Patient’s characteristics
The baseline characteristics of the cohorts (Figure 1) and pathology data are shown in Table 1. Briefly, the patient mean age was 66 (standard deviation (SD) 8.2) years in the NAT group and 62 (SD; 9.1) years in the UFS group. In the NAT group 33% of patients (n=6) presented pathological complete response and 67% (n=12) were classified T1 or T2 according to the eighth edition of the American Joint Committee on Cancer TNM staging system (19). In the UFS group most tumors were T3 or T4 (88% of cases, n=15) and a minority were T2 (n=2, 12%). Three patients (17%) received adjuvant chemotherapy in NAT group versus 12 patients (71%) in the UFS group.
Table 1
Clinicopathologic Characteristics of the Study Patients
Characteristics | Neo-Adjuvant treatment group (n = 18) | Up-front surgery group (n = 17) |
| No of patients (%) | No of patients (%) |
Age, y | | |
≤60 | 4 (22) | 2 (12) |
>60 | 14 (88) | 15 (88) |
Sex | | |
Male | 12 (67) | 10 (59) |
Female | 6 (33) | 7 (41) |
Differentiation | | |
Well | 8 (44) | 5 (29) |
Moderate | 7 (39) | 7 (42) |
Poor | 3 (17) | 5 (29) |
Lymph node invasion | | |
Yes | 0 (0) | 17 (100) |
No | 18 (100) | 0 (0) |
T category | | |
T0 | 6 (33) | 0 |
T1-T2 | 12 (67) | 2 (12) |
T3-4 | 0 | 15 (88) |
Adjuvant treatment | | |
Yes | 3 (17) | 12 (71) |
No | 15 (83) | 5 (29) |
GEMCITABINE | 2 (11) | 4 (24) |
mFOLFIRINOX* | 1 (6) | 13 (76) |
*modified Folfirinox |
Residual molecular disease in tumor tissues
The patient populations included exclusively patients undergoing PDs. We evaluated whether molecular quantification of mutant KRAS paralleled the pathological response in the NAT group or was predictive of worse prognosis in both groups.
First, we determined that KRAS mutant allele frequency (MAF) positivity threshold was 1.02%, using non tumor DNA. An example of positive and negative KRAS mutation detection is presented in Figure 2A. Positivity threshold was calculated as the mean + 3 SD of MAFs from 5 healthy pancreatic and 2 healthy duodenum tissue samples (a total of 7 KRAS wild type DNA controls, Figure 2B).
Next, we quantified KRAS MAFs in tumor tissues from patients who underwent UFS, and found an average value of 23% ±20.5% (Figure 3A). In resected tumor tissues from patients receiving NAT, MAFs were significantly higher in the ypT1 group than the ypT0 group (p=0.04; Figure 3B), as expected. Noticeably, and confirming the validity of the 1.02% positivity threshold, we observed a mean MAF value of 1.1%±0.2% in the ypT0 group, close to the threshold. By contrast the average values of the ypT1 group were 3-fold higher (3.5% ±2.62%), showing concordance between the pathological response and the molecular residual disease.
Thus, molecular detection and quantification of mutant KRAS in tumor tissues is in agreement with expected results.
Tumor and venous margin KRAS MAF assessment
Within the course of patient follow-up after PD, pathological R0 tumor or venous margins do not carry reliable prognostic value since the majority of patients display disease relapse (20). We hypothesized that considering the fibrotic nature of PDAC, the identification of residual tumor cells after NAT is very challenging for the pathologist. It is even more difficult after UFS since tumor cells might blend into the surrounding healthy tissue. The same difficulties might arise in venous margin pathological analysis. We tested the possibility to detect residual tumor cells in tumor and venous margins by the molecular presence of KRAS mutations in the resected margins.
The average MAFs in the resected margins of UFS samples was 1.0 % ±0.99, in agreement with the molecular positivity threshold (Figure 3C). However, detailed analysis showed that only 2 out of 17 tumor margins (12%) had MAFs>1.02%, suggesting that most of the resection margins were tumor cell free. Venous margins tended to have higher MAFs although without statistical difference compared to resection margins (mean MAF of 2.08 % ±3.9, p=0.07, Figure 3D). Again, although 7 out 17 (41%) of venous margin were positive, it was not statistically different from the positive tumor margins (p=0.26).
In the NAT group, the average MAF in the resected margins was 1.0 % ±0.25 and 9 out 18 samples (50%) were above the positivity threshold (Figure 3E). KRAS positivity in venous margins was 1.01% ±0.38 (p>0.05 as compared to resected margins), and 5 out 18 (28%) were above the positivity threshold. The mean MAFs in resection or venous margins were not different in the UFS and NAT groups (Figure 3F).
Survival analysis based on residual molecular disease
Mean MAFs or proportions of KRAS-positivity in margins were not different in the UFS and in the NAT groups. We evaluated whether they were associated with patients’ survival. The mean follow-up period was 33 months ± 17 months (median = 27 months; range: 7-64 months). First, the NAT group showed longer recurrence free survival (RFS) and overall survival (OS) as compared to the UFS group (58.9 months vs 13.9 months, p<0.01 and 64.4 vs 21.2 months, p<0.01, respectively, Supplementary Figure 1).
Expectedly, in the UFS group, high KRAS MAFs in tumor tissues tended to be associated with shorter recurrence free survival (RFS, 6.7 months for MAFs>23.3% versus 16.5 months for MAFs <23.3, p=0.09, Supplementary Figure 2A), even if the analysis did not reach significance probably because of the small size of the cohort. This trend was less marked for overall survival (OS, median survival of 21.2 months for MAFs>23.3% versus 18.9 months for MAFs <23.3, p=0.08, Supplementary Figure 2B).
Combined analysis of both groups showed no statistical difference in the UFS for positive versus negative tumor margins (p = 0.13; Figure 4A) or for positive versus negative venous margins (p=0.28; Figure 4B). In the same way, negative margins did not correlate with longer OS (Figure 4C and 4D, p=0.52 for resection margins and p=0.17 for venous margins).
Double negative margins (i.e. patients for whom both margins were negative) did not correlate with longer RFS (Figure 4E, p=0.5) or OS (Figure 4F, p=0.12).
Finally, in the NAT group, double pathological (ypT1) and margins (either resection or venous) positivity did not identify patients with shorter RFS as compared to single positives (Figure 4G, n=9 patient, p=0.86) or OS (Figure 4H, p=0.81).
Together, these results show that the detection of molecular residual disease in the resection or venous margins of UFS or NAT PDAC patients does not carry prognostic value.
In the same way, we tested whether carrying double-positive margins (resection and venous) or double-negative margins did not correlate with local or distant recurrence.