MD G Canavese

MD P Bruzzi

MD A Catturich

MD C Vecchio

D Tomei

MD F Carli

MD M Truini

MD G B Andreoli

MD V Priano

PhD Beatrice Dozin
email: beatrice.dozin@istge.it

Intra-operative evaluation of the sentinel lymph node for T1-N0 breast-cancer patients : always or never? A risk/benefit and cost/benefit analysis

Keywords: breast cancer, sentinel lymph node, intra-operative evaluation, axillary lymph node dissection

Aim : To investigate whether omitting intra-operative staging of the sentinel lymph node (SLN) in T1-N0 breast-cancer patients is feasible and convenient because it could allow a more efficient management of human and logistic resources without leading to an unacceptable increase in the rate of delayed axillary lymph node dissection (ALND).

Methods : According to the experimental procedure, T1a-T1b-patients were to not receive any intra-operative SLN evaluation on frozen sections (FS). In all T1c-patients, the SLN was macroscopically examined; if the node appeared clearly free of disease, no further intra-operative assessment was performed; if the node was clearly metastatic or presented a dubious aspect, the pathologist proceeded with analysis on FS. T2-patients, enrolled in the study as reference group, were treated according the institutional standard procedure; they all received SLN staging on FS.

Results : The study included 395 T1-N0 patients. Among the 118 T1a-T1b-patients whose SLN was not analyzed at surgery, 12 (10.2%) were recalled for ALND. In the group of 258 T1c-patients, 112 received SLN analysis on FS and 146 did not. A SLN falsely negative either at macroscopic or FS examination was found in 33 (12.8%) cases. Overall, the rate of recall for ALND was 11.6% as compared to 8.4% in T2patients. Using the experimental protocol, the institution reached a 9.6% cost saving, as compared to the standard procedure.

Conclusions : Omission of SLN intra-operative staging in T1-N0 patients is rather safe.

It provides the institution with both management and economical advantages.

Introduction

Resection of the primary tumor together with axillary lymph node dissection (ALND) has long been the standard surgical management of early-stage breast cancer.

However, axillary metastases are found in only ~40% of the cases [START_REF] Kim | Lymphatic mapping and sentinel lymph node biopsy in early-stage breast carcinoma: a metaanalysis[END_REF][START_REF] Krag | National Surgical Adjuvant Breast and Bowel Project. Technical outcomes of sentinel-lymph-node resection and conventional axillary-lymphnode dissection in patients with clinically node-negative breast cancer: results from the NSABP B-32 randomised phase III trial[END_REF]; the remaining patients derive no benefit from axillary surgery but unnecessarily face common morbidities from ALND [START_REF] Mansel | Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC Trial[END_REF]. For patients with early-stage tumor (T1-T2) and clinically negative lymph nodes (N0), this problem is now obviated by the use of the sentinel lymph node biopsy (SLNB). This minimally invasive, safe and acceptably accurate procedure has a high negative predictive value for axillary status [START_REF] Veronesi | Sentinel-node biopsy to avoid axillary dissection in breast cancer with clinically negative lymph-nodes[END_REF][START_REF] Giuliano | Sentinel lymphadenectomy in breast cancer[END_REF][START_REF] Krag | The sentinel node in breast cancer-a multicenter validation study[END_REF] and is nowadays the recommended method for axilla staging [START_REF] Schwartz | Proceedings of the consensus conference on the role of sentinel lymph node biopsy in carcinoma of the breast[END_REF][START_REF] Lyman | American Society of Clinical Oncology.American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer[END_REF][START_REF] Silverstein | Image-detected breast cancer: state of the art diagnosis and treatment[END_REF]. Nevertheless, several aspects need further evaluation: 1) is ALND mandatory for all patients with metastatic SLN?; 2) what is the prognostic significance of SLN micrometastases, particularly those evidenced only by immunohistochemistry, and of isolated tumor cells? 3) can patients presenting both SLN micrometastasis and smallsize tumor be spared ALND? 4) is it necessary to always perform SLNB, and when needed, ALND during the initial surgery or could these procedures be postponed in specific instances?

This last question emerges from several considerations [START_REF] Lyman | American Society of Clinical Oncology.American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer[END_REF][START_REF] Weiser | Is routine intraoperative frozen-section examination of sentinel lymph nodes in breast cancer worthwhile?[END_REF][START_REF] Chan | Does the benefit of sentinel node frozen section vary between patients with invasive duct, invasive lobular, and favorable histologic subtypes of breast cancer?[END_REF]: screening mammography has increased the proportion of patients presenting a ≤1cm tumor; these patients have alow probability of nodal disease as the incidence of positive SLN increases with tumor size; the presence of SLN micrometastases is predominantly associated with pT1a-1b tumors; intra-operative false-negative SLN staging on frozen sections (FS) is largely due to the failure to detect micrometastasis by hematoxylin- Based on these considerations, the present study was designed to investigate whether intra-operative SLN staging on FS could be omitted in T1-N0-patients and deferred to definitive evaluation on paraffin sections (PFS). As for risk/benefit and cost/benefit, we hypothesized that this approach could provide patients with shorter surgery duration and hospital stay without substantially increasing the rate of second surgery for ALND, but allowing more efficient use of human resources and logistic facilities.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Patients and methods

Study hypothesis

The main hypothesis was that intra-operative SLN evaluation on FS could be omitted, with measurable efficiency gains, in all patients clinically staged T1a-T1b, and in the subgroup of T1c-patients presenting a SLN macroscopically free of disease. We investigated whether this approach was associated with an unacceptably high rate of recall for ALND, thereby counterbalancing/nullifying the advantages deriving from the reduced duration of the initial surgery. The rate of delayed ALND in T2-patients due to false-negative SLN on FS, was used as reference. Indeed, an overview of our whole case series across the period 1998-2004 has given an acceptable false-negative rate of 8-9%.

A second objective was to assess the accuracy and negative predictive value of the macroscopic evaluation performed by the pathologist in T1c-patients.

A third objective was to estimate the economical and organizational implications of the experimental procedure.

Patient population

This single-center, uncontrolled study was conducted at the National Cancer Research Institute of Genoa, Italy. The protocol, which introduced a modification in our current surgical procedures, that is intra-operative SLN evaluation on FS for all patients, was approved by Institutional Ethical Committees and conducted in accordance with International Good Clinical Practice Guidelines. Patients satisfying the following criteria were selected: primary infiltrating breast carcinoma (cytohistologically C5), small-size tumor (T1-T2) as estimated by mammography and/or ecography, clinically

Surgical protocol (Figure 1)

In all patients, SLN was identified and retrieved as previously described [START_REF] Canavese | Sentinel node biopsy compared with complete axillary dissection for staging early breast cancer with clinically negative lymph nodes: results of randomized trial[END_REF].

Patients clinically staged T1a, T1b or T1c represent the experimental group undergoing the intra-operative protocol under investigation. Information on T2-patients treated according to our standard intra-operative protocol is also provided to allow comparisons with surgical practices in other institutions.

Experimental intra-operative protocol

No intra-operative SLN evaluation was performed in T1a-T1b-patients (tumor size ≤1cm); SLN diagnosis was deferred to the definitive examination on PFS and no axillary dissection was performed in concomitance with surgery.

In T1c-patients (tumor size>1cm but ≤2cm), SLN was subjected to intraoperative macroscopic evaluation as follows: the pathologist first appraised SLN dimension and consistency as well as the status of the capsula. The SLN was then bisected along its major axis and both cut surfaces were assessed for color, nodularity, hemorrhage and necrosis. If none of these parameters was suspicious, the SLN was considered negative and embedded in paraffin for definitive post-operative evaluation;

Standard intra-operative protocol

All T2-patients (tumor size >2cm but ≤5cm) underwent SLNB on FS, which was immediately followed by ALND if the SLN was metastatic.

Intra-operative SLN evaluation on frozen sections

The SLN was bisected along its major axis. Five frozen sections, each 4-µm thick, were cut every 50 µm in each half and stained with hematoxylin-eosin.

Definitive post-operative SLN evaluation on paraffin sections

The bisected SLN was fixed in formalin for 24 hours and embedded in paraffin.

In each half, 10 sections, each 4-µm thick, were cut at every 50 µm (first 5) and every 150 µm (next 5). All sections, but the second and the ninth, were stained with hematoxylin-eosin. If the diagnosis resulted negative or ambiguous, the second and the ninth sections were tested by immunohistochemistry for the presence of cytokeratins, as previously described [START_REF] Canavese | Sentinel node biopsy compared with complete axillary dissection for staging early breast cancer with clinically negative lymph nodes: results of randomized trial[END_REF].

Economical and organizational evaluations

We considered 4 types of surgical procedures and estimated their respective costs including surgery room occupation, medical and nursing staff time, histological SLN assessment and patient care: procedure #1) tumor excision with SLN retrieval but no evaluation on FS; procedure #2) tumor excision with SLNB on FS but no ALND; procedure #3) tumor excision with SLNB on FS and ALND; procedure #4) second

Results

Patient characteristics

Between January 2005 and December 2007, 558 T1-T2-N0 patients were admitted for surgery at our institution. Among them, 33 were not eligible for the study because they had undergone previous surgery on the same breast (n=26), or presented a bilateral (n=5) or multifocal (n=2) tumor. The study thus included 525 patients whose characteristics are summarized in Table 1 Compliance with protocol Considering all 525 patients, a total of 989 SLNs were identified and removed (median 2.0 node/patient, range 1 to 6). Table 2 reports both intra-operative SLN evaluation and ALND performed during surgery, as well as the compliance with the protocol assigned according to the clinical T.

Experimental protocol

Among the 137 T1a-T1b-patients, 118 did not undergo any intra-operative SLN evaluation and, consequently, no concomitant ALND was performed. SLN status was assessed only at definitive examination on PFS, in compliance with the protocol. For the other 19 T1a-T1b-patients, the surgeon specifically requested intra-operative SLN examination on FS because of the dubious macroscopic appearance of the node. Six of those patients presented SLN metastases and underwent concomitant ALND.

For all 258 T1c-patients, SLN was macroscopically evaluated. In 146 cases, it appeared negative, and neither further SLN analysis nor ALND was performed. Ten other patients had a clearly metastatic SLN, which was histologically confirmed, and

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
10 received immediate ALND. For the remaining 102 patients, the macroscopic evaluation resulted unclear and was thus followed by histological analysis on FS. Metastases were found in 43 cases and concomitant ALND was performed. The other 59 patients who presented a histologically negative SLN were spared ALND.

Standard protocol

Among the 130 T2-patients, 126 received histological SLN analysis. Fifty-one of them presented metastases and underwent immediate ALND. Other 75 patients had a negative SLN. For the last 4 patients, SLN assessment on FS was unfeasible due to the exceeding adiposity of the node. The SLN was thus macroscopically evaluated and resulted negative in all 4 cases. None of these 79 patients with negative SLN received ALND

SLN status at definitive diagnosis

Table 3 summarizes the results of the definitive diagnosis of the SLN on PFS as compared with the intra-operative evaluation. 3A) Among the 118 T1a-T1b-patients whose SLN was not evaluated during surgery, 12 resulted positive on PFS. Six of them presented SLN micrometastases (foci > 0.2 mm but ≤ 2 mm) and other 6 had SLN macrometastases (foci >2mm). All 12 patients were thus recalled to undergo ALND. For the remaining 106 patients, the SLN was free of disease, which obviated the need for ALND. cases with macrometastasis and 72 cases free of disease. The 51 macrometastases were all confirmed at definitive examination while 2 of the 8 micrometastases resulted to be macrometastases. Among the 72 SLNs diagnosed disease-free on FS, 9 were found positive on PFS. Seven of these SLNs presented micrometastases and 2 had macrometastases. Thus, for this subgroup of 131 patients, the prevalence of metastatic SLN was 51.9% (95% CI 48.8 -55.0). The accuracy and the negative predictive value of the intra-operative SLN evaluation on FS were 93.1% (95% CI 91.4 -94.5) and 87.5% (95% CI 85.3 -89.4), respectively. The rate of recall for delayed ALND was 6.9% (95% CI 5.5 -8.7).

Experimental protocol (Table

Accuracy of intra-operative macroscopic SLN examination for the subgroup of T1patients

Overall, among the 258 T1c-patients, 86 presented SLN metastases, (24 micrometastases and 62 macrometastases). Fifty-three of these positive SLNs were identified by macroscopic evaluation for a sensitivity of 61.6% (95% CI 58.6 -64.6), while 51 SLNs were false-positive at macroscopic evaluation for a specificity of 70.3% (95% CI 67.4 -73.1). Positive and negative predictive values of SLN macroscopic evaluations were 54.5% (95% CI 51.4 -57.6) and 82.9% (95% CI 80.4 -85.1), respectively.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 12

Standard protocol (Table T3B)

All T2-patients, except for the 4 cases with lipomatous SLN, received intraoperative SLN analysis on FS, without preliminary macroscopic evaluation.

Micrometastases and macrometastases were found in 6 and 46 cases, respectively. SLN positivity was confirmed on PFS for all these 52 patients. Among the remaining 78 patients diagnosed SLN negative on FS, 67 were truly negative while 11 presented micrometastatic (n=8) or macrometastatic (n=3) at definitive evaluation. Prevalence of metastatic SLN in this subgroup was 48.5% (95% CI 45.4 -51.6). Accuracy and negative predictive value of the diagnosis on FS were 91.5% (89.6 -93.1) and 85.9% (95% CI 83.6 -87.9), respectively. The rate of second surgery for ALND was 8.4% (95% CI 6.8 -10.3), which is in line with the ≈8 to 9% rate of recall we observed at our institution across the 1998-2004 period.

Risk/benefit and cost/benefit evaluations

Benefit evaluation

Table 3A shows that, overall, 46 out of the 395 patients treated according to the experimental procedure had to undergo a second surgery for ALND. Thus, the risk of not identifying metastatic SLN(s) in this settings is slightly higher as compared with the expected risk of having false-negative SLN(s) on standard FS (11.6% versus the ≈8.0% estimated from our 1998-2004 institutional report).

Cost evaluation

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

13 Table 4 compares, within this group of 395 patients, the actual costs of the experimental protocol with the theoretical costs the institution would have if all patients were treated according to the standard protocol. Table 4A summarizes the cost of each of the 4 surgical procedures used in the study. . All 118 T1a-T1b-patients not receiving any intra-operative SLN evaluation underwent procedure #1 (Table 4B). Twelve of them were also recalled for second surgery. The total cost was thus 157,600 euros. If these patients were treated with the standard protocol, 106 would have procedure #2. Considering a ≈8% false-negative rate on FS, 1 of the other 12 patients would receive both procedure #2 and recall for ALND.

The remaining 11 patients would undergo procedure #3. Overall, the theoretical cost of the standard procedure would be 186,900 euros. For this subgroup of T1a-T1b-patients, the actual cost was thus ≈15.7% less than the theoretical one.

When we analyzed on the same line the subgroup of 146 T1c-patients who underwent only macroscopic SLN evaluation, we obtained an actual cost of 212,800 euros (Table 4C), which includes 146 procedures #1 and 25 recalls for ALND. The standard protocol would foresee, for a total cost of 242,700 euros, 123 procedures #2 including 2 cases of false-negative SLN needing delayed ALND, and 23 procedures #3.

The actual cost thus represented 87.7% of the theoretical one.

The remaining 131 T1c-patients who underwent both macroscopic and histological SLN evaluations were treated essentially as the standard protocol. Actual and theoretical costs are thus equivalent.

M A N U S C R I P T A C C E P T E D

(procedure #2), 59 with positive SLN underwent concomitant ALND (procedure #3) and 46 with false-negative SLN had delayed ALND (procedure #4). The total cost of the experimental procedure was thus 629,100 euros. If the standard protocol were applied, 299 of these patients, that is those with a negative SLN, would have procedure #2, 59 patients with positive SLN would receive procedure #3 and other 9 patients with false-negative SLN would have procedure #4. Among the remaining 37 patients who did not have intra-operative histological SLN assessment, 3 would have a delayed ALND based on the expected 8% rate of false-negative node, which raises the number of procedure #4 to 12. The other 34 patients would be correctly diagnosed with a positive SLN and undergo concomitant ALND. The total theoretical cost would be 697,400 euros. Using the experimental protocol, the institution thus reached an overall 9.8% saving, corresponding to ≈70.000 euros.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Discussion

Rate of delayed ALND

We investigated whether intra-operative SLN evaluation could be omitted for patients clinically staged T1-N0, without excessively jeopardizing their post-surgery course while allowing more efficient use of human resources and logistic facilities. The main parameter under assessment was the rate of delayed ALND as compared to that we usually observe when applying standard surgical procedures. This rate of 8-9% is provided by the group of T2-patients here included as reference and is similar to that observed across 69 other studies [START_REF] Lyman | American Society of Clinical Oncology.American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer[END_REF].

For the group of T1a-T1b-patients whose SLN was not evaluated at surgery, the rate of delayed ALND was 10.2%, which is quite acceptable as compared to the 8.4% rate observed for the T2-patients. Among the 146 T1c-patients whose SLN was evaluated only macroscopically, the node was falsely negative in 25 cases, thus giving a rate of delayed ALND as high as 17.1%. However, in most cases, the pathologist could not have detected these metastases as they were either micrometastases <0.2mm (n=12), or sparse metastatic cells originating from infiltrating lobular carcinomas (n=4), or metastases embedded in a lipomatous SLN (n=3).

Risk and benefit considerations a) in the present settings

Overall, 46 of the 395 patients of the experimental group needed a second surgery for completion of axillary lymph node dissection. This corresponds to a 3.2% increase of delayed ALND as compared to the 8.4% reference rate. In terms of

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

16 cost/benefit, the overall cost of these 395 experimental procedures was 9.8% less than if all patients were treated according to the standard procedures.

The limited, though non-negligible, risk of delayed ALND and the economical implications are not the sole factors to be taken into account when considering our experimental protocol as a possible routine option for T1-N0-patients. Indeed, this protocol warrants both shorter surgery duration and hospital stay for the majority of patients; in turn, this allows more efficient management of human resources and use of surgery rooms. When intra-operative SLN examination is not performed, surgery duration is reduced by 23 % or 34% as compared to a surgery that includes SLNB with negative result or SLNB with positive result and subsequent ALND, respectively. Thus, more patients can be treated by the same staff and waiting lists can be shortened. by simply scheduling surgery, on fixed days, for only T1-N0 patients.

b) in the absence of any intra-operative SLN evaluation

We are aware that at some institutions, intra-operative SLN evaluation is never performed for T1-T2-N0 patients but is straightly deferred to the definitive diagnosis on PFS. Considering the T1-patients of our study, those with a T1c-tumor and a SLN macroscopically unclear or positive received intra-operative SLN examination. If this intra-operative evaluation were not performed, the management of human resources and logistic facilities would further improve. However, the additional economical advantage would be insignificant (less than 1.5%) and patients would suffer major distress knowing that the possibility to be recalled for a second surgery is greater than 1 over 10.

Conclusions

The experimental protocol here evaluated (no SLN evaluation or macroscopic examination) seems to be rather safe for the subgroup of T1-N0-patients. Furthermore, it is beneficial for the institution, in terms of efficiency and costs. Alternatively, the possibility not to perform any intra-operative evaluation but to always defer SLN assessment to post-surgery definitive diagnosis might be considered for all T1-N0patients. A third option is to let the patients, fully informed on the risks and implications of the procedure, freely choose whether to undergo intra-operative SLN evaluation and, if needed, immediate ALND. Intra-operative SLN examination should remain the standard procedure for all T2-N0-patients.

Conflict of interest statement

None of the authors has any of interest regarding the manuscript.

  pT1c-pT2-patients mostly benefit from FS examination since the sensitivity of the procedure increases with tumor size.

  nodes. Eligible patients who had undergone previous surgery on the same breast and/or the ipsilateral axilla, or presented a bilateral, multifocal or multicentric tumor were excluded. All enrolled patients were informed on study aims and implications and signed a consent form.

  no axillary dissection was performed in concomitance with surgery. If the SLN presented areas possibly metastatic, FS were analyzed. If metastatses were found, the surgeon proceeded with ALND. If the SLN was clearly metastatic, FS were anyway M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 analyzed to confirm the diagnosis of node disease and ALND was immediately performed.

  ALND. Based on costs of personnel and hospital care at our institution, the estimates of procedures #1, #2, #3 and #4 were 1150, 1467, 2550 and M A N U S C R I P T A C C E P T E D

  Upon intra-operative macroscopic evaluation, the SLN of 146 T1c-patients was considered negative. The diagnosis was correct in 121 of the cases. By contrast, the M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 SLN was falsely negative for 25 patients, resulting at definitive diagnosis micrometastatic or macrometastatic in 12 and 13 of the cases, respectively. All these 25 patients thus received delayed ALND. For the 131 patients (112 T1c and 19 T1a-T1bpatients) whose SLN was macroscopically either clearly positive or dubious, additional intra-operative examination on FS evidenced 8 cases with micrometastasis, 51 (39.8%)

  On the same line, if the SLN of the T2-patients were evaluated only at definitiveM A N U S C R I P T A C C E P T E Danalysis, time and cost savings would be negligible due a rate of delayed ALND as high as 40%.

.

Figure 1 :

 1 Figure 1 : Study design and protocol both for intra-operative evaluation of the sentinel

Table 4D

 4D summarizes the global cost evaluation for all 395 patients of the experimental group. Overall, 264 patients did not have histological SLN examination (procedure #1) , 63 presented a histologically negative SLN and did not receive ALND

Table 1 . Patient characteristics (n = 525)

 1

	Characteristic	n	(% )
	Age (years)
	Median (range)	63 (23 -85)
	Age by category
	≤ 45 y	126	(24.0)
	46 -55 y	102	(19.4)
	56 -65 y	163	(31.0)
	> 65 y	134	(25.5)
	Clinical T T1a
	T1b	28	( 5.3)
	T1c	109	(20.8)
	T2	258	(49.1)
		130	(24.8)
	Pathological T
	pT1a	27	(5.1)
	pT1b	106	(20.2)
	pT1c	250	(47.6)
	pT2	141	(26.9)
	pT3	1	(0.2)
	Tumor site
	Right	248	(47.2)
	Left	277	(52.8)
	Quadrant
	QSE	215	(41.0)
	QSI	60	(11.4)
	QII	47	(9.0)
	QIE	31	(5.9)
	QC	24	(4.6)
	>1 Quadrant	148	(28.1)
	Tumor histology
	Ductal	431	(82.1)
	Lobular	60	(11.4)
	Other	34	(6.5)

Table 2 : Compliance to the protocol established for both intra-operative SLN evaluation and axillary lymph node dissection, stratified by clinical T.

 2 intra-operative histological diagnosis performed upon explicit request from the surgeon. § macroscopic evaluation only (intra-operative histological assessment not feasible).

	SLN evaluation	Axillary				T1a -1b (total=137)		T1c (total=258)	T2 (total=130)
	(H.E. histological	dissection	n		compliance	n	compliance	n	compliance
	staining)
	no	no	118	(as protocol)	146	(as protocol -macroscopic negative)	4 §	fatty SLN
	yes	no	13	#	SLN dubious	59	macroscopic unclear -(as protocol -	75	(as protocol)
								H.E. negative)
	yes	yes	6	#		SLN dubious	43	macroscopic unclear -(as protocol -	51	(as protocol)
								H.E. positive)
	yes	yes	-		-	10	(as protocol -	-	-
								macroscopic positive)

#

Table 3 : SLN status at definitive diagnosis as compared to the intra-operative assessment. A. Experimental protocol (T1a, T1b and T1c patients)

 3

	SLN status	No evaluation	Macroscopic	Macroscopic evaluation -positive or unclear followed
				evaluation -negative	by intra-operative H.E .histological staining
		T1a -T1b	T1c	T1c	T1a -T1b
			(n=118)	(n=146)	(n=112)	(n=19)
		Intra	Definitive	Intra	Definitive	Intra	Definitive	Intra	Definitive
	Micrometastasis	-	6	-	-	8	neg 0	-	-
							pos 8
							-micro 6
				-	-		-macro 2
	Macrometastasis	-	6			45	neg 0	6	neg 0
							pos 45		pos 6
	Negative	-	106	146	neg 121	59	neg 51	13	neg 12
					pos 25		pos 8		pos 1
				-micro 12		-micro 6		-micro 1
				-macro 13		-macro 2
	Test in % (95% CI)					T1c	Total	T1a-T1b
	Recall for ALND	10.2 (8.5-12.2)	17.1 (14.9-19.6)
	Accuracy		-	-
	Negative Predictive Value		-	82.9 (80.4-85.1)
	Prevalence of metastatic SLN	10.2 (8.5-12.2)	17.1 (14.9-19.6)

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

including the 19 T1a-1b patients who underwent SLN intra-operative histological evaluation upon explicit request from the surgeon

B. Standard protocol (T2 patients)

SLN status

Intra-operative H.


M.D. Frank Benedix
email: frankbenedix@gmx.de

A Reimer

I Gastinger

P Mroczkowski

H Lippert

R Kube

epidemiology, surgery and survival: results of a German multi-center study

Keywords:

Background. While carcinoma of the colon is a common malignancy, primary carcinoma of the appendix is rare. Many retrospective reviews outlined experience from different centers on appendiceal neoplasms. However, the study population is often small because it is so rare. The aim of this study was to analyze the type of surgery and survival of patients with appendiceal malignancies using data from a German multi-center observational study (31 341 patients).

Methods. During a five-year period, 196 consecutive patients with malignant appendiceal tumors were distributed into four groups: appendiceal carcinoids, adenocarcinoma, mucinous adenocarcinoma and adenosquamous carcinoma. The following parameters were analyzed: demographics, clinical presentation, comorbidities, type and appropriateness of surgery, final pathology and survival.

Results. Adenocarcinoma had the highest incidence (50.5%). The most common presentation was that of acute appendicits. Mean age at presentation was youngest for carcinoid tumors. Carcinoid tumors had lowest tumor size and localized disease was present in 72.9%. Metastatic spread at presentation was highest for adenosquamous and mucinous adenocarcinoma and each had a distinct pattern.

Right hemicolectomy was performed in 71.4%, limited resection in 11.7%. Overall 5year survival was 83.1% for carcinoid vs. 49.2% for non-carcinoid tumors. Histological subtype and tumor stage significantly affected survival.

Conclusions.

Long-term outcome of carcinoid tumors is superior to noncarcinoid neoplasms. Among all appendiceal neoplasms, adenosquamous carcinoma is the rarest histological subtype which is most commonly associated with advanced tumor stage and worst prognosis. Appropriate oncologic resection is being performed in a significant percentage of cases in Germany. However, the high rate of right hemicolectomy in patients with small carcinoid tumors needs to be critically discussed.

Introduction

Primary appendiceal tumors are rare entities, occuring in < 2% of all appendectomies with an age-adjusted incidence of 0.12 cases per 1,000,000 people per year [START_REF] Connor | Appendiceal tumors: retrospective clinicopathologic analysis of appendiceal tumors from 7,970 appendectomies[END_REF][START_REF] Bucher | Appendix tumors in the era of laparoscopic appendectomy[END_REF][START_REF] Iwuagwu | Primary carcinoma of the appendix -Hull series[END_REF][START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Von Suchodoletz | Distinctive features of appendicitis in childhood[END_REF].

The vast majority of these tumors are not diagnosed preoperatively -rather, they present with acute appendicitis or are detected as an incidental finding during exploration for another surgical pathology. Endocrine appendiceal tumors make up to 35 -85% of all appendiceal neoplasms [START_REF] O'donnell | Malignant neoplasms of the appendix[END_REF][START_REF] Sandor | A retrospective analysis of 1570 appendiceal carcinoids[END_REF]. Most of these tumors are located at the tip of the appendix with a maximal diameter of < 1cm in 60 -80% of cases [START_REF] O'donnell | Malignant neoplasms of the appendix[END_REF][START_REF] Stinner | Neuroendocrine tumours (carcinoids) of the appendix[END_REF].

Metastatic disease in patients with tumors < 2cm is rare. Five-year survival is 83% for all stages [START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. Primary adenocarcinoma of the appendix is a rare malignancy that constitutes less than 0.5% of all neoplasms of gastrointestinal origin [START_REF] Chang | Adenocarcinoma of the appendix[END_REF]. These tumors arise in pre-existing adenomas, either by a cystic and colonic growth pattern.

Cystic type is a mucin-producing tumor and is comparable to cystadenocarcinoma of the ovary, tending to rupture and spread throughout the abdominal cavity, resulting in pseudomyxoma peritonei. Colonic type is a polypoid or ulcerative tumor that develops from a tubular or tubulovillous adenoma [START_REF] O'donnell | Malignant neoplasms of the appendix[END_REF][START_REF] Deans | Neoplastic lesions of the appendix[END_REF]. It is comparable to adenocarcinoma of the colon. Mucinous histology has a better survival than colonic histology for appendiceal adenocarcinomas [START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF][START_REF] Cortina | Management and prognosis of adenocarcinoma of the appendix[END_REF]. Right hemicolectomy has been recommended for all adenocarcinomas. Surveillance for synchronous or metachronous tumors is warranted because the risk of a second primary malignancy in the gastrointestinal tract is significantly increased [START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF].

Adenosquamous carcinoma of the colon is defined as a neoplasm comprising adenocarcinoma and squamous cell carcinoma. The epithelium near the dentate line is anatomically capable of differentiating into both glandular and squamous epithelium. Primary adenosquamous carcinoma of the colon is rare at sites other than the lower rectum and anal canal and has a poor prognosis [START_REF] Yokoi | Case of adenosquamous carcinoma of the ascending colon[END_REF].

Adenosquamous carcinoma originating from the vermiform appendix has not been reported so far.

A number of retrospective reviews have outlined experience from different centers on appendiceal neoplasms. However, due to its rarity the number of patients in most of the reports is small. The purpose of this study was to examine all patients with neoplasms of the appendix in a German multi-center observational study. We evaluated epidemiology, patient characteristics, type of surgery, final pathology and survival for four distinct histological types of appendiceal tumors. Finally, we determined appropriateness of resection for appendiceal tumors according to current treatment guidelines in Germany.

Material and Methods

Within the German multi-center observational study "Colon/Rectal Carcinoma (Primary Tumor)", data for 31 341 patients with colon malignancy was acquired prospectively for a five year period.

A standardized questionnaire was completed at the hospital where the patient was treated, to record information about the patient, tumor, operation, final pathology and perioperative course. A follow-up questionnaire was completed by the patient's GP or, by the primary hospital itself to gather information on the last patient contact, life status, recurrence and if the patient died, the cause of death (tumor-related, tumorindependent). The study was conducted according to the Declaration of Helsinki for Biomedical Research. Participation was voluntary, evaluation was based on anonymous data, and the study involved observation only -it had no influence upon the choice or course of therapy. For these reasons, an Ethics Committee vote was not necessary. All patients gave written consent for data collection and anonymous evaluation.

Inclusion criteria for the present analysis were:

-Patients with malignant appendiceal tumors -All patients recruited between January 1, 2000 and December 31, 2004 -Patients with surgical tumor-resection to ensure final pathology.

Exclusion criteria were as follows:

-Benign and non-epithelial tumors of the vermiform appendix -Palliative surgery without resection of the tumor (i.e. bypass procedure).

Patients were distributed into four groups according to the histological subtype:

1) Appendiceal Carcinoids (AC)
2) Appendiceal Adenocarcinoma (aA)

3) Mucinous Appendiceal Adenocarcinoma (mAA)

4) Appendiceal Adenosquamous carcinoma (AAS)

The following parameters were analyzed for all patients: age, sex, body mass index (BMI), reason for admission, comorbidities, presence of synchronous distant metastases, type of surgery, necessity of multivisceral resection, final pathology, 5year-overall and disease-free survival (DFS). Histopathologically, all non-carcinoid neoplasms were classified according to the TNM and UICC-classification. All carcinoid tumors were categorized by maximum tumor size as < 1cm, 1 to 2cm and > 2cm. Furthermore, they were subdivided into three groups for staging purposes: 1) localized (the neoplasm confined entirely to the appendix); 2) regional (extension beyond the limits of the appendix directly into surrounding tissue and/or regional lymph nodes; 3) distant (tumors with metastatic spread). Overall and disease-free survivals were compared.

Statistics

All data was entered into an MS ACCESS data base and was processed by using the statistical package SPSS (version 13.0; SPSS Ltd., Chicago, IL, USA). Data is expressed as arithmetic means ± standard deviation. The statistical significance of differences between the subgroups was estimated by one-factor analysis of variance (ANOVA) with subsequent multiple comparison according to Tukey or by Pearson's χ 2 test. Kaplan-Meier curves for survival were compared by the log-rank test.

Differences between the groups were to be regarded as significant if p < 0.05 was found in a two-sided test.

Results

Between January 1, 2000 and December 31, 2004, all consecutive patients with colon malignancy (n=31 341) were analyzed for this study. Of those, 196 (0.63%) had histologically confirmed malignant appendiceal tumors. Among all appendiceal neoplasms, the highest incidence in this study was demonstrated for aA (n = 99; 50.5%), followed by AC (n = 48; 24.5%), mAA (n = 45; 23%) and AAS (n = 4; 2%).

Study population / Demographics

Mean age at presentation for AC (mean 51.1 ± 17.4 years) was significantly lower than for the other subtypes (p < 0.01). mAA had the highest proportion of female patients (51.1%). A male predominance was noted for all other groups. Mean BMI did not differ significantly among all groups. (p = 0.396) (Table 1)

Clinical presentation / characteristics

Overall, 57 patients (29.1%) were referred to the surgical department for secondary procedure after an appendiceal neoplasm was confirmed histologically following primary appendicectomy. Seventy-three patients (37.2%) presented with signs and symptoms of an acute abdomen suggestive of acute appendicits. In 45 patients (23%), the appendiceal lesion was discovered incidentially during another surgical procedure. The remaining 21 patients (10.7%) were initially referred to the hospital with nonspecific abdominal pain or tumor-related symptoms for further investigation (Table 1). Comorbidities were significantly more common in patients with mAA (77.8%), predominantly cardiovascular (53.3%) and diabetes mellitus (15.5%) (p < 0.01).

Operative procedure

Right hemicolectomy was performed in 71.4% of the cases (140 patients; AC: 35; aA: 67; mAA: 35; AAS: 3). This type of surgery was performed either due to apparent or suspected tumor intraoperatively or as intendedprocedure for patients admitted to the hospital with confirmed malignancy or as complementary procedure during the same hospital stay after primary appendicectomy. Twenty-three patients (11.7%) underwent primary ileocecal resection, all for apparent or suspected tumor during initial surgery (AC: 3; aA: 18; mAA: 2). Limited resection (cecal resection or appendicectomy) was performed in 11.7% (23 patients, AC: 8; aA: 8; mAA: 6; AAS: 1). An extended right hemicolectomy became necessary in 9 patients (4.6%, AC: 2; aA: 5; mAA: 2), a subtotal colectomy in one patient (0.5%, aA: 1) due to synchronous colonic pathology. For the same reason, 5 patients had simultaneous sigmoid colon resection (AC: 1; aA: 3; mAA: 1), 3 patients anterior resection (aA: 1; mAA: 2) and one patient transverse colon resection (mAA: 1).

Multivisceral resection due to locally advanced tumor growth was performed in 10.4% of AC cases (2 small bowel, 2 ovary, 1 abdominal wall) and 16.2% of aA (4 small bowel, 3 ovary, 8 abdominal wall, 2 omentum, 1 uterus, partial resection of urinary bladder / liver / stomach: 1/2/1). Extended resection was most frequently (33.3%; p = 0.012) required in mAA patients (15 patients; 2 small bowel, 3 ovary, 1 abdominal wall, 3 omentum, 4 uterus, partial resection of urinary bladder 2). Complementary carcinologic resection was planned in a further 8 (4.1%) patients (5 with primary appendicectomy, 3 with right hemicolectomy but inadequate lymph node dissection).

Based on final pathology, adjuvant treatment with chemotherapy was initiated in 12.2% (aA: 15, mAA: 9 patients).

Histopathologic staging / pathological analysis

The mean tumor size at presentation was significantly lower for AC (1.8 cm) compared to all other tumor subtypes (p < 0.01). (Table 2). In 41.6% of all AC patients, tumor size was < 1cm. There were 14 patients (29.1%) in each of the remaining groups: tumor diameter of 1-2 cm and > 2cm, respectively. Lymph node positive disease was diagnosed in 16.6%. Stage distribution of localized appendiceal carcinoids, regionalized lesions and distant metastatic disease was 72.9%, 22.9% and 4.2%, respectively. Lymphatic invasion was present in 10 AC patients and was associated with advanced tumors. In all AC patients with distant metastastic disease and 62.5% of patients with lymph node positive disease, lymphatic invasion was found. Eight of the ten patients had a tumor size > 2cm. None of the patients with a tumor size < 1cm had lymph node positive disease or lymphatic invasion. For noncarcinoid tumors, the highest proportion of locally advanced tumors was found for AAS (100%), followed by mAA (88.9%). Lymph node positive disease was most frequently diagnosed in AAS (50%), followed by aA (36.4%). The lowest rate of lymphatic tumor spread was found for AC (16.6 %) (p = 0.052). Analysis of tumor differentiation revealed the highest proportion of poorly differentiated carcinomas for AAS (75%), whereas mAA had the highest number of well differentiated tumors (24.4%; p = 0.042) (Table 2). Incidence of synchronous metastases at presentation was highest for AAS (50%), followed by mAA (28.9%) and aA (23.2%). In AC, presence of distant metastatic disease was found in 4.2% and only for tumors with a diameter > 2cm. Distinct results were also found regarding the pattern of metastatic spread. Among all patients with metastatic disease, hepatic metastases were more commonly associated with AC (50%) and aA (43.5%) compared with mAA (7.7%) and AAS (0%) (p = 0.049). By contrast, peritoneal carcinomatosis was found in both cases of metastatic AAS and 76.9% of the metastatic mAA cases (Table 3). Rate of synchronous colon neoplasms was 6.2%, 10.1% and 8.9% for AC, aA and mAA, respectively.

Appropriateness of surgical resection/therapy

Appendicectomy or cecal resection alone as definitive surgical procedure was performed on 23 (11.7%) patients (AC: 8, aA: 8, mAA: 6; AAS: 1). Seven patients (aA: 4, mAA: 2, AAS: 1) had palliative appendicectomy/cecal resection due to distant metastastic disease. Six patients had AC with a tumor diameter of < 1cm. In five ( 2AC with a tumor size > 2 cm, 1 T2 aA, 1 T2 mAA, and 1 T4 mAA) complementary carcinologic resection was planned. The remaining 5 patients with appendiceal tumors (AC > 2cm; T1&T2 aA; T2&T3 mAA) and local resection alone had no oncological surgery.

Ileocecal resection was performed in 23 (11.7%) patients. Of those, 8 patients had distant metastatic disease and two AC with a tumor diameter < 2cm. The remaining 13 patients had AC > 2cm (1 patient) and aA (12 patients, T1 x 1, T2 x 4, T3 x 6; T4 x 1; N1 x 2).

According to current guidelines, right hemicolectomy should be performed for all noncarcinoid invasive tumors and AC tumors > 2 cm. Eighteen patients (9.2%) in this study fulfilled these requirements, but did not undergo oncologic surgery. For small (≤ 1cm) carcinoid tumors, appendicectomy is recommended, however, of the 20 patients with an AC tumor < 1cm in this study, 60% underwent unnecessary right hemicolectomy. None of these patients was diagnosed with concomitant lymphatic invasion.

According to current guidelines, all patients with UICC stage III colon adenocarcinoma should receive adjuvant chemotherapy. In this study, only 24 out of 29 (82.7%) patients with lymph node positive disease had chemotherapy following curative surgical resection.

Survival

Complete and plausibility-checked follow-up data was available for 89.3% of all 196 patients. The median follow-up time was 40.6 months. Perioperative mortality rate was 2.5% (5 patients). Overall 5-year survival was 83.1% for carcinoid vs. 49.2% for non-carcinoid tumors (aA: 47.5%, mAA: 50.9%) (p=0.01) (Fig. 1). Two of four patients with AAS died within 25.9 months of diagnosis. Tumor stage (localized, regionalized and distant metastatic disease) and tumor size significantly affected survival in AC with overall 5-year survival rates of 92.2%, 80% and 50% (tumor stage), and 100%, 80% and 50% (tumor size), respectively (p < 0.01). Patients with mAA fared better than those with aA when analyzed with respect to UICC stage except for stage III (100% (I), 79.3% (II), 25.4% (III), 17.9% (IV) vs. 87.5%, 58.7%, 38.8%, 12.5%).

Similarly, histological subtype significantly affected DFS with 5-year survival rates of 87.7% for carcinoid vs. 53.5% for noncarcinoid tumors (p < 0.01) (Fig. 2). Of the four patients with AAS, two were diagnosed with primary metastatic disease. In the remaining two, there is no evidence of tumor recurrence 19 and 29 months, respectively, after curative resection. To determine the influence of type of surgery on DFS, all patients with limited resection were compared with those who underwent appropriate oncologic resection with curative intent. Surprisingly, of the latter group, 42% developed recurrent disease whereas only 22.2% of the patients with limited resection had a local recurrence or distant metastatic spread. However, these results may not be representative because median follow-up for patients with limited resection was shorter (31.0 months) and sample size was significantly smaller.

Discussion

While carcinoma of the colon is a common malignancy, primary carcinoma of the appendix is rare and is a somewhat different entity, particularly in presentation and histological type. It constitutes < 0.5% of all gastrointestinal neoplasms [START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. The lifetime incidence of malignant appendiceal tumors ranges from 0.2 to 0.5% [START_REF] Cerame | A 25-year review of adenocarcinoma of the appendix. A frequently perforating carcinoma[END_REF].

The current study evaluates the epidemiology, type of surgery, final pathology and survival outcome for four distinct histological types of appendiceal carcinoma using data from a multi-centered observational study. Among all colon carcinomas, the incidence of malignant appendiceal tumors in this report was 0.63%. Incidence of noncarcinoid appendiceal neoplasms was higher than that of carcinoid tumors, which is in line with the results of the two largest series of appendiceal tumors [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. Mean age at presentation for noncarcinoid appendiceal neoplasms ranged from 59 to 63 years in most reports -consistent with the findings in this study [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Sandor | A retrospective analysis of 1570 appendiceal carcinoids[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. Although patients with malignant carcinoids were significantly younger at the time of diagnosis, mean age was higher than reported by others [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Sandor | A retrospective analysis of 1570 appendiceal carcinoids[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. This can be attributed to the fact that goblet cell carcinoids, which present at a higher age, were not separately analyzed in this study. Gender distribution for noncarcinoid tumors was comparable with other reports [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. By contrast, there was a male predominance for carcinoid tumors, which has also been observed in smaller series [START_REF] Tchana-Sato | Carcinoid tumor of the appendix: a consecutive series from 1237 appendectomies[END_REF][START_REF] Hof | Carcinoid tumour of the appendix: an analysis of 1,485 consecutive emergency appendectomies[END_REF][START_REF] Graham | Primary epithelial tumours of the appendix in a black population: a review of cases[END_REF]. This is possibly related to analysis of both, classical carcinoids and adenocarcinoids as one group and to the relatively low number of patients. Clinical presentation varied in this study.

Most commonly, patients presented with symptoms and signs suggesting acute appendicitis, nonspecific pain or tumor-related symptoms, similar to results of other studies [START_REF] Bucher | Appendix tumors in the era of laparoscopic appendectomy[END_REF][START_REF] O'donnell | Malignant neoplasms of the appendix[END_REF][START_REF] Cortina | Management and prognosis of adenocarcinoma of the appendix[END_REF][START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. However, the number of patients with a preoperative diagnosis of acute appendicitis was lower compared with other reports, which may be attributed to the fact that nearly one third of the patients were referred for complementary surgery.

In these patients, initial presenting symptoms were not recorded. The higher rate of concomitant medical disease for patients with noncarcinoid neoplasms in this study is most likely related to the higher age at the time of diagnosis.

For appendiceal carcinoids, the stage distribution was 72.9% for localized, 22.9% for regionalized lesions and 4.2% for distant metastatic disease, which is comparable with those reported by others [START_REF] Sandor | A retrospective analysis of 1570 appendiceal carcinoids[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. Lymphatic invasion was found to be associated with advanced tumor growth and metastatic disease and thus, may be of predictive value. Therefore, based on these results, in every patient with lymphatic invasion on final pathology, complementary surgery should be strongly considered. For noncarcinoid tumors, mAA had a higher proportion of locally advanced tumors, whereas aA was more frequently diagnosed with lymph node positive disease. This is in agreement with the results of other studies [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF][START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. Analysis of tumor grade revealed the significantly highest rate of well differentiated carcinoma for mAA, which is not being reflected in proportion to locally advanced and metastatic tumors. The results provide further evidence that there are specific differences in the molecular biological pattern between mAA and aA which subsequently cause a different carcinogenesis and development of biologically different cancers.

Appendiceal carcinoid tumors exhibit little metastatic potential and therefore, rarely present with metastases [START_REF] Roggo | Carcinoid tumors of the appendix[END_REF][START_REF] Lehner | Liver resection for noncolorectal, non-neuroendocrine liver metastases--is hepatic resection justified as part of the oncosurgical treatment?[END_REF]. They usually metastasize to the regional lymph nodes rather than to the liver. The tumor size is of predicting value for metastatic disease. The calculated risk of metastases from tumors < 1cm is zero, while a definite increase occurs with a tumor size > 2cm, with a rate of metastases of 20% [START_REF] Tchana-Sato | Carcinoid tumor of the appendix: a consecutive series from 1237 appendectomies[END_REF][START_REF] Thompson | Carcinoid tumors of the gastrointestinal tract: presentation, management, and prognosis[END_REF]. In this report, only tumors > 2cm had distant metastatic disease at the time of diagnosis with a rate of 14.3%. By contrast, noncarcinoid neoplasms of the appendix exhibit a more aggressive behaviour and are more likely to develop metastatic disease. mAA were reported to have a higher rate of metastases than aA, which is in agreement with our results [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. In mAA, perforation will often result in pseudomyxoma peritonei, that is, peritoneal tumor implantation with free intraperitoneal mucous containing cellular elements. Apart from AAS, mAA had the highest rate of peritoneal carcinomatosis in this study. Peritoneal cavity was also the most common location for metastases of mAA in a study by Nitecki et al. [START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. In contrast to our results, no lymphatic or blood-borne metastases were found in that report. It has been recognized that patients with pseudomyxoma peritonei and mucinous tumors of the ovaries often have mucinous tumors of the appendix and that the ovarian tumors are probably secondary to the appendiceal tumor [START_REF] Young | Mucinous tumors of the appendix associated with mucinous tumors of the ovary and pseudomyxoma peritonei. A clinicopathological analysis of 22 cases supporting an origin in the appendix[END_REF]. This is in agreement with our results, with the highest rate of metastases to the ovaries for mAA. In a study by Cortina et al., ovaries were the second most common location of metastases in patients with mAA [START_REF] Cortina | Management and prognosis of adenocarcinoma of the appendix[END_REF]. In contrast, patients with appendiceal adenocarcinoma were more frequently found to have liver metastases in this study, suggesting a different pattern of metastatic spread, and thus supporting the notion that mAA and aA are two distinct morphologies with different biological behavior.

According to the current recommendations, all noncarcinoid tumors should undergo right hemicolectomy, whereas the type of surgical intervention for carcinoid tumors of the appendix depends on size, high grade or high mitotic index, mesoappendiceal invasion, location, and presence of goblet cell histology [START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF][START_REF] Goede | Carcinoid tumour of the appendix[END_REF][START_REF] Plöckinger | Consensus guidelines for the management of patients with digestive neuroendocrine tumours: well-differentiated tumour/carcinoma of the appendix and goblet cell carcinoma[END_REF]. Local resection is adequate for small carcinoid tumors (< 2cm) at the appendiceal tip, with no evidence of local spread [START_REF] Goede | Carcinoid tumour of the appendix[END_REF]. However, adequate therapy of patients with tumor size from 1 to 2cm in diameter remains a matter of controversy since there is a risk of regional metastases. On the other hand, right hemicolectomy is a significant abdominal procedure with an associated risk, especially in the elderly or infirm [START_REF] Goede | Carcinoid tumour of the appendix[END_REF]. Comparing the risk of right hemicolectomy with the survival figures for patients with lesions smaller than 2cm, a more conservative approach seems appropriate in most circumstances. In those with tumors of 1 -2cm towards the tip of the appendix, with typical carcinoid histology, no mesoappendiceal invasion and low proliferative index, a right hemicolectomy does not appear to be justified [START_REF] Goede | Carcinoid tumour of the appendix[END_REF]. Roggo et al. concluded that it would be reasonable to offer right hemicolectomy only to younger patients [START_REF] Roggo | Carcinoid tumors of the appendix[END_REF].

Goede et al. described acceptable indications for re-intervention: lesion larger than

2cm in diameter, histological evidence of of mesoappendiceal extension, tumors at the base of the appendix with positive margins or involvement of the cecum, highgrade malignant carcinoid and goblet-cell adenocarcinoids [START_REF] Goede | Carcinoid tumour of the appendix[END_REF]. In addition, angiolymphatic invasion was demonstrated in this study to be associated with advanced tumor growth and metastatic disease. Therefore, we would also recommend complementary surgery in these patients providing they are in a reasonable medical condition. Timing of secondary hemicolectomy should be within 3 months after appendicectomy and can safely be performed by laparoscopic approach. There is no data to support that a two-step approach may negatively affect the prognosis [START_REF] Plöckinger | Consensus guidelines for the management of patients with digestive neuroendocrine tumours: well-differentiated tumour/carcinoma of the appendix and goblet cell carcinoma[END_REF].

Adenocarcinoma of the appendix should be treated with right hemicolectomy due to the high rate of invasion and nodal metastases [START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. Patients with primary appendicectomy are best managed by reoperation and a formal right hemicolectomy because of the risk of overlooked nodal metastases which have been reported to be approximately 38% [START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. Adjuvant therapy is similar to what is recommended for colon adenocarcinomas. Thus, all patients with lymphatic involvement should receive adjuvant chemotherapy [START_REF] O'donnell | Malignant neoplasms of the appendix[END_REF]. The treatment of appendiceal mucinous-type tumors appears to be more controversial. However, a few studies reported a survival advantage between right hemicolectomy and simple appendicectomy [START_REF] Cortina | Management and prognosis of adenocarcinoma of the appendix[END_REF][START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF].

Moreover, the results of this study support a more aggressive approach even if metastatic disease is present due to a better survival compared to patients with adenocarcinoma. Although there is a tumor spread throughout the peritoneal cavity with superficial invasion in up to 65% in patients with pseudomyxoma peritonei, deep visceral invasion is rare [START_REF] Gough | Pseudomyxoma peritonei. Long-term patient survival with an aggressive regional approach[END_REF][START_REF] Paulman | Pseudomyxoma peritonei[END_REF]. Therefore, oncologic right hemicolectomy, tumor debulking and removal of all mucinous ascites seem to be justified in these patients.

We agree that routine oophorectomy, especially if post-menopausal, should at least be considered in all cases with adenocarcinoma, particularly in mAA due to a high rate of ovarian metastases.

The results of this study suggest that appropriate oncologic resection and adjuvant chemotherapy are being performed for patients with appendiceal carcinoma in a significant percentage of cases in Germany. However, undertreatment of patients was still apparent in some cases as well as overtreatment in a high proportion of patients with carcinoid tumors < 1cm who underwent unnecessary right hemicolectomy, significantly increasing perioperative morbidity.

It is worth mentioning that patients with appendiceal carcinoma have a notable risk of developing a synchronous colorectal neoplasm which was highest in this study for patients with mucinous adenocarcinoma. Therefore, careful diagnostic work-up is required before complementary surgery is performed.

Most patients with AC have a favorable prognosis with a 5-year survival of 83% for all stages [START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. Patients with local disease are reported with a 5-year survival rate ranging from 92 to 94%, those with regional metastases 81 to 84%, and the few with distant metastases 31 to 33% [START_REF] Sandor | A retrospective analysis of 1570 appendiceal carcinoids[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF][START_REF] Modlin | A 5-decade analysis of 13,715 carcinoid tumors[END_REF]. This was confirmed by our study, but with a better outcome for patients with metastatic disease, possibly related to the relatively small number of cases. Tumor size was also found to be significantly related to survival with a 5-year survival rate of 100% for tumors < 1cm. Accordingly, histological subtype also affected DFS with the highest survival rate for patients with AC.

The excellent survival of appendiceal carcinoids may reflect the specific histopathogenesis (neuroectodermal origin) [START_REF] Sandor | A retrospective analysis of 1570 appendiceal carcinoids[END_REF]. Carcinoids of the appendix mostly develop from the subepithelial-endocrine cells and are immunoreactive for S-100, a protein normally present in Schwann cells, indicating that anatomically they belong rather to the peripheral nervous system and the ectoderm [START_REF] Roggo | Carcinoid tumors of the appendix[END_REF]. The outcome of patients with noncarcinoid tumors is much worse with a 5-year survival rate ranging from 44 to 55% for all stages in most of the reports including the current study [START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF][START_REF] Nitecki | The natural history of surgically treated primary adenocarcinoma of the appendix[END_REF]. Among all noncarcinoid tumors, mAA was demonstrated to have highest survival rate. The difference was most pronounced in patients with metastatic disease in both our series and other reports [START_REF] Mccusker | Primary malignant neoplasms of the appendix: a population-based study from the surveillance, epidemiology and end-results program, 1973-1998[END_REF][START_REF] Mcgory | Malignancies of the appendix: beyond case series reports[END_REF]. This underlines the importance and potential benefit of an aggressive approach to the management of mAA -even if peritoneal carcinomatosis is present -with right hemicolectomy, and debulking and removal of all mucinous ascites where appropriate.

Adenosquamous carcinoma of the colon is extremely rare and represents 0.025% to 0.1% of all colorectal malignancies [START_REF] Kiran | Adenosquamous carcinoma of the colon: a rare tumor[END_REF]. They occur most frequently in the lower rectal area. To the best of our knowledge, there are no reports in the English literature about adenosquamous carcinoma arising from the vermiform appendix. Among all colon malignancies and appendiceal tumors, incidence of this rare histological subtype in this study was 0.01% and 2.04%, respectively. Mean age at presentation for colorectal AAS ranges from 62.5 to 63.3 years, which is higher than observed for appendiceal AAS in this report [START_REF] Yokoi | Case of adenosquamous carcinoma of the ascending colon[END_REF][START_REF] Dong | Primary adenosquamous carcinoma of the colon: report of five cases[END_REF][START_REF] Frizelle | Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study[END_REF]. We observed a male predominance, which was also demonstrated for colorectal AAS [START_REF] Yokoi | Case of adenosquamous carcinoma of the ascending colon[END_REF][START_REF] Frizelle | Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study[END_REF]. Presenting symptoms and signs of colorectal AAS are similar to those for patients with adenocarcinoma of the colon, whereas half of the patients with appendiceal AAS -as most of the patients with appendiceal neoplasm -presented with symptoms suggestive of acute appendicitis. All patients with AAS in this study had locally advanced tumors at the time of diagnosis with 50% having lymph node positive disease and 50% distant metastatic spread to the peritoneum. There was also a high rate of poorly differentiated carcinoma (75%). Comparable rates of lymph node and distant metastases have been reported for colorectal AAS [START_REF] Yokoi | Case of adenosquamous carcinoma of the ascending colon[END_REF][START_REF] Frizelle | Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study[END_REF]. Accordingly, prognosis is much worse than that of adenocarcinoma alone with 52% not surviving beyond one year [START_REF] Frizelle | Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study[END_REF]. In this study, 50% of all patients died within 26 month after initial presentation.

There are some limitations to the study that should be acknowledged. The data used for this study was collected from many hospitals in Germany and include diagnoses of numerous pathologists. Thus, there are no effective means of establishing the reliability of the tissue diagnoses. Secondly, no data regarding location of appendiceal carcinoid, presence of mesoappendiceal invasion and proliferative index were recorded. Furthermore, carcinoid tumors were analyzed as one group without distinguishing between classical carcinoid and goblet-cell carcinoid which may have influenced results, especially in terms of mean age and survival.

Primary carcinoma of the appendix remains a rare entity, which is difficult to diagnose preoperatively and classically presents as inflammatory disease. Long-term outcome of carcinoid tumors is superior to noncarcinoid neoplasms, and is significantly affected by tumor stage and tumor size. Lymphatic invasion should be considered as an additional risk factor. Among all appendiceal neoplasms, adenosquamous carcinoma is the rarest histological subtype, which is most commonly associated with advanced tumor stage at the time of diagnosis and thus, with worst prognosis.

The results of the study suggest that appropriate oncologic resection is being performed in a significant percentage of cases in Germany. However, the high rate of unnecessary right hemicolectomy in patients with small carcinoid tumors as well as the undertreatment of appendiceal cancer patients needs to be critically assessed and should encourage surgeons to improve adherence to current treatment guidelines.

Table legends

Table 1 Demographic data and clinical presentation for all histological subtypes of appendiceal carcinoma Table 2 Histopathological characteristics of appendiceal carcinoma according to histological subtype Table 3 Pattern of metastatic spread for all histological subtypes of appendiceal carcinoma

Figure 1 Overall 5 - 2 5

 152 Figure 1Overall 5-year survival according to histological subtype

  Figure 2 5-year DFS survival of carcinoid vs. noncarcinoid appendiceal tumors

Table 1

 1 Demographic data and clinical presentation for all histological subtypes of appendiceal

	carcinoma
					AC	aA	mAA	AAS
	Number (n/%)	48 (24.5)	99 (50.5)	45 (23)	4 (2)
	Age (years)	1	51.1 ± 17.4	64.7 ± 12.0	62.4 ± 15.4	56.2 ± 8.5
	Male / female ratio	1.17 : 1	1.67 : 1	0.96 : 1	3 : 1
	BMI (kg/m)	1	25.6 ± 4.33	26.3 ± 4.56	26.3 ± 5.47	24.3 ± 2.17
	Clinical presentation (%)
	-	referral for complementary
		surgery	22.9	34.3	24.4	25
	-	acute appendicitis	37.5	36.4	37.8	50
	-	incidential finding	29.1	22.2	20	-
	-nonspecific /tumor-related
		symptoms	10.4	7.0	17.8	25
	AC, appendiceal carcinoids; aA, appendiceal adenocarcinoma; mAA, mucinous appendiceal
	adenocarcinoma; AAS, appendiceal adenosquamous carcinoma

1

Mean; standard deviation in parentheses

Table 2

 2 Histopathological characteristics of appendiceal carcinoma according to histological subtype

			AC	aA	mAA	AAS
	Overall cases (n)	48	99	45	4
	Tumor size (cm)	1	1.8 (± 1.64)	3.27 (± 2.01)	4.0 (± 3.05)	3.1 (± 1.41)
	T status
	-T1 / T2 (n/%)		-	27 (27.3)	5 (11.1)	-
	-T3 / T4 (n/%)		-	72 (72.7)	40 (88.9)	4 (100)
	N Status (pos. /%)	8 (16.6)	36 (36.4)	12 (26.7)	2 (50)
	Grading (n/%)
	-G1		-	9 (9.1)	11 (24.4)	-
	-G2		-	61 (61.6)	21 (46.7)	1 (25)
	-G3		-	29 (29.3)	13 (28.9)	3 (75)
	UICC stage (n/%)
	-I		-	15 (15.1)	3 (6.7)	-
	-II		-	42 (42.4)	20 (44.4)	1 (25)
	-III		-	19 (19.2)	9 (20)	1 (25)
	-IV		-	23 (23.2)	13 (28.9)	2 (50)
	AC, appendiceal carcinoids; aA, appendiceal adenocarcinoma; mAA, mucinous appendiceal
	adenocarcinoma; AAS, appendiceal adenosquamous carcinoma

1

Mean; standard deviation in parentheses

Table 3

 3 Pattern of metastatic spread for all histological subtypes of appendiceal carcinoma

	Anatomical site of	AC	aA	mAA	AAS
	distant metastases
	Overall cases (n)	48	99	45	4
	Metastases (n /%)	2 (4.2)	23 (23.2)	13 (28.9)	2 (50)
	Site:
	-Hepatic (n)	1	10	1	-
	-Ovaries (n)	-	2	3	-
	-Peritoneal carcinomatosis (n)	1	16	10	2
	AC, appendiceal carcinoids; aA, appendiceal adenocarcinoma; mAA, mucinous appendiceal
	adenocarcinoma; AAS, appendiceal adenosquamous carcinoma

AC, appendiceal carcinoids; aA, appendiceal adenocarcinoma; mAA, mucinous appendiceal adenocarcinoma; AAS, appendiceal adenosquamous carcinoma time (years)

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest.