Database and samples

The data from the Surveillance, Epidemiology, and End Results (SEER) database was acquired from the SEER*Stat 8.3.6 Software (https://seer.cancer.gov/data/), and we accessed the SEER database on March 1, 2020. A total of 2875 AVC patients who underwent surgical resection and lymph node dissection (RLNs ≥ 1) from January 2004 to December 2013 were identified by that primary site of tumor was C24.1-Ampulla of Vater, year of diagnosis was from 2004 to 2013, surgery of primary tumor was encoded from 20 to 90 (such as simple/partial surgical removal of primary site, total surgical removal of primary site, radical surgery), number of lymph nodes examined ≥ 1, diagnostic confirmation was positive histology, and type of follow-up expected was active follow-up. A number of 510 patients who < 18 years old or > 80 years old, died within 1 month, and had M1, MX, or NX diseases were excluded. Finally, 2347 AVC patients with M0 disease were included in the study.

The data collected in this study included age at diagnosis, gender, race, marital status at diagnosis, tumor size, tumor grade, lymph node metastasis, number of RLNs and PLNs, adjuvant radiotherapy and chemotherapy.

Statistical analysis

Continuous variables and categorical variables were presented as median (range) and frequency (percentage). Overall survival (OS) and disease-specific survival (DSS) rates were calculated from diagnosis to death due to any reason and from diagnosis to death due to VAC, respectively. The last follow-up time was November 2018. To get the optimal cut-off value of RLNs, the survival analysis performed by Kaplan-Meier method (log-rank test) was compared the 5-year OS and DSS rates of different cut-off values of RLNs in N0, N1 and entire cohorts, respectively, and it was used the 5-year survival rates and P values of different cut-off values of RLNs as line charts. The optimal number of PLNs was gained by X-tile software (Version 3.6.1, Yale University) and verified by the survival analysis. A two-tailed P < 0.05 was considered statistically significant. Statistical analysis was using STATA 16.0 software. Ethics statement was not required for this study, because SEER database was publicly available. In addition, Data-Use Agreements for the 1975–2017 SEER Research Data File and SEER Radiation Therapy and Chemotherapy Information were signed and the database can be accessed.

Baseline characteristics

During the period from January 2004 to December 2013, 2347 AVC patients with M0 disease who underwent surgical resection and lymph node dissection from the SEER database were included in this study (Table 1). The median age at diagnosis was 65 (20–80) years, and male accounted for about 56.8% (1333/2347). The vast majority (78.7%) were white, 7.3% (172/2347) blcak, and 13.9% (327/2347) other. A total of 33255 lymph nodes were examined with a median number of 13 (1–67), and a total of 4105 positive lymph nodes were found with a median number of 1 (0–31). Tumor size for 145 (6.2%) patients was unknown, and the median tumor size for the remaining patients (93.8%) was 21 (1–87) mm. The median follow-up was 39 (2–155) months. As of November 2018, 981 (41.8%) patients were alive and 1366 (58.2%) patients had died. In the entire cohort (n = 2347), the 1-, 3-, and 5-year OS rates were 85.1%, 56.4%, and 45.5%, respectively, and the corresponding DSS rates were 86.8%, 60.0%, and 50.8%, respectively.

Table 1

Demographics and clinical characteristics for AVC patients with M0 disease (n = 2347).

Demographics	Ampulla of vater cancer (n = 2347)
Age at diagnosis (years)	65(20–80)
Gender, male/female (%)	1333(56.8)/1014(43.2)
Race, n (%)
white	1848(78.7)
black	172(7.3)
other	327(13.9)
Marital status at diagnosis, n (%)
married	1509(64.3)
single/widowed/divorced	751(32.0)
unkown	87(3.7)
Tumor size (mm)	21(1–120)
Tumor grade, n (%)
well differentiated	272(11.6)
moderately differentiated	1206(51.4)
poorly differentiated	741(31.6)
undifferentiated	19(0.8)
unkown	109(4.6)
Lymph node metastasis, yes/no (%)	1211(51.6)/1136(48.4)
Number of retrieved lymph nodes	13(1–67)
Number of positive lymph nodes	1(0–31)
Adjuvant radiotherapy, yes/no or unkown (%)	609(25.9)/1738(74.1)
Adjuvant chemotherapy, yes/no or unkown (%)	1073(45.7)/1274(54.3)

The other comprises American Indian/Alaska Native, Asian/Pacific Islander.

Exploration for the optimal cut-off value of retrieved lymph nodes

For exploring the optimal cut-off value of RLNs, survival analysis was conducted to compared the 5-year OS and DSS rates of different cut-off values of RLNs in N0, N1 and entire cohorts for AVC patients with M0 disease was compared by survival analysis (Table 2), and it was used the 5-year survival rates and P values of different cut-off values of RLNs as line charts (Fig 1). In N0 cohort (n = 1136), the optimal cut-off value of RLNs was 18 due to the highest 5-year OS rate (71.4%) and DSS rate (77.7%) and separation of survival curves between patients with RLNs ≥ 18 and RLNs < 18 (Fig 1A and 1B; all P < 0.001). In N1 cohort (n = 1211), the optimal cut-off value of RLNs was 16 due to the highest 5-year OS rate (34.3%) and the third highest 5-year DSS rate (37.7%) and separation of survival curves between patients with RLNs ≥ 16 and RLNs < 16 (Fig 1C, P = 0.004; Fig 1D, P = 0.005). In the entire cohort (n = 2347), the optimal cut-off value of RLNs was 16 due to the highest 5-year OS rate (49.0%) and DSS rate (53.6%) and separation of survival curves between patients with RLNs ≥ 16 and RLNs < 16 (Fig 1E, P = 0.008; Fig 1F, P = 0.015).

Fig 1

Survival analysis comparing the different numbers of PLNs.

a: Survival analysis exploring the 5-year OS rates and P values for the optimal cut-off value of RLNs in N0 cohort (n = 1136). b: Survival analysis exploring the 5-year DSS rates and P values for the optimal cut-off value of RLNs in N0 cohort (n = 1136). c: Survival analysis exploring the 5-year OS rates and P values for the optimal cut-off value of RLNs in N1 cohort (n = 1211). d: Survival analysis exploring the 5-year DSS rates and P values for the optimal cut-off value of RLNs in N1 cohort (n = 1211). e: Survival analysis exploring the 5-year OS rates and P values for the optimal cut-off value of RLNs in entire cohort (n = 2347). f: Survival analysis exploring the 5-year DSS rates and P values for the optimal cut-off value of RLNs in entire cohort (n = 2347).

Table 2

Survival analysis comparing the 5-year OS and DSS rates of different cut-off values of RLNs in N0, N1 and entire cohorts for AVC patients with M0 disease (n = 2347).

Retrieved lymph nodes	N	Overall Survival (OS)		Disease-specific survival (DSS)
		5-year OS rate (%)	P value	5-year DSS rate (%)	P value
In N0 cohort	1136
< 2	44	49.8	0.007	57.8	0.029
≥ 2	1092	62.7		69.5
< 4	136	46.9	<0.001	55.1	<0.001
≥ 4	1000	64.4		71.0
< 6	241	51.1	<0.001	59.5	0.001
≥ 6	895	65.3		71.7
< 8	361	54.4	<0.001	61.7	<0.001
≥ 8	775	65.9		72.5
< 10	479	57.0	<0.001	63.9	0.002
≥ 10	657	66.1		72.9
< 12	585	57.1	<0.001	64.3	<0.001
≥ 12	551	67.7		74.2
< 14	692	58.2	<0.001	65.6	<0.001
≥ 14	444	68.8		74.6
< 16	785	58.4	<0.001	65.5	<0.001
≥ 16	351	71.0		77.2
< 18	862	59.4	<0.001	66.4	<0.001
≥ 18	274	71.4		77.7
< 20	929	61.0	0.018	67.8	0.010
≥ 20	207	67.8		75.0
< 22	987	61.4	0.051	68.2	0.037
≥ 22	149	67.8		75.2
< 24	1030	61.6	0.097	68.6	0.102
≥ 24	106	67.9		74.0
In N1 cohort	1211
< 2	7	14.3	0.192	19.0	0.311
≥ 2	1204	29.8		33.6
< 4	48	15.1	0.001	18.7	0.001
≥ 4	1163	30.3		34.2
< 6	113	21.7	0.019	27.3	0.035
≥ 6	1098	30.5		34.3
< 8	198	21.3	0.002	25.5	0.005
≥ 8	1013	31.4		35.1
< 10	304	25.7	0.062	29.9	0.075
≥ 10	907	31.0		34.8
< 12	434	24.9	0.003	29.3	0.007
≥ 12	777	32.5		36.0
< 14	557	25.2	0.001	29.8	0.002
≥ 14	654	33.7		36.9
< 16	684	26.4	0.004	30.5	0.005
≥ 16	527	34.3		37.7
< 18	787	27.8	0.062	31.5	0.031
≥ 18	424	33.5		37.4
< 20	877	28.2	0.079	31.9	0.020
≥ 20	334	33.7		38.1
< 22	951	28.5	0.063	32.1	0.015
≥ 22	260	34.2		38.8
< 24	1019	28.9	0.123	33.0	0.101
≥ 24	192	33.9		36.6
In entire cohort	2347
< 2	51	44.9	0.440	52.6	0.953
≥ 2	2296	45.5		50.8
< 4	184	38.6	0.006	45.6	0.093
≥ 4	2163	46.1		51.3
< 6	354	41.7	0.075	49.2	0.416
≥ 6	1993	46.2		51.1
< 8	559	42.8	0.057	49.1	0.278
≥ 8	1788	46.3		51.4
< 10	783	44.9	0.441	50.9	0.932
≥ 10	1564	45.8		50.8
< 12	1019	43.4	0.027	49.6	0.104
≥ 12	1328	47.1		51.8
< 14	1249	43.4	0.017	49.7	0.077
≥ 14	1098	47.9		52.2
< 16	1469	43.5	0.008	49.2	0.015
≥ 16	878	49.0		53.6
< 18	1649	44.3	0.061	49.8	0.074
≥ 18	698	48.4		53.4
< 20	1806	45.1	0.334	50.4	0.156
≥ 20	541	46.7		52.3
< 22	1938	45.2	0.405	50.5	0.200
≥ 22	409	46.4		52.3
< 24	2049	45.4	0.579	50.9	0.611
≥ 24	298	46.0		50.1

Exploration for the optimal cut-off value of positive lymph nodes

The media numbers of PLNs were 1 (0–31) in the entire cohort (n = 2347) and 1 (0–31) in patients with RLNs ≥ 16 (n = 878). Whether in the entire cohort or in patients with RLNs ≥ 16, the optimal cut-off values of PLNs were 0 and 2 obtained by X-tile software. In the entire cohort (n = 2347), patients with PLN = 0 demonstrated significantly better 5-year OS and DSS rates (62.1% and 68.9%) compared to those with PLNs = 1–2 (36.4% and 40.4%; Fig 2A and 2B; all P < 0.001), and patients with PLNs = 1–2 demonstrated significantly better 5-year OS and DSS rates (36.4% and 40.4%) compared to those with PLNs ≥ 3 (21.7% and 25.3%; Fig 2A and 2B; all P < 0.001). In patients with RLNs ≥ 16 (n = 878), patients with PLN = 0 demonstrated significantly better 5-year OS and DSS rates (70.9% and 77.1%) compared to those with PLNs = 1–2 (41.6% and 44.7%; Fig 2C and 2D; all P < 0.001), and patients with PLNs = 1–2 demonstrated significantly better 5-year OS and DSS rates (41.6% and 44.7%) compared to those with PLNs ≥ 3 (24.3% and 28.0%; Fig 2C and 2D; all P < 0.001).

Fig 2

Survival analysis comparing the different numbers of PLNs.

a: Survival analysis comparing the 5-year OS rates of the optimal cut-off value of PLNs (0, 2) in entire cohort (n = 2347). b: Survival analysis comparing the 5-year DSS rates of the optimal cut-off value of PLNs (0, 2) in entire cohort (n = 2347). c: Survival analysis comparing the 5-year OS rates of the optimal cut-off value of PLNs (0, 2) in patients with RLNs ≥ 16 (n = 878). d: Survival analysis comparing the 5-year DSS rates of the optimal cut-off value of PLNs (0, 2) in patients with RLNs ≥ 16 (n = 878).