) MRI: (−)
8	Female, age 56 Intercostal Neuralgia	+	Breast/ colorectal cancer	MRI, colonoscopy	Negative
9	Female, age 61	+	Breast cancer	Mammography, breast U.S.	Negative
10	Female, age 46	+	Breast cancer	Breast U.S.	Negative Breast U.S.: (−) Abdominal U.S.: a right hepatic cyst and a left functional ovarian cyst
No diagnostic test
11	Female, age 62	+	Breast cancer	–	Patient delays taking the diagnostic test
12	Female, age 48	+	Colorectal/ Breast cancer	–	Patient delays taking the diagnostic test
13	Male, age 51	+	Liver/Gastric	–	Patient delays taking the diagnostic test

CT: Computed tomography; MRI: Magnetic resonance imaging; U.S.: ultrasound; FNA: fine needle aspiration.

Table 5. SPOT-MAS performance and tumor origin accuracy.

	Total (N = 2795)
ctDNA signal detection	n (%)
Detected	13 (0.47)
True positive (confirmed cancer lesion)	6 (0.21)
False positive (confirmed cancer-free)	4 (0.14)
No diagnostic confirmation	3 (0.11)
Not detected	2782 (99.5)
PPV for cancer signal detection	%
	60
Prediction accuracy of tumor origin	%
Overall prediction accuracy	83.3

Figure 5. SPOT-MAS and confirmed diagnosis tests result of a case with detectable ctDNA in K-DETEK study. (A) A 60-year-old male (case#5) with unknown cancer status or symptoms was enrolled in K-DETEK study. His SPOT-MAS test was “ctDNA detected” and predicted to have liver cancer or colorectal cancer with probabilities of 75% and 25%, respectively. (B) After receiving the SPOT-MAS test result, the patient was recommended to undertake an ultrasound test, which revealed a hypo-echoic mass of 3.5 × 3.3 cm located in right lobe segment VIII of the liver, suspecting of hepatocellular carcinoma. His following CT-scan confirmed the presence of tumor in the same location.

Data availability statement

Sequencing data will be deposited in a public portal database (NCBI SRA) upon acceptance and are available on request from the corresponding author, LST. The data are not publicly available due to ethical restrictions.