Impact of resection margin status on survival in advanced N stage pancreatic cancer – a multi-institutional analysis

Background The present study aimed to examine the impact of microscopically tumour-infiltrated resection margins (R1) in pancreatic ductal adenocarcinoma (PDAC) patients with advanced lymphonodular metastasis (pN1–pN2) on overall survival (OS). Methods This retrospective, multi-institutional analysis included patients undergoing surgical resection for PDAC at three tertiary university centres between 2005 and 2018. Subcohorts of patients with lymph node status pN0–N2 were stratified according to the histopathological resection status using Kaplan-Meier survival analysis. Results The OS of the entire cohort (n = 620) correlated inversely with the pN status (26 [pN0], 18 [pN1], 11.8 [pN2] months, P < 0.001) and R status (21.7 [R0], 12.5 [R1] months, P < 0.001). However, there was no statistically significant OS difference between R0 versus R1 in cases with advanced lymphonodular metastases: 19.6 months (95% CI: 17.4–20.9) versus 13.6 months (95% CI: 10.7–18.0) for pN1 stage and 13.7 months (95% CI: 10.7–18.9) versus 10.1 months (95% CI: 7.9–19.1) for pN2, respectively. Accordingly, N stage–dependent Cox regression analysis revealed that R status was a prognostic factor in pN0 cases only. Furthermore, there was no significant survival disadvantage for patients with R0 resection but circumferential resection margin invasion (≤ 1 mm; CRM+; 10.7 months) versus CRM-negative (13.7 months) cases in pN2 stages (P = 0.5). Conclusions An R1 resection is not associated with worse OS in pN2 cases. If there is evidence of advanced lymph node metastasis and a re-resection due to an R1 situation (e.g. at venous or arterial vessels) may substantially increase the perioperative risk, margin clearance in order to reach local control might be avoided with respect to the OS. Supplementary Information The online version contains supplementary material available at 10.1007/s00423-021-02138-4.


Introduction
Current multimodal treatment strategies of pancreatic ductal adenocarcinoma (PDAC) have resulted in a significant survival benefit for patients with localized or even locally advanced tumours. High-impact randomized results of surgical resection followed by modern adjuvant multi-agent chemotherapy for localized PDAC have shown an overall survival of 28 to 54 months (using modified FOLFIRINOX) [1,2]. Of note, the best outcome was achieved within the R0 (microscopically tumourfree resection margin) subgroup of patients compared with cases in which the resection margins harboured microscopic tumour invasion (R1) [2]. Intraoperative surgical clearance of the resection margin is an ongoing matter of debate [3]. Radical R0 resection, especially in vulnerable and critical anatomic structures (e.g. the portal vein, arterial vessels, or the pancreatic neck margin), may significantly increase the intra-and perioperative risk for the patient and result in attempts of re-resection of the portal vein anastomosis, arterial reconstruction or completion pancreatectomy.
Besides R status, regional lymph node metastases (pN) are a strong prognostic survival determinant after resection of PDAC. This formed the basis for the recent revision of the N classification [4,5]. In accordance with the 8 th edition of the American Joint Committee on Cancer (AJCC) guidelines, PDAC is now classified based on the total number of infiltrated lymph nodes as pN1 (1-3 lymph nodes) and pN2 (>3 lymph nodes) [6]. Extended lymphadenectomy has still not shown any significant survival advantage compared with standard lymph node dissection so far; however, selected patients may benefit from this approach [7,8].
Therefore, although there is evidence that both the R and pN stage are significant prognostic factors, the question remains of whether local control (R0 status) matters in advanced lymphonodular positive disease (e.g. pN2 according to the new N classification). In gastric cancer surgery, for instance, a positive proximal resection margin is associated with advanced N stage; however, it is not independently associated with survival in these cases and recurrence is mainly systemic [9,10].
The present study therefore aimed to examine the clinical impact of the R status during PDAC resections in a multimodal treatment era including the use of neoadjuvant treatment on patients' survival with respect to the different lymph node stages (pN0-N2).

Study design, patients, and pathology reporting
The present study was designed as a retrospective German multicentre analysis. Data were collected from 3 different pancreatic cancer databases of the following tertiary university centres: Department of Visceral, Thoracic and Vascular Surgery, University Hospital Dresden (2005-2017), Department xof Surgery, University Hospital Erlangen (2010-2018), and Department of Surgery, University Medical Centre Schleswig-Holstein, Campus Lübeck (2013-2018). All patients undergoing surgical resection for PDAC within the indicated timeframes were retrospectively analyzed. The surgical procedures included pylorus-preserving (PPPD) or classic (cPD) pancreatoduodenectomy, total pancreatectomy (TP), distal pancreatectomy (DP), completion pancreatectomy, and the Appleby procedure (Appleby). During the study period, the three participating centres had increasingly utilized neoadjuvant chemotherapy protocols (e.g. FOLFIRINOX) for borderline resectable cases according to the international classification [11]. Tumours with unilateral narrowing (superior mesenteric or portal vein contact [SMV] of less than 180°) of the SMV/portal vein were usually classified resectable and resected upfront. Intraoperative resection margins of the pancreatic neck and bile duct were routinely sent for frozen section analysis. A re-resection for margin clearance was aimed at in cases of tumour cell infiltration. Pathology reports were retrospectively screened and the pN category was adjusted to pN1 (1-3 infiltrated lymph nodes) and pN2 (>3 infiltrated lymph nodes) according to the latest 8 th AJCC classification. All subjects were stratified into three subgroups with respect to pN category and resection status (R0 or R1) as follows: pN0-R0/1, pN1-R0/1, and pN2-R0/1. R0 status was defined as microscopic absence of tumour cells at the resection margin in all centres. Beginning in the year 2010, R0 resections were subdivided into CRM+ (tumour cells within 1 mm of the circumferential resection margin) and CRM− (no tumour cells within 1 mm of the CRM) in all three participating centres according to the national consensus guidelines. The CRM classification was considered for subsets of patients. The R0 CRM+ status corresponds to the redefined R1 resection margin involvement introduced by Verbeke et al. [12]. In addition, para-aortic lymph nodes of the Ln16b1 station (PALN) were analyzed separately if they were unequivocally labelled. Kaplan-Meier survival curves were generated with respect to the pN and R status.
Follow-up was defined as the time between the index operation and the last contact with a physician, which was obtained from the respective hospital information system or by contacting the patient's general practitioner.

Statistical analysis
The open source software package R Studio was used (Version 1.2.1335; https://rstudio.com/products/rstudio/) for the statistical calculations and creating data plots. Data are presented as median with interquartile range (IQR) or 95% confidence interval (95% CI) unless otherwise indicated. For comparing the patient subgroups, the t-test (continuous variables, normal distribution), Wilcoxon-Mann-Whitney (continuous variables, not normal distribution), or chi-square tests (categorical variables) were used. The statistical significance level was set a priori to P = 0.05 for all calculations. Kaplan-Meier survival curves for OS were generated considering the indicated parameters. OS was defined as the time between index operation and death or last appointment (censored). The logrank test was performed for testing differences between survival curves. For R-dependent survival analysis, Rx (unclear resection margin) and R2 (macroscopic residual tumour) resection status were excluded. Univariate Cox proportional model analysis was calculated including the indicated variables. The lymph node ratio (LNR) was calculated as the quotient between infiltrated and resected lymph nodes. Statistically significant parameters in the univariate model were included in the multivariate analysis model. The hazard ratio was calculated and is shown.
Most of the patients were resected with microscopically tumour-free resection margins (R0, n = 469, 75.6%). Within this R0 subgroup, the CRM classification was available for 379 cases: 191 (40.7%) cases did not show any tumour infiltration within 1 mm of the resection margins (CRM−), whereas tumour cells were found within 1 mm of the margin in 188 cases (CRM+; 40.1%). In 90 patients, a retrospective analysis of the microscopic specimen and reclassification according to CRM status was not possible because they were analyzed before the implementation of the CRM classification. An R1 resection status was found in 124 cases (20%). The majority of these cases (n = 42, 33.9%) had remaining PDAC cells at the pancreatic resection margin, followed by 28 patients (22.6%) having residual tumour within the medial (vessel) margin and 25 patients with a retroperitoneal R1 situation  Fig. S1).
We further aimed to investigate procedure-specific analysis for tumours localized in the pancreatic head or tail. Patients who underwent resections for head and body tumours (cPD, PPPD, or TP) showed a significant OS benefit of 34.4 months for R0 resections compared with 14.4 months for R1 resections in the pN0 subcohort (P < 0.001; n = 179). However, in the pN2 group (n = 132), no significant survival benefit could be detected (R0: 13.7 months vs. R1: 9.6 months, P = 0.3).
There were only 94 patients with DP for tumours localized in the pancreatic tail (pN0 = 42 cases, pN1 = 43 cases, pN2 = 9 cases). In this subgroup, a median OS benefit was observed for patients with R0 resections compared with R1 resected patients (R0: 24.2 months vs. R1: 17.7 months, P = 0.03). A subanalysis for pN2 patients after DP was not possible because there was only 1 patient with a R1 resection in this subgroup.
Recently, para-aortic lymph node station Ln16b1 (PALN) has re-emerged in the discussion of standard lymphadenectomy for PDAC [8,15]. Tumour invasion into this lymph node station is considered a distant metastasis. Furthermore, it is also an indicator of more advanced lymph node spread and aggressive tumour biology. Therefore, PALN metastases seem to clinically bridge the tumour spread from local (pN2) to distant metastasis (pM1). Currently, there is no consensus recommendation that these lymph nodes should be part of a standard oncologic lymphadenectomy. The present study cohort included 156 patients, where a PALN resection could be traced and analyzed based on the separately labelled lymph nodes in this area. Twenty-four (15.4%) of these cases displayed PALN tumour metastases (PALN+). PALN+ patients had a significantly worse OS of 8.5 (95% CI: 3.2-12.7) months compared with 19.6 months in PALN− patients (95% CI: 17.8-27; P < 0.001). The percentage of pN2 stages within the PALN+ patients was 63.0%. Subdivision according to R status was not applicable because of the low number of PALN+ cases with R1 status (n = 4).

Prognostic survival determinants
Portal vein and arterial resection, complications ≥ grade 3, haemoglobin ≤7 mmol/l, lymph node ratio (LNR), and the lymph node status pN were found to be independent negative prognostic OS factors in the entire patient cohort (Table 3). Adjuvant systemic treatment was associated with a longer OS survival rate. The likelihood ratio for the calculated multivariate analysis revealed a comprehensive model (P < 0.001). Moreover, the pN status-dependent univariate Cox analysis showed R1 resection to be a significant prognostic factor for

Discussion and conclusion
In the present study, resection margin status seems to lose prognostic relevance in patients with advanced lymphatic tumour cell spread. This is relevant because the analyzed data when obtained from an era when multimodal treatment with neo-or adjuvant (poly-) chemotherapy for PDAC was standard, and advanced lymph node metastasis stages (pN2) according to current guidelines were considered. Very recently, the 8 th edition of the AJCC manual for cancer staging was published, which includes a new TNM classification system for pancreatic cancer [6]. The former N1 stage was subdivided into the N1 (1-3 positive lymph nodes) and N2 (>3 positive lymph nodes) stages.
In patients with positive resection margins (R1), Morales-Oyarvide and colleagues demonstrated that lymph node metastases have a weaker prognostic value for the patients' disease-free and overall survival [16]. More importantly, several recent studies point to the fact that PDAC recurrence patterns (time or localization) are similar between R1 and R0 groups, especially in patients with N1 disease (7 th edition AJCC), and that lymph node status was predictive for time to recurrence, but not location of recurrence [17,18]. The OS of patients with distant or local recurrence was not significantly different, which confirms that PDAC behaves as a systemic disease [19]. It is accepted that R1 resection provides a survival advantage over palliative treatment and R0 resection offers improved survival compared with R1 resections when all cases (including lymph node negative and positive patients) are considered [20]. Our group also found that resection margin clearance at the pancreatic neck margin using frozen section analysis leads to improved survival if all lymph node stages are included in the analysis [3]. In contrast, advanced lymph node metastasis stages probably reflect more advanced systemic tumour burden, which determines the further course and prognosis of the disease. At least in pN2 stages, systemic lymphonodular spread seems to outweigh the survival difference between local R0 and R1 margins, because the presented data revealed no significant difference in OS rate between R0 and R1 status in pN2 staged patients. However, our retrospective data set did not qualify for a differentiated recurrence pattern analysis in pN0, pN1, and pN2 stages and recent studies on the recurrence patterns after PDAC resection did not consider the advanced pN2 stages separately [17,18,21].
Given the heterogeneity of the pathological assessment of resection margins, the circumferential resection margin (CRM) concept was incorporated into national treatment guidelines several years ago [12,22]. Therefore, an R0 Significant values are shown in bold resection is further classified as R0 CRM− (R0 wide) if microscopic tumour cells are found more than 1 mm from the resection margin. CRM+ cases (R0 narrow) show tumour cells within 1 mm of the resection margin but not within the margin itself. It has been shown that this concept comprehensively reflects the OS rate of PDAC patients [23]. To date, the classification of CRM status is still a matter of debate and no international consensus has been reached so far [24]. The CRM concept at least allows fine discrimination of the resection margin status or tumour cell density at the tumour margins. Patients with tumour cells within 1 mm from the resection margin (R0 CRM+) have a worse prognosis compared with R0 CRM− patients in pN0 or pN1 stages. Here again, no significant improvement in OS was observed for pN2 patients (P = 0.5). Surgical margin clearance during pancreatic resection can be demanding and is associated with increased intraoperative risk. As more and more patients are explored with locally advanced tumours and involvement of the portal/superior mesenteric vein or visceral arteries, intraoperative frozen section analysis is a routine procedure for intraoperative assessment of surgical margin clearance at these critical vascular structures. This may be the case if the portal vein anastomosis is accomplished and the final venous resection margins (e.g. analyzed by frozen section) at the vein or at the visceral arteries display tumour cells. Both re-resection of the vein (e.g. superior mesenteric vein close to the mesenteric root) with subsequent re-anastomosis of the portal vein and arterial resection with reconstruction in the case of R1 situation at the hepatic or superior mesenteric artery can substantially increase the intra-and postoperative risk for the patient. This can also be true, if a re-resection of a R1-pancreatic neck margin frozen section requires a total pancreatectomy and there is evidence of advanced lymph node metastasis. Some centres even perform a completion pancreatectomy if an arterial resection is performed. The present data has clinical implications. According to the present data, attempts to radically clear these critical resection margins can be avoided if intra-or postoperative signs of advanced lymph node metastasis are evident or probable (e.g. by frozen section analysis of suspected lymph nodes or PALN). The presented data on PALN metastasis demonstrate that intraoperative evidence of PALN metastasis may be such an indicator for advanced lymph node metastasis (pN2).
In addition, adjuvant (chemo)radiation is recommended by some US centres after margin positive (R1) resection in lymph node positive patients [25]. The present results indicate that potential effects of adjuvant (chemo)radiation (versus modern systemic treatment alone) on disease-free or overall survival after R1 resection may be recapitulated with respect to the novel N-stages (pN1 and pN2). Several trials including the randomized ESPAC trials have shown that adjuvant chemotherapy is critical for control of the systemic burden of this disease. Only 38.4% (n = 238) of the entire cohort in the present study received a documented adjuvant chemotherapy, additional 12.6% of the patients (n = 78) received adjuvant treatment of unknown protocol (the data could not be obtained because of the retrospective multicentre design). The fact that almost half of the patients did not receive adjuvant chemotherapy is oncologically suboptimal and multifactorial (patient fitness, denial, etc.). The participating centres agree in the need for an increased rate of adjuvant treatment.
In order to increase the R0 resection rate, neoadjuvant therapy in borderline or non-resectable PDAC patients is at the centre of current clinical research. The latest data demonstrated a benefit in OS as well as R0 resection rates compared with upfront surgery. Moreover, neoadjuvantly treated patients showed less lymph node infiltration after surgical resection [26][27][28][29][30]. This further highlights the importance of achieving local tumour control (R0) during resection following neoadjuvant chemotherapy. The present data also demonstrate a reduction of lymph node metastasis after neoadjuvant treatment. This can be interpreted as a partial response to the neoadjuvant chemotherapy. Thus, local control in neoadjuvantly treated patients probably becomes more critical (less proportion of pN2 stages) and special attempts should be made to achieve R0 resections (including vascular reconstruction) in this patient subgroup for optimal outcome.
In conclusion, margin status (local control) seems not to be associated with OS in patients with advanced lymph node spread. A simultaneous or postoperative surgical re-resection in an R1 situation may be avoided for oncological reasons if there is evidence of advanced lymph node metastasis and the re-resection is associated with a substantially increased perioperative risk. These patients should be evaluated for modern adjuvant systemic treatment.
Authors' Contributions TW, CT, RG, UW, TK, and JW are responsible for the study concept. The study design was developed by CT, TW, MD, and JW and the data acquisition was performed by CT, RS, and LB. CT, TW, UW, LB, and SM performed the quality control of data and algorithms. The data were analyzed and interpreted by CT, TW, MD, RG, TK, DEA, and JW. The statistical analysis was done by CT, LB, and SM. The manuscript was prepared by CT and TW, and edited/ reviewed by UW, DEA, RG, TK, and JW.
Funding Open Access funding enabled and organized by Projekt DEAL. The study was funded from the authors' institution only.
Data availability Data supporting the results can be requested by the senior authors upon request.

Declarations
Ethics approval The study was performed in accordance with the Declaration of Helsinki. The study protocol was approved by the local ethics committee of the Technical University of Dresden (EK 196042019).

Conflict of interest The authors declare no competing interests.
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