MD Roberto Biffi
email: roberto.biffi@ieo.it

E Botteri

S Cenciarelli

F Luca

S Pozzi

M Valvo

A Sonzogni

A Chiappa

T Leal Ghezzi

N Rotmensz

V Bagnardi

Leal Ghezzi

Impact on survival of the number of lymph nodes removed in patients with nodenegative gastric cancer submitted to extended

Keywords: gastric cancer, gastrectomy, extended lymph node dissection, TNM classification, preoperative chemotherapy

published or not. The documents may come     Impact on survival of the number of lymph nodes removed in patients with node-negative gastric cancer submitted to extended lymph node dissection

INTRODUCTION

Background

Despite a declining incidence in the Western world, gastric cancer still is a major malignant disease and the second leading cause of cancer mortality worldwide [START_REF] Forman | Gastric cancer: global pattern of the disease and an overview of environmental risk factors[END_REF]. The prognosis of a locally advanced tumour is generally poor in Western surgical and population-based series, with a 5-year overall survival of 25% or less [START_REF] Jemal | Cancer statistics[END_REF], in spite of apparently radical excision of the gastric and nodal components of the disease.

The extent of lymphadenectomy in the treatment of gastric cancer still is a matter of worldwide debate, since the introduction of extended lymphadenectomy (D2) by Japanese surgeons in the 1960s. While Japanese surgeons routinely perform a D2 dissection, the rationale being that the orderly centrifugal pattern of lymphatic tumour dissemination allows for the prediction of the risk of metastasis to specific nodal groups (3), many Western surgeons perform more limited (D1) lymph node dissection, not having found advantages with extensive lymphadenectomy in two large European randomized clinical trials [START_REF] Hartgrink | Extended lymph node dissection for gastric cancer: who may benefit? Final results of the randomized Dutch gastric cancer group trial[END_REF][START_REF] Cuschieri | Patient survival after D1 and D2 resections for gastric cancer: long-term results of the MRC randomized surgical trial. Surgical Co-operative Group[END_REF] and one meta-analysis [START_REF] Yang | An evidence-based medicine review of lymphadenectomy extent for gastric cancer[END_REF]. Current evidence is compatible with no benefit from D2 resection, but also with substantial benefit in a subgroup of patients with advanced N2 disease, or in all patients after a long-term (15-year) follow-up [START_REF] Songun | Surgical treatment of gastric cancer: 15-year follow-up results of the randomised nationwide Dutch D1 D2 trial[END_REF]. Furthermore, the benefit from D2 resection is evident only when operative mortality is less than 2%, a result that is achievable only by centres of excellence with a high-volume experience in the resection of gastric cancer [START_REF] Kodera | Radical surgery for gastric carcinoma: it is not an issue of whether to perform D1 or D2. Dissect as many lymph nodes as possible and you will be rewarded[END_REF][START_REF] Biffi | Extended lymph node dissection without routine spleno-pancreatectomy for treatment of gastric cancer: low morbidity and mortality rates in a single centre series of 250 patients[END_REF][START_REF] Degiuli | Morbidity and mortality after D1 and D2 gastrectomy for cancer: interim analysis of the Italian Gastric Cancer Study Group (IGCSG) randomised surgical trial[END_REF]. Previous studies have shown that survival of patients with locally advanced gastric adenocarcinoma is related to the number of regional lymph nodes (LNs) with metastases [START_REF] Kodera | The number of metastatic lymph nodes: A promising prognostic determinant for gastric carcinoma in the latest edition of the TNM classification[END_REF]. As the probability of identifying node-positive cancers increases with the number of LNs removed and examined, the UICC-TNM Classification of Malignant Tumours recommends the removal and examination of at least 16 LNs for adequate staging.

Purpose of the study

Extent of lymphadenectomy could play an important role also in less advanced disease. While prospective randomized studies have shown that the lymph node yield is much greater with D2 than with D1 resection, the effect of the number of surgically removed nodes on survival in node-negative gastric cancers has not been established. disease-free or overall survival benefit in this clinical setting. This was the aim of the present study, which evaluated the contribution of the number of removed cancer-negative nodes to the long-term outcome of patients with gastric cancer submitted to D2 dissection.

PATIENTS AND METHODS

Patient population

We analyzed data of 100 consecutive patients admitted to the Department of General Surgery of the European Institute of Oncology (IEO) in Milan for adenocarcinoma of the stomach between 2000 and 2005. They all underwent gastrectomy (total or subtotal) and extended lymph node dissection, resulting in node-negative disease. Fourteen additional patients, who were referred to IEO for a second opinion, were taken into consideration; they had undergone the same surgical procedure elsewhere and a comprehensive assessment of their clinical records showed that the pathologic stage of their tumours was the same. Data for this study were extracted from the IEO Tumour Registry (TR), an Institutional database activated in March 2006, with the objective of collecting data on all individuals consulting the IEO, independently of whether they actually received treatment at the Institute or not [START_REF] Botteri | Case mix at the European Institute of Oncology: first report of the Tumour Registry, years 2000-2002[END_REF]. The inclusion criteria for the present study population were: no previous diagnosis of other cancers, radical resection (R0), pathologic diagnosis of adenocarcinoma of the stomach, no LN involvement and no synchronous distant metastases (any pT, pN0 and M0, according to the UICC-TNM Classification). No age limits were defined. This study was approved by the Scientific Research Committee of IEO.

Diagnosis and staging procedures

All patients affected by pathologically-proven adenocarcinoma of the stomach were submitted to a preoperative staging work-up in the attempt to perform curative surgery. It included a physical examination, a CT scan of the abdomen and pelvis, a chest X-ray (or Thorax CT scan in selected cases), a liver US scan, a gastric Endoscopic Ultrasound scan (in selected cases), and upper GI endoscopy and confirmatory biopsies, if they had not already been performed at our Institution. Preoperative PET scan was not routinely used in this clinical series, as at that time it was not available for routine use. Patients were considered eligible for gastrectomy and extended lymph node D2 dissection when they had no evidence of metastatic spread according to preoperative investigations.

Surgical technique

First of all, careful intraoperative staging of disease was performed, in order to rule out peritoneal seeding, ovarian involvement, "drop" metastasis in the pelvis, or gross periaortic adenopathy.. The extent of gastrectomy depended on the proximal distance of the tumour from the cardias: total gastrectomy was performed in all patients with cardias involvement and in the presence of antral and/or body tumours, when a 6-cm gross proximal margin could not be obtained.

Lymphadenectomy included excision of all N1 and most N2 stations (stations 7, 8, 9 and station 11), according to the classification of the Japanese Research Society for the Study of Gastric Cancer (JRSGC) [START_REF]Japanese classification of gastric carcinoma[END_REF][START_REF]Japanese Classification of Gastric Carcinoma-2nd English Edition-Gastric Cance[END_REF]. Hepatoduodenal ligament nodes (station 12) were also dissected, limiting the lymphadenectomy to station 12a (left side of the hepatic artery), and leaving parts b and p of the station (right side of the ligament and the part immediately posterior to the portal vein, respectively) undissected. LNs of the surgical specimen were routinely dissected by experienced pathologists with standard techniques.

Caudal pancreas was always preserved, according to Maruyama's technique, even when splenectomy was performed, unless direct involvement of the tumour was clinically evident [START_REF] Maruyama | Pancreas preserving total gastrectomy for proximal gastric cancer[END_REF].

Statistical methods

Kruskal-Wallis Test, Chi-square test and Mantel-Haenszel Chi-Square for trends were used to test differences in distributions between patients who had >15 LNs dissected and those who had ≤15 .

Distant disease-free survival (DDFS) was defined as the time free of both tumour recurrence at distant sites and death from any cause. Other primary tumours were considered as censoring events. Disease-free survival (DFS) was calculated from the date of surgery to any loco-regional or distant recurrence, other primary tumour and death from any cause. Overall survival (OS) was calculated from the date of surgery to death from any cause. In the absence of any of the above-mentioned events, survivals were censored at last follow-up visit. Survival curves were estimated using the Kaplan-Meier method and compared by the Log-rank test. The prognostic impact of the number of extracted LNs on survival was evaluated using the multivariate Cox proportional hazard regression model and expressed as hazard ratio (HR) with 95% Confidence Intervals (CI).

The functional form of the relationship between DDFS and the number of dissected

LNs analysed as a continuous variable was evaluated fitting a restricted cubic spline multivariable Cox regression model [START_REF] Durrleman | Flexible regression models with cubic splines[END_REF]. Three knots were placed at quartile values [START_REF] Sierra | Role of the extended lymphadenectomy in gastric cancer surgery: experience in a single institution[END_REF]22 and 29 LNs). Other numbers and positions of knots were examined, and the curves were similar. The model was adjusted for pT, neoadjuvant treatment and place of surgery.

All analyses were carried out with the SAS software (SAS Institute, Cary, NC) and the R software (The R Development Core Team 2004; Free Software Foundation, Boston, MA). All tests were two-sided.

RESULTS

Patient characteristics

Sixty-seven patients out of 114 (58.8%) were males. Median age was 63 years (range 31-87). Median number of dissected LNs was 22 (range 2-73). One hundred patients (87.7%) underwent surgery at the IEO, 14 at other Institutes. Table 1 shows patient and tumour characteristics, stratified by number of dissected LN (≤15 LNs versus >15 LNs). Twenty-four patients had ≤15 LNs removed, 90 patients had >15 LNs harvested (45 of them >25 lymph nodes). The only significant difference between the two groups was the number of patients who received neoadjuvant chemotherapy: it was significantly higher in the limited lymphadenectomy group (P=0.03), as expected.

Survival findings

Median follow-up was 76 months (range 5-129). No patients had a local or regional recurrence, 8 had distant metastases and 11 experienced a second primary cancer at a site other than the stomach. Overall, 9 patients died.

At the univariate survival analysis (Table 2), pT and the number of dissected LNs were found to be associated with DDFS, while the latter was the only statistically significant variable associated with OS. Figure 2 shows OS with stratification by number of dissected LN. Once again, patients with a lower number of nodes had a worse prognosis than patients with a number of nodes exceeding 15, even after stratifying for pT . Multivariable analysis was not performed in the analysis of OS due to the low number of deaths. DDFS, DFS and OS were significantly associated with the number of dissected LNs even after the exclusion of patients receiving neoadjuvant chemotherapy and patients not operated at IEO (N=87). pT-adjusted HRs of LNs ≤ 15 versus LNs > 15 were 8.4 (95% C.I. 1.8-38.2) and 3.9 (95% C.I. 1.2-12.2) for extracted LNs ≤ 15 had a worse prognosis than other patients (Log-rank P<0.001).

Figure 3 shows the DDFS risk by number of LNs analyzed as a continuous variable.

Fifteen LNs were chosen as reference (HR=1). The risk of the event was inversely proportional to the number of dissected LNs, even over 15 LNs, up to about 20 LNs; thereafter it slightly increased as the number of dissected LNs increased..

DISCUSSION

Our study was primarily aimed at answering the question whether the number of surgically removed cancer-negative nodes has an effect on distant disease-free (DDFS) and overall survival (OS) in patients with any pT, pN0 TNM-staged gastric cancer, who underwent gastrectomy with extended (D2) lymphadenectomy. The results obtained indicate that patients who have > surgically removed 15 LNs have a statistically significant survival advantage, both in terms of DDFS and OS. Moreover, the evaluation of DDFS risk by number of harvested LNs -analyzed as a continuous variable -suggested that the risk of events was inversely proportional to the number of dissected LNs up to about 20 LNs.

Interestingly, the results persisted even when pT1 ("early gastric cancer") and pT2-3 cancers (locally advanced) were considered separately, thus indicating the need to remove at least 15 nodes also in patients with early stage disease.

Extent of lymphadenectomy in the treatment of locally advanced and early gastric cancer. Its role in pN0 disease.

The extent of lymphadenectomy in the treatment of gastric cancer is still a matter of debate. The first large multicentre randomized study that compared D1 and D2 dissection was conducted in the Netherlands by the Dutch Gastric Cancer Group (4). A total of 711 patients underwent a curative resection (380 in the D1 group and 331 in the D2 group).

Morbidity (25 vs. 43%) and mortality (4 vs. 10%) were higher following D2 dissection. Fiveyear survival rates were similar in the two groups: 45% for the D1 group and 47% for the D2 group. Improved survival was seen in patients with N2 cancer who underwent extended lymph node dissection, but unfortunately it remains difficult to correctly identify patients with N2 disease in preoperative or intraoperative settings. Moreover, as the authors identified splenectomy and pancreatectomy as contributing risk factors for increased morbidity and mortality, they concluded that extended lymph node dissections may be beneficial if morbidity and mortality can be avoided (4). In the most recent update, after 15 years of follow-up, D2 lymphadenectomy was associated with lower locoregional recurrence and tumour-related death rates than D1 surgery [START_REF] Songun | Surgical treatment of gastric cancer: 15-year follow-up results of the randomised nationwide Dutch D1 D2 trial[END_REF]. Because a safer, spleenpreserving D2 resection technique is available in many centres, authors reached the conclusion that D2 lymphadenectomy is the recommended surgical approach for patients with resectable disease. D2 gastrectomy is a safe treatment for gastric cancer in Western patients, at least when it is performed at experienced centres (9, 10); moreover, gastric cancer resection should probably be added to the growing list of procedures that are safer when they are performed at high-volume institutions [START_REF] Sierra | Role of the extended lymphadenectomy in gastric cancer surgery: experience in a single institution[END_REF]. A second large randomized multicentre trial comparing D1 with D2 dissection was conducted by the British Medical Research Council (MRC) [START_REF] Cuschieri | Patient survival after D1 and D2 resections for gastric cancer: long-term results of the MRC randomized surgical trial. Surgical Co-operative Group[END_REF]. Four hundred patients were randomly assigned to treatment (200 to D1 and 200 to D2). The results showed that the D2 group had greater postoperative hospital mortality (13 vs. 6.5%; p = 0.04) and higher overall postoperative morbidity (46 % vs. 28%; p < 0.001). The five-year survival rates were 35% for D1 resection and 33% for D2 resection. The authors concluded that D2 resection offered no survival advantage over D1 surgery. A more recent single-centre randomized study in Taiwan [START_REF] Wu | Randomized clinical trial of morbidity after D1 and D3 surgery for gastric cancer[END_REF][START_REF] Wu | Nodal dissection for patients with gastric cancer: a randomised controlled trial[END_REF], where 110 patients were randomly assigned to D1 surgery and 111 patients to D3 surgery, demonstrated a survival benefit from extended lymphadenectomy.

The morbidity rate was higher after D3 than after D1 resection (17.1 vs. 7.3%). The overall five-year survival was significantly higher in patients in the D3 group than in the D1 group (59.5 vs. 53.6%; p = 0.041). A recent meta-analysis ( 6) reviewing 14 randomized clinical trials (3,432 patients overall), published in 2009 before the acquisition of the 15year results of the Dutch Trial, confirmed that operative mortality and postoperative morbidity were higher in the D2 group than in the D1 group, and the three-and five-year survival rates were not statistically different.

The Japanese Research Society for the Study of Gastric Cancer (JRSGC) strongly recommends extended lymphadenectomy, although it is still unclear, in terms of high level scientific evidence, whether this procedure improves survival or merely refines staging [START_REF] Bunt | Surgical/Pathologic stage migration confounds comparisons of gastric cancer survival rates between Japan and western countries[END_REF][START_REF] Yoshikawa | Stage migration caused by D2 dissection with para-aortic lymphadenectomy for gastric cancer from the results of a prospective randomized trial[END_REF]. It is well known that overall survival rates after radical surgical treatment for gastric cancer at Western and Japanese institutions differ substantially. When new lymph node metastases are identified as a consequence of more extended lymphadenectomy and/or more accurate pathologic techniques, nodal staging determines a stage migration of the disease [START_REF] Wu | Stage migration influences on stage specific survival comparison between D1 and D3 gastric cancer surgeries[END_REF]. This is a possible explanation of the frequent underestimation of tumour stage in Western series, due to incomplete removal of the LNs necessary for accurate staging. This could partly explain also our findings. We were not able to ascertain whether the difference in the number of lymph nodes found is related to differences in surgical technique or in the quality of histopathological dissection; as a matter of fact, 14 patients were operated on elsewhere and strict adherence to surgical rules of D2 dissection could not be verified. However, in our study DDFS (Fig. 1) was adjusted also by surgical centre, together with pT stage and preoperative chemotherapy occurrence. From the U.S.

National Cancer Data Base Report of 2000, we know that only 18% of U.S. patients have 15 LNs analyzed [START_REF] Hundahl | The National Cancer Data Base Report on poor survival of U.S. gastric carcinoma patients treated with gastrectomy: Fifth Edition American Joint Committee on Cancer staging, proximal disease, and the "different disease" hypothesis[END_REF]. It is unlikely that selective lymphadenectomy, based upon the gross appearance of LNs, is feasible or appropriate, as 30% of LN metastases occur in nodes less than 3 mm in diameter [START_REF] Keller | Lymph node staging in 872 patients with carcinoma of the stomach and the presumed benefit of lymphadenectomy[END_REF] and surgeons are probably able to correctly diagnose metastatic involvement by macroscopic appearance in only 20% of patients [START_REF] Okamura | Lymphadenectomy for cure in patients with early gastric cancer and lymph node metastasis[END_REF].

Intraoperative assessment of frozen lymph node sections seems practically unfeasible and largely invalidated by the occurrence of the ''skip metastasis'' phenomena, as 11% of patients with negative perigastric nodes show metastatic deposits in more distant nodes, even in pT1 tumours [START_REF] Yoshikawa | Is D2 lymph node dissection necessary for early gastric cancer?[END_REF].

This adds further points of discussion related to the issue of lymph node dissection in T1 tumours (adenocarcinoma confined to submucosa, EGC: Early Gastric Cancer). The incidence of early gastric cancer has recently increased as advances in diagnostic techniques have made it possible to detect small lesions at an early stage. It was reported that radical lymph node dissection should be limited in patients with EGC, because extended lymph node dissection in these patients offers modest survival benefits [START_REF] Yoshikawa | Is D2 lymph node dissection necessary for early gastric cancer?[END_REF][START_REF] Kasakura | Gastrectomy with D2 lymph node dissection in gastric cancer: a retrospective study at a single institution[END_REF][START_REF] Nitti | Extended lymphadenectomy (D2) in patients with early gastric cancer[END_REF]. Consequently, new treatment modalities with minimally invasive techniques, such as endoscopic resection, laparoscopic gastrectomy, and robotic surgery, are becoming more frequent in EGC patients. These minimally invasive techniques are focused on enhanced recovery after surgery (ERAS) and improving quality of life, as the survival rates of EGC patients are high [START_REF] Kitano | Laparoscopyassisted Billroth-I gastrectomy (LADG) for cancer: our 10 years' experience[END_REF][START_REF] Soetikno | Endoscopic mucosal resection[END_REF][START_REF] Song | Robot-assisted gastrectomy with lymph node dissection for gastric cancer: lessons learned from an initial 100 consecutive procedures[END_REF][START_REF] Kehlet | Anaesthesia, surgery, and challenges in postoperative recovery[END_REF]. A very recent paper [START_REF] Jung | The long-term results of distal gastrectomy by mini-laparotomy in Early Gastric Cancer patients[END_REF] assessed the feasibility of radical distal gastrectomy with a mini-laparotomic approach in EGC patients in terms of long-term survival. A mean of approximately 30 LNs were harvested, and no difference in terms of DFS and OS arose from the comparison with a matched series of patients undergoing conventional surgery with a laparotomic approach through a long midline incision. As in our study, no locoregional recurrence to the regional LNs or carcinomatosis peritonei occurred.

Another recent paper [START_REF] An | Assessment of open versus laparoscopy-assisted gastrectomy in lymph node-positive early gastric cancer: a retrospective cohort analysis[END_REF] assessed oncological outcomes following laparoscopyassisted versus open gastrectomy in patients with lymph node-positive EGC. Mean number of retrieved LNs did not differ between the two groups. During a median follow-up of 35 months, 14 patients (8.6%) developed recurrence after open surgery, compared with 4 patients (9.5%) after laparoscopy procedure. Overall 5-year disease-free survival was 89.9% and 89.7% respectively. Lymph node metastatic status was the only independent prognostic factor for disease-free survival. These data reinforce the assumption that an adequate number of LNs must be harvested, independently of the technique adopted.

Findings obtained by another Italian group (Italian Research Group for Gastric

Cancer) were somewhat different. This Group collected and analyzed the clinical and pathological characteristics of 301 lymph node-negative advanced gastric carcinoma patients in a multicentre retrospective study on gastrectomy and D2 lymphadenectomy.

The factors associated with better disease-free survival at univariate analysis were age <60, T2 tumours, distal location, intestinal histotype, and number of retrieved nodes >25, whereas in our study disease-free and overall survival were similar in patients having 16-25 or >25 nodes removed. In this study depth of infiltration and histotype were the only 2 independent predictors of 5-year recurrence-free survival at multivariate analysis [START_REF] Baiocchi | A multicentric Western analysis of prognostic factors in advanced, node-negative gastric cancer patients[END_REF].

Future perspectives

Worthy of a final comment is the potential value of sentinel node technique in the identification of the nodes to be removed. A recent study compared lymphatic mapping in early gastric cancer with indocyanin green (IG) and infrared ray electronic endoscopy (IREE) to IG alone [START_REF] Kelder | Sentinel node mapping with indocyanine green (ICG) and infrared ray detection in early gastric cancer: an accurate method that enables a limited lymphadenectomy[END_REF]. The sentinel node identification rate and sensitivity for IREE versus ICG alone were 99.5 and 85.8% versus 97.0 and 48.4%, respectively. The Authors concluded that dissection of the lymphatic basin, based on IREE, is a safe method of nodal dissection in patients with T1 or limited T2 tumours. These interesting findings need confirmation by larger well-designed studies.

Limitations of this study

Our study suffers from some limitations. The main limitation is that it was a retrospective study. notwithstanding the added value provided by the accurate methodology used for the statistical analyses. Moreover, the low number of patients with a very large number of LNs removed (i.e. only 8 patients had more than 40 dissected LNs) may have led to unreliable risk estimates for that group of patients. Sampling error is a likely explanation for the slight increase in the risk of recurrence when many LNs were removed, as shown in Fig. 3.

Thirteen patients received neoadjuvant chemotherapy; this is a potential confounding factor that could influence the pathology assessment of lymph node status, as well as patient survival. As shown in Table 1, preoperative neoadjuvant treatment had an impact on the number of harvested lymph nodes, which was significantly lower.

Nevertheless, survival curves were adjusted in our study also for this variable, and the protective effect of the number of removed lymph nodes on survival was maintained (Fig. 1).

Finally, we did not stratify the study population in terms of histological type (intestinal vs diffuse/mixed adenocarcinoma, according to Lauren's classification).

However, we analyzed tumor grading (1 to 4) and did not find significant differences in its distribution in the groups (Table 1).

Conclusion

In conclusion, more extended lymph node resection resulted in survival benefit, both in terms of DDFS and OS, for patients suffering from node-negative gastric cancer; moreover, evaluation of DDFS risk by number of harvested LNs demonstrated that the risk of events is inversely proportional to the number of dissected LNs, up to 20.

Interestingly, the results did not change when pT1 ("early gastric cancer") and pT2-3 cancers (locally advanced) were analyzed separately, suggesting the need to remove at least 15 nodes even in patients with early stage disease. HR is plotted on a log scale. HR was adjusted for pT, surgical centre and neoadjuvant chemotherapy. 15 lymph nodes is the reference (HR=1).

Lymph Nodes

Hazard Ratio

Figure 1

 1 Figure 1 shows DDFS by number of dissected LNs. Patients with extracted LNs ≤ 15 had a worse prognosis than other patients: adjusted HR 8.1 (95% C.I. 2.5-25.7). This result remained statistically significant even after stratifying for pT (data not shown). The number of dissected nodes had a significant impact also on DFS: adjusted HR of LNs ≤ 15 versus LNs > 15 was 3.1 (95% C.I. 1.3-7.6).

cFigure 1 :

 1 Figure 1: Distant disease free survival with stratification for number of dissected lymph nodes

Figure 2 :

 2 Figure 2: Overall survival with stratification for number of dissected lymph nodes

Figure 3 :

 3 Figure 3: Distant disease free survival according to number of dissected lymph nodes

Table 1 :

 1 Patient characteristics, with stratification for number of dissected lymph nodes

				All patients No. 114	Dissected lymph nodes ≤ 15 No. 24	Dissected lymph nodes > 15 No. 90	χ P-value 2 test
	Age		Median (Range)	63 (31-87)	62 (39-78)	63 (31-87)	0.88 b
			<50	17	2	15
	Age		50-64	50	13	37	0.45
			≥65	47	9	38
	Sex		M F	67 47	18 6	49 41	0.10
	Type of Adenocarcinoma	Intestinal Diffuse	50 64	10 14	40 50	0.80
			1	52	12	40
	pT		2 3	46 13	9 1	37 12	0.25 c
			X	3	2	1
			1	16	6	10
			2	37	5	32
	Grading	a	3	36	5	31	0.79 d
			4	6	2	4
			NA	13	6	7
	Neoadjuvant treatment	Yes No	13 101	6 18	7 83	0.03
	NA: Not Applicable.

a Information is missing for some patients; b Kruskal-Wallis Test

Acknowledgements

The authors would like to thank Elena Albertazzi, Marina Francesca Alfieri, Barbara Bazolli, Nadia Burzoni, Marco Martinetti, Laura Manghi and Bruno Montanari for their precious contribution to data entry and quality control of the IEO Tumour Registry data.


N Serres

F Hlawka

S Costil

C Langlade

F Machi

A Cornet

Combined plasma spray and in situ laser melting treatment of NiCrBSi powder

Keywords: Plasma-laser hybrid process, Mechanical properties, Wear, NiCrBSi

Recently, mechanical industry has been affected by many environmental regulations. For example, hexavalent chromium was prohibited due to its toxicity. Thus it is essential to find new coatings which are able to replace hard chromium plating. In this paper a technique, based on the association between APS (Atmospheric Plasma Spray) and laser melting using a 3kW diode laser is described. This process allows the production of denser coatings with finer structures than as-sprayed coatings. Depending on the processing parameters the mechanical properties, studied by nanoindentation, microhardness measurement and tribological ball-on disc experiments, were improved in terms of hardness and wear resistance.

Introduction

In order to protect the environment, mechanical engineering as all other industries has to comply with new environmental regulations. Particularly, hexavalent chromium was recently prohibited in the European automotive industry by the end-of-life vehicles directive of the European Parliament. This regulation which applies to the surface treatment industry, reclassified the chromic anhydride among very toxic substances.

Surface coatings by thermal spraying were already developed in industry and some authors obtained good results to replace hard chromium plating [1,2]. However, mechanical applications are not always satisfactory for reasons of porosity and microstructure. Furthermore, the materials used are often expensive, especially WC-Co. The applications of laser cladding were also studied and allowed the synthesis of denser coatings [3]. But this process presents also a slow velocity of treatment and its efficiency was often criticized. Nowadays, diode lasers, such as the laser used in this study, arouse a great interest. Also, the two processes appear very complementary. In this paper, a technology which combines Atmospheric Plasma Spraying (APS) and laser re-melting in an in situ process is described. In order to modify the structure without generating too high level of stress incompatible with mechanical resistance properties, in situ coating remelting appeared as an interesting alternative. In addition, this technology could be another alternative to hard chromium plating, if its mechanical properties are high. A first study was carried out on ceramics coatings [START_REF] Antou | Améliorations de revêtements barrières thermiques par un procédé de refusion laser in situ utilisant un laser à diodes[END_REF], and the treatment changed structure from lamellar to dendritic without modifying the elastic response of the deposit. Additionally to decrease the cost of materials, NiCrBSi coatings are broadly used to improve the quality of components whose surface is subjected to wear and/or corrosion [START_REF] Planche | [END_REF], i.e. hard chromium plating well-known properties.

Thermal spraying, such as APS presents the advantage of carrying out coatings very quickly (velocity ranging from 60 to 80 m/min depending on the application). Thus it seems interesting to preserve the treatment velocity, if at the same time the microstructure of the coating will be improved by using a laser. The greatest difficulty is to find an agreement between the very high APS process velocity and the laser, in order that the laser can redesign the surface. Indeed, the coating elaboration by laser cladding is carried out at a very low velocity. For example, to cover a steel surface with NiCrBSi powder using laser cladding process, velocity was equal to 1.5x10 -1 m/min according to González et al. [6].

The aim of this paper is to find appropriate parameters to develop this in situ process for industrial application. Furthermore, a mechanical investigation is carried out by nanoindentation and microhardness measurements to evaluate the improvement of effective Young's elastic modulus and hardness of selected samples. Then, tribological ball-on-disc experiments are also carried out to calculate the wear rate of selected samples and compare them with as-sprayed NiCrBSi coatings.

Experimental procedure

Feedstock materials

A self-fluxing alloy of high chromium and boron content was investigated in this study. NiCrBSi powder from Höganäs, referenced as grade 1160-00 was used as feedstock material. The sprayed powder composition, morphology and size distribution are listed in Table I. The powder was sprayed on a C38 steel pin with a diameter of 25 mm and a height of 10 mm. This kind of substrate was used in the experiments. Indeed, the coatings are carried out on the flat surface of the pin (S = 491 mm²).

Processing parameters

The experimental device of the hybrid plasma spray process was constituted by the association of a F4 plasma spray gun from Sulzer-Metco and a diode laser (3kW, average power) of 848 nm wavelength, from Laserline (Figure 1). Before the spraying operations, samples were degreased with acetone and alcohol and grit blasted with Al 2 O 3 to obtain a surface with a mean roughness of about 5 µm. This operation improves the mechanical bonding between the coating and the substrate. Then samples are placed on a holder which has a linear movement and they are swept by the plasma gun and remelted in situ by the laser. The coating feedstock material is injected vertically into the plasma jet by Ar carrier gas. The process is carried out at a very high velocity for a laser treatment (v = 75 m/min). Thus, in order to obtain a suitable laser contribution of the process, the area exposed to combined treatment must be small in order to increase the power density at a maximum. Consequently, the laser contribution will be significant. A fixed-focus lens of 0.8 × 2 mm² is used, which correspond to the laser beam dimensions. In order to increase the temperature of the sample, the air cooling system is placed at the rear of the holding device. This positioning allows for conservation of the thermal effect provided by the APS. Each sample is treated on all surface area, i.e. 491 mm². In this paper three representatives samples are studied. It is assumed that the laser power density and the number of steps have a significant influence on the coating properties. The holding device is fixed on a robot: it moves laterally according to the relative motion and from top to bottom according to the spray step. Therefore, some variations are evaluated, regarding the evolution of the properties of different samples according to the selected processing parameters. The identification of the samples and the processing parameters are given in Tables 2 and3.

Microstructure observation

The samples were cut parallel to the spray direction using a diamond saw in an oil medium and mounted in bakelite. Then the cross-sections were polished following standard metallographic techniques i.e., prepolishing and diamond slurry polishing to achieve a final coating roughness of Ra ~ 0.2 µm. Then, the samples were observed using SEM, coupled with energy dispersive spectroscopy (EDS).

The mean roughness, Ra (µm) data of samples was calculated by an extended field confocal microscope (STIL's Micromesure station) with a vertical resolution of 0.01 µm and lateral resolution of 2 µm. The topography was measured on a large surface, with a sampling step of 2 µm in the horizontal plan.

Moreover, an assessment of the coating density was made by accurately weighing the samples before and after spraying and then calculating from the volume of the coating deposited. This procedure was undertaken on twelve samples to yield an average value of the layer density.

Berkovich nanoindentation

Effective Young's elastic modulus of selected samples was determined using an ultra-low load indentation system equipped with a Berkovich indenter tip (Nano Indenter ® XP, MTS Systems Corporation). This microprobe records continuously both the load and the displacement as indents are made on a sample, with resolutions up to 75mN and 0.04 nm, respectively. Thirty indentations were made on selected samples at different loads in the range of 7.5-30mN on coated polished cross-sections. The effective Young's modulus was then calculated from the Oliver-Pharr model [7] and the results were averaged. The Poisson's ratio of different samples was assumed equal to 0.32.

It is also possible to determine the hardness of the coatings with this system, but the samples are thick enough to make standard microhardness measurements. Thus, Vickers hardness measurements were performed on a section perpendicular to the spraying direction with a 100 gf load. These tests provide more reliable information on the influence of laser treatment on the coating hardness.

Wear tests

To compare the wear performance of different samples, tribological tests were done. A pin-on-disc type tribometer from CSM Instruments was used for the wear experiments with a sliding distance of 10 km, a sliding speed of 50 cm/s, a ball of 100Cr6 (Ø = 6 mm), under drysliding conditions, at atmospheric pressure and room temperature. The normal load, F N , applied on the sample was 2 N. These tests are done on the flat surface of the pin. Four measurements per sample were carried out to ensure the reproducibility of the tests. Before the experiments, the sample surfaces were polished following standard metallographic techniques, i.e. prepolishing and diamond slurry polishing. Indeed it is essential to work with a smooth surface in order to decrease the friction efforts between the sample and the counterbody surfaces.

Wear rate was characterized by observing wear scars by microtopography and using SEM. The microtopographic data of wear tracks were obtained by an extended field confocal microscope with a vertical resolution of 0.01 µm and lateral resolution of 2 µm. The topography was measured on a large surface, with a sampling step of 2 µm in the horizontal plan. Finally, the wear coefficient (K) was calculated by means of the equation ( 1) proposed by Archard [8], where V is the total wear volume loss (mm 3 ), P the applied normal load (N) and S the total accumulated displacement (m).

S P V K  

(1)

Results and discussion

Coating structure characterization

The coating general microstructures are shown in Fig. 2. Differences in morphology are observed. APS coatings have a lamellar structure and exhibit cracks, pores and unmelted particles, which are emblematic defects for this kind of treatment. The average porosity is about 3.4 ± 0.3 % for plasma sprayed coatings [START_REF]ASTM International, Test Methods for Determining Area Percentage Porosity in Thermal Sprayed Coatings[END_REF]. On the contrary, as-sprayed remelted coatings are denser with a dendritic structure and the absence of porosity and microcracks shows that the selected processing parameters have ensured good quality coatings. The thickness of both coatings is comparable, i.e. 330 µm for as-sprayed samples and 460 µm for remelted samples. However the surface roughness was changed by the laser treatment. Indeed, there is a reduction of the mean roughness due to laser remelting: Ra without laser = 5.24 µm and Ra with laser = 2.91 µm. As we can see in Fig. 3, the main result is that the re-melting of the coating reduces porosity and creates a metallurgical bond between the coating and the substrate, which also enhances the cohesion between the particles of the coating, without propagation of cracks. This result is observed for all selected processing parameters. As a consequence, the adhesion will probably strongly increase in comparison with the APS process. Note that the black points in Figure 3b are not porosity but chromium precipitates in a Ni/Si/Fe matrix, such as presented in Figure 4. Furthermore, all remelted samples present finer structure and higher density than APS coating (Fig. 5). Indeed, as-sprayed sample has a density of 6.64 g/cm 3 in comparison with remelted sample which has a density of 8.23 g/cm 3 . Thus, whatever the irradiance, the microstructure resulting from laser treatment is a dendritic structure.

Mechanical properties

Vickers hardness measurements clearly indicate that the remelted coatings have a higher hardness than the APS coating because of their finer structure and higher density (Fig. 6). Remelted areas with a dendritic structure seem to exhibit a slightly enhanced toughness compared to the lamellar structure of the as-sprayed coating. Indeed, plasma sprayed coatings exhibit lower hardness because of the effect of porosity. The specific comparison between remelted samples demonstrates that the hardness values are increased at the same time as the laser power density and the number of steps.

Load -displacement curves performed with a 30mN load for non-treated and laser treated NiCrBSi allow for comparison of their elastic properties (Figure 6). A little increase in Young's modulus can be verified after in situ laser treatment. So, in a first approximation, remelted samples present the same elastic properties as non-treated samples, especially because all samples have a similar chemical composition (Table IV). Indeed, the main difference deals with the presence of oxygen for APS sample due to oxidation during spraying in plasma. The dimensions of the indented area are several hundred times lower than the average layer cross-section (i.e., ~1µm² versus 750µm², approximately). Thus, the Young modulus determination derived from these measurements is related to the intrinsic or effective modulus, depending on the NiCrBSi structure exclusively and not on the coating architectures. There is also the effect of porosity in case of APS sample, which decreases the cohesion of the coating, improving its elasticity. As a conclusion, the Young's modulus of laser treated samples is higher, with few differences according to the selecting processing parameters.

Wear experiments

Tribological ball-on-disc experiments are carried out on the APS and remelted samples. Figure 7 shows the wear profiles after ball-on-disk testing for all of the samples. The wear track of the APS coating contains smooth cavities and it seems that some un-melted particles are torn off the coating during the wear test [START_REF] Serres | [END_REF]. According to [11], fatigue is the main wear mechanism for this kind of coating and this process is called splat delamination, which is common in thermal sprayed coatings with poor cohesion energy. The porosity of the APS coating, equal to 3.4 ± 0.3 % [START_REF]ASTM International, Test Methods for Determining Area Percentage Porosity in Thermal Sprayed Coatings[END_REF], as well as the cohesion between splats may induce this wear mechanism. Moreover, the wear track profiles of the remelted samples (Fig. 7) reveal that the scars are less deep compared to the APS scar. This result is due to improved mechanical properties of remelted coatings as discussed in the previous section. The wear rate of the samples was next calculated using Archard's model (Fig. 8). All values of wear rate are advantageous in case of potential tribological industrial application of these coatings. However the remelted sample types present a better behavior, of high hardness levels that improve the wear resistance of coatings [11]. Moreover, corresponding to the selected processing parameters the wear rates of the remelted samples are modified. Also, better results are obtained with the sample labeled H2. Thus, the increase of the laser power density coupled with a high number of steps seems to give better results, like with the hardness values observed in Fig. 6.

Conclusions

The in situ process (laser re-melting after plasma spraying) allows the synthesis of denser coatings than APS with finer structures and without cracks and porosity. The coating is metallurgically bonded to the substrate, which probably will increase the adhesion. Moreover, mechanical properties, especially the hardness are increased and better tribological behavior of the remelted samples can be observed, especially concerning the wear rate of selected samples. Finally, there are some variations according to the selected processing parameters and better mechanical results are obtained with a higher laser power density and elevated number of steps. So this process could be a good alternative to electrolytic hard chrome process due to its good mechanical properties and a specific comparison between these two processes will be carried out soon.

Fig. 1 .

 1 Fig. 1. Experimental device constituted by the association of a plasma spray gun and a diode laser.

Fig. 2 .

 2 Fig. 2. General view of NiCrBSi coatings produced by APS (a) and laser melting treatment (b).

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. Typical interface morphologies of (a) as-sprayed and (b) in situ remelted coating.

Fig. 6 .

 6 Fig. 6. Elastic modulus determined using an ultra-low load (30 mN) indentation system equipped with a Berkovich indenter tip and microhardness values of coatings.

Fig. 7 .

 7 Fig. 7. Wear profiles after ball-on-disk testing.

Fig. 8 .

 8 Fig. 8. Wear rates after ball-on-disk testing.

Table 1 .

 1 Chemical composition (wt. %) of spray powder.

	Chemical composition (wt. %)
	C	Ni	Fe	Cr	Si	B	O	Particles size distribution	Morphology
	0.76 72.33 3.87 15.16 4.65 3.19 0.043 ranging from 20 to 63 µm (average 45 µm) Spherical

Table 2 .

 2 Selected processing parameters.

	Parameter	Value
	Nozzle diameter (mm)	6
	Arc current intensity (A)	580
	Argon flow rate (L/min)	50
	Hydrogen flow rate (L/min)	8
	Feedstock injector diameter (mm)	1.8
	Feedstock injector tip location from gun centerline axis (mm)	6
	Feedstock carrier gas flow rate (Ar) *	1.4 -1.8
	Powder feed rate (g/min)	25
	Spray step (mm)	3 -6
	Area exposed to plasma spray (mm²)	30
	Area exposed to combined treatment	1.6
	(mm²)
	Spray distance (mm)	120
	Irradiation distance (mm)	70
	Distance between powder impact and	22
	laser irradiation (mm)
	α (°)	13
	Spray velocity (m/min)	75

* Dependant on the spray step

Table 3 .

 3 Nomenclature of selected samples.

	Sample	Laser power density (W/mm²)	Number of steps (-)
	APS	0	8
	H1	1875	4
	H2	1875	8
	H3	1250	8
	2.3. Coatings characterization

Table 4 .

 4 Chemical composition of the coatings obtained by EDS.

	Wt. (%) as-sprayed	laser treated
	O 3.37	-
	Si 4.92	5.25
	Cr 11.77	13.71
	Fe 5.19	7.56
	Ni 74.76	73.47

Acknowledgments

The authors gratefully acknowledge the "Région Alsace" for financial assistance in the Ecosurf project and the "Véhicule du Futur" Pole which has sponsored this project.