MD Baki Topal
email: baki.topal@med.kuleuven.be

Comparative study of microwave ablation and radiofrequency ablation of liver metastases

Keywords: malignancy, liver, microwave, surgery

Aims: Microwave ablation (MWA) is the most recent development in the field of local ablative therapies. The aim of this study was to evaluate the variability and reproducibility of single-probe MWA vs. radiofrequency ablation (RFA) of liver metastases smaller than 3 cm in patients without underlying liver disease.

Methods: Sixteen liver metastases were treated using MWA, and matched for size and localisation with 13 metastases treated by RFA. Tumour diameters and postoperative ablation diameters were recorded (D1 transverse; D2 antero-posterior; D3 cranio-caudal; mm) on computed tomography scans.

Results: Median D1, D2, and D3 ablation diameters after MWA vs. RFA were 18.5 (12-64) vs. 34 (16-41) mm (p=0.003), 26 (14-60) vs. 35 (28-40) mm (p=0.046), and 20 (10-73) vs. 32 (20-45) mm (p=0.025), respectively. As compared to RFA, the variability between the lesions after MWA was significantly higher for D2 (p<0.0001) and D3 (p=0.002) but not for D1 (p=0.15). The ablation diameters were less uniform after MWA than after RFA (p<0.001).

Conclusion:

Ablation diameters after single-probe MWA of metastatic liver tumours are highly variable and suboptimal. Improvements are needed before MWA can be implemented routinely. ClinicalTrials.

INTRODUCTION

Surgical resection is the gold standard for treatment of primary or metastatic liver cancer. However, most patients are not candidate for hepatic resection due to anatomic limitations, multifocal nature of the disease, insufficient functional liver reserve, extrahepatic metastases or co-morbidities. Consequently, a number of interstitial or local ablative therapies (LAT) have been developed and gained popularity for the treatment of unresectable liver tumours. Radiofrequency ablation (RFA) has become an established treatment modality due to its efficacy, reproducibility, low complication rates and availability [START_REF] Bilchik | Cryosurgical ablation and radiofrequency ablation for unresectable hepatic malignant neoplasms: a proposed algorithm[END_REF][START_REF] Sutherland | Radiofrequency ablation of liver tumors: a systematic review[END_REF]. However, high local recurrence rates particularly for lesions larger than 3 cm in diameter are a point of concern [START_REF] Sutherland | Radiofrequency ablation of liver tumors: a systematic review[END_REF][START_REF] Van Duijnhoven | Factors influencing the local failure rate of radiofrequency ablation of colorectal liver metastases[END_REF]. Further criticism has focused on the potential for incomplete tumour ablation near large vessels due to the heat-sink effect, slow rate of heating and the small zone of active heating requiring multiple overlapping probe placements to achieve larger ablation zones [START_REF] Kim | Radiofrequency thermal ablation of hepatic tumors: pitfalls and challenges[END_REF][START_REF] Chin | Effect of vascular occlusion on radiofrequency ablation of the liver: results in a porcine model[END_REF][START_REF] Goldberg | Radiofrequency tissue ablation: increased lesion diameter with a perfusion electrode[END_REF]. Therefore, technological advancements resulted in the development of new local ablative modalities such as the microwave.

Microwave ablation (MWA) has several theoretical advantages over RFA, which may make it more attractive than RFA to treat hepatic tumours. The benefits of MWA are an improved convection profile, higher constant intra-tumoural temperatures, faster ablation times and the ability to use multiple probes to treat multiple lesions simultaneously. Moreover, MWA does not require the placement of grounding pads that can result in skin burns. The size and shape of the MWA zone may be more consistent and less dependent on the heat-sink effect from vascular structures in proximity of the lesion [START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF][START_REF] Awad | ACCEPTED MANUSCRIPT hepatic porcine model: correlation of CT and histopathologic findings[END_REF]. Thus MWA may overcome some known limitations of RFA and may result in higher efficacy.

Any LAT should provide long-term complete tumour destruction with predictable and reproducible results. There are few studies available, all on animal models, reporting

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conflicting results on the size and geometry of the coagulation zone obtained with MWA vs. RFA [START_REF] Wright | Radiofrequency versus microwave ablation in a hepatic porcine model[END_REF][START_REF] Shibata | Comparison of the effects of in-vivo thermal ablation of pig liver by microwave and radiofrequency coagulation[END_REF]. The aim of the current study was to evaluate the variability and reproducibility of ablation diameters obtained after single-probe MWA vs. RFA in patients with liver metastases smaller than 3 cm in patients without underlying liver disease.

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PATIENTS and METHODS

Patients and tumours

Liver metastases measuring less than 3 cm in diameter were considered for the current study. From August 2008 until November 2008, single-probe MWA was used to treat 16 liver metastases in 6 patients without underlying liver disease (4 female and 2 male; median age 64 yr, range 47-82). Four patients underwent MWA for 10 liver metastases from colorectal cancer. Patients with colorectal liver metastases were considered for MWA but not for surgical resection since they had a high clinical risk score (CRS 3 or more) [START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer. Analysis of 101 consecutive cases[END_REF],

showed none to minimal response to systemic chemotherapy, and suffered from severe systemic disease (ASA 3 or more).

One patient was treated for a solitary liver metastasis from lung cancer, and another patient for 5 metastases from cancer of the ampulla Vateri. Segmental localisation of liver metastases was as follows: 1 in Sg4 (segment), 6 in Sg5, 2 in Sg6, 3 in Sg7, and 4 in Sg8.

Within our liver database, metastases treated with MWA were matched for size and localisation with 12 liver metastases from colorectal cancer and 1 from cervical cancer, which were treated with RFA in 13 patients (6 female and 7 male; median age 58 yr.). Segmental localisation of liver metastases in the RFA group was as follows: 1 in Sg4, 4 in Sg5, 3 in Sg7, and 5 in Sg8.

Surgical procedure

MWA was performed via laparoscopy in 5 patients to treat 15 liver metastases and percutaneously in 1 patient with a solitary lesion. In 1 patient with colorectal liver metastases laparoscopic MWA was combined with segmental hepatectomy. A single cooled percutaneous/laparoscopic microwave antenna of 22 cm length and 3.7 cm active radiating tip (VT2237) was used with a 915 MHz Valleylab MW ablation generator (VTSYS3;

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Covidien, Europe NV). Microwave energy was applied for 10 minutes at 40 W, according to the manufacturer's guidelines.

RFA was performed for 15 minutes per tumour using a monopolar 200W RF generator and a single cool-tip laparoscopic electrode of 25 cm length and 3 cm activating tip (Covidien, Radionics Europe NV) as described earlier [START_REF] Topal | Laparoscopic radiofrequency ablation of unresectable liver malignancies: feasibility and clinical outcome[END_REF]. Radiofrequency ablation was performed by laparoscopy in 7 patients, by laparotomy in 4 (with simultaneous colorectal surgery), and percutaneously in 2 patients. At all times, intra-operative ultrasound was used for positioning the MWA antenna or the RFA needle electrode. Continuous ultrasound monitoring was performed to track the progress of the ablation. The extent of the ablated coagulation could be roughly approximated on the basis of the appearance of the transient hyperechoic zone.

Assessment of outcome measures

Pre-operative tumour diameters and postoperative ablation diameters, assessed by dual-phase contrast enhanced helical computed tomography (CT) liver scan, were recorded (D1 transverse; D2 antero-posterior; D3 cranio-caudal; mm). Microwave ablation diameters were compared with those of matched RF ablated tumours. Postoperative measurements were performed within one week and at 3 month after surgery. Irregular peripheral contrast enhancement and a multilobular shape at the ablation margin were considered as a criterion for residual tumour on 3-phase CT-scan of the liver. Local recurrence was defined as cancer recurrence at the site of ablated liver metastasis.

Statistical analysis

MWA and RFA were compared with respect to the increase in lesion diameters measured before and after treatment, the variability between the lesions after ablation, and the variability within the lesion diameters.

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Mann-Whitney U tests were used to compare measured diameters. A linear model was used to compare the changes between pre-operative diameters and postoperative ablation zones between both groups. A logarithmic transformation was needed to meet the statistical assumptions of the model. To assess differences in variability of the posttreatment measurements, likelihood-ratio tests were used (using maximum likelihood estimation) comparing linear models fitted on the post-treatment measurements. Included lesions were considered as independent units. All analyses were performed using the statistical software SAS (version 9.2). A p-value ≤ 0.05 was considered statistically significant.

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RESULTS

Tumour and ablation diameters

The median transverse (D1), antero-posterior (D2), and cranio-caudal (D3) tumour diameters before MWA vs. RFA were 12 (range 6-18) vs. 12 (7-24) mm, 12 [START_REF] Chin | Effect of vascular occlusion on radiofrequency ablation of the liver: results in a porcine model[END_REF][START_REF] Goldberg | Radiofrequency tissue ablation: increased lesion diameter with a perfusion electrode[END_REF][START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF][START_REF] Awad | ACCEPTED MANUSCRIPT hepatic porcine model: correlation of CT and histopathologic findings[END_REF][START_REF] Wright | Radiofrequency versus microwave ablation in a hepatic porcine model[END_REF][START_REF] Shibata | Comparison of the effects of in-vivo thermal ablation of pig liver by microwave and radiofrequency coagulation[END_REF][START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer. Analysis of 101 consecutive cases[END_REF][START_REF] Topal | Laparoscopic radiofrequency ablation of unresectable liver malignancies: feasibility and clinical outcome[END_REF][START_REF] Mulier | Radiofrequency ablation versus resection for resectable colorectal liver metastases : time for a randomized trial ?[END_REF][START_REF] Lau | The current role of radiofrequency ablation in the management of hepatocellular carcinoma: a systematic review[END_REF][START_REF] Curley | Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies: results in 123 patients[END_REF][START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF][START_REF] Kuvshinoff | Radiofrequency ablation of liver tumors: influence of technique and tumor size[END_REF](19)[START_REF] Siperstein | Local recurrence after laparoscopic radiofrequency thermal ablation of hepatic tumors[END_REF][START_REF] Mulier | Local recurrence after hepatic radiofrequency coagulation : multivariate meta-analysis and review of contributing factors[END_REF][START_REF] Topal | Cancer cell dissemination during curative surgery for colorectal liver metastases[END_REF][START_REF] Adam | A comparison of percutaneous cryosurgery and percutaneous radiofrequency for unresectable hepatic malignancies[END_REF][START_REF] Livraghi | Hepatocellular carcinoma: radio-frequency ablation of medium and large lesions[END_REF] vs. 12 (7-17) mm, and 10.5 [START_REF] Chin | Effect of vascular occlusion on radiofrequency ablation of the liver: results in a porcine model[END_REF][START_REF] Goldberg | Radiofrequency tissue ablation: increased lesion diameter with a perfusion electrode[END_REF][START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF][START_REF] Awad | ACCEPTED MANUSCRIPT hepatic porcine model: correlation of CT and histopathologic findings[END_REF][START_REF] Wright | Radiofrequency versus microwave ablation in a hepatic porcine model[END_REF][START_REF] Shibata | Comparison of the effects of in-vivo thermal ablation of pig liver by microwave and radiofrequency coagulation[END_REF][START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer. Analysis of 101 consecutive cases[END_REF][START_REF] Topal | Laparoscopic radiofrequency ablation of unresectable liver malignancies: feasibility and clinical outcome[END_REF][START_REF] Mulier | Radiofrequency ablation versus resection for resectable colorectal liver metastases : time for a randomized trial ?[END_REF][START_REF] Lau | The current role of radiofrequency ablation in the management of hepatocellular carcinoma: a systematic review[END_REF][START_REF] Curley | Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies: results in 123 patients[END_REF][START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF][START_REF] Kuvshinoff | Radiofrequency ablation of liver tumors: influence of technique and tumor size[END_REF](19)[START_REF] Siperstein | Local recurrence after laparoscopic radiofrequency thermal ablation of hepatic tumors[END_REF] vs. 11 [START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF][START_REF] Awad | ACCEPTED MANUSCRIPT hepatic porcine model: correlation of CT and histopathologic findings[END_REF][START_REF] Wright | Radiofrequency versus microwave ablation in a hepatic porcine model[END_REF][START_REF] Shibata | Comparison of the effects of in-vivo thermal ablation of pig liver by microwave and radiofrequency coagulation[END_REF][START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer. Analysis of 101 consecutive cases[END_REF][START_REF] Topal | Laparoscopic radiofrequency ablation of unresectable liver malignancies: feasibility and clinical outcome[END_REF][START_REF] Mulier | Radiofrequency ablation versus resection for resectable colorectal liver metastases : time for a randomized trial ?[END_REF][START_REF] Lau | The current role of radiofrequency ablation in the management of hepatocellular carcinoma: a systematic review[END_REF][START_REF] Curley | Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies: results in 123 patients[END_REF][START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF][START_REF] Kuvshinoff | Radiofrequency ablation of liver tumors: influence of technique and tumor size[END_REF](19)[START_REF] Siperstein | Local recurrence after laparoscopic radiofrequency thermal ablation of hepatic tumors[END_REF] mm, respectively (p>0.792). The median D1, D2, and D3 ablation diameters after MWA vs. RFA were 18.5 (12-64) vs. 34 (16-41) mm (p=0.003), 26 (14-60) vs. 35 (28-40) mm (p=0.046), and 20 (10-73) vs. 32 (20-45) mm (p=0.025), respectively (Figure 1). The increase in all 3 diameters was more pronounced after RFA as compared to that after MWA, and most apparent for the transverse diameter (Table 1). For the 3 dimensions taken together, the diameter increased 2.6 times (95% confidence interval CI: 2.3-3.1) after RFA compared to 2.1 times (95% CI: 1.8-2.5) after MWA (p=0.058).

As compared to RFA, the variability between the lesion diameters after MWA was significantly higher for D2 (p<0.0001) and D3 (p=0.002) but not for D1 (p=0.15). The degree of uniformity, i.e. the variability between the three diameters within a lesion, was significantly higher after MWA than after RFA (p<0.001).

Clinical outcome and radiological assessment

No peri-operative mortality was observed. After MWA, one patient developed haemobilia that resolved conservatively. According to the CT-scan performed within 1 week after MWA, the overall tumour destruction was complete. In all these patients the zone of ablation extended into the liver capsule at the site of MWA-probe insertion. This phenomenon, known as the 'comet effect', is illustrated in Figure 2. The median follow-up time after MWA was 6.3 (4.9-7.8) months, and was complete. One biopsy proven local recurrence was observed after MWA in a patient treated for a solitary colorectal liver metastasis. No recurrences were observed in the RFA group.

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DISCUSSION

Local recurrence after ablation of liver tumours

The majority of patients with hepatic malignancies are not candidates for surgical resection. For selected patients with unresectable disease LAT has become an accepted therapeutic modality to provide local tumour control, with the best opportunity for longterm survival [START_REF] Mulier | Radiofrequency ablation versus resection for resectable colorectal liver metastases : time for a randomized trial ?[END_REF][START_REF] Lau | The current role of radiofrequency ablation in the management of hepatocellular carcinoma: a systematic review[END_REF]. Currently, radiofrequency energy is the most widely used modality for the thermal ablation of liver tumours. The main criticism on RFA has focused on recurrence rates varying from 2% to 60% [START_REF] Curley | Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies: results in 123 patients[END_REF][START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF][START_REF] Kuvshinoff | Radiofrequency ablation of liver tumors: influence of technique and tumor size[END_REF]. Local recurrence is related to the lack of complete coagulative necrosis and the persistence of residual tumour cells in or around the ablation zone [19,[START_REF] Siperstein | Local recurrence after laparoscopic radiofrequency thermal ablation of hepatic tumors[END_REF]. Independent risk factors for local recurrence after LAT are tumour size larger than 3 cm, percutaneous approach, and the lack of a curative intention to treat [START_REF] Mulier | Local recurrence after hepatic radiofrequency coagulation : multivariate meta-analysis and review of contributing factors[END_REF][START_REF] Topal | Cancer cell dissemination during curative surgery for colorectal liver metastases[END_REF].

The limits in size of the coagulation zone that can be created by LAT, the irregular borders of both the tumour and the LAT zone, and the presence of satellite lesions around tumours, all contribute to incomplete tumour ablation [START_REF] Adam | A comparison of percutaneous cryosurgery and percutaneous radiofrequency for unresectable hepatic malignancies[END_REF][START_REF] Livraghi | Hepatocellular carcinoma: radio-frequency ablation of medium and large lesions[END_REF][START_REF] Shirabe | Analysis of prognostic risk factors in hepatic resection for metastatic colorectal carcinoma with special reference to the surgical margin[END_REF][START_REF] Maeda | Small hepatocellular carcinoma of single nodular type: a specific reference to its surrounding cancerous area undetected radiologically and macroscopically[END_REF]. Blood vessels in the proximity of liver tumours can cause a heat-sink effect of local blood flow, resulting in local recurrence [START_REF] Kim | Radiofrequency thermal ablation of hepatic tumors: pitfalls and challenges[END_REF][START_REF] Mulier | Local recurrence after hepatic radiofrequency coagulation : multivariate meta-analysis and review of contributing factors[END_REF]. The importance of coagulating a peri-tumoural margin of 1 cm has been highlighted in histological and clinical studies [START_REF] Mulier | Local recurrence after hepatic radiofrequency coagulation : multivariate meta-analysis and review of contributing factors[END_REF][START_REF] Topal | Cancer cell dissemination during curative surgery for colorectal liver metastases[END_REF]. Not only the size of the ablation zone matters but also the geometry of the lesion [START_REF] Mulier | Size and geometry of hepatic radiofrequency lesions[END_REF]. Viable tumour cells can be present at the margin when the coagulation zone is asymmetric or eccentric [START_REF] Shirabe | Analysis of prognostic risk factors in hepatic resection for metastatic colorectal carcinoma with special reference to the surgical margin[END_REF]28]. Unfortunately, size and shape of radiofrequency-induced ablations of liver tissue cannot be objectively evaluated intra-operatively [START_REF] Raman | Creation of radiofrequency lesions in a porcine model: correlation with sonography, CT, and histopathology[END_REF]. Indeed, usually a LAT device is chosen, according to the size of the tumoural lesion, which is supposed to yield an ablation zone of a determined size greater than the malignant lesion. In this regard, it is important that variability of size and shape of necrosis induced by any LAT technique is low in order to achieve

Microwave ablation of liver tumours

The application of microwave energy is the most recent development in the field of tumour ablation. The technique can be used percutaneously, via laparoscopy or via open surgical access. Microwave ablation offers many of the benefits of RFA but has some theoretical advantages that may result in improved performance. Compared with RFA, microwave ablation has a broader field of power density, resulting in a larger zone of active heating [START_REF] Skinner Mg | A theoretical comparison of energy sources -microwave, ultrasound and laser -for interstitial thermal therapy[END_REF]. In some reports the MWA zone can be up to 6 cm surrounding the MW antenna, allowing for a large volume of cell destruction within the targeted zone [START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF][START_REF] Skinner Mg | A theoretical comparison of energy sources -microwave, ultrasound and laser -for interstitial thermal therapy[END_REF][START_REF] Strickland | Experimental study of large-volume microwave ablation in the liver[END_REF][START_REF] Strickland | Rapid microwave ablation of large hepatocellular carcinoma in a high-risk patient[END_REF].

Unlike RF energy, MW energy does not appear to be limited by charring and tissue desiccation [START_REF] Skinner Mg | A theoretical comparison of energy sources -microwave, ultrasound and laser -for interstitial thermal therapy[END_REF], while the size and shape of the MWA zone may be more consistent and less dependent on the heat-sink effect from vascular structures in proximity [START_REF] Wright | Radiofrequency versus microwave ablation in a hepatic porcine model[END_REF][START_REF] Meredith | Microwave ablation of hepatic tumors using dual-loop probes:results of a phase I clinical trial[END_REF][START_REF] Simon | Intraoperative triple antenna hepatic microwave ablation[END_REF]. The knowledge on size and geometry of the coagulation zone is another determinant of local recurrence after LAT [START_REF] Poon | Learning curve for radiofrequency ablation of liver tumors: prospective analysis of initial 100 patients in a tertiary institution[END_REF]. Reports on the size of lesions obtained with MWA vs. RFA are conflicting and little is known about the geometry of the lesions [START_REF] Wright | Radiofrequency versus microwave ablation in a hepatic porcine model[END_REF][START_REF] Shibata | Comparison of the effects of in-vivo thermal ablation of pig liver by microwave and radiofrequency coagulation[END_REF].

Ablation diameters and variability

In the present study, the comparison between MWA vs. RFA lesions was based on the analysis of the increase in lesion diameters measured before and after the ablation, the variability between the lesions after ablation, and the variability within a lesion diameter.

The increase in all 3 diameters was more pronounced after RFA as compared to that after MWA. The lowest increase was measured for the transverse diameter after MWA, resulting in a final transverse diameter of 18.5 mm. A final coagulation diameter of the MWA zone of less than 2 cm will inevitably lead to local cancer recurrence, as was observed in one patient in the current study, unless the tumour to be treated is smaller than 1 cm. The variability between the lesions as well as the non-uniformity within one lesion diameter was

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higher after MWA as compared to RFA, making MWA less reproducible. Thus, largest ablation diameters were obtained after RFA, with minimal variability in diameters between different lesions and high uniformity of the diameters within each lesion. The 'comet effect' of MWA was demonstrated by radiologic findings and confirmed by statistical comparison of the cranio-caudal diameters obtained after MWA vs. RFA. This dimension was the largest diameter obtained after MWA, and strongly dependent on the distance between the tumour and the site of probe insertion through the liver capsule.

In the current study, a single-probe MWA or RFA procedure was performed in all patients. Several authors reported that larger ablations can be obtained with multiple or clustered MW antennas [START_REF] Strickland | Experimental study of large-volume microwave ablation in the liver[END_REF][START_REF] Meredith | Microwave ablation of hepatic tumors using dual-loop probes:results of a phase I clinical trial[END_REF][START_REF] Simon | Intraoperative triple antenna hepatic microwave ablation[END_REF][START_REF] Iannitti | Hepatic tumor ablation with clustered microwave antennae: the US phase II trial[END_REF]. Simultaneous multiple-probe MWA could result in more uniform coagulation zones and better performance near blood vessels, and might lead to a more adequate treatment and decreased recurrence rates after tumour ablation [START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF][START_REF] Strickland | Experimental study of large-volume microwave ablation in the liver[END_REF]. However, while multiple-probe MWA is able to achieve higher ablation volumes, it can also cause an increased 'comet effect' and unwanted collateral damage to neighbouring organs or structures [START_REF] Wright | Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis[END_REF].

Ablation energy source

Limitations of the current study are the short follow-up time and its nonrandomized nature. Nevertheless, before a randomized controlled trial should be conducted to assess the oncologic outcome of MWA vs. RFA, their local effectiveness in terms of complete tumour destruction and complication rate should be evaluated. In the current study, the small transverse diameter obtained with MWA clearly raises concerns about the completeness of destruction even of small liver tumours. This finding was supported by the occurrence of a local recurrence within 6 months after MWA. One patient developed haemobilia after MWA of a solitary liver metastasis, which might be related to the unpredictable collateral damage caused by the 'comet effect' rather than to

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surgical expertise [START_REF] Topal | Laparoscopic radiofrequency ablation of unresectable liver malignancies: feasibility and clinical outcome[END_REF][START_REF] Topal | Cancer cell dissemination during curative surgery for colorectal liver metastases[END_REF][START_REF] Topal | Morbidity and[END_REF]. One could also debate the use of different energy sources.

Since both ablative techniques were used for a finite time at a specific setting, the amount of energy supplied by RFA was about eight times as high as the energy supplied by MWA.

However, we believe that this reflects the difference in the physics of microwaves and radiofrequency. Thus a direct relation between energy supply and tissue damage is not clear. It is possible that increased power or longer times of MW application may create greater of more uniform lesions, but this was not addressed by our study. Second, we only looked at the performance of the new 915 MHz Valleylab MW ablation system. An extensive experience with MWA comes from Asia where most microwave antennae operate at 2,450 MHz. However, the 915 MHz system that we used is more tuned to the dielectric properties of human tumour tissue. A comparative study of MWA zones between antennae operating at 2,450 MHz and 915 MHz, showed larger ablation zones with 915 MHz microwaves [38]. Another aspect of LAT modalities is their cost, provided they can ascertain similar short-term clinical and long-term oncologic outcomes.

CONCLUSION

Ablation diameters after single-probe MWA of liver metastases are highly variable and suboptimal. The small transverse diameter after MWA raises concerns about the completeness of destruction in tumours larger than 10 mm, while the 'comet effect' may cause unwanted collateral damage to neighbouring organs or structures. Improvements are needed before MWA can be implemented routinely in clinical practice.

Figure 1 .Figure 2 .

 12 Figure 1. Diameters after microwave (a) and radiofrequency ablation (b) of liver tumours

Table 1 .

 1 The increase in diameters after microwave vs. radiofrequency ablation of liver

	tumours
	Dimension (mm)	RFA	MWA	p-value
	Transverse	22.0 (4-27)	9.0 (2-47)	0.006
	Antero-posterior	24.0 (18-30)	13.7 (5-42)	0.058
	Cranio-caudal	18.0 (8-31)	11.0 (0-53)	0.034

The increase in diameters is presented as median (range) in mm; MWA microwave ablation; RFA radiofrequency ablation

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Brandberg

MD Helena Sackey
email: helena.sackey@ki.se

K Sandelin

J Frisell

M Wickman

Y Brandberg

Ductal carcinoma in situ of the breast. Long-term follow-up of health-related quality of life, emotional reactions and body image

Ductal carcinoma in situ of the breast. Long-term follow-up of health-related quality of life, emotional reactions and body image

Introduction

Health-related quality of life (HRQoL) has been studied extensively in patients with invasive breast cancer, but longterm follow-up studies of HRQoL, emotional reactions, and body image after treatment for ductal carcinoma in situ of the breast (DCIS) are sparse. The yearly incidence of DCIS in Sweden is approximately 10% of all diagnosed cases of breast cancer [1]. Since the 1980s, breast-conserving surgery for DCIS has been recommended whenever feasible.

Several randomized trials, including the Swedish National DCIS study, have unanimously shown a decreased rate of ipsilateral in situ or invasive breast cancer recurrence through the addition of adjuvant radiotherapy [START_REF] Julien | Radiotherapy in breast-conserving treatment for ductal carcinoma in situ: first results of the EORTC randomised phase III trial 10853. EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group[END_REF][START_REF] Holmberg | Absolute risk reductions for local recurrence after postoperative radiotherapy after sector resection for ductal carcinoma in situ of the breast[END_REF][START_REF] Emdin | SweDCIS: Radiotherapy after sector resection for ductal carcinoma in situ of the breast. Results of a randomised trial in a population offered mammography screening[END_REF][START_REF] Fisher | Lumpectomy compared with lumpectomy and radiation therapy for the treatment of intraductal breast cancer[END_REF][START_REF] Fisher | Lumpectomy and radiation therapy for the treatment of intraductal breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-17[END_REF][START_REF] Houghton | Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial[END_REF]. Today, national guidelines include radiotherapy up to a total of 50 Gray after breast-conserving therapy for high-risk subgroups of patients with DCIS. Mastectomy is still recommended for women with multifocal DCIS, an unfavourable proportion between tumor size and breast volume, and non-clear surgical margins after breast conservation. For these women immediate breast reconstruction (IBR) may be an alternative in order to maintain a breast contour.

In DCIS, the tumour cells do not penetrate the basal membrane and, by definition, do not metastasize. Women should therefore be expected to be cured by surgical treatment with clear margins. Nevertheless, both invasive and non-invasive recurrences do occur [START_REF] Warnberg | Prognosis in women with a carcinoma in situ of the breast: a population-based study in Sweden[END_REF][START_REF] Innos | Risk of second primary breast cancers among women with ductal carcinoma in situ of the breast[END_REF][START_REF] Franceschi | Second cancers following in situ carcinoma of the breast[END_REF]. Owing to the complexity of the disease, there is still no international consensus on how extensively DCIS should be treated. The fact that the majority of DCIS is not life-threatening does not necessarily imply that women with DCIS experience less psychological distress than those with invasive breast cancer. Two qualitative studies report that women experience confusion upon the information that DCIS is not a life-threatening disease, while they are recommended a similar treatment to patients with invasive breast cancer [START_REF] De Morgan | Well, have I got cancer or haven't I?" The psycho-social issues for women diagnosed with ductal carcinoma in situ[END_REF][START_REF] Kennedy | The challenge of being diagnosed and treated for ductal carcinoma in situ (DCIS)[END_REF]. In two American studies, women with DCIS highly overestimated their future risk of recurrence and the risk that their breast cancer would metastasize [START_REF] Bluman | Knowledge, satisfaction, and perceived cancer risk among women diagnosed with ductal carcinoma in situ[END_REF][START_REF] Partridge | Risk perceptions and psychosocial outcomes of women with ductal carcinoma in situ: longitudinal results from a cohort study[END_REF]. A Canadian study reported similar levels of anxiety and depression in women with early-stage invasive breast cancer and those with DCIS. No differences were found between the two groups regarding perceptions of the risk of developing local recurrence, distant recurrence, or breast cancer death [START_REF] Rakovitch | A comparison of risk perception and psychological morbidity in women with ductal carcinoma in situ and early invasive breast cancer[END_REF]. In a Dutch study, HRQoL was compared between women with DCIS and those with early-stage invasive breast cancer [START_REF] Van Gestel | A comparison of quality of life, disease impact and risk perception in women with invasive breast cancer and ductal carcinoma in situ[END_REF]. No differences were found regarding HRQoL, but women with DCIS reported better physical health, sex life, and relationships with friends and acquaintances than women with invasive breast cancer.

The majority of women with DCIS have a very good prognosis. As surgery is the primary treatment for this disease, it is essential to increase current understanding of its long-term consequences. To our knowledge, there is no study comparing long-term HRQoL, body image, and emotional reactions in women with DCIS treated with different surgical

Study population

All women who took part in the Swedish National DCIS study during the years 1991-1999 in the County of Stockholm were eligible [START_REF] Emdin | SweDCIS: Radiotherapy after sector resection for ductal carcinoma in situ of the breast. Results of a randomised trial in a population offered mammography screening[END_REF]. These women had been randomized to sector resection alone or followed by radiotherapy. In addition, all women with DCIS who underwent mastectomy and IBR with an implant during the same period at the Karolinska University Hospital, Stockholm, were asked to participate in the present study. These three treatment options for DCIS defined our study groups. Exclusion criteria were: Paget´s disease of the nipple, invasive breast carcinoma, lobular cancer in situ, ongoing tamoxifen treatment, and a history of present or previous malignancy (except basalioma and cervical cancer). Furthermore, women with dementia, severe brain damage, and without sufficient knowledge of the Swedish language were excluded as were those with an invasive recurrence after primary DCIS surgery. Immediate flap reconstruction was indicated in very few women and they were not included in the study. Women in the sector resection alone group and the sector resection and postoperative RT group were excluded if they had a DCIS recurrence that led to mastectomy. Prior to inclusion, all data were controlled against the Regional Oncology Centre to validate the DCIS diagnosis, and the Cause of Death Register, to ensure that patients were still alive. To further validate data from the Regional Oncology Centre, all patient charts from their respective hospitals in the County of Stockholm were reviewed.

Only women with DCIS proven by final histopathology were included in the study.

In the mastectomy and IBR group, three women were excluded due to invasive breast cancer recurrence and two women due to previous malignancy. In the sector resection alone group, ten women were deceased, seven were excluded due to invasive breast cancer recurrence, nine due to DCIS recurrence that led to mastectomy, two due to previous malignancy, one due to dementia, two due to ongoing tamoxifen treatment, one did not speak Swedish, and one woman had undergone a delayed breast reconstruction. In the sector resection and postoperative RT group, six women were deceased, eight were excluded due to invasive breast cancer recurrence, one due to DCIS recurrence and re-operation by mastectomy, four due to previous malignancy, and one due to severe brain damage. In total, 162 women were included in the study.

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Surgical procedures

According to prevailing national and regional guidelines, the breast-conserving procedure was performed as a sector resection as previously defined [START_REF] Aspegren | Standardization of the surgical technique in breast-conserving treatment of mammary cancer[END_REF]. Women opting for mastectomy and IBR consulted with a breast surgeon and a reconstructive plastic surgeon for information about the procedure. The reconstructive method was implant-based with expander prostheses. According to previously published data, these women underwent on average 3 corrective procedures in order to complete the reconstruction [START_REF] Lagergren | Technical aspects of immediate breast reconstruction with implants: five year follow-up[END_REF]. Several patients later underwent minor and major adjustments to achieve a better functional and cosmetic result.

Study questionnaires

All women included in the study were asked to complete three questionnaires 4-15 years after surgery which were sent to the patients together with an information letter and a prepaid return envelope. If no response was obtained within two months, a reminder was sent.

The Swedish SF-36 Health Survey (SF-36) is used to asses HRQoL, and is a standardized questionnaire that has been widely used in international studies. The Swedish version has been validated, and normative data for Swedish women are available [START_REF] Sullivan | Swedish manual and interpretation guide. International Quality of Life Assessment (IQLA) Project[END_REF][START_REF] Sullivan | The Swedish SF-36 Health Survey--I. Evaluation of data quality, scaling assumptions, reliability and construct validity across general populations in Sweden[END_REF]. The SF-36 consists of 36 items constituting eight domains: physical functioning (PF), role limitations as a result of physical problems (RP), bodily pain (BP), general health perception (GH), vitality (VT), social functioning (SF), role limitation due to emotional problems (RE), and mental health (MH). The first three domains (PF, RF, and BP) measure physical well-being and the last three domains (SF, RE, and MH) relate to emotional well-being. The two remaining domains (GH and VT) are associated with both physical and emotional dimensions. A high score on the subscales signifies a higher level of function and HRQoL.

The Hospital Anxiety and Depression (HAD) scale consists of 14 items, seven assessing anxiety and seven depression [START_REF] Zigmond | The hospital anxiety and depression scale[END_REF]. The possible sum score of each scale ranges from 0 to 21, with each item scoring 0 to 3 points. A high summated score represents high levels of problems. Cut-off points for each of the scales are: < 8 (within normal range), 8-10 (possible clinical case), and ≥ 11 (clinical case). The HAD scale is considered a reliable and valid instrument for the assessment of anxiety and depression in somatic, psychiatric, and primary care patients, as well as in the general population [START_REF] Bjelland | The validity of the Hospital Anxiety and Depression Scale. An updated literature review[END_REF]. The Swedish version has been validated in breast cancer patients against diary recordings [START_REF] Arving | Four weeks of daily assessments of anxiety, depression and activity compared to a point assessment with the Hospital Anxiety and Depression Scale[END_REF].

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The Body Image Scale (BIS) was designed for the assessment of body image in cancer patients [START_REF] Hopwood | A body image scale for use with cancer patients[END_REF] and consists of 10 items concerning the impact of surgery on self-consciousness, physical and sexual attractiveness, femininity, satisfaction with body and scars, body integrity, and avoidance behavior. The BIS items score from 0 (not at all) to 3 (very much). A high score represent problems with body image. The BIS scale has a high reliability and good clinical validity [START_REF] Hopwood | A body image scale for use with cancer patients[END_REF]. No formal validation or reliability testing of the Swedish version has been performed. The translation to Swedish was performed by a group of five professionals (three nurses, one sociologist, and one psychologist) at the Department of Oncology, Karolinska University Hospital, Stockholm, in 1997.

Statistical methods

The scores derived from the SF-36 questionnaire were linearly transformed into a 0 to 100 scale according to the SF-36 manual [START_REF] Sullivan | The Swedish SF-36 Health Survey--I. Evaluation of data quality, scaling assumptions, reliability and construct validity across general populations in Sweden[END_REF]. A high score on the subscales signifies a higher level of function and HRQoL. The results were compared with normative data from an age-matched reference group consisting of 920 women from the general Swedish population [START_REF] Sullivan | The Swedish SF-36 Health Survey--I. Evaluation of data quality, scaling assumptions, reliability and construct validity across general populations in Sweden[END_REF]. The mean HAD subscale values were compared between groups, as well as proportions of patients in each clinical stage, as suggested by the original authors [START_REF] Zigmond | The hospital anxiety and depression scale[END_REF]. In order to determine the proportion of women who reported problems on each of the BIS items, a score of 0 was categorized as "no problem" and of 1 to 3 as "problem".

Statistical analyses were performed using Statview version 4.5. ANOVA repeated measurement was used to evaluate differences between the three study groups on HAD subscales and SF-36 subscales. Chi2 test was used for categorical data (HAD and BIS). Student's t-test was used to evaluate differences between the study sample and normative data (SF-36). Differences between groups were considered statistically significant when p <0.01.

The ethics committee of the Karolinska University Hospital, Sweden approved the study (Dnr 2002-018).

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Results

In total, 162 women were included in the study and 131 (81%) responded to the questionnaires. The number of patients and reasons for attrition are presented in Figure 1. The median age was 58.5 years (range 40 to 77) for women treated with mastectomy and IBR, 65 years (range 55 to 83) for those treated with sector resection alone, and 64 years (range 48 to 89) for women treated with sector resection and postoperative RT. The mean time from surgery to the completion of the questionnaires was 7 years (SD 2.4) in the mastectomy and IBR group, 9.8 years (SD 2.8) in the sector resection and postoperative RT group and 9.9 years (SD 2.7) in the sector resection alone group.

Health-related quality of life

Mean values and standard deviations for the SF-36 subscales for the three study groups and their respective age-adjusted norm values are presented in Table 1. The scores for physical functioning (PF) and bodily pain (BP) were statistically significantly higher among the women in the mastectomy and IBR group as compared with the other two groups. The score for mental health (MH) was statistically significantly higher in the sector resection alone group than in the other two groups. Women in the mastectomy and IBR group scored statistically significantly higher on physical functioning (PF) and bodily pain (BP) than their age-adjusted norm group. No other statistically significant differences were found between the study groups and their respective age-adjusted norm groups.

Anxiety and Depression

Mean scores and standard deviations for the HAD anxiety and depression subscales and the proportion of patients scoring ≥ 11(clinical case) is presented in Table 2. No statistically significant between-group differences were found for the mean scores on the HAD anxiety or depression subscales. No differences were found between the study groups regarding proportions of patients scoring in the clinical categories on the anxiety or depression subscale.

Body image

Numbers and proportions of women reporting problems are shown in Table 3. Overall, statistically significant differences between the three study groups were found for six of the items (self-conscious, less physically attractive, less feminine, less sexually attractive, dissatisfied with body, dissatisfied with scars) with larger proportions of women in the mastectomy and IBR group reporting problems.

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Overall, women in all three study groups appeared to have a very satisfactory HRQoL in the long term, similar to women in the general population. These findings are consistent with three other studies of long-term HRQoL among women with DCIS [START_REF] Van Gestel | A comparison of quality of life, disease impact and risk perception in women with invasive breast cancer and ductal carcinoma in situ[END_REF][START_REF] Nekhlyudov | Prospective changes in quality of life after ductal carcinoma-in-situ: results from the Nurses' Health Study[END_REF][START_REF] Amichetti | Quality of life in patients with ductal carcinoma in situ of the breast treated with conservative surgery and postoperative irradiation[END_REF].

Women who underwent mastectomy and IBR scored higher on physical functioning and bodily pain than the other two study groups as well as compared to their age-adjusted norm groups. The physical functioning subscale concerns problems in daily life caused by physical restraints. It includes items about everyday life activities, for example carrying groceries, walking, climbing stairs, bowling, participating in strenuous sports. The bodily pain subscale includes items about pain levels during the past four weeks and to what extent pain has interfered with normal activities and work. The finding that women who underwent mastectomy and IBR reported better physical functioning and less bodily pain than the other two study groups as well as their age-adjusted norm groups is surprising. It might be explained by response shift. Response shift is an adaptation process in which patients with a severe disease undergo to accommodate their illness. A response shift involves changing internal standards and values and the conceptualization of quality of life [START_REF] Sprangers | Integrating response shift into health-related quality of life research: a theoretical model[END_REF]. Thus, it is possible that the more extensive surgical procedures and probably, a more prolonged rehabilitation period that these women have experienced, resulted in a response shift with respect to bodily pain and physical functioning. A Dutch study also showed that women with DCIS score higher than the norm group on the bodily pain subscale and the physical functioning subscale of the SF-36 [START_REF] Van Gestel | A comparison of quality of life, disease impact and risk perception in women with invasive breast cancer and ductal carcinoma in situ[END_REF].

The addition of postoperative radiotherapy to breast-conserving therapy did not seem to have any negative impact on HRQoL in the long term. This is consistent with the only prior study that we could find evaluating the impact of radiation therapy on long-term HRQoL in women with DCIS [START_REF] Amichetti | Quality of life in patients with ductal carcinoma in situ of the breast treated with conservative surgery and postoperative irradiation[END_REF]. However, women in the sector resection alone group scored higher on the mental health subscale than the other two study groups, probably reflecting less extensive treatment.

Our results show differences between the three study groups for six of ten of the BIS items, with a greater proportion of women in the mastectomy and IBR group reporting problems. To our knowledge, this is the first study comparing different surgical treatments with respect to long-term body image among women with DCIS. Three previous studies on long-term postoperative body image among women with invasive breast cancer reported a more favourable body image among women treated with breast-conserving surgery than those treated with mastectomy alone [START_REF] Hartl | Impact of medical and demographic factors on long-term quality of life and body image of breast cancer patients[END_REF][START_REF] Arndt | Quality of life over 5 years in women with breast cancer after breastconserving therapy versus mastectomy: a population-based study[END_REF][START_REF] Rowland | Role of breast reconstructive surgery in physical and emotional outcomes among breast cancer survivors[END_REF]. Contradictory

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10 results have been found in studies that address whether mastectomy with reconstruction results in a better long-term body image than mastectomy alone [START_REF] Rowland | Role of breast reconstructive surgery in physical and emotional outcomes among breast cancer survivors[END_REF][START_REF] Parker | Short-term and long-term psychosocial adjustment and quality of life in women undergoing different surgical procedures for breast cancer[END_REF][START_REF] Al-Ghazal | Comparison of psychological aspects and patient satisfaction following breast conserving surgery, simple mastectomy and breast reconstruction[END_REF]. In addition, one long-term follow-up study failed to show any difference in body image between women treated with breast-conserving surgery and mastectomy with reconstruction [START_REF] Schover | Partial mastectomy and breast reconstruction. A comparison of their effects on psychosocial adjustment, body image, and sexuality[END_REF]. It is important to consider that sector resection was not an option for most women in the mastectomy and IBR group and that the alternative -mastectomy alone -is not represented in this study. Given that mastectomy and IBR is the surgical option that provides a superior esthetical result, emphasis on preoperative information about expected postoperative changes in body image might have improved these results. During the study period two thirds of all women in Stockholm treated with mastectomy for DCIS also underwent IBR. In our unit we have long experience of IBR with implants which has thus become our method of choice. Women with DCIS are suitable candidates for implant based reconstructions as they are seldom treated with radiation therapy which is known to increase the risk for capsular contracture. If the cosmetic outcome becomes unsatisfactory, women are offered other modes of reconstructive techniques. Immediate flap reconstructions were performed in only a few cases during the study period and were not the purpose of the study. We cannot however exclude the possibility that the use of other immediate reconstruction techniques could result in a better body image. Further, among women treated with mastectomy, it is possible that those who are more concerned about their bodily appearance were overrepresented in the IBR group One might have expected that mastectomy and IBR, a treatment implying a lower risk of recurrence, would result in less anxiety than the two other treatment options in the study. In our study, no statistically significant difference in anxiety and depression was found between the three study groups. However, our results show a trend towards increased levels of anxiety in the mastectomy and IBR group in comparison with the other two study groups. Women in the mastectomy and IBR group also scored statistically significantly lower than the other two study groups on the mental health subscales of the SF-36, although there were no statistically significant differences to their age-adjusted norm group. In an American study, perception of risk of recurrence did not diminish in women who underwent mastectomy [START_REF] Partridge | Risk perceptions and psychosocial outcomes of women with ductal carcinoma in situ: longitudinal results from a cohort study[END_REF]. A Dutch study found that women with DCIS had comparable perceptions of the risk of recurrence and breast cancer death to women with invasive breast cancer, despite their better prognosis [START_REF] Van Gestel | A comparison of quality of life, disease impact and risk perception in women with invasive breast cancer and ductal carcinoma in situ[END_REF]. We speculate that these trends, with increased anxiety and lower mental health, might reflect the more extensive and longer treatment that mastectomy and IBR implies when compared with sector resection alone or followed by radiotherapy, and that these women may have become more severely marked by their disease.

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ACCEPTED MANUSCRIPT 11 This study has a number of limitations. No women treated with mastectomy alone were included and better body image might have been expected with IBR than without. In addition, no preoperative data on our study variables were collected. Therefore, it was not possible to ascertain differences in the studied variables before surgery.

The study has a number of strengths. To our knowledge, there is no previous study evaluating long-term HRQoL and body image in women with DCIS treated with different surgical methods, including mastectomy with IBR and sector resection with or without radiotherapy. The response rate to the questionnaires was 81%, which is high considering the long time between operation and follow-up. Standardised questionnaires employed in many previous studies were used.

Conclusions:

Women treated for DCIS have a satisfactory long-term HRQoL. The addition of radiotherapy to breastconserving surgery did not seem to have any negative impact on HRQoL in the long term. However, body image appeared to be affected in mastectomy and IBR patients. Our results indicate that these women need more preoperative information about what changes in body image to expect after surgery. 2 Mean scores and standard deviations (SD) for the HAD anxiety and depression subscales according to treatment, and proportions of women in the three study groups in the three categories "no case" (0-7), "possible clinical case" (8-10) and "clinical case"( ≥11) for the HAD.

  aim of the present study was to investigate and compare long-term anxiety and depression, body image, and HRQoL in women operated for DCIS by mastectomy and IBR, sector resection alone, or sector resection and postoperative RT. All women in the study would have a retained breast contour after the treatment for DCIS.

Figure 1

 1 Figure1shows inclusion and attrition.

Table 1 .

 1 Mean values and standard deviations for the SF-36 subscales for the three study groups and their respective age-adjusted norm values. Range 0 -100 (high values represent high functioning)2 No statistically significant differences were found between the other two study groups and their norm values

		Mastectomy and IBR	Sector resection alone	Sector resection	P-values for
	Variables					and radiotherapy	P-values for	differences
		Mean 1 (SD)		Mean 1 (SD)		Mean 1 (SD)	differences	between
		N=40-42		N=32-36		N=45-52	between the	mastectomy and IBR
							study samples	and the norm 2
		Study sample Norm	Study sample Norm	Study sample Norm
	Physical functioning PF	92.4 (10.4)	80.8 (20.8) 73.4 (26.6)	72.1 (24.3) 74.1 (25.5)	72.1 (24.2) 0.002	0.002
	Role physical RP	90.2 (23.0)	77.8 (35.0) 75.0 (38.4)	67.2 (40.1) 79.5 (36.0)	65.5 (39.6) NS	NS
	Bodily pain BP	86.5 (19.7)	68.3 (27.4) 70.2 (28.0)	65.9 (28.4) 70.7 (28.1)	65.3 (28.5) 0.006	<0.001
	General health GH	72.7 (20.5)	70.6 (23.1) 70.6 (22.4)	65.4 (23.8) 66.0 (22.4)	65.2 (23.6) NS	NS
	Vitality VT	56.4 (16.0)	67.0 (24.0) 63.6 (12.0)	65.6 (25.0) 57.8 (16.0)	63.6 (25.2) NS	NS
	Social functioning SF	86.6 (20.4)	87.0 (21.1) 93.2 (13.0)	85.3 (22.4) 85.2 (21.3)	84.4 (22.4) NS	NS
	Role emotional RE	78.9 (36.3)	83.3 (31.2) 82.9 (32.7)	77.6 (35.9) 83.7 (34.8)	76.9 (35.6) NS	NS
	Mental health MH	73.0 (20.0)	79.5 (20.3) 84.9 (13.3)	78.5 (21.3) 76.0 (18.7)	77.6 (21.3) 0.012	NS

1

Acknowledgements

This study was supported by grants from the Johan and Jakob Söderberg's Foundation, The Paola Brunetti-Jacovone's Bequest, The Swedish Cancer Foundation in Stockholm and The Swedish Cancer Society. The Swedish National DCIS study group is acknowledged for contributing the cohort of Stockholm patients.

Conflict of interest statement

We have no conflicts of interest.

Role of the funding source

The study sponsors had no involvement in the study.


R Jeevan

D A Cromwell
email: dcromwell@rcseng.ac.uk

J P Browne

M Trivella

J Pereira

C M Caddy

C Sheppard

J H P Van Der Meulen

Regional variation in use of immediate breast reconstruction after mastectomy for breast cancer in England

Keywords: Breast Neoplasms, Mastectomy, Mammaplasty, England

Regional variation in use of immediate breast reconstruction after mastectomy for breast cancer in England

INTRODUCTION

Each year, 38 000 women are diagnosed with breast cancer in England. [START_REF]Cancer statistics registrations: registrations of cancer diagnosed in 2006[END_REF] 40% of those treated within the National Health Service (NHS) undergo a mastectomy as part of their treatment. [START_REF] Jeevan | First Annual Report of the National Mastectomy and Breast Reconstruction Audit 2008[END_REF] Advances in plastic surgical techniques have made immediate breast reconstruction a safe option for most women undergoing mastectomy. [START_REF] Malata | Immediate breast reconstruction after mastectomy for cancer[END_REF] The United Kingdom National Institute for Health and Clinical Excellence (NICE) stated in 2002 that "reconstruction should be available [to all women with breast cancer] at the initial surgical operation", [START_REF]Guidance on cancer services. Improving outcomes in breast cancer -manual update[END_REF] and in 2009 that clinicians should "discuss immediate breast reconstruction with all patients who are being advised to have a mastectomy, and offer it except where significant comorbidity or (the need for) adjuvant therapy may preclude this option". [START_REF]Early and locally advanced breast cancer: diagnosis and treatment[END_REF] In England, 30 regional Cancer Networks have responsibility for service delivery and resource allocation. [START_REF]A policy framework for commissioning cancer services: A report by the Expert Advisory Group on Cancer to the Chief Medical Officers of England and Wales. London: The Expert Advisory Group on Cancer to the Chief Medical Officers of England and Wales[END_REF]7 Cancer Networks were created to overcome service fragmentation by bringing together health service commissioners and providers. A principal aim is for them to ensure patients within their regions have equitable access to high quality cancer care.

In 2009, the National Mastectomy and Breast Reconstruction Audit, a national prospective cohort study of women undergoing mastectomy with or without immediate reconstruction in England, suggested wide regional variation in immediate reconstruction rates. [START_REF] Jeevan | Second Annual Report of the National Mastectomy and Breast Reconstruction Audit 2009[END_REF] However, only three quarters of eligible patients were included, mastectomy-only patients were underreported, and regional rates were based on relatively small samples.

In this paper we used Hospital Episode Statistics (HES) 9 data to derive rates of immediate breast reconstruction after mastectomy for breast cancer within the English Cancer Networks.

The HES database captures all surgical procedures in NHS hospitals, and is less prone to differential reporting of mastectomy and reconstruction procedures. It also provides larger

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patient samples because it is not restricted to activity within a limited timeframe. We investigated the effect of patient characteristics on immediate reconstruction use, and the extent to which they could explain variation in rates across Networks.

METHODS

Hospital Episode Statistics

The HES database contains demographic, geographical and clinical information on all patients admitted to NHS hospitals in England. Surgical procedures are described using the UK Office for Population Censuses and Surveys classification, 4th revision (OPCS4) 10 while medical conditions are described using the International Classification of Diseases, 10th revision (ICD10). [START_REF]World Health Organisation. International Classification of Diseases and Related Health Problems, Tenth Revision[END_REF]

Patient population

We extracted the HES records of all women diagnosed with invasive carcinoma of the breast (ICD10: C50) who underwent a mastectomy (OPCS4: B27) between 1 April 2006 and 28 February 2009. A woman was considered to have undergone an immediate breast reconstruction if reconstruction occurred on the same day as the mastectomy. The OPCS4 codes that defined breast reconstruction were B29 (excluding B29.5 revision), B38, B39 as well as B30.1 (insertion of prosthesis) and S48.2 (insertion of skin expander).

For each woman, we also calculated age on admission, level of comorbidity, ethnicity, and regional socioeconomic deprivation. Comorbidity was derived using the RCS Charlson comorbidity score. [START_REF] Armitage | on behalf of the Royal College of Surgeons Co-morbidity Consensus Group. Identifying co-morbidity in surgical patients using administrative data with the Royal College of Surgeons Charlson Score[END_REF] This identifies whether a women has one or more comorbidities, from a list of 22 conditions, by counting the number recorded in the index admission and in admissions during the 12 months before the index admission date. Ethnicity was coded as white, non-white, unknown or not stated. Socioeconomic deprivation was characterised using the overall combined score of the Index of Multiple Deprivation (IMD). 13

Statistical methods

Unadjusted rates of immediate reconstruction were derived as a percentage of all mastectomies and were calculated for the 30 English Cancer Networks that existed on 1 January 2008. Women were allocated to Networks by their postcode of residence. A multivariable logistic regression model was used to adjust the Network rates for age on admission, comorbidity, ethnicity, and regional deprivation. In the regression model, age and IMD score were included as continuous variables because the trend was approximately linear.

For the unadjusted descriptive analyses, rates of immediate reconstruction are presented for categories of age and IMD score to aid interpretation.

The model was used to estimate the probability of a woman having an immediate reconstruction. These probabilities were then summed for women resident in a Network to calculate the predicted number of immediate reconstructions for that Network. Risk-adjusted rates of reconstruction for each Network were produced by dividing the observed number of immediate reconstructions by the predicted number, and multiplying this ratio by the national rate of reconstruction. Estimates are presented with 95% confidence intervals (CI). The statistical significance of the variation in the Network-level reconstruction rates was assessed using the likelihood ratio test.

RESULTS

Between 1 April 2006 and 28 February 2009, 44 837 mastectomies were performed in women with breast cancer. Their average age was 60.9 years (SD 13.9) and 6,297 (14.0%) had one or more co-morbidities. Ethnicity was known for 87.2%, of whom 94.1% were white.

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6 Overall, 16.5% of women had an immediate reconstruction. Unadjusted immediate reconstruction rates among the 30 English Cancer Networks varied from 8.4% to 31.9% (p<0.001). Excluding the Networks with the three highest and lowest values, the rates still ranged from 11.5% to 26.9% (see Figure 1). Tables 1 and2 summarise the relationship between immediate reconstruction rates and patient characteristics. The reconstruction rate was highest in women under 50 years (32.7%) and lowest in women aged 70 years or over (1.5%). The rate was also lower in women with more comorbidities independent of age. After adjustment, reconstruction rates were lower in women of non-white ethnicity and also decreased with deprivation (Table 2). However, differences in these patient characteristics between Network populations explained only a limited amount of the observed variation. There was still significant regional variation (p<0.001), with adjusted rates varying from 8.0% to 29.4%. After excluding the Networks with the three highest and lowest values, the adjusted rates varied from 11.7% to 25.4% (see Figure 1).

DISCUSSION

There is considerable variation in the use of immediate breast reconstruction between English Cancer Networks which persists after adjusting for patient characteristics. In addition, women who are non-white or from deprived areas are less likely to undergo immediate reconstruction.

Rates of reconstruction were highest amongst women under 50 years.

Limitations of the study

There are known inaccuracies in HES coding, [START_REF]PbR data Assurance Framework 2007/08[END_REF] but it is unlikely that coding errors could explain the four-fold variation observed. Validation work undertaken on HES data for women having breast cancer surgery suggests that HES procedure codes are accurate, with 90-93%

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7 agreement with surgeon-provided data. [START_REF]Breast Cancer Clinical Outcome Measures Project Newsletter[END_REF] To minimise the chance of including prophylactic mastectomy, we selected women with both a diagnosis code for breast cancer and a procedure code for mastectomy. We also used the full range of OPCS codes for breast reconstruction (except for revision surgery) to take account of potential differences in coding between Networks and to limit the likelihood of missing an immediate reconstruction.

The HES database does not include women diagnosed with breast cancer in independent hospitals. These women are sometimes referred back to the NHS if they decide to have immediate reconstruction. [START_REF] Jeevan | First Annual Report of the National Mastectomy and Breast Reconstruction Audit 2008[END_REF] This may have increased NHS reconstruction rates in the Networks with relatively greater independent sector activity, such as those in and around London.

We did not have information on patient characteristics such as smoking and obesity, which are relative contraindications to reconstruction. Their prevalence is linked to deprivation [START_REF] Adams | Effects of area deprivation on health risks and outcomes: a multilevel, cross-sectional, Australian population study[END_REF] which was included as a confounding factor in the logistic regression model. While this would only partially eliminate their effect, differences in the prevalence of these characteristics between Networks are unlikely to account for the large regional variation observed. The National Audit collected information on smoking and obesity and found that adjusting for these characteristics did not reduce the observed Network-level variation. [START_REF] Jeevan | Second Annual Report of the National Mastectomy and Breast Reconstruction Audit 2009[END_REF]

Sources of regional variation

There are various sources for this regional variation. First, women's preferences for the types of breast cancer treatment will vary. In particular, preferences will depend on the information women are given about immediate reconstruction and it has been shown that clinicians are less likely to discuss reconstruction with women from deprived backgrounds. [START_REF] Polacek | Breast cancer disparities and decision-making among U.S women[END_REF][START_REF] Greenberg | Do variations in provider discussions explain socioeconomic disparities in postmastectomy breast reconstruction?[END_REF] Second, post-mastectomy radiotherapy use varies across Networks. [START_REF] Jeevan | First Annual Report of the National Mastectomy and Breast Reconstruction Audit 2008[END_REF] Radiotherapy may impair the aesthetic outcomes of immediate reconstruction and women who are expected to

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have post-operative radiotherapy may therefore prefer a delayed reconstruction. This may also apply for other adjuvant therapies.

Third, in regions with high rates of breast conserving surgery, women undergoing mastectomy would be expected to have a relatively high grade and stage. This should make them less suitable for immediate reconstruction due to the increased likelihood of post-mastectomy radiotherapy. After additional modelling (results not shown), we found that regions with high rates of breast conserving surgery also had high rates of immediate reconstruction after mastectomy. This suggests that clinicians in some regions are more inclined to offer breast conservation or immediate reconstruction than in others, irrespective of cancer stage at treatment.

Finally, there may be variation in service provision. In Networks with inadequate breast reconstruction capacity, women may be dissuaded from having an immediate reconstruction as this could delay their primary cancer treatment beyond national cancer waiting time targets. 21

Findings of the National Mastectomy and Breast Reconstruction Audit

The National Audit provides some insight into the influence of these factors. [START_REF] Jeevan | Second Annual Report of the National Mastectomy and Breast Reconstruction Audit 2009[END_REF] It reported that immediate reconstruction offer rates varied substantially between Networks and were not strongly associated with the actual rates. This was thought to reflect differences in both how the offer was communicated and to which women an offer was made.

The Audit also asked clinicians to give reasons for why women were not offered immediate reconstruction (more than one could be given). [START_REF] Jeevan | Second Annual Report of the National Mastectomy and Breast Reconstruction Audit 2009[END_REF] Anticipated radiotherapy to the chest wall and other adjuvant therapy issues were cited as a reason for around one-third of women but the proportion fluctuated from 15% to 65% between Networks.

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

Availability of immediate reconstruction was give as a reason for 9% of women, although clinicians in four Networks gave this as the reason for not offering reconstruction for over 20% of women. This suggests that some regions suffer from resource constraints. But, overall, women were most commonly deemed inappropriate due to their age, comorbidities, lifestyle factors such as smoking, and concerns about local recurrence. The NICE guidance suggests that, among the various patient characteristics, only comorbidity should legitimately preclude an offer of immediate reconstruction.

Conclusion

We have found considerable variation in the regional use of immediate breast reconstruction which persists after adjusting for patient characteristics. The variation in the HES-derived rates is comparable with the findings of the National Mastectomy and Breast Reconstruction Audit and demonstrates that the differences have not existed for a short period of time.

The analysis does not presuppose that there is an optimal rate for immediate breast reconstruction. However, the wide regional variation suggests that women with breast cancer have unequal access to immediate breast reconstruction. Cancer Networks in England need to examine local reconstructive services, both in terms of provision and how choices are offered, and act to remove barriers to its uptake.

It is likely that the issues about access to reconstruction affect breast cancer services in other countries. Our findings suggest that, when informing and counselling women diagnosed with breast cancer, clinicians should examine their local decision-making processes and ensure that, where safe and appropriate, immediate reconstruction is offered to those women undergoing mastectomy.

  IMD scores are M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 calculated for 32 482 geographical areas (population around 1 500) and were allocated to patients based on their postcode of residence. Greater levels of deprivation are indicated by higher IMD scores.

ACKNOWLEDGEMENTS

We would like to thank Lynn Copley of the Clinical Effectiveness Unit for preparing the extract used in this work, and the NHS Information Centre for Health and Social Care for providing the Hospital Episode Statistics data.

FUNDING

The National Mastectomy and Breast Reconstruction Audit is funded by the Healthcare Commission and the Healthcare Quality Improvement Partnership.

CONTRIBUTORS

RJ, JB, JP, CC, CS and JvdM conceived and designed the study; MT and DC conducted the statistical analyses; RJ, DC, JB and JvdM drafted the manuscript; MT, JP, CC and CS commented on and critically revised drafts. RJ, DC, JB, MT, JP, CC, CS and JvdM all approved the final version for publication. DC is guarantor.

NATIONAL MASTECTOMY AND BREAST RECONSTRUCTION AUDIT

The Audit is managed by the National Clinical Audit Support Programme of the NHS Information Centre for Health and Social Care in partnership with the Association of Breast Surgery, the British Association of Plastic, Reconstructive and Aesthetic Surgeons, the Royal College of Nursing and the Clinical Effectiveness Unit of The Royal College of Surgeons of England.

CONFLICT OF INTEREST STATEMENT

The authors report no conflicts of interest.


MD Erika Richtig
email: erika.richtig@medunigraz.at

MD Peter Komericki

MD Michael Trapp

PhD Arthur Ott

MD ; Bor- Jana Bisail

MD Josef W Egger

PhD Iris Zalaudek

Ratio of marked and excised sentinel lymph nodes and scintigraphic appearance time in melanoma patients with negative sentinel lymph node

Keywords:

Aim: Metastases can occur in up to 15% of all melanoma patients with negative sentinel lymph node examination (SN -). We retrospectively investigated the number of preoperatively marked sentinel lymph nodes (SNs) with lymphoscintigraphy and effectively surgically removed SNs in SN -patients with cutaneous melanoma > 1mm. Ratio of these parameters was calculated and impact of this ratio as well as impact of scintigraphic appearance time (SAT) on disease progression was studied.

Materials and Methods: Data on 122 SN -patients -70 women (58%), mean age 56.5 years -were analysed. Mean follow-up time was 58 months.

Results: Mean tumour thickness of all patients was 2.3 mm. In 51 patients (42%) the number of SNs marked in lymphoscintigraphy was higher than excised in surgery, in 47 patients (38%) the same number as marked was excised and in 24 patients (20%) a lower number was marked than excised. Metastases occurred in 17 patients (14%) after a mean time of 24.8 months. Mean tumour thickness (5.4 mm) was significantly higher in these patients than in the other patients (p=0.000). Ratio of marked and excised SNs had no influence on disease progression; the only parameter influencing outcome was tumour thickness (p=0.000). Short SAT was significantly associated with higher tumour thickness (p=0.004).

Conclusion:

Our study indicates that, in routine clinical practice, it suffices to harvest the first SN, as the ratio of marked and excised SNs has no impact on disease progression.

Introduction

Sentinel node biopsy (SNB) has become a routine surgical procedure to improve staging of patients with primary melanoma ≥ 1.0 mm tumour thickness, although some authors also suggest SNB for thinner minimally invasive melanomas. [START_REF] Morton | Technical details of lymphatic mapping for early stage melanoma[END_REF][START_REF] Garbe | Deutsche Leitlinie: Malignes Melanom[END_REF][START_REF] Zakrzewski | Meta-analysis of sentinel node positivity in thin melanoma (≤ 1 mm)[END_REF][START_REF] Zapas | The risk of regional lymph node metastases in patients with melanoma less than 1.0 mm thick : recommendations for sentinel node biopsy[END_REF][START_REF] Roulin | Prognostic value of sentinel node biopsy in 327 prospective melanoma patients from a single institution[END_REF] Positive sentinel lymph nodes (SN +) are detected in approximately 20% of patients without palpable lymph nodes; these patients have a shorter disease-free survival than those with negative sentinel lymph nodes (SN -). [START_REF] Morton | Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases[END_REF][START_REF] Rousseau Dl | Revised American Joint Committee on Cancer staging criteria accurately predict sentinel lymph node positivity in clinically node-negative melanoma patients[END_REF][START_REF] Berk | Sentinel lymph node biopsy for cutaneous melanoma: the Stanford experience, 1997-2004[END_REF] For the latter group of patients, tumour thickness of the primary melanoma and ulceration remain the strongest prognostic factors. 9 The course of disease in melanoma patients after negative SN biopsy has long been the subject of closer investigation, as up to 15% of SN -patients will develop disease progression. 9-14 Various theories, including primary haematogenic metastatic pathways or falsenegative SNs, have been proposed to explain this constantly reported event. [START_REF] Morton | Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases[END_REF][START_REF] Nieweg | What is a sentinel node and what is a false-negative sentinel node?[END_REF] Little attention has been directed to the correlation of SNs detected by lymphoscintigraphy and the number of effectively surgically removed SNs and its effect on disease progression.

The aim of our study was to compare the number of SNs marked in lymphoscintigraphy with those effectively removed during surgery in a cohort of SN -patients and to investigate the impact of this ratio on disease progression. Furthermore scintigraphic appearance time (SAT) and its possible influence on the ratio of marked and excised SNs was studied. Only patients for whom sex, age, location, melanoma type, tumour thickness in mm, special histological features including regression, ulceration, microsatellites and vascular invasion, number of SNs marked in lymphoscintigraphy and number of surgically removed SNs, and scintigraphic appearance time (SAT) were available were included for further analysis.

Analysis of follow-up comprised the occurrence of the first loco-regional or distant metastasis, time to progression and death of disease. Protocol for recurrence was identical for all patients and included regular physical examinations, sonography of the lymph nodes and abdomen and chest radiography.

Differences in number of marked (SNm) and excised (SNe) SNs were documented as ratio and patients were divided into three groups: SNm>SNe: higher number of SNs marked in lymphoscintigraphy than excised during surgical procedure, SNm=SNe: same number as marked was excised and SNm<SNe: lower number marked than excised. SAT was divided into 2 groups: ≤ 30 minutes and > 30 minutes. Ethics commission approval was obtained.

All institutional rules governing clinical investigation of human beings were strictly fol- All patients underwent the same standardized procedure. [START_REF] Alazraki | Procedure guideline for lymphoscintigraphy and use of intraoperative gamma probe for sentinel lymph node localization in melanoma of intermediate thickness 1.0[END_REF] SN lymphoscintigraphy was performed using Tc-99m nanocolloid (Nanocoll®, GE Healthcare Buchler GmbH, Braunschweig, Germany) with 0.2 ml injected 4 times (4x 37 MBq Tc-99m nanocolloid) intracutaneously around the primary tumour or excisional biopsy wound. Image acquisition (Elscint Apex SPX, GE Healthcare, Milwaukee, USA) started after 3 -5 minutes with sequential static images up to 1 hour. When SN could not be identified within 1 hour, further images were performed every 15 minutes up to 2 hours and then every hour till SN was detected.

SAT was defined as time from injection to appearance of the first lymph node in lymphoscintigraphy and measured in minutes.

One to three hours after appearance of the SN in lymphoscintigraphy, surgery was carried out by two surgeons experienced in SN biopsies. During surgery, identification of SN was performed according to the guidelines of Alazraki et al. [START_REF] Alazraki | Procedure guideline for lymphoscintigraphy and use of intraoperative gamma probe for sentinel lymph node localization in melanoma of intermediate thickness 1.0[END_REF] with injection of Patent Blue V (Bleu Patenté V Sodique Guerbet 2.5%; Guerbet, Roissy CdG, France). An intra-operative gamma-detecting probe was used to find sentinel nodes (Gamma Finder II, World of Medicine AG, Ludwigsstadt, Germany). All blue nodes, the node with the highest radioactive count and any node with > 10% count rate of the most radioactive node were harvested.

Histopathological procedure:

For histopathologic examination, the excised SNs entered pathologic SN protocol according to Cochran et al. [START_REF] Cochran | Current practice and future directions in pathology and laboratory evaluation of the sentinel node[END_REF] 1.

Characteristics of primary melanomas:

The most common sites of primary melanoma were the extremities and the trunk with no differences in age, sex and location between patients with and without progression.

Mean tumour thickness for all 122 SN -patients was 2.3 mm (SD ± 2.5 mm); 57 (46%) were Clark level IV, followed by level III, V and II in 45 (37%), 6 (5%) and 3 (3%) melanomas, respectively. Clark level was missing in 11 patients (9%). Histopathologically, ulceration was present in 20 melanomas (16%), widespread regression in 16 melanomas (13%), focal regression in 5 melanomas (4%) and 2 melanomas (2%) had microsatellites. None of the melanomas showed vascular invasion.

At time of diagnosis 25 patients (20%) suffered from melanoma clinical stage IA according to the American Joint Committee on Cancer Staging System (AJCC) criteria 18 , 55 (45%) from stage IB, 19 (16%) from stage IIA, 18 (15%) from IIB and 5 (4%) from IIC.

Melanoma characteristics in patients developing metastases:

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8

Melanoma metastases occurred in 17 SN-patients (14%). The mean tumour thickness was 5.4 mm (SD ± 5.4 mm), which was significantly higher than in patients without disease progression (p=0.000). Ulceration was seen in 41% of melanomas (7 patients), focal regression was present in only 6% (1 patient) of melanomas, and none of the cases showed microsatellites. In this progression group, melanomas on the trunk and upper arm prevailed in men, whereas location on the lower leg and acral sites dominated in women. A histopathologic diagnosis of primary nodular melanoma (PNM) was more common in the progression group (p=0.008) compared with patients without progression. Eight of the 17 patients with disease progression suffered from melanoma clinical stage IIB according to the AJCC criteria 18 and only 3 patients suffered from melanoma stage IIC.

First sign of metastasis and death from disease:

Mean time to progression was 24.8 months (SD ± 19.4, range from 3 to 73 months). The lungs were the most common site for metastases, followed by regional lymph node and distant lymphatic metastases (Table 1). Further metastatic sites included brain, liver, bone and abdomen. Death from disease occurred in 14 of the 17 patients after a mean time of 10.9 months (SD ± 12.8, range between 1 and 52 months).

Sentinel lymph node results:

Total numbers of SNs marked and excised are given in Table 1. In 51 patients (42%) the number of marked SNs in lymphoscintigraphy was higher than the number effectively surgically removed (SNm>SNe). In 47 patients (38%) the same number as marked was excised (SNm=SNe) and in 24 patients (20%) a lower number was marked than excised (SNm<SNe). No difference was seen in total number of SNm and SNe between SN -patients with or without metastases (p=0.943 and 0.638 respectively, Table 2).

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

9 For all patients, short SAT (≤ 30 minutes) was more common than long SAT (> 30 minutes).

Patients with long SAT were significantly older than patients with short SAT (p=0.023), but SAT had no impact on the ratio of marked and excised SNs (p=0.199). However, short SAT was associated with a higher tumour thickness (p=0.004).

Comparing patients without and with progression, no differences in the ratio of marked and excised SNs or SAT were seen (Table 2). The only significant parameter associated with disease progression was tumour thickness (p=0.000).

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

10 Discussion SNB has a staging and prognostic value in patients with melanoma, but to date its curative impact remains to be determined. [19][20] Although a SN -is associated with a better prognosis, it does not rule out metastases as disease progression occurs in up to 15% of patients. 9- 14 Possible reasons for this event include false-negative results related to technical limitations, errors during scintigraphic mapping or false-negative interpretation of histopathology. [21][22][23] Schmidt and al. 24 showed that the total number of removed SNs had no influence on relapse-free survival and overall survival in SN -patients. With respect to these results, we also found no association between the total number of excised SNs and disease progression, but we additionally focused on the question whether the ratio of marked and excised SNs could have an impact on the development of metastases. Although one would expect that the number of SNm is equally to those excised, we found the same number of marked and excised SNs in only 38% of our patients, while in 20% the number of marked SNs was lower, than effectively surgically removed. A reason for the latter could be the coincidental removal of small lymph nodes adjacent to the SN when removed "en bloc". Furthermore, surgeons are prone to remove all radioactive nodes without distinguishing between firsttier nodes from second-tier nodes or to excise also lymph nodes marked with blue dye without radioactivity, as it is reported to happen in 15%-30% of patients with breast cancer. [START_REF] Nieweg | What is a sentinel node and what is a false-negative sentinel node?[END_REF] More importantly, in 42% of our patients the number of surgically removed SNs was lower than those marked in lymphoscintigraphy. A possible explanation could be an inadequate timing between lymphoscintigraphy and effected surgery in the case of short SAT, in which the rapid lymphatic flow "clears" the SN even within the recommended time to surgery. This scenario is however unlikely as in all our SN -patients associated with short

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
11 SAT, SAT had no impact on the total number of marked and excised SNs and on disease progression. However, we observed a prolonged SAT especially in elderly patients, suggesting an age-related lymphatic dysfunction which may play a more important role than previously considered. This is in line with a study by Conway et al. 25 who showed an inverse association between radioactive count rate in SNs and age. Interestingly despite the lower radioactive count rates in the SNs of elderly patients, the percentage of SN + patients in their cohort was 21%, which corresponds well with the rates given in the literature. [START_REF] Morton | Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases[END_REF][START_REF] Rousseau Dl | Revised American Joint Committee on Cancer staging criteria accurately predict sentinel lymph node positivity in clinically node-negative melanoma patients[END_REF][START_REF] Berk | Sentinel lymph node biopsy for cutaneous melanoma: the Stanford experience, 1997-2004[END_REF] The lack of influence of the ratio of marked and effectively excised SNs on disease progression in our study questions the need of re-excision in patients, in whom fewer SNs were surgically removed that marked. Despite the certainty that metastatic disease is more common in SN + patients, patterns of recurrence are similar between SN -and SN + patients with distant sites more likely to be involved. [START_REF] Dalal | Patterns of first-recurrence and post-recurrence survival in patients with primary cutaneous melanoma after sentinel lymph node biopsy[END_REF] In the light of these data, it seems plausible that some melanomas metastasize via haematogenic rather than lymphogenic routes as also discussed in the incubator hypothesis by Morton et al. [START_REF] Morton | Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases[END_REF] Alternatively, biologically highly aggressive thick tumours could enrich the lymph fluid with tumour cells that rapidly cross the sentinel lymph node basin, as outlined by Mahieu-Renard et al. 26 Our finding of distant metastases as the most common event in SN -patients with disease progression supports these hypotheses.

However metastases to the regional SN dissected nodal basin also occurred in 35% of our patients. This number is nearly equal to previous data 9, [START_REF] De Giorgi | Outcome of patients with melanoma and histologically negative sentinel lymph nodes: a one institutions´s experience[END_REF][START_REF] Yee | Outcome of 846 cutaneous melanoma patients from a single center after a negative sentinel node biopsy[END_REF][START_REF] Fincher | Patterns of recurrence after sentinel lymph node biopsy for cutaneous melanoma[END_REF]27 and favours the immunosuppressive theory, [START_REF] Morton | Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases[END_REF] which suggests that increased expression of factors resulting in immunosuppression allows sudden growth of initially silent melanoma cells in the lymph node. Finally, it seems also reasonable that melanomas could simultaneously metastasize to both the SNs and the distant sites, as proposed by the marker hypothesis. [START_REF] Morton | Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases[END_REF] Even in cases of recurrence of melanoma in the SN lymph node basin, differences between marked and excised SNs did not play a role in our study.

Limitations of our study are the small cohort of patients and the small number of metastatic patients. False-negative results related to technical limitations or errors during scintigraphic mapping are unlikely, as lymphoscintigraphic mapping is a well-established procedure performed according to defined protocols [START_REF] Alazraki | Procedure guideline for lymphoscintigraphy and use of intraoperative gamma probe for sentinel lymph node localization in melanoma of intermediate thickness 1.0[END_REF] for more than 10 years at our Department of Nuclear Medicine. Furthermore we routinely perform more than the usually recommended 10 serial sections by Cochran et al. [START_REF] Cochran | Current practice and future directions in pathology and laboratory evaluation of the sentinel node[END_REF] at our histopathologic laboratory as well as immunohistochemistry staining, which make false negatives related to histopathologic examination also unlikely.

In conclusion, our study supports that thickness of the primary tumour remains the most important prognostic marker for progression of patients with melanoma. From a practical point of view it seems sufficient to harvest the first SN, as the ratio of marked and excised SNs has no impact on disease progression.

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

  For this retrospective study, we searched the databases of the Department of Dermatology, Medical University of Graz, Austria and two Departments of Pathology in Styria (Medical University of Graz and Leoben General Hospital) between 2001 and 2003 for records of patients with melanoma > 1mm, SN -status and a follow-up time of > 10 months. A SN -status was considered when both routine hematoxylin/eosin stains and immunohistochemistry failed to detect metastases.

  Helsinki Declaration with respect to human subjects in biomedical research was conformed.Technical procedure:Lymphoscintigraphic mapping and surgical procedure:

7 Results

 7 with a slight modification in that at least 16 serial sections instead of the M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 recommended ten were performed. Negativity was assessed when both routine hematoxylin/eosin stain and immunohistochemistry using S100, HMB-45 and Melan-A failed to detect macro-or micrometastases. Statistical methods Data were entered into a computerized database and analyzed. All computations yielding statistical inferences were conducted with the statistical packages SPSS for Windows (SPSS, version 15.0.1; SPSS Inc., Chicago, Illinois, USA) and SAS (SAS version 9.2; Copyright (c) 2002-2008 by SAS Institute Inc., Cary, NC, USA). The regular distribution analysis was performed according to Kolmogorov-Smirnov and to Shapiro-Wilk. To describe the relationship between the dichotomous response variable (1 = metastases; 0 = no metastases) and different explanatory variables, a Binary Logistic Regression Model was used. To explain metric but non-normal response variables a Generalized Linear Model was used. Categorical variables where calculated by Chi-Square-Test. A p-value ≤ 0.05 was considered statistically significant. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Patient demographics: A total of 158 sentinel node biopsies were performed from 2001-2003; they were positive in 35 patients (22.2%) and negative in 123 patients (77.8%). From the 123 SN -patients, one patient was excluded because of missing data regarding the lymphoscintigraphic protocol resulting in a total of 122 patients, 69 women (57%) and 53 men (43%), included in the study. Mean age of all 122 patients was 56.5 years (Standard deviation=SD ± 15.3, range 15-81 years). Mean follow-up time of all patients was 58 months (SD ±16.0, range 10-85 months). Patients' demographics, as well as information on their primary melanoma and SN, are summarised in Table

Acknowledgments

We thank Ralf Ludwig, MD, and Emad Arbab, MD, for their help of data acquisition, Walter H. C. Burgdorf, MD, for English editing and Tanja Strimitzer, MSc, from data, statistics and risk assessment of AGES for her independent control of our statistical results.

A C C E P T E D