**3. Pilot study**

388 Advances in the Biology, Imaging and Therapies for Glioblastoma

and/or other pathologies which were normally not visible created a complete new field of gastrointestinal diagnosis followed by specific treatment before classical histology was accessible (Bojarski et al., 2009). For the use in humans intravenous applied fluorescein sodium distributes throughout the mucosa, however, most of the serum albumin bound

Fig. 1. Distal tip of the confocal endomicroscope EC-3870CIFK (Pentax, Europe) used in gastrointestinal endoscopy. As a protrusion on the end of a conventional endoscope a microscopic lens is mounted (diameter of the lens is 5 mm). All other features of the distal

Fig. 2. (With kind permission of Peter Delaney, Optiscan, Australia and Ralf Kiesslich, University Medicine Mainz, Germany). The use of confocal endomicroscopy in human gastrointestinal tract. When the confocal lens was hold in gentle contact with the

gastrointestinal mucosa, blue laser light generated a series of images every 7µm up to an imaging depth of 250µm. Typical contrast dyes were fluorescein and acriflavine as indicated.

fraction of fluorescein highlights the blood vessels and the capillaries.

tip are standard in routine endoscopy.

In our pilot study NLM was performed in an ex vivo approach on small tissue samples of patients suspicious to suffer from GBM (WHO IV, n=9) or meningeoma (grade I n=2, grade II n=1) after diagnostic radiology. Open tumor resection was performed in the neurosurgical OR in all patients. No prior histological diagnosis was available before neurosurgery. One sample of tumor tissue was used for direct comparison of NLM and histopathology of the same area. After the examination with NLM the specimen was transferred to neuropathology for conventional tissue examination. Additional fragments of the tumor center and border were analysed by NLM. Different additional fragments were sent for conventional pathological diagnosis. These were not marked as study material and therefore assessment and diagnosis was blinded.

Fig. 3. (Removed with approval Schlosser, Cen Eur Neurosurg, 2010, 71(1):13-9) Histopathology and Neurolasermicroscopy (NLM) of a GBM (a-f same patient, a-c center; df infiltration zone, bar = 100 µm). a, d: H&E staining of tumor border (a) and infiltration zone (d). H&E staining of tumor center shows a high density of tumor cells; b, e: immunohistochemical staining with MIB-1antibody to assess proliferation in tumor center (b) and infiltration zone (e). c, f: NLM of tumor center (c) and infiltration zone (f) with detection of a high density of tumor cells in the center and markedly reduced cell density in the infiltration zone. The margin of the tumor appears similar to a parabolic curve.

Confocal Neurolasermicroscopy - Modern Perspectives for Glioma Resection on a Cellular Level 391

border depicting the differences of both areas. The overall image quality was good. In one patient with GBM the scanning images directly correspond to conventional histopathology in all five diagnostic aspects. The WHO criteria for the diagnosis of GBM - cell density, cell pleomorphy, mitoses, microvascular proliferation and pseudopalisading or ischemic

Identification of the infiltration zone or the center of the tumor was possible in any patient by determination of the cell density comparing center and border resection bloc (Fig 3). Further WHO criteria for tumor classification to identify GBM were compared to histology

The NLM images of nine patients with histologically proven GBM were analysed for the presence of WHO criteria of GBM. In all 9 patients cell density and cell pleomorphy were clearly visible with NLM. In 44% of the patients (4/9) microvascular proliferation was visible and in 22% of the patients (2/9) mitosis and stroke like necrosis was identified. Additional aspects, e.g. apoptotic figures in perinecrotic palisading tumor cells, and important histological structures such as giant cells, fibrillary tumor matrix and blood vessels were also visible with NLM (Fig. 5). Typical features of NLM in patients with

Fig. 5. (Removed with approval Schlosser, Cen Eur Neurosurg, 2010, 71(1):13-9) Additional findings of GBM with NLM (bar = 100 µm). a: giant cell within the tumor formation. b: pathological blood vessel, the thickened wall of the vessel is clearly visible (arrows). c: necrosis formation in the near of tumor cells. d: apoptotic figure within the tumor center (square), magnification shows characteristic chromatin fragments at the inner side of the

in the same resection bloc and were identified in some but not all patients (Fig. 4).

necrosis - were all detectable by NLM.

nuclear membrane (inset).

histologically proven meningeoma are shown in Fig. 6.

Frozen sections were done in 4 out of the 9 patients with GBM and all patients with meningeoma (n=3). It was beyond the scope of the present study to directly compare the diagnostic outcome of frozen sections, conventional histology and NLM even because all patients had a final histological confirmation of glioblastoma or meningeoma. However, we see some future indications for NLM to contribute to a rapid diagnosis intraoperatively and to reduce the proportion of frozen sections reasonably.

After the tissue sample was coated with acriflavine 0.05% the NLM device was hold in gentle contact with the surface of the tumor and confocal scanning process was initiated. Histological processing included staining with hematoxylin and eosin (HE), periodic acidic Schiff (PAS), silver-impregnation for reticulin or immunohistochemical staining.

Histopathology was performed according to the criteria for GBM of the recent WHO classification respecting the exceptions and subtypes (Louis et al., 2007). In nine patients with histologically proven GBM, in which no case of "small cell glioblastoma" or glioblastoma with oligodendroglial component" occurred, the presence of the following WHO criteria for GBM were analysed in the NLM obtained images: a) cell number and density, b) cell pleomorphy, c) mitotic figures and rate of mitosis (high, moderate, low), d) microvascular proliferation and e) pseudopalisading necrosis. Diagnosis and specific findings of NLM images and conventional histopathology were compared when all data were available. In three patients with histologically proven meningeoma typical features were visible with NLM including pleomorphy, onion-skin shaped appearance of tumor cells and single apoptotic cells.

Fig. 4. (Removed with approval Schlosser, Cen Eur Neurosurg, 2010, 71(1):13-9) Histology (H&E; a-d, bar = 100 µm; c, bar = 50 µm) and NLM (e-h, bar = 100 µm) of WHO criteria for the diagnosis of GBM. a, e: tumor center with a high density of pleomorphic tumor cells. b, f: microvascular proliferation within the tumor formation (arrow). c, g: mitotic figures (arrows in c, square in g), higher magnification in g shows the mitosis within the nucleus (inset). d, h: pseudopalisading necrosis.

NLM was an easy to handle tool and revealed in all nine patients with GBM and three patients with meningeoma typical characteristics of tumor architecture. This unique technique provided evaluable scans for all patients with GBM from the center and from the

Frozen sections were done in 4 out of the 9 patients with GBM and all patients with meningeoma (n=3). It was beyond the scope of the present study to directly compare the diagnostic outcome of frozen sections, conventional histology and NLM even because all patients had a final histological confirmation of glioblastoma or meningeoma. However, we see some future indications for NLM to contribute to a rapid diagnosis intraoperatively and

After the tissue sample was coated with acriflavine 0.05% the NLM device was hold in gentle contact with the surface of the tumor and confocal scanning process was initiated. Histological processing included staining with hematoxylin and eosin (HE), periodic acidic

Histopathology was performed according to the criteria for GBM of the recent WHO classification respecting the exceptions and subtypes (Louis et al., 2007). In nine patients with histologically proven GBM, in which no case of "small cell glioblastoma" or glioblastoma with oligodendroglial component" occurred, the presence of the following WHO criteria for GBM were analysed in the NLM obtained images: a) cell number and density, b) cell pleomorphy, c) mitotic figures and rate of mitosis (high, moderate, low), d) microvascular proliferation and e) pseudopalisading necrosis. Diagnosis and specific findings of NLM images and conventional histopathology were compared when all data were available. In three patients with histologically proven meningeoma typical features were visible with NLM including pleomorphy, onion-skin shaped appearance of tumor cells

Fig. 4. (Removed with approval Schlosser, Cen Eur Neurosurg, 2010, 71(1):13-9) Histology (H&E; a-d, bar = 100 µm; c, bar = 50 µm) and NLM (e-h, bar = 100 µm) of WHO criteria for the diagnosis of GBM. a, e: tumor center with a high density of pleomorphic tumor cells. b, f: microvascular proliferation within the tumor formation (arrow). c, g: mitotic figures (arrows in c, square in g), higher magnification in g shows the mitosis within the nucleus (inset). d,

NLM was an easy to handle tool and revealed in all nine patients with GBM and three patients with meningeoma typical characteristics of tumor architecture. This unique technique provided evaluable scans for all patients with GBM from the center and from the

Schiff (PAS), silver-impregnation for reticulin or immunohistochemical staining.

to reduce the proportion of frozen sections reasonably.

and single apoptotic cells.

h: pseudopalisading necrosis.

border depicting the differences of both areas. The overall image quality was good. In one patient with GBM the scanning images directly correspond to conventional histopathology in all five diagnostic aspects. The WHO criteria for the diagnosis of GBM - cell density, cell pleomorphy, mitoses, microvascular proliferation and pseudopalisading or ischemic necrosis - were all detectable by NLM.

Identification of the infiltration zone or the center of the tumor was possible in any patient by determination of the cell density comparing center and border resection bloc (Fig 3). Further WHO criteria for tumor classification to identify GBM were compared to histology in the same resection bloc and were identified in some but not all patients (Fig. 4).

The NLM images of nine patients with histologically proven GBM were analysed for the presence of WHO criteria of GBM. In all 9 patients cell density and cell pleomorphy were clearly visible with NLM. In 44% of the patients (4/9) microvascular proliferation was visible and in 22% of the patients (2/9) mitosis and stroke like necrosis was identified.

Additional aspects, e.g. apoptotic figures in perinecrotic palisading tumor cells, and important histological structures such as giant cells, fibrillary tumor matrix and blood vessels were also visible with NLM (Fig. 5). Typical features of NLM in patients with histologically proven meningeoma are shown in Fig. 6.

Fig. 5. (Removed with approval Schlosser, Cen Eur Neurosurg, 2010, 71(1):13-9) Additional findings of GBM with NLM (bar = 100 µm). a: giant cell within the tumor formation. b: pathological blood vessel, the thickened wall of the vessel is clearly visible (arrows). c: necrosis formation in the near of tumor cells. d: apoptotic figure within the tumor center (square), magnification shows characteristic chromatin fragments at the inner side of the nuclear membrane (inset).

Confocal Neurolasermicroscopy - Modern Perspectives for Glioma Resection on a Cellular Level 393

Realizing in vivo histology during neurosurgery would contribute to a better definition of the histological borders of the tumor. This would improve the definition of the resection margins significantly. However, due to the infiltrative growth of many primary brain tumours it is not possible to clearly define the exact margins of a tumor mass in all cases, neither by conventional histology nor by NLM. The in vivo look on these areas from tumor to intact brain tissue (probably by using a histopathological NLM classification) could

On a cellular basis the excision could be performed as much as necessary but as little as possible which could be beneficial for patients suffering from a brain tumor (Lacroix et al., 2001) (Ammirati et al., 1987). The amount of residual tumor mass after surgery is one of the most important prognostic factors (Burger and Green, 1987) (Wood et al., 1988). NLM scans on a cellular and subcellular level could be more accurate than performing the whole

Regarding those aspects one has to focus on the appearance of the NLM scans depicting tumor pathologies. Cell types, cell division, neovascularisation and boarder zones have to become acquainted to the observer as well as th possibility for dynamic investigation. This histology is different from the appearance in classical histopathology. So neurosurgeon and pathologist have to share their insight and practically an atlas for defining all pathologies seen in NLM with regard for the process in the theatre has to be developed in the near

Compared to conventional "random" brain tissue biopsies with the possibility of sampling errors NLM allowed "targeted" biopsies which could increase the reliability of the diagnosis

By using NLM the process of frozen sections could be influenced considerably. On the one hand thee cellular findings could be discussed between surgeon and pathologist demonstrating different areas and shifting the focus depending on the microscopic results which can affect the further direction of the procedure, on the other hand the NLM scans could be transferred directly to the pathologist via a network with marked reduction of the processing time for serial-cuts. This would also eliminate transfer time of the tissue bloc

The presented scans show also vascular and important subcellular aspects contributing to the final diagnosis by predicting typical disease features. The options for biochemical or immunologic in vivo imaging by using antibodies or cell surface markers can be evaluated in the future after establishing NLM as an in vivo tool for neurosurgery providing a cellular and subcellular view. This subcellular view already enabled physiologic investigations in

provide new insights towards a standardized diagnosis during neurosurgery.

investment of brain navigation even with shift correction (Asthagiri et al., 2007).

**5. Definition of histological borders** 

future.

**6. Targeted biopsies** 

**7. Potential indications** 

from the OR to the laboratory.

**8. Molecular imaging** 

when multiple cell types contribute to a tumor.

skin (Lademann et al., 2007) (Suihko et al., 2005).

Fig. 6. Representative NLM images from patients with histologically proven meningeoma (n=3, bar = 100 µm). a: tumor center with a high density of pleomorphic tumor cells in grade II meningeoma, b: undirected tumor cells with a pathological blood vessel (in between the spotted line) and neoangiogenesis (arrows) in the same patient; c: typical onion-shaped tumor cell configuration in grade I meningeoma (circle), d: single apoptotic cells (arrows) in the tumor border in the same patient.
