Patient Characteristics and Follow-Up

Patient characteristics are shown in Table 1. A total of 7882 person-years of follow-up were obtained (mean 8.8 years, SD 6.5) on the 875 patients followed for > 1 month. Of the 875 patients, 251 were followed until death. Disease status at last follow-up was known on 822 (94%) of the 875 patients.

Table 1 Descriptive summary of patient characteristics at the time of surgery and in follow-upMethods of Determining Ki-67 Labeling Index

One hundred of the 534 in which the pathologist’s assessment was used to determine the Ki-67 labeling index were freshly stained for Ki-67 (to avoid assessing the labeling of the original slides which may have faded over time), and at least 500 cells in a hot spot were manually recounted. As has been reported in other tumor types, a strong positive linear relationship (r=0.78) was observed when the Ki-67 labeling index determined by manual counting of these 100 PanNETs was compared to the original pathologist’s count [57].

Prognosticators in the Entire Cohort

OS for the entire cohort is shown in Fig. 1A, and a sub-analysis of OS by stage in Supplemental Fig. 1. Vascular invasion (HR 3.00, 95% CI: [2.31, 3.91], p<0.0001), tumor size ≥ 2 cm (HR 2.88, 95% CI: [2.10, 3.93], p<0.0001), perineural invasion (HR 2.42, 95% CI: [1.88, 3.12], p<0.0001), positive margins (HR 2.18, 95% CI: [1.56, 3.04], p<0.001 for the overall cohort and HR 3.10, 95% CI: [2.18, 4.43], P≤0.0001 excluding enucleations), age ≥60 years (HR 2.08, 95% CI: [1.62, 2.68], p <0.0001), and male sex (HR 1.46, 95% CI: [1.14, 1.89], p=0.003) were all associated with worse OS (Table 2). Insulinoma (HR 0.34, 95% CI: [0.19, 0.62], p<0.001), sclerosing variant (HR 0.47, 95% CI: [0.22, 1.00], p=0.05), and cystic variant (HR 0.61, 95% CI: [0.38, 0.99], p=0.05) were associated with improved OS. Although DAXX and ATRX mutation status has been shown previously to correlate with prognosis, DAXX and ATRX mutations or immunolabeling were not specifically examined in these tumors. Grade, T stage, N stage and M stage, as defined by the World Health organization (WHO) and Union for International Cancer Control (UICC), were also all statistically significant classifiers of outcome [7, 43, 58].

Fig. 1

A Kaplan-Meier curves for survival for the entire cohort. B Changes in the hazard ratio for death with increases in tumor size, estimated with restricted cubic splines. The plotted results represent the hazard ratio for different values of the continuous predictor (tumor size), visualizing any non-linear effects captured by the restricted cubic spline. C Kaplan-Meier curves for overall survival, separately according to grade. D Changes in the hazard ratio for death with increases in Ki-67, estimated with restricted cubic splines. The plotted results represent the hazard ratio for different values of the continuous predictor (Ki-67), visualizing any non-linear effects captured by the restricted cubic spline. E Predicted probability [95% CI] of being diagnosed with stage I, II, III, or IV disease (y-axis) by year of surgery grouped into quintiles (x-axis). Probabilities and corresponding odds ratios for the average relative change in the odds of a stage I, II, or III diagnosis compared to stage IV with every increasing quintile of year of surgery, adjusted for age and sex, are estimated from a multinomial regression model

Table 2 Overall Survival: Estimates of survival after surgery for the whole cohort and according to patient subgroups defined at the time of surgery, from univariate Cox proportional hazards models

Non-linear models for the association between tumor size and survival revealed a stronger positive association between 0 and 4 cm that reduces but remains positive without an additional inflection point for tumors >4 cm (Fig. 1B).

Subdividing Grade 2 PanNETs

We found that patients with G1 PanNETs had 10-year OS of 81%, 95% CI: [77%, 86%], G2a PanNETs 68%, 95% CI: [61%, 76%], G2b PanNETs 44%, 95% CI: [29%, 66%], and G3 PanNETs 23%, 95% CI: [8%, 61%] (Fig. 1C and Table 2). When compared to patients with G2b disease, patients with G2a had a better overall survival (HR = 0.35, 95% CI: [0.22–0.56], p < 0.001) while patients with G3 disease did worse (HR = 1.91, 95% CI: [1.01–3.59], p = 0.05).

Plotting Ki-67 versus the HR (Fig. 1D) revealed a slight change to the slope of the curve at around a Ki-67 of 10%, supporting the introduction of a 10% threshold in the grading system.

Prognosticators in M0, N0M0 and T1N0M0 Cohorts

As the most significant surgical decisions present in patients free of metastases at diagnosis, we estimated OS in lower stage subgroups, including patients who were M0 at surgery (Supplemental Table 1), N0M0 at surgery (Supplemental Table 2) and T1N0M0 at surgery (Table 3). In particular, 251 patients had tumors < 2 cm and N0M0 disease. These patients lived a median of 27 years after surgery, and their ten-year overall survival was 88%, 95% CI: [83%, 93%]. Both positive margins (excluding enucleations, HR 19.5, 95% CI: [5.49, 66.8], p <0.0001) and vascular invasion, HR 5.7, 95% CI: [1.65, 19.8], p=0.006) were associated with poor outcome in this group.

Table 3 Overall Survival in Patients with T1N0M0 Disease: Estimates of survival after surgery for patients with M0, N0, and T1 disease, overall and according to patient subgroups defined at the time of surgery, from univariate Cox proportional hazards models

The differences in OS between G2a and G2b persisted for patients with M0 or N0M0 disease, but they were not statistically significant for these disease subgroups for comparing G3 to G2b. There were too few (N=2) patients with G2b disease among the T1N0M0 cohort to make any comparisons.

Multivariate Analyses

Multivariate analyses of OS were performed on four cohorts (all patients, patients with M0 disease, patients with N0M0 disease, and patients with T1N0M0 disease) (Supplemental Table 3) using the LASSO method. For each cohort, the following candidate variables were offered to the model selection: age, sex, year of surgery, tumor size, Ki67 (measured continuously), presence of insulinoma, sclerosing variant, cystic variant, perineural invasion or positive margins, T stage, N stage, and M stage. For the whole cohort, M stage (M1 vs M0, HR 2.46, 95% CI: [1.85, 3.30], p<0.001), perineural invasion (HR 1.62, 95% CI: [1.24, 2.09], p=0.002), N stage (N1 vs N0, HR 1.56, 95% CI: [1.19, 2.02], p=0.005), age (per 5 years) (HR 1.26, 95% CI: [1.20, 1.32], p <0.001), Ki-67 (HR 1.06, 95% CI: [1.04, 1.09], p <0.001), tumor size (per 1 cm) (HR 1.04, 95% CI: [1.01, 1.12], p=0.023), and year of surgery (per 1 year) (HR 0.96, 95% CI: [0.93, 0.97], p<0.001) were selected by the LASSO. Sex, vascular invasion and positive margins were also retained but not statistically significant. For the T1N0M0 cohort, age (per 5 years) (HR 1.38, 95% CI: [1.18,1.59], p<0.001), year of surgery (HR 0.97, 95% CI: [0.91, 1.25], p = 0.556) and Ki-67 labeling (HR 1.15, 95% CI: [1.08,1.23], p<0.001) were retained.

Cause-Specific Survival

Information on cause-specific survival for patients with M0 disease and M0N0 disease are presented in Supplemental Tables 4 and 5 respectively. With the exception of patient age (HR 1.29, 95% CI: [0.79, 2.11], p=0.31) and patient sex (HR 1.30, 95% CI: [0.80,2.10], p=0.30), all variables identified as statistically associated with OS remained significant. These results suggest that the patient age and sex findings observed with OS were due to the long follow-up obtained in this study, and not tumor-specific biological drivers.

Cumulative Incidence of Relapse

Next, we estimated the cumulative probabilities of disease relapse at 2, 5, and 10 years after surgery for the M0, M0N0 and T1N0M0 cohorts (Table 4 and Supplemental Tables 6 and 7). In the M0 cohort, tumor size (HR 6.19, 95% CI: [3.90, 9.85], p<0.001), vascular invasion (HR 4.41, 95% CI: [3.18, 6.12], p<0.001), and perineural invasion (HR 3.17, 95% CI: [2.31, 4.35], p<0.001) remained poor prognosticators, while insulinoma (HR 0.26, 95% CI: [0.11, 0.63], p=0.003), sclerosing variant (HR 0.27, 95% CI: [0.09, 0.84], p=0.02), and cystic variant (HR 0.29, 95% CI: [0.13, 0.65], p=0.003) were associated with improved outcome. T stage and N stage were also significantly associated with relapse.

Table 4 Cumulative Incidence of Relapse: Estimates of cumulative probability of relapse at 2, 5 and 10 years after surgery, accounting for death before relapse as a competing event, for the M0 cohort and according to patient subgroups defined at the time of surgery

As with OS, the cumulative incidence of relapse was associated with refined tumor grade. In the M0 cohort, patients with G1 PanNETs had 5-year cumulative incidence of relapse of 8%, 95% CI: [5%,11%], those with G2a PanNETs 25%, 95% CI: [19%, 32%], those with G2b PanNETs 62%, 95% CI: [43%, 80%], and those with G3 PanNETs 69%, 95% CI: [44%, 94%] (Table 4). These results again support the inclusion of a 10% threshold in the grading system.

For the 251 patients with T1N0M0 disease, 10 were missing information on recurrence, leaving 241 patients. Of these 241, 15 recurred and 29 died without relapse. The cumulative probability of relapse by 10 years was 6%, 95% CI: [3%, 10%] (Supplemental Table 7). Only three of the 251 patients died of their disease, and the disease-specific survival at 20 years was 96%, 95% CI: [92%, 100%].

Year and Age of Surgery

A comparison of the distribution of patients diagnosed with stage I, II, III, or IV disease by year of surgery grouped into quintiles revealed a down shift in stage at diagnosis over time (Fig. 1E). Over the study period, patients were more likely to be diagnosed with stage I (Odds Ratio (OR) 1.61, 95% CI: 1.34, 1.93, p <0.001), stage II (OR 1.30, 95% CI: 1.08, 1.55, p =0.005), or stage III disease (OR 1.2, 95% CI: 1.00, 1.45, p=0.054) compared to stage IV disease.

Hypothesizing that a down shift in stage over time would be associated with an earlier age at diagnosis, we next examined trends in age at surgery by year of surgery (Supplemental Fig. 2). Surprisingly, and perhaps reflecting regional referral practices, we observed a slight increase in the mean age at diagnosis over time, with mean age increasing 1.08 years every five years (95% CI: 0.51, 1.65, p<0.001).