A Sommariva

P M Donisi

B Gnocato

R Vianello

V Stracca

G Zaninotto

FACTORS AFFECTING FALSE-NEGATIVE RATES ON EX VIVO SENTINEL LYMPH NODE MAPPING IN COLORECTAL CANCER

PURPOSE: Despite the increasing use of sentinel lymph node (SLN) mapping after colorectal cancer resection, reported node identification and false-negative rates vary considerably. The main aim of this prospective study was to quantify the false-negative rates on SLN mapping after resection and to evaluate factors influencing them. METHODS: Sixty-nine patients with biopsy-proven cancer of the colon and rectum underwent SLN mapping according to a protocol involving the ex vivo submucosal and peritumoral injection of 2-4 ml of Patent Blue V dye. All lymph nodes visualized were marked as SLN and totally embedded, then two 4 μm sections were cut for hematoxylin and eosin staining, and cytokeratin (AE1/AE3) immunostaining. A standard examination of the whole specimen and of the regional non-sentinel lymph nodes was also performed. RESULTS: SLNs were identified in 97.3% of the evaluable cases. A mean 5.0 SLNs were removed per patient (SD ±4.2). Nine false negatives were identified. Rectal cancer, tumor size > 60 mm, number of metastatic non-sentinel lymph nodes, and mucinous tumors were associated with falsenegative SLNs. At multivariate analysis, a rectal location and mucinous differentiation were independently associated with false-negative SLNs.

CONCLUSIONS: Ex-vivo SLN mapping after colorectal cancer surgery is technically feasible with a high identification rate. Tumor size and stage, rectal involvement and a mucinous histology seem to interfere with the reliability of SLN staging. It is mandatory to standardize the procedure and selection criteria in order to deal with the question of the reliability of SLN mapping in colorectal cancer.

Introduction

Lymph node metastases are the most important predictors of survival in patients with potentially curable colorectal cancer (CRC). Adjuvant chemotherapy is given only to patients with positive nodes, whereas node-negative patients are considered cured by surgery [START_REF] Moertel | Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma[END_REF][START_REF]Impact B2Investigators. Efficacy of adjuvant fluorouracil and folinic acid in B2 colon cancer. International Multicentre Pooled Analysis of B2 Colon Cancer Trials (IMPACT B2)Investigators[END_REF]. Up to 30% of patients with negative nodes will recur, however, in the course of their lives [START_REF] Voyer | Colon cancer survival is associated with increasing number of lymph nodes analyzed: a secondary survey of intergroup trial INT-0089[END_REF]. This is at least partially due to understaging of the regional lymph nodes: patients with metastatic nodal disease that goes unnoticed are not given adjuvant chemotherapy and are at higher risk of recurrence.

There are two possible reasons why regional nodes go understaged:

1.

not all metastatic nodes are identified due to an inadequate extension of the surgical resection, or some are overlooked during standard pathological dissection; 70% of metastatic lymph nodes are <5 mm in size and may escape notice because of their small size [START_REF] Rodriguez-Bigas | Clinical significance of colorectal cancer: metastases in lymph nodes < 5 mm in size[END_REF]. Cawthorn et al [START_REF] Cawthorn | Clearance technique for the detection of lymph nodes in colorectal cancer[END_REF] recommended using a xylene alcohol clearance technique to increase the number of nodes identified, but the procedure is time-consuming and has not been widely adopted;

2. some metastatic deposits in harvested nodes are too small to be detected in 1-2 sections stained with hematoxylin and eosin (H&E) at standard pathological analysis. Stepwise sectioning of the nodes and the extensive use of immunohistochemistry and molecular analysis (reverse transcription polymerase chain reaction, RT-PCR) on the lymph nodes may improve the detection threshold for such metastases but, here again, such methods are labor-intensive and expensive, and they cannot be used routinely on all lymph nodes detected [START_REF] Braat | Lymphatic staging in colorectal cancer: pathologic, molecular, and sentinel node techniques[END_REF].

Sentinel lymph node mapping (SLNM) has been recommended in cases of CRC to help overcome these problems and improve disease staging [START_REF] Joosten | Intraoperative lymphatic mapping and the sentinel node concept in colorectal carcinoma[END_REF][START_REF] Saha | Technical details of sentinel lymph node mapping in colorectal cancer and its impact on staging[END_REF][START_REF] Wood | Validation of lymphatic mapping in colorectal cancer: in vivo, ex vivo, and laparoscopic techniques[END_REF]. Unlike the situation in breast cancer and melanoma, where the SLN concept has been used to lower the morbidity of regional lymphadenectomy [START_REF] Veronesi | Sentinel-node biopsy to avoid axillary dissection in breast cancer with clinically negative lymph-nodes[END_REF][START_REF] Morton | Technical details of intraoperative lymphatic mapping for early stage melanoma[END_REF], SLNM for CRC enables the pathologist to focus on the node(s) at

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greatest risk of spread and examine these nodes with multiple slices and immunohistochemistry, and/or RT-PCR, and thereby detect metastatic tumor cells that would otherwise go unrecognized.

The results achieved with SLNM in CRC vary, however, in terms of the identification, falsenegative and upstaging rates [START_REF] Saha | A multicenter trial of sentinel lymph node mapping in colorectal cancer: prognostic implications for nodal staging and recurrence[END_REF][START_REF] Bertagnolli | Sentinel node staging of resectable colon cancer: results of a multicenter study[END_REF] and several questions remain to be addressed. It is still not clear which patients benefit most from this procedure (case selection), the best timing of the injection, the best tracer, the ultrastaging protocol and the prognostic value of micrometastatic cells detected in SLN.

The aim of the present study was to quantify the detection and false-negative rates achieved by ex vivo sentinel node mapping in CRC, and to assess the factors influencing these rates.

Patients and Methods

Patient selection

All patients were prospectively selected for SLNM according to a preoperative and intraoperative staging protocol, if they met the following criteria: All patients gave their written, informed consent. The standard surgical procedure included:

high division of the mesenteric vessels (for colon cancer), or division at the origin of the inferior

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mesenteric vessels and total mesorectal excision (for rectal malignancies). Every suspect lymph node outside the resection field was retrieved and prepared for frozen section and pathological examination. Tumors were staged according to the AJCC/TNM [START_REF] Greene | AJCC Cancer Staging Handbook: TNM Classification of Malignant Tumors[END_REF]. The tumor's site, macroscopic features, maximal size, T and N stages, grade, mucinous features, and lymphatic or venous invasion were also recorded.

Sentinel lymph node mapping procedure and pathological analysis

Dye was injected by two surgeons (AS and BG) within 15 minutes of resecting the specimen, which was opened along the antimesenteric (or antimesorectal) border and 1-4 ml of Patent Blue V dye in 2.5% solution (Monico SpA, Mestre, Italy) were injected into the submucosa along the edge of the tumor.

Statistical analysis

The identification rate (IR) was defined as the number of mapping procedures finding at least one blue node out of the total number of mapping procedures performed. False negative cases (FN) were defined as those where the SLNs were negative but the NSLNs were positive. True positive cases (TP) were considered as those where the SLNs were positive and the NSLNs were positive or negative. The false-negative rate (FNR) was calculated using the formula (FN/FN+TP).

Continuous data were expressed as mean ± standard deviation (SD). Continuous variables were compared using the Wilcoxon test. Categorical data were compared using Fisher's exact test. Risk factors with a univariate p value below 0.05 were included in a multiple regression analysis. A ROC curve analysis was performed to identify the cut-off for tumor size.

Results

Study population and demographics

From

Discussion

The aim of this study was to establish the sentinel node detection rate and the prognostic factors influencing the false-negative rate in colorectal cancer. Ex-vivo SLN identification was done in the vast majority of patients by injecting patent blue dye into the submucosa of the tumor. The procedure was simple and inexpensive, and took only a few minutes for one of the surgeons to complete in the operating room, immediately after resecting the specimen.

The in-depth search on the few SLN identified and retrieved from each specimen enabled the pathologist to perform an extensive search for metastatic tumor cells in these nodes. By means of multiple sections and immunostaining, SLNs were identified as the sole site of metastases in 8 patients: most of these were micrometastases and would probably have gone undetected using a normal procedure, meaning that patients would have been denied adjuvant chemotherapy.

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The second relevant finding of the study was the number of negative SLNs, despite metastatic disease being found in the NSLNs, -9 in the group as a whole -leading to a falsenegative rate of 31%. The main factors associated with false-negative findings in ex-vivo SLNM were rectal location, a mucinous histology, tumor stage (both T and N) and tumor size > 60mm.

A rectal tumor site is reportedly associated with higher false-negative SLN rates [START_REF] Braat | Sentinel node detection after preoperative short-course radiotherapy in rectal carcinoma is not reliable[END_REF]. The mesocolon is much easier to inspect on both sides than the mesorectum, which may be fat and bulky. The rectal lymphatics are also more complex than their mesocolic counterpart: it may be that, in rectal cancer, a radioguided search (rather than a dye-driven search) might help to overcome these problems. There is no clear explanation for the diversity of behavior between non-mucinous and mucinous tumors: perhaps the presence of a mucinous lake alters normal lymphatic drainage.

Although patients with macroscopically-evident metastatic nodes were excluded from this study, the finding of numerous (> 3.9) metastatic NSLNs was associated with false-negative SLNs:

this confirms previous reports that positive SLNs were less likely to be found in tumors with extranodal growth and gross lymph node invasion [START_REF] Wong | Ex vivo sentinel node mapping in carcinoma of the colon and rectum[END_REF]. Extensive metastasis via lymphatic vessels may clog some lymphatic pathways, leading to the primary draining nodes being skipped and more distant nodes being invaded via secondary routes. Similarly, a bulky or locally-advanced tumor may interfere with sentinel node mapping, as shown by Patten and Saha [START_REF] Saha | Technical details of sentinel lymph node mapping in colorectal cancer and its impact on staging[END_REF][START_REF] Patten | A prospective evaluation of radiocolloid and immunohistochemical staining in colon carcinoma lymphatic mapping[END_REF]. False-negative SLNs in large tumors may also depend on the amount of dye injected, as shown by Viehl et al. [START_REF] Viehl | Identification of sentinel lymph nodes in colon cancer depends on the amount of dye injected relative to tumor size[END_REF]. In the present series, the amount of dye injected sufficed to color the submucosa around the tumor (up to 4 ml). It may be that using even larger amounts of dye could overcome this problem.

The search for SLNs in colon cancer has a completely different clinical goal from the case of breast cancer or melanoma (when SLNM is done to avoid any unnecessary extensive lymphadenectomy and to reduce the morbidity by yielding 1-2 prognostic nodes rather than the regional basin): in colon cancer, regional lymphadenectomy with en-bloc tumor resection remain the standard treatment, and the clinical problem is to yield the identification of any metastatic cells in retrieved nodes. The results of the present study confirm that pathological examination of ex-

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  Sufficient dye was injected to color the submucosal layer around the tumor. No massage was performed. A few minutes later, the pericolic and perirectal tissue was systematically searched and all blue lymph nodes were retrieved and classified as SLNs. For rectal cases, the specimen was incised longitudinally along the antimesenteric border for posterior tumors and along the lateral edge for anterior tumors. The mesorectum was opened, paying attention to maintain the anatomical relationship between rectum, tumor and mesorectal fascia. A standard examination of the whole specimen, and of the regional non-sentinel lymph nodes (NSLNs) was subsequently performed. The SLNs were embedded in paraffin and two consecutive 4 µm thick tissue sections were cut on two levels 200 µm apart. The sections were stained with H&E and immunostained with mononuclear antibody for cytokeratin (Keratin AE1/AE3, Pan Ab-1 NeoMarkers, Inc Fremont CA, USA). Standard H&E-stained sections (2 per node) were obtained from the NSLNs. Micrometastases and isolated tumor cells were defined according to the guidelines of the International Union Against Cancer[START_REF] Hermanek | International Union Against Cancer. Classification of isolated tumor cells and micrometastasis[END_REF]. .

Table 1 .

 1 Patients and pathological results

	1 1

Table 2 .

 2 Factors relating to false-negative results on univariate analysis. ^Wilcoxon test* Positive cases only.

	M A N U S C R I P T

Table 3 .

 3 Multiple logistic regression analysis for factors related to false negative results

Acknowledgments:

In memory of Prof. Carlo Tremolada

Funding source

The work was supported by a grant from the Regione Veneto, Ricerca sanitaria finalizzata, 2007.

vivo SLNs cannot be used as a short-cut for determining the pathological status of colon cancer since the method carries an unacceptable false-negative rate, ranging from 20% to 50% [START_REF] De Haas | Sentinel lymph node mapping in colon cancer: current status[END_REF] (31% in this study). SLN identification enables the pathologist to focus on just a few nodes, and search these nodes extensively for metastatic cells, enabling the identification of metastatic cells in the category of patients who can benefit most from this more detailed search, i.e. those with small T1/T2 colon cancer. On the other hand, there is no point in searching for SLN in patients with advanced tumors because of the high false-negative rate and because finding positive SLN in such patients is unlikely to change the therapeutic strategy.

Two questions are still open and need to be investigated: 1. whether a more sophisticated radiolabeled SLN searching technology could make it easier to find SLN than using simple dye injection in rectal cancer; and 2. whether the random extensive examination of NSLNs might be as effective as examining SLNs. This issue was not addressed specifically in this study, but other authors recently showed that SLNs were significantly more likely to harbor micrometastases than NSLNs. [START_REF] Stojadinovic | Prospective randomized study comparing sentinel lymph node evaluation with standard pathologic evaluation for the staging of colon carcinoma: results from the United States Military Cancer Institute Clinical Trials Group Study GI-01[END_REF][START_REF] Bembenek | Sentinel lymph node biopsy in colon cancer: a prospective multicenter trial[END_REF].

In conclusion, sentinel lymph node mapping is feasible in colon cancer with a high identification rate: it enables metastases or micrometastases to be identified in a relevant number of patients, consequently inducing changes in treatment strategies. Since large tumors, extensive nodal metastases and a tumor location in the extraperitoneal rectum are associated with false-negative findings in SLNs, such patients could be excluded from the SLNM process, which appears to be more useful in the less-advanced stages of colon tumors. Further studies are needed to clarify the association between the mucinous variant and false negative findings in SLNs.


MD Makoto Nakamura
email: nakamura.makoto@mh-hannover.de

MD J K Krauss

Nakamura Krauss

Image-guided resection of small lesions in the cavernous sinus and Meckel's cave

Keywords: neuronavigation, computer assisted surgery, cavernous sinus, Meckel

Objective:

The microsurgical resection of tumors or vascular lesions in the cavernous sinus and the neighbouring Meckel's cave has been considered as hazardous because of often associated cranial nerve morbidity. Despite increasing consent that many of such tumors should not undergo surgical therapy, the cavernous sinus and Meckel's cave may harbour small lesions of various origin, which are amenable for surgical resection. Surgery in this anatomical area needs a well directed approach. In this setting, neuronavigational guidance may provide a useful tool. We report on a series of patients operated on guided by neuronavigation.

Methods: Five patients underwent a pterional approach for microsurgical resection. The procedures were planned and assisted by a pointer based neuronavigation system (Medtronic Stealth Station). Pathological entities included schwannoma, epidermoid, cavernoma and capillary hemangioma. Three lesions were located in the Meckel's cave and two lesions in the cavernous sinus.

Results:

Intraoperative guidance by neuronavigation appeared to be particularly instrumental in identification of the appropriate site of dural incision over the target region for microsurgical resection. Except of a mild increase of facial hypesthesia in one patient, there were no new cranial nerve deficits. In three patients, preoperative symptoms improved immediately after surgery.

Conclusion:

The surgical resection of small tumors or vascular lesions within the Meckel's cave or cavernous sinus is facilitated by neuronavigational guidance with sufficient intraoperative reliability and safety. In consideration of well known anatomical landmarks, targeted entry into the cavernous sinus or Meckel's cave directed by neuronavigation enables a tailored approach for microsurgical resection.

Introduction

The cavernous sinus, located at the central cranial base, and the neighbouring Meckel's cave represent a complex anatomical region, which is surrounded by dural walls containing important neurovascular structures [START_REF] Rhoton | Microsurgical Anatomy of the Sellar Region and Cavernous Sinus[END_REF][START_REF] Yasuda | Microsurgical Anatomy and Approaches to the Cavernous Sinus[END_REF] . It may harbour various neoplastic and vascular lesions of different origin, which are increasingly found in an early stage of growth because of the advent and availability of modern imaging studies [START_REF] Razek | Imaging Lesions of the Cavernous Sinus[END_REF] . The issue concerning the best treatment option for such lesions and preferable surgical approaches to this region are still a matter of debate [START_REF] Mefty | Surgery of Tumors Invading the Cavernous Sinus[END_REF][START_REF] Sepehrnia | Management of Intracavernous Tumours: an 11-Year Experience[END_REF][START_REF] Dolenc | Surgery of Vascular Lesions of the Cavernous Sinus[END_REF][START_REF] Sekhar | Surgical Management of Lesions Within the Cavernous Sinus[END_REF] .

Since the pioneering work of Parkinson 8 various surgical approaches have been described to approach the cavernous sinus or Meckel's cave including either an intra-or extradural transcranial approach [START_REF] Sepehrnia | Management of Intracavernous Tumours: an 11-Year Experience[END_REF][START_REF] Dolenc | Surgery of Vascular Lesions of the Cavernous Sinus[END_REF][START_REF] Sekhar | Surgical Management of Lesions Within the Cavernous Sinus[END_REF][START_REF] Parkinson | A Surgical Approach to the Cavernous Portion of the Carotid Artery. Anatomical Studies and Case Report[END_REF][START_REF] Hakuba | A Combined Orbitozygomatic Infratemporal Epidural and Subdural Approach for Lesions Involving the Entire Cavernous Sinus[END_REF][START_REF] Inoue | Surgical Approaches to the Cavernous Sinus: a Microsurgical Study[END_REF] or more recently the endoscopic transsphenoidal approach [START_REF] Alfieri | Endoscopic Endonasal Approaches to the Cavernous Sinus: Surgical Approaches[END_REF][START_REF] Jho | Endoscopic Endonasal Skull Base Surgery: Part 2--The Cavernous Sinus[END_REF] . Although the microsurgical anatomy of this region has been extensively described in the past [START_REF] Parkinson | A Surgical Approach to the Cavernous Portion of the Carotid Artery. Anatomical Studies and Case Report[END_REF][START_REF] Inoue | Surgical Approaches to the Cavernous Sinus: a Microsurgical Study[END_REF][START_REF] Kawase | Meningeal Architecture of the Cavernous Sinus: Clinical and Surgical Implications[END_REF][START_REF] Krisht | The Blood Supply of the Intracavernous Cranial Nerves: an Anatomic Study[END_REF][START_REF] Van Loveren | The Dolenc Technique for Cavernous Sinus Exploration (Cadaveric Prosection). Technical Note[END_REF][START_REF] Umansky | The Lateral Wall of the Cavernous Sinus. With Special Reference to the Nerves Related to It[END_REF] , surgical exposure is still challenging. Because of the complex anatomy and density of critically important neurovascular structures, many lesions in this area have been considered as unresectable [17][START_REF] Iwai | Gamma Knife Radiosurgery for the Treatment of Cavernous Sinus Meningiomas[END_REF][START_REF] Nicolato | Radiosurgical Treatment of Cavernous Sinus Meningiomas: Experience With 122 Treated Patients[END_REF] . The microsurgical exposure itself may often lead to high cranial nerve morbidity. In addition, the advent of radiosurgery has resulted in a decrease of patients undergoing microsurgical resection of lesions in the cavernous sinus 17-19 . Despite increasing consent that many tumors with intracavernous infiltration, for example cavernous sinus meningiomas, should not undergo surgical resection, the cavernous sinus and Meckel's cave may harbour small lesions of other origin, which are amenable for a cure with surgical therapy. In consideration of other therapeutic options nowadays, microsurgical resection in this anatomical area needs a well directed approach with avoidance of any cranial nerve morbidity. In this setting, intraoperative image guidance may provide a useful tool. We have reported on our experience with navigated approaches to the clivus and the anterior skull base elsewhere [START_REF] Nakamura | Neuronavigational Guidance in Craniofacial Approaches for Large (Para)Nasal Tumors Involving the Anterior Skull Base and Upper Clival Lesions[END_REF] . In this report, we present a series of patients

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Nakamura and Krauss 4 operated on small lesions of the cavernous sinus and Meckel's cave guided by neuronavigation. Lesions included a cavernous angioma and an epidermoid tumor in the cavernous sinus. Three lesions were confined to the Meckel's cave including a capillary hemangioma [START_REF] Montibeller | Capillary Hemangioma of the Meckel Cave in an Adolescent. Case Illustration[END_REF] , schwannoma and a tumor of unconfirmed origin (histologically most consistent with schwannoma) (Table 1). There were 4 women and 1 man, mean age at surgery was 42,4 years (16-71 years).

Patients and Methods

Our

Imaging Studies

Preoperative CT and MRI scans were obtained in all patients with and without intravenous administration of a contrast agent.

Surgery

Surgeries were planned and assisted by advanced Computer-assisted surgical techniques with image fusion of preoperative CT and MRI (Treon, Medtronic Stealth Station). Registration for the neuronavigation system was achieved via surface matching. Surgery was performed in general anaesthesia using the operating microscope and microsurgical instrumentation in all cases. The lesion was approached by a

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Nakamura and Krauss 5 standard pterional craniotomy. The surgical corridor to the cavernous sinus or Meckel's cave was determined by the location of the lesion and its extension along the dural surface. Once the lesion was identified with the help of image guidance, entry into the cavernous sinus was performed over the lesion starting with superficial incision of the meningeal layer of the cavernous sinus dura directing the incision in parallel to the extended intracavernous course of the entering cranial nerves. Dural incision was never performed perpendicular to the presumed course of the nerves. Incision of the dura over the Meckel's cave was performed in an analogous fashion according to the trigeminal nerve. These measures enabled us to enter the cavernous sinus or Meckel's cave without damage to the nerves at risk and cautious dissection was possible in small lesions.

In general, the set up and application of neuronavigation during the surgical procedure required 30-45 minutes additionally per case.

Results

Clinical features

The presenting clinical symptoms depended on the anatomical location of the lesions. Double vision was claimed in both patients with a cavernous sinus lesion (Figures 1 A andB) and symptoms of trigeminal affection were reported in all three patients harbouring a lesion in the Meckel's cave.

The clinical symptoms and signs were summarized in Table 1.

Neuronavigational guidance

The accuracy of neuronavigation was verified preoperatively and several times intraoperatively comparing landmarks such as the pterion, anterior clinoid process and other fixed structures. There were no problems with shift of anatomical structures at the skull base during the course of surgery.

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Nakamura and Krauss 6

Margins of the lesions were defined by using neuronavigation in the coronal and axial plane on gadolinium enhanced MRI scans (Figures 1 A andB).

Neuronavigational guidance revealed to be useful in defining the anterior and posterior margins of the lesion. Particularly, it was instrumental in defining the depth of each lesion into the cavernous sinus or Meckel's cave. There was no interacting shift of assumed borders of the lesion both in the cavernous sinus and Meckel's cave.

Radicality of tumor removal

Total tumor removal was achieved in 3 out of 5 patients. In one patient with an interdural epidermoid tumor of the cavernous sinus, the medial capsule of the tumor was left in situ because of severe adherence to the oculomotor nerve. In one patient with a tumor of the Meckel's cave (patient no. 5), a subtotal resection was performed because of significant tumor adherence to the trigeminal nerve (Table 1).

Clinical outcome

In three patients, preoperative cranial nerve deficits improved immediately after surgery. In one patient, facial numbness increased postoperatively and in another patient there was no improvement of preoperative abducens nerve palsy after surgery.

No new postoperative deficits of cranial nerves occurred and there was no procedure related morbidity or mortality.

The mean follow-up time was 13.4 months (7-20 months). A recurrence of the lesion was not found during this follow up period.

Discussion

Treatment strategies for lesions of the cavernous sinus and Meckel's cave

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Nakamura and Krauss 7

Treatment strategies for neoplastic and vascular lesions of the cavernous sinus and Meckel's cave have undergone considerable changes since the introduction of cavernous sinus surgery decades ago by Bowder and Parkinson [START_REF] Parkinson | A Surgical Approach to the Cavernous Portion of the Carotid Artery. Anatomical Studies and Case Report[END_REF] . Detailed microanatomical studies of the cavernous sinus and Meckel's cave have initially contributed to the development of various surgical approaches to this critical region [START_REF] Sepehrnia | Management of Intracavernous Tumours: an 11-Year Experience[END_REF][START_REF] Dolenc | Surgery of Vascular Lesions of the Cavernous Sinus[END_REF][START_REF] Sekhar | Surgical Management of Lesions Within the Cavernous Sinus[END_REF][START_REF] Parkinson | A Surgical Approach to the Cavernous Portion of the Carotid Artery. Anatomical Studies and Case Report[END_REF][START_REF] Hakuba | A Combined Orbitozygomatic Infratemporal Epidural and Subdural Approach for Lesions Involving the Entire Cavernous Sinus[END_REF][START_REF] Inoue | Surgical Approaches to the Cavernous Sinus: a Microsurgical Study[END_REF] . Intra-or extradural approaches usually via a frontotemporal or pterional craniotomy with different amount of additional osteotomy (including the orbit or zygoma) were described [START_REF] Parkinson | A Surgical Approach to the Cavernous Portion of the Carotid Artery. Anatomical Studies and Case Report[END_REF][START_REF] Hakuba | A Combined Orbitozygomatic Infratemporal Epidural and Subdural Approach for Lesions Involving the Entire Cavernous Sinus[END_REF][START_REF] Inoue | Surgical Approaches to the Cavernous Sinus: a Microsurgical Study[END_REF] in order to attempt exposure of cranial nerves and the carotid artery within the cavernous sinus. In neoplastic lesions, like meningiomas of the cavernous sinus, the main aim to enter the cavernous sinus was to achieve radical tumor removal and lower the risk of early tumor recurrence. Some authors favoured aggressive surgical removal [START_REF] Demonte | Outcome of Aggressive Removal of Cavernous Sinus Meningiomas[END_REF][START_REF] George | Cavernous Sinus Exenteration for Invasive Cranial Base Tumors[END_REF] even with intracavernous neural and vascular reconstruction [START_REF] Sekhar | Anatomical Study of the Cavernous Sinus Emphasizing Operative Approaches and Related Vascular and Neural Reconstruction[END_REF] , while others have shown that there is no oncological benefit in performing radical surgery within the cavernous sinus [START_REF] Sindou | Long-Term Follow-Up of Meningiomas of the Cavernous Sinus After Surgical Treatment Alone[END_REF] . Instead, radical removal of big tumors in the cavernous sinus has increasingly shown to be associated with a very high risk of injury to the carotid artery and almost always lead to cranial nerve deficits with inacceptable impairment of life quality after surgery [START_REF] Sindou | Long-Term Follow-Up of Meningiomas of the Cavernous Sinus After Surgical Treatment Alone[END_REF] . Reports have consecutively emphasized that the surgical exploration of the cavernous sinus itself is often associated with cranial nerve deficits or excessive bleeding, even when meticulous microsurgical dissection within the cavernous sinus was performed. Although techniques to approach the cavernous sinus have been extensively described [START_REF] Yasuda | Microsurgical Anatomy and Approaches to the Cavernous Sinus[END_REF][START_REF] Dolenc | Surgery of Vascular Lesions of the Cavernous Sinus[END_REF][START_REF] Umansky | The Lateral Wall of the Cavernous Sinus. With Special Reference to the Nerves Related to It[END_REF] , the visualization of intracavernous cranial nerves and the carotid artery requires elaborate extradural bone removal and hazardous dissection between the two dural layers of the cavernous sinus.

Therefore, there are still controversial opinions concerning the optimal treatment options for tumors like meningiomas, which lead to extensive infiltration of the cavernous sinus. Some authors prefer a more conservative approach with

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Nakamura and Krauss 8 combination of limited extracavernous tumor resection and adjuvant radiosurgery [START_REF] Froelich | Large Sphenoid Wing Meningiomas Involving the Cavernous Sinus: Conservative Surgical Strategies for Better Functional Outcomes[END_REF][START_REF] Maruyama | Proposed Treatment Strategy for Cavernous Sinus Meningiomas: a Prospective Study[END_REF][START_REF] Pamir | Changing Treatment Strategy of Cavernous Sinus Meningiomas: Experience of a Single Institution[END_REF] or radiosurgery alone [START_REF] Iwai | Gamma Knife Radiosurgery for the Treatment of Cavernous Sinus Meningiomas[END_REF][START_REF] Kuo | Gamma Knife Radiosurgery for Benign Cavernous Sinus Tumors: Quantitative Analysis of Treatment Outcomes[END_REF][START_REF] Roche | Gamma Knife Radiosurgery in the Management of Cavernous Sinus Meningiomas[END_REF] .

Surgery of small vascular or nonmeningeal neoplastic lesions

Surgery for nonmeningeal tumors of the cavernous sinus are considered safer than surgery for meningiomas and total tumor removal may often be achieved [START_REF] Yasuda | Microsurgical Anatomy and Approaches to the Cavernous Sinus[END_REF] . Due to the density of important neurovascular structures within the cavernous sinus and Meckel's cave, small vascular or other nonmeningeal neoplastic lesions can lead to early compression syndromes of cranial nerves. Even when dealing with small nonmeningeal lesions of the cavernous sinus or Meckel's cave, the mainstay of cavernous sinus surgery was to visualize most of the intracavernous cranial nerves along with the intracavernous carotid artery in an early step of surgery to gain initial control over these critical structures. It was often recommended that proximal and distal control of flow through the internal carotid artery should be achieved before proceeding to the cavernous sinus [START_REF] Yasuda | Microsurgical Anatomy and Approaches to the Cavernous Sinus[END_REF] . With this approach, the surgical preparation until entrance into the cavernous sinus would require time consuming bone work (for example drilling of the anterior clinoid or exposure of the carotid artery along its petrous segment) and the surgical exposure of these structures itself may present an additional risk of morbidity.

Image-guidance in surgery of the cavernous sinus or Meckel's cave

In limited and small symptomatic lesions of the cavernous sinus or Meckel's cave, a direct intradural exposure of the lesion through the dural walls of the cavernous sinus or Meckel's cave can be feasible without superfluous anatomical dissection.

However, for this purpose, a well directed approach to the lesion with opening of the dural layer precisely over the target needs to be accomplished. Small lesions lying within the cavernous sinus or Meckel's cave may not always be visible through inward protrusion or bulging into the parasellar cisterns. In those cases,

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Nakamura and Krauss 9 the use of neuronavigation revealed to be useful to guide the surgeon to the lesion, allowing a targeted opening of the dura over the cavernous sinus or Meckel's cave. In such lesions, which were large enough to be recognized due to bulging of the dura, still the determination of tumor extension, especially to the anterior, posterior and medial direction, was helpful to estimate the grade of resection and particularly the distance to the intracavernous carotid artery. The application of a pointer based neuronavigation system with surface matching of the data revealed to be sufficient for a reliable and safe image guidance at the base of the skull, particularly the cavernous sinus and Meckel's cave. With imageguidance, the site of the lesion may be delineated on the surface of the cavernous sinus wall or Meckel's cave, however, the method of entrance into the cavernous sinus will be crucial to avoid any nerve injury during exposure of the lesion. It is not possible to identify the nerves directly using the image-guidance pointer, but the operative measures described previously, enabled us to enter the cavernous sinus or Meckel's cave without damage to the nerves at risk. The overall outcome of cranial nerve function after surgery was excellent and there was no new cranial nerve deficit related to the surgical opening of the cavernous sinus or Meckel's cave. However, larger numbers of patients, who undergo image-guided resection of such lesions, need to be further investigated and the results compared with cases, which underwent surgery without the application of image-guidance.

Image-guided skull base surgery

Intracranial neuronavigation and image guided surgery is already well established in neurosurgery and in ENT surgery, while there is still relatively little experience in skull base surgery. Although rarely used, image guided surgery for skull base lesions appears to be an ideal application as no or only minimal intraoperative shift of relevant structures is to be expected compared to its application to surgery of intracerebral or other lesions. The application of image guidance and its

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Nakamura and Krauss 10 advantages for a targeted resection of lesions in the cavernous sinus or Meckel's cave have not been reported so far. In our series, selected small lesions of the cavernous sinus and Meckel's cave could be resected with image guidance without any new cranial nerve deficit, which reportedly is the most common morbidity after cavernous sinus surgery.

In our experience, the use of neuronavigation may help to achieve a targeted exposure of selected small lesions in the cavernous sinus and Meckel's cave, obviating the need for extensive surgical dissection of this densely packed and critical anatomical region. Although image guidance at the cavernous sinus or Meckel's cave seems to be precise enough to accurately guide the surgeon to the lesion, it cannot replace detailed microanatomical knowledge of this complex region, which every skull base surgeon needs to acquire beforehand.

Conclusion

The microsurgical resection of small lesions of the cavernous sinus and Meckel's cave can be enhanced by image-guidance allowing targeted exposure of the lesion with safe removal under preservation of important neurovascular structures within this highly sophisticated region.

  surgical series included 5 patients, who underwent neuronavigation guided microsurgical resection of a small neoplastic or vascular lesion in the cavernous sinus or Meckel's cave between 2007 and 2008 at the Department of Neurosurgery, Hannover Medical University, Germany.

Figure legends Figure 1 :

 legends1 Figure legends

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MD. PhD M U Schuhmann
email: martin.schuhmann@med.uni-tuebingen.de

H D Zucht

R Nassimi

G Heine

C G Schneekloth

H J Stuerenburg

H Selle

Peptide Screening of Cerebrospinal Fluid In Patients With Glioblastoma Multiforme

Keywords: glioblastoma, neurosurgery, cerebrospinal fluid, peptidomics, biomarker

Aims: To apply modern mass spectrometry based technology to identify possible CSF peptide markers of glioblastoma multiforme (GBM).

Methods

: Mass spectrometry based peptidomics ® technology enables a systematic and comprehensive screening of cerebrospinal fluid (CSF) with regard to its peptide composition. Differential Peptide Display ® (DPD) allows the identification of single marker peptides for a target disease. Using both, we analysed CSF samples of 11 patients harbouring a glioblastoma multiforme in comparison to 13 normal controls.

Results: Four CSF peptides which significantly distinguished GBM from controls in all applied statistic tests could be identified out of more than 2000 detected CSF peptides.

They were specific C-terminal fragments of alpha-1-antichymotrypsin, osteopontin, and transthyretin as well as a N-terminal residue of albumin. All molecules are constituents of normal CSF, but none has previously been reported to be significantly elevated in CSF of GBM patients.

Conclusion:

The study showed that peptidomics technology is able to identify possible biomarkers of neoplastic CNS disease. It remains to be determined if the identified elevated CSF peptides are specific for GBM. With regard to GBM, however, the more important role of CSF peptide biomarkers than aiding initial diagnosis might be early recognition of disease recurrence or monitoring of efficacy of adjuvant therapy protocols.

Introduction

The peptide composition of cerebrospinal fluid (CSF) has been examined in recent years following the development of modern mass spectrometry techniques combined with suitable software solutions for multiplex analysis. Our approach in the search for marker peptides and small proteins in body fluids was termed peptidomics, defined as "the technology for the comprehensive qualitative and quantitative description of peptides in biological samples" [START_REF] Schulz-Knappe | Peptidomics: the comprehensive analysis of peptides in complex biological mixtures[END_REF]. The adaptation of this peptide display technology to CSF samples enables to analyze and visualize more than 6,000 peptide signals in a sample of human CSF [START_REF] Lamerz | Correlation-associated peptide networks of human cerebrospinal fluid[END_REF]. The peptidomics approach and its performance characteristics have been published before [START_REF] Tammen | Prerequisites for Peptidomic Analysis of Blood Samples: II. Analysis of Human Plasma after Oral Glucose Challenge -A Proof of Concept[END_REF][START_REF] Tammen | Prerequisites for Peptidomic Analysis of Blood Samples: I. Evaluation of Blood Specimen Qualities and Determination of Technical Performance Characteristics[END_REF].

Currently there is no CSF biomarker in clinical use in malignant brain tumours, neither for diagnostic nor for follow-up purposes (like detection of recurrence or response to adjuvant therapy). The usual approach searching for CSF tumour markers is to use a technique like ELISA that is specifically directed against a particular molecule known to be involved in tumour metabolism. With regard to broader screening techniques, the proteomics approach combining 2D gel electrophoresis with mass spectrometry analysis has been applied to CSF of patients with different types of primary brain tumours [START_REF] Zheng | Identification of tumor-related proteins by proteomic analysis of cerebrospinal fluid from patients with primary brain tumors[END_REF].

The aim of this pilot study was to explore the potential of peptidomics technologies for the identification of possible peptide biomarkers of malignant gliomas in CSF. An extended knowledge of changes in CSF peptide composition could be used for the identification not only of diagnostic markers, but also of possible target pathways for therapy. Up to our knowledge this study represents the first attempt of a systematic peptide screening in CSF of patients suffering from glioblastoma multiforme.

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Patients and Methods

Patient enrolment, sample collection and preparation

All CSF samples were obtained prospectively as part of a protocol for the development and evaluation of peptidomics technologies in CSF, approved by the Human Investigation Committee of Hanover Medical School (No. 1952/98). CSF was obtained for peptide analysis from tumour patients without contraindication for lumbar puncture and who gave written consent. Control lumbar CSF was acquired during the same period from patients with spinal nerve root compression syndrome undergoing a diagnostic myelography. For this pilot study we selected patients only of there were i) normal CSF cell counts, ii) normal serum lab results and iii) sterile CSF culture. According to these criteria 11 patients with GBM and 13 patients from the control group entered the study. In tumour patients the pre-operative magnetic resonance imaging (MRI) information was used to estimate the lesion volume according to the formula "volume = height x length x width x π /6". Furthermore tumours were described as solid, partial cystic or cystic. Uptake of contrast agent, amount of surrounding oedema and brain atrophy was graded as none, moderate, or severe.

We included CSF samples from further nine patients with various non-neoplastic conditions, resulting in different degrees of blood brain barrier disruption, to investigate the influence of blood-brain-barrier disturbance on the CSF peptide composition. The data from these samples were not used for biomarker detection but for correlating massspectrometric signal intensity with the function of the blood-brain-barrier.

After lumbar puncture 5 ml of CSF were immediately mixed with 0.1 ml 0.5M EDTA, centrifuged at +4ºC with 2000 x g for 10 minutes and the supernatant stored at -80 ºC. All samples were processed as described below.

Chromatographic separation with reversed phase HPLC

The samples were thawed gently, 550 μL CSF were diluted 1:3.75 with water and pH was

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adjusted to 2-3. Using the Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) samples were loaded onto reversed phase columns (250 x 4 mm column, source RPC, Amersham, Freiburg, Germany) and eluted with an acetonitrile gradient (4 to 80 % in 48 min) in 0.05 % trifluoroacetic acid at a flow rate of 500 µL/min. From each sample 96 fractions (250 µL each) were collected. The retention times of major peptide peaks were used to control the reproducibility of the method. The collected fractions were lyophilized and stored at -80 ºC before further analysis.

Mass-spectrometric analysis

Peptide displays

All spectra were transferred to a database. An in-house developed software suite was used for data pre-processing and visualization, data mining and interpretation of peptide displays. This comprised baseline correction (RAZOR Library 4.0, Spectrum Square Associates, Ithaca, NY, USA), normalization of the mass spectra to a constant integral value, transformation of signal heights into color intensity for visualization and binning of spectra to 1 Dalton channels.

All 96 processed mass spectra were visualized in a 2D-gel like format, an averaged peptide display: Each peak is depicted as a bar with its colour intensity corresponding to the signal

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intensity of the related mass peak. The data matrix of an individual peptide display consisted of 16 million data points. The x-, y-and z-axes represent the mass to charge ratios (m/z) within a range of 850 to 15000, the chromatographic fraction and the mass spectrometric signal intensity, respectively (Fig. 1). For all samples, an identical set of signal coordinates (fraction number and m/z value) was extracted and used for statistical analysis of the signal intensities. The underlying peptides comprise the peptidome of CSF, i.e. all peptides extracted and measured from CSF.

Differential peptide display ® and statistical analysis

The individual datasets were combined to two master displays representing the peptidomes of GBM patients and of normal controls: Within each of the two the individual data sets were averaged for each chromatographic fraction visualized as a peptide master display. These averaged spectra were used as template for the definition of peak coordinates, which were distinct from the baseline noise. More than 6000 peak positions were identified and the signal intensities of each of these peaks were extracted into a database and entered in spreadsheets for explorative statistical analysis.

To detect possible marker candidates the two peptidomes were compared using the differential peptide display approach [START_REF] Schrader | Peptidomics technologies for human body fluids[END_REF]. Subtraction maps were calculated for visual inspection and plausibility control (see Figure 1). Each measured peptide signal in the data matrix was tested for its discriminatory power by computing absolute and relative difference of the signal intensity, the Mann-Whitney-U-Test, and receiver operating characteristic (ROC) integrals. An absolute and relative difference of > 2500 units and > 40 %, respectively, and a ROC integral > 80% were required to reject the null hypothesis of equivalence. Peptide signals with the highest discriminatory power were subjected to identification of the amino acid sequence.

Patient data including age, duration of symptoms and the morphological MRI data were correlated to the mass-spectrometric signal intensity (SI) of the peptides in CSF (correlating with a peptide's concentration) using Spearman's rank correlation coefficient.

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The latter was also used for a subset of 9 samples correlating the signal intensity (SI) to the CSF/serum albumin quotient (Q Alb ) and to the CSF/serum total protein quotient (Q Prot ).

Peptide identification by nanoESI-qTOF-MS/MS

Peptides were identified by nanoESI-qTOF-MS/MS (QSTAR pulsar, Sciex, Toronto, Canada) with subsequent protein database searches. Data processing included charge-state deconvolution and deisotoping. The resulting spectra were saved in MASCOT (Matrix Science, London, UK) generic file format and submitted to the MASCOT search engine. Cascading searches including several posttranslational modifications in Swiss-Prot (Version 39.6, www.expasy.ch) and MSDB (Version 030212 or higher, EBI, Cambridge, UK) were conducted. Results of peptide identification were accepted in automated database searches if the probability based MOWSE score for individual peptides was below a significance threshold of p = 0.05 indicating an absolute probability of less than 5 % for a random result from the database search.

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Results

Enrolled patients

4 female and 7 male patients with a single supratentorial GBM manifestation and a tumour volume ranging from 3.6 cm 3 to 96.4 cm 3 were enrolled. Duration of symptoms ranged from 2-26 weeks. In the control group 4 female and 9 male patients with mostly spinal canal stenosis were enrolled. There was no difference in body mass index between the GBM and the control group, however GBM patients were significantly older than control patients (median 68 y, range 43-79 y vs. median 50 y, range 21-64 y, p<0.02).

Peptide identification

Over 6000 peptide signals corresponding to an estimated number of at least 2000 different peptides were distinguished in the peptide master displays of the two peptidomes. Figure 1A shows the difference in signal intensities (SI) of the two groups. In statistical analysis eight peptide signals surpassed the indicated thresholds of significance. Five of eight candidates were successfully identified. They were specific C-terminal fragments of the proteins alpha-1-antichymotrypsin (AACT), transthyretin (TTHY), osteopontin (OPN) and an N-terminal fragment of albumin (ALB), with the abbreviations referring to these specific fragments within this article. OPN was detected twice, as single as well as double charged ion. AACT, TTHY and OPN are short C-terminal peptide residuals with 39, 28, and 65 amino acids, respectively. ALB is a 24-residue N-terminal fragment of albumin. Three candidate peptides were not identified. In two cases a glycosylation of the peptide backbone prevented the acquisition of a suitable MS/MS spectrum. A third candidate occurred with a low concentration only and no MS/MS spectrum could be acquired.

Peptide characteristics

Figure 2 shows the ROC chart for the top ranked AACT. When peptide SI in CSF was correlated to tumour volume, a negative correlation with increasing tumour volume was found for AACT (r=-0.65/p=0.03). We did not observe correlations between any other

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radiological feature of the tumours and SI of any of the peptides. The SI of all peptides did neither depend on direct contact of the tumour to the CSF space nor correlate to age of patients or duration of symptoms.

Correlation to blood-brain-barrier function

In five female and four male patients with non-neoplastic blood-brain-barrier disruption the SI of ALB in CSF, which corresponds to the ALB peptide concentration in the CSF sample, was correlated to the Q Alb . There was a significant correlation between both parameters (r 2 = 0.503, p<0.05). For GBM patients Q Alb was not available, therefore the Q Prot was used as a surrogate parameter for the barrier status. In GBM patients the SI of ALB correlated to Q Prot even better as it did in the patients with non-neoplastic barrier disruption (r 2 = 0.824, p<0.01 ). No correlation to Q Prot was found for TTHY, AACT and OPN.

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Discussion

We identified four peptides cleaved from different protein precursors that were elevated in CSF of GBM patients as compared to normal controls, thus carrying a potential as possible biomarkers in GBM diagnosis and follow-up. None of these peptides or their precursors has been reported in CSF of GBM patients so far. The presented unique approach of comparative CSF peptide profiling represents a new screening tool for analysis and visualisation of CSF peptides in an individual sample. The sensitivity of this approach was found to be as low as the mid picomolar level [START_REF] Tammen | Prerequisites for Peptidomic Analysis of Blood Samples: II. Analysis of Human Plasma after Oral Glucose Challenge -A Proof of Concept[END_REF]. A comparable comprehensive profiling approach in CSF so far could only be employed on the protein level with gelelectrophoresis techniques [START_REF] Sickmann | Towards a high resolution separation of human cerebrospinal fluid[END_REF] or by the combination of ultrafiltration, liquid chromatography and mass spectrometry [START_REF] Noben | Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry[END_REF].

Difference to Control group

We chose CSF of normal control patients without brain disease as a control. Only in a second step, which is beyond the scope of this paper, aspects of differential diagnosis versus other brain tumour entities can be considered. There was a distinct age difference between the tumour patients (median 68 years) and the control group (median 50 years). Therefore the higher age could have contributed to an increase in concentration of the 4 investigated peptides. The CSF proteome was analyzed with specific reference to the age of the patients/participants addressing the problem of ageing in interpretation of CSF proteomic results [START_REF] Zhang | Quantitative proteomic analysis of agerelated changes in human cerebrospinal fluid[END_REF]. The authors compared older individuals with an age between 66-85 years and younger subjects aged 22-36 years. In 30 out of more than 300 identified proteins they found a greater than 20 % change in the concentration. The proteins relevant to our study (albumin, transthyretin, antichymotrypsin and osteopontin) were identified as unchanged by age. Furthermore, we correlated the signal intensities of all 4 peptides from our experiments to the age of the corresponding subjects within each group. No significant correlation was found. Thus, an age related effect on our findings seems unlikely.

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Influence of blood brain barrier function

Only peptides with statistical significance in all performed statistical procedures were considered. Eight of approximately 2,000 peptides fulfilled these criteria. Therefore, a first observation is that of high stability of the CSF peptide composition in the presence of a malignant and fast growing brain tumour. The tumour induced in 10 out of 11 patients a marked surrounding brain oedema and a strong contrast enhancement following gadolinium application in magnetic resonance imaging. Both, brain oedema and contrast uptake are directly related to a defective barrier function, which leads to an increased transcellular and paracellular passage of molecules [START_REF] Papadopoulos | Emerging molecular mechanisms of brain tumour oedema[END_REF][START_REF] Roberts | ARTICLE IN PRESS microvascular permeability in human brain tumors achieved using dynamic contrastenhanced MR imaging: correlation with histologic grade[END_REF][START_REF] Schneider | Glioblastoma cells release factors that disrupt blood-brain barrier features[END_REF]. This increase in barrier permeability in and around a GBM, however, does not seem to lead to a significant alteration of the usual passage mechanisms for blood peptides, which occurs under normal conditions mainly by non-saturable trans-membrane diffusion or selective saturable transport systems [START_REF] Banks | Passage of peptides across the blood-brain barrier: pathophysiological perspectives[END_REF][START_REF] Kastin | Peptides crossing the blood-brain barrier: some unusual observations[END_REF].

Otherwise, a more extensive change of the overall peptide composition in CSF of GBM patients should have been found.

To investigate a possible influence of the intra-or peritumoral BBB dysfunction on the results we correlated the concentration of the peptides to the extent of BBB dysfunction.

In GBM, where tumour cells take up albumin in high quantities [START_REF] Kremer | Laser-induced fluorescence detection of malignant gliomas using fluorescein-labeled serum albumin: experimental and preliminary clinical results[END_REF][START_REF] Ichioka | Enhanced detection of malignant glioma xenograft by fluorescein-human serum albumin conjugate[END_REF], the increased presence of ALB in CSF could either be due to a disturbance of the BBB or due to a release of ALB or the complete albumin from GBM cells to the extracellular compartment. The first hypothesis is supported by our data. The signal intensity of ALB in tumour patients was correlated to Q Prot , as this was shown for the Q Alb in the group of patients with a nonneoplastic blood-brain-barrier disruption (Figure 3). The increase in CSF concentrations of TTHY, AACT and OPN seems to be independent of barrier function, since no correlation to Q Prot was found.

Role of osteopontin

Osteopontin is a phosphorylated non-collageneous matrix protein, found in all body fluids and synthesized in all epithelial tissues. Osteopontin is over-expressed by almost all types

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of human cancer as well as by cancer-infiltrating macrophages [START_REF] Rittling | Osteopontine function in pathology: Lessons from osteopontine-deficient mice[END_REF][START_REF] Coppola | Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies[END_REF]. In human malignant glioma cells and in the tumour microvasculature osteopontin gene expression and protein levels are significantly elevated [START_REF] Markert | Differential gene expression profiling in human brain tumors[END_REF][START_REF] Takano | Tissue factor, osteopontin, alphavbeta3 integrin expression in microvasculature of gliomas associated with vascular endothelial growth factor expression[END_REF]. Osteopontin functions in cell attachment and promotes remodelling of the extracellular matrix [START_REF] Ding | Promotion of malignant astrocytoma cell migration by osteopontin expressed in the normal brain: differences in integrin signaling during cell adhesion to osteopontin versus vitronectin[END_REF][START_REF] Wang | Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA-protein interactions[END_REF]. Therefore the increase of OPN in CSF of GBM patients can be best explained by a tumour-induced upregulation of osteopontin. Neither OPN, which represents a cleavage product of a Lys-Arg cleavage site, nor osteopontin so far have been used as a tumour marker in CSF of GBM patients. OPN might not be specific for GBM, since osteopontin was reported to be significantly higher in CSF of patients with atypical teratoid/rhabdoid tumours as compared to medulloblastoma cases [START_REF] Kao | Elevation of plasma and cerebrospinal fluid osteopontin levels in patients with atypical teratoid/rhabdoid tumor[END_REF].

Role of alpha-1-antichymotrypsin

Alpha-1-antichymotrypsin is part of the acute-phase response of the host in the acute phase of inflammation, infection, malignancy and trauma. In the brain, astrocytes are a main source of alpha-1-antichymotrypsin [START_REF] Abraham | Alpha 1-antichymotrypsin is associated solely with amyloid deposits containing the beta-protein. Amyloid and cell localization of alpha 1-antichymotrypsin[END_REF]. Brain tumours, especially high-grade gliomas, can stain positively in immunohistochemistry for alpha-1-antichymotrypsin, however, this is not limited to GBM [START_REF] Ng | Immunostaining for alpha 1-antichymotrypsin and alpha 1-antitrypsin in gliomas[END_REF][START_REF] Kroh | Alpha-1-antichymotrypsin in human glioblastomamultiforme cells and its relation to GFAP immunostaining[END_REF]. There are no reports, which show alpha-1antichymotrypsin, or a fragment of it, elevated in CSF in GBM patients. Proteases and their inhibitors are synthesized in cancer-affected tissues since they are important in tumour invasion. A correlation between the level of protease inhibitors and the prognosis was described for plasminogen activator-1 and -2 in breast cancer patients [START_REF] Foekens | Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer[END_REF][START_REF] Umeda | Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues[END_REF]. Thus, an increased protease inhibitor activity in and around a malignant glioma can be reflected by an increased level of relevant cleavage products in CSF, like the identified AACT.

Role of transthyretin

Transthyretin has a function in the transport of both thyroxine and retinol in plasma and CSF. The disproportionately high concentration of soluble monomeric transthyretin (14 kDa) in human ventricular CSF is related to the fact, that it is synthesized in high M A N U S C R I P T

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quantities within the choroid plexus cells [START_REF] Herbert | Transthyretin: a choroid plexus-specific transport protein in human brain. The 1986 S. Weir Mitchell award[END_REF] but not within the brain parenchyma. In an analysis of different brain tumours, transthyretin mRNA and protein were not found in 23 anaplastic astrocytomas and glioblastomas, but were exclusively limited to choroid plexus papillomas [START_REF] Lignelid | Cystatin C and transthyretin expression in normal and neoplastic tissues of the human brain and pituitary[END_REF]. A dependency of TTHY to brain barrier dysfunction could not be found by correlating its concentration to Q Prot as discussed above. We suspect, that a tumourinduced increase in protease activity at constant transthyretin levels is the most likely cause of the observed increase in the transthyretin fragment (TTHY).

Conclusion

This pilot study investigated the application of peptidomics analysis in the unbiased global screening for tumour-related peptides in CSF of GBM patients. The identified up-regulated peptides have not yet been reported in previous CSF studies of glioblastoma patients. They can either be attributed to tumour cell metabolism, induced changes in protease activity, induced functional changes of brain barriers, or an altered elimination. It seems possible that none of the peptides is specific for GBM. This study, however, was neither designed to validate these peptides as potential tumour markers for malignant glioma in the differential diagnosis to metastases or primary CNS lymphomas, nor investigated their utility in monitoring the response to surgery, radiotherapy or chemotherapy. The reason is that the peptidomics technique is not primarily suitable for high throughput screening in clinical routine but is designed for marker detection. The value of the peptides identified with the peptidomics approach herein needs to be determined in a trial using cheaper assays like ELISA, which are directed specifically against the identified peptides. B) The average MALDI-TOF spectra of the fragment "AACT" from all datasets of the GBM group is plotted in red versus the corresponding data of control group in blue. The y-axis shows the SI of the peptide, which corresponds the peptides concentration in CSF. The xaxis displays the range of the relative molecular masses. Obviously, the SI of the AACT fragment is higher in the GBM group. The bottom arrow in panel A labels the peptide AACT.

Figure 2: Receiver Operating Characteristic (ROC) chart of AACT, which determines the discriminatory power (ROC area) of AACT using the ROC method, plotting sensitivity versus specificity for any given AACT value. This peptide has a high discriminatory power, since there is only a minimum loss of specificity with increasing sensitivity, resulting in a ROC integral of 92%. A sensitivity of about 82 % is given at a specificity of 100 %, with the latter one depicted as 100 -specificity = 0 %

  All 96 chromatographic fractions of a sample were analyzed with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) generating 96 mass spectra per sample. Each individual HPLC fraction was resuspended in matrix solution consisting of α-cyano-4-hydroxycinnamic acid and L-fucose (co-matrix) in 0.1 % acetonitrile/ trifluoroacetic acid (1:1 v/v). A 15 µL equivalent of CSF in 0.3 µL matrix was transferred onto a MALDI target. Sample ionization was performed by multi-position acquisition of 6 x 20 laser shots/spot over a representative area of the sample spot. The ions were analyzed in a time-of-flight mass spectrometer (Voyager-DE STR, Applied Biosystems, Framingham, MA, USA) in linear mode.

Figure 1 :

 1 Figure 1: A) The "differential peptide display" is shown, depicting the differences between the averaged (mean) signal intensities (SI) of all mass-spectrometric intensities obtained of all GBM samples in comparison with the corresponding data of the control patients. Red spots indicate peptide signals with a higher SI in the CSF of GBM patients, whereas blue spots indicate signals with a lower SI. Peptide signals, which had a comparable SI in the GBM and the control group, are depicted in grey. Arrows indicate peptide signals, which were identified by statistical testing as signals with significant discriminatory power. Not marked is the signal corresponding to the second double charged osteopontin fragment.

Acknowledgements

The authors would like to express their gratitude to the entire Digilab BioVisioN Team involved in the CSF project for their continuous support, especially to Thomas Möring and Jens Lamerz. Matthias Zumkeller, MD has contributed to the set-up of the study in the clinical environment. Differential Peptide Display and peptidomics are registered trademarks of Digilab BioVisioN GmbH.

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M.D. Florian H Ebner
email: florianebner@virgilio.it

cand.med J S Marquardt

M.D B Hirt

M.D G C Feigl

M.D M Tatagiba

M.D M U Schuhmann

Broadening Horizons of Neuroendoscopy with a Variable-View Rigid Endoscope: An Anatomical Study

Aim: To test the versatility and usefulness of a prototype rigid endoscope with a continuously variable-view-angle tip (Endochamaeleon, Karl Storz, Tuttlingen, Germany) with regard to field of vision and applicability for endoscopic assisted neurosurgery.

Methods:

In five formaldehyde fixed specimens frontolateral and retrosigmoid approaches were prepared on both sides and five target positions of the endoscopes tip were defined. A rigid 4 mm endoscope, which offers in one plane a viewing range of approximately -10º to +120º by turning a proximal knob coupled to fine distal optomechanics was compared to 0º, 45º, and 70º rigid endoscopes. The visualisable neuroanatomical structures were assessed for each position, endoscope angle, and plane of view as well as the over-all visibility of neuroanatomical structures and the time factor.

Results: 1905 recorded images of 1800 different views were analyzed. The EC offers a variable angle of view in one plane without need to change the endoscope position. This feature is well suited for inspection of functionally delicate areas at the skull base. The maximum number of visible structures for each position was only seen with the EC. Endoscopic exploration was significantly less time consuming with the EC than with the other endoscopes.

Conclusion:

The EC provided superior usability and visualization potential compared to standard rigid endoscopes with fixed angulation. It combines the ergonomic and safety advantage of not having to insert endoscopes with different angles with the opportunity to "scan" the surgical field with a variable angle of 0-120º within one plane of view.

Introduction:

The roots of endoscopy are originally in urology and internal medicine. Thus, for many decades endoscopes and instruments have significantly been influenced by these disciplines. The real breakthrough of endoscopy in neurosurgery occurred in the 1980s [START_REF] Levy | Robotic virtual endoscopy: development of a multidirectional rigid endoscope[END_REF]. Since then, the specific problems of illumination, viewing-and working-angles, and visualization have been solved stepwise. Continuous improvement of lens systems has enhanced the optical quality, and the introduction of flexible endoscopes has enlarged the angle of view. However, in endoscopeassisted neurosurgery of the skull base, flexible endoscopes harbour an unacceptable risk of injury to the delicate neurovascular or nervous structures within the small working spaces of the cisterns. The insertion of angled optics into the surgical field under microscopic vision, which is current practice, is cumbersome and time consuming and does not enable an easy 360º view and orientation within the field of surgery. The ideal solution in terms of optimal visualization, safety, economy of time and ergonomics would be a rigid endoscope with a continuously variable tip, enabling a multidirectional 180º view angle, to cover a 360º horizon.

The aim of this study was to test in the anatomical lab a prototype rigid endoscope with a continuously variable-view-angle tip (Endochamaeleon, Karl Storz, Tuttlingen, Germany) with regard to field of vision and applicability for endoscope-assisted neurosurgery.

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Methods:

Endochamaeleon -Technical description

The Endochamaeleon (EC) is a rigid 4 mm endoscope that is capable of changing its angle of view while remaining stationary and shape-invariant. By turning a proximal knob, which is coupled to fine distal optomechanics, the user can change the endoscope's angle of view without moving the endoscope shaft or the attached camera. This, however, is only possible in one plane, since the tip of the endoscope cannot be rotated around the shaft axis without turning the shaft (and the attached camera) itself. Nevertheless, this provides the operator with a greater viewing mobility: instead of having to insert a different endoscope with a different viewing angle, the operator can actuate the knob (and thus the angle of view) and keep the endoscope in place. The EC provides in one plane a viewing range of approximately -10º to +120º, which effectively means that it combines the capabilities of 0º, 12º, 30º, 45º, 70º, and 90º fixed-angle rigid endoscopes into a single instrument and goes beyond by allowing views "backwards" up to 120º.

Set-up of Anatomical study

In five 4% transfemoral whole body formaldehyde fixed, arterially injected head specimens, frontolateral and retrosigmoid skull base approaches [START_REF] Samii | Surgery of the Skull Base -An Interdisciplinary Approach[END_REF] were performed on both sides in standard technique and size. After meticulous microsurgical dissection of the intradural neurovascular structures, 0º, 45º, and 70º rigid endoscopes (Karl Storz, Tuttlingen, Germany) and the Endochamaeleon (Karl Storz, Tuttlingen, Germany) were inserted. For each approach, five target positions of the endoscope tip were defined (Figures 1 A andB). In preparation of the study, high-resolution computed tomography scans of all specimens with acquisition of dataset for neuronavigation were performed. Thus, the topographic position of the endoscope's tip could be

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defined additionally by neuronavigation (Cbyon, Sunnyvale, USA). Images were taken with the 0º optic in the straight plane and with the 45º and 70º optics in cranial, medial, caudal, and lateral rotation (by 90º each). Digital image documentation of the specific view per tip position, angle of rotation, and used endoscope was carried out with the AIDA-system (Karl Storz, Tuttlingen, Germany). For each image, specimen number, type and side of approach, angle of endoscope optics, endoscope position and viewing direction were recorded. The EC was positioned with 0º straight view at the desired tip position and the viewing angle was continuously changed from 0 to 120º. Images were taken so to cover the whole horizon of 120º. Then the endoscope was rotated by 90º in the next plane of view.

To investigate the supposed time advantage of the Endochamaeleon compared to a set of different fixed angle optics a checklist guided inspection of surgically relevant structures in the supra-and parasellar cistern was performed with both options, and the time necessary to visualize all check-list structures was recorded. Fixed-angle optics with 0º, 45º, and 70º were used in comparison to the EC. The inspection included view of the parasellar area from both the right and the left frontolateral approach in each specimen (10 sides) and included insertion and extraction of the endoscopes, white balance when the optic was changed, exploration of the anatomical check-list structures and pivoting the operation microscope for the controlled insertion of fixedangle endoscopes (45º and 70º optics) compared to the direct insertion of the EC and continuously variable inspection.

Analysis

For efficient analysis of the large amount of recorded images (n=1905) two software tools were developed: A first tool for the systematic naming of the image files, which initially were serially

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numbered, and as second tool an image browser, allowing categorized selection and comparison of up to four images by accessing the previous parameterized files.

The parameterization with the objective of explicit identification of every single image includes information with reference to specimen number, type and side of approach, angle of endoscope optics, endoscope position and viewing direction, encoded as a sequence of numbers.

On the basis of these parameters images can be viewed in the image browser whose user interface contains, besides the control elements for the selection of the image source folder on the right, four image panels, each with the corresponding drop-down menu for the selection of the required parameters.

For minimization of operation effort, the drop-down menus can by synchronized in such a way, that changes in the left upper image panel also affect the other panels, so that analogous images in terms of the synchronized parameters are shown. This concept provided for a convenient analysis and comparison of the graphical material coming from the four different endoscopes.

Hence, according to each specific endoscope tip position, the visualizable neuroanatomical structures were assessed for each endoscope angle and plane of view. Further, the quality of view was evaluated.

In addition, the pictures were used to compare the anatomical preparations with each other regarding the over-all visibility of neuroanatomical structures without consideration of the endoscope model.

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Results:

At least one image in all five positions per surgical approach was taken, in four or five possible directions with each of the four endoscopes in each of the five specimens. So a number of 1800 different views resulted. The variable optic of the EC allowed to take images at different angles from the same target position. The visible neuroanatomic structures of interest are itemized in Table 1 regarding the frontolateral and the retrosigmoid approach. Summarizing the data of all specimens, the maximum number of visible structures for each position was defined as 100% and in relation to that, percentages of visualized structures per endoscope were reported (Table 2A and Table 2B). Table 3 shows a representative extraction of visible structures with the EC in absolute terms. Furthermore, the EC allows to "follow" a structure of interest, e.g. a cranial nerve or vessel during its course through a cistern, within the degree of its viewing angle (Figure 2), while the rigid, not variable endoscopes visualized only a certain section of the structure. However, used in combination, the 0º, 45º, and 70º optics complement one another and cover almost the full range of the structure of interest compared to the EC Exploring the parasellar region and identifying pituitary stalk, the diaphragma sellae, the ipsi-and contralateral medial border of the ICA, the ophalmic artery and the infero-medial border of both optic canals was much less time consuming with the EC (15-30 s; mean 22 ± 9 s) than with the standard, invariant rigid endoscopes (120-150 s; mean 134 ± 5 s). The difference is statistically significant according to the non-parametric Man-Whitney U-test (p<0.0001).

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Discussion:

The Endochamaeleon (EC) offers a variable angle of view in one plane without the need to change the endoscope. This feature is well suited for inspection of functionally delicate areas at the skull base and for example within the third ventricle. The Endochamaeleon is inserted into the operating field with a straight axis of view (0º), which is an important safety feature preventing the collision of the endoscope with neurovascular structures. The optic is fixed at the target point by the mounting device and the surrounding structures may be explored continuously between 0º and 120º in the preselected plane of view without any external movement of the endoscope's tip.

Since the surgeon knows the viewing direction and is able to modify the viewing angle continuously by manual operation of the turning knob coupled to the optomechanics, the sense of orientation is preserved at any time.

Thus, a tailored and controlled visualization of the anatomical structures of interest is possible (Figure 2). The benefits become apparent when the EC is applied e.g. in endoscopic assisted skull base surgery: After microsurgical tumor removal, the operating site may be explored in detail for tumor remnants even in the depth, below and behind delicate neurovascular structures. In this situation the surgeon will benefit from the advanced characteristics of the EC since viewing angles outside the straight microscopic view can be explored in a safe and little time consuming way compared to the current way of inserting different angled rigid endoscopes under microscopic control. This feature may be of particular aid in removing tumours that grow inside the internal auditory canal, like acoustic neuromas and meningiomas, or tumours located ventrally to delicate cranial nervous, like petrous apex meningiomas, where every retraction and new insertion of the endoscope harbours the danger of injuring important structures. Endoscopic extended approaches [START_REF] Ebner | Endoscopeassisted retrosigmoid intradural suprameatal approach to the middle fossa: anatomical and surgical considerations[END_REF] in oncologic skull base surgery become even more feasible with this

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS variable-view rigid device.
It is a limitation of the device, however, that the endoscope shaft still has to be rotated in order to change the plane of view. In addition, the camera, which is attached in a 90º angle, turns around which makes the procedure of rotating the shaft at least inconvenient. With regard to a skull base environment a 180º rotation with a camera attached at 90º is even impossible. The goal of further technical development of the EC endoscope is to achieve multiplane viewing in addition to the variable viewing angle without rotating the entire shaft and the attached camera. Then the endoscope can be fixed in the holding-device and the operating field explored in a range of 360º with the tip position immobile. One of the technical challenges to be solved is the preservation of the image horizon, which is an essential requirement for the surgeon's ability to orientate his instruments in the operation field.

Furthermore, the results of our study clearly show, that the fixed 0º optic gives a panoramic view with advantages at greater distance, whereas the angled optics allow for a better exploration of the deeper located neurovascular structures. As demonstrated by the data in Table 1, the EC combines with its continuously adjustable optic the advantages of both. This characteristic facilitates inspection, delicate dissection and tumour removal in deep-located areas such as the perisellar region or petrous apex. However, individual anatomical conditions still can limit the feasibility to visualize specific structures even with this multivariable endoscope (Table 3).

The next intermediate-term goal in endoscopically assisted skull base surgery will be the step from seeing to working around the corner. To visualize residual tumour creates the need to remove those tumour parts, which are, after exploration, known to exist. If targeted displacement of covering structures, like optic nerve, brain stem or major arteries, to remove tumour remnants

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is not possible, then the angulated view has to be combined with angulated instruments for tumour removal. The requirements to do so are preserved horizontal/ vertical orientation and a frame fixed endoscope in the surgical field that does not hinder the introduction and manipulation of angulated surgical instruments. The EC endoscope is the first important step to broaden the horizons and thus, in the long run, the possibilities of endoscope assisted skull base surgery.

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and interpretation of data; The decision to submit the manuscript for publication was taken exclusively by the authors, which have no financial and personal relationship to Karl Storz and its representatives.

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Figure legend:

Figure 1 A: The intradural target positions for endoscopic exploration in the frontolateral approach are at the level of 1. the craniotomy, 2. the planum sphenoidale, 3. the anterior clinoid process, 4. the sella turcica, 5. the oculomotor nerve.

Figure 1 B: The intradural target positions for endoscopic exploration in the retrosigmoid approach are at the level of 1. the craniotomy, 2. the inner auditory canal, 3. the plane between tentorium and trigeminal nerve, 4. the plane between trigeminal nerve and facial/vestibulocochlear nerve, 5. the plane between facial/vestibulocochlear nerve and caudal cranial nerves. Anterior cerebral artery

• • • • • • • • • • CN III • • • • • • • • • • • • • • • Pituitary stalk • • • • • • • • • • • • Exit zone CN VI • • • • • • • • Meckel's cave • • • • • • • • • • • • • PICA • • • • • • • • • • • • • • visible
Table 3: Representative extraction of visible structures with the EC through the frontolateral approach (light grey) and through the retrosigmoid approach (dark grey). Abbreviations: CN = Cranial nerve; PICA = Posterior inferior cerebellar artery

Figure 2 :

 2 Figure 2: Endoscopic images taken with the EC demonstrating the variable view by focusing on the tip of basilar artery (A), cisternal course of CN III (B) and PCA/ PcomA (C) from a fixed endoscope position (frontolateral approach on the right side, position 5). An anatomic particularity in this specimen is the splitted basilar artery. Abbreviations: BA = Basilar artery; CN III = Cranial nerve III; PCA = Posterior cerebral artery; PcomA = Posterior communicating artery; SCA = Superior cerebellar artery

Table 2 A

 2 : Percentages of visualized structures per endoscope at the frontolateral approach.

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Table 2 B

 2 : Percentages of visualized structures per endoscope at the retrosigmoid approach.

Acknowledgments

The authors are thankful to Mr.H.Schramm, Foto-Repro-Grafik CRONA, Eberhard-Karls-University Tübingen, for preparing the illustrations.

Conclusion:

The EC provided superior usability and visualization potential compared to standard rigid endoscopes with fixed angulation. It combines the ergonomic advantage of not having to retract and insert scopes with different angles with the opportunity to "scan" the surgical field with a variable angle of 0-120º within one plane of view. Currently, the device limits risks, saves operating time and broadens the applicability of endoscopy in skull base surgery. It is the first important step towards a multiplane, variable view endoscope for skull base surgery.

Conflict of interest statement

The present study was sponsored by Karl Storz GmbH, Tuttlingen, Germany on request of the authors with regard to the costs of injection and preparation of the anatomical specimens. Karl Storz supplied as well the Endochamaeleon as the fixed view endoscopic equipment for the time of laboratory investigation. Karl Storz did not influence the study design and collection, analysis

Table 1 : Visible neuroanatomic structures with the EC, the O°, 45° and 70° rigid optics through the frontolateral approach (light grey) and through the retrosigmoid approach (dark grey). The column of the Endochamaeleon (EC) is highlighted in grey.

Abbreviations: CN = Cranial nerve; SF = sylvian fissure