Journal of Clinical Neuroscience xxx (2014) xxx–xxx

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Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn

Clinical Study

Fast cell cycle analysis for intraoperative characterization of brain tumor margins and malignancy George A. Alexiou a,d,⇑, George Vartholomatos b, Anna Goussia c, Anna Batistatou c, Konstantinos Tsamis d, Spyridon Voulgaris a, Athanasios P. Kyritsis d,e a

Department of Neurosurgery, University Hospital of Ioannina, Ioannina, Greece Haematology Laboratory-Unit of Molecular Biology, University Hospital of Ioannina, Ioannina, Greece Department of Pathology, University Hospital of Ioannina, Ioannina, Greece d Neurosurgical Institute, University of Ioannina School of Medicine, Ioannina, Greece e Department of Neurology, University Hospital of Ioannina, Ioannina, Greece b c

a r t i c l e

i n f o

Article history: Received 15 January 2014 Accepted 11 May 2014 Available online xxxx Keywords: Cell cycle Flow cytometry Glioma Meningioma

a b s t r a c t Flow cytometry, although indispensable for the characterization of hematologic malignancies, has not been extensively evaluated in solid tumors. To date intraoperative pathology evaluation of frozen sections of tissue obtained during surgery is the gold standard for intraoperative diagnosis. We investigated the value of a modified rapid protocol for cell cycle analysis for the intraoperative characterization of intracranial lesions and their surgical margins. We investigated patients who underwent surgery for an intracranial lesion suspicious for a tumor. DNA analysis and frozen sections were performed on tumor samples that were taken during surgery. Thirty-one patients met the inclusion criteria for the study. There was a significant difference in G0/G1 phase between high-grade and low-grade tumors. Receiver operating characteristic (ROC) analysis provided 75% of G0/G1 fraction as the optimal cutoff value thresholding the discrimination between low and high-grade tumors. There was a significant difference in S-phase and mitoses fraction between high-grade and low-grade tumors. ROC analysis indicated 6% of S-phase and 9.7% of mitoses as the optimal cutoff values thresholding the discrimination between these two groups. In the glioblastoma patients, we also analyzed the perilesional tissue and found significant differences between tumor mass and margins regarding the G0/G1 phase, the S-phase and mitoses fraction. In conclusion rapid cell cycle analysis is a method capable of differentiating low from high-grade tumors and delineating tumor margins in gliomas. Thus, the role of cell cycle analysis in brain tumors warrants further investigation. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Flow cytometry immunophenotyping has become a standard practice for the diagnosis, classification, staging, and monitoring of patients with hematologic neoplasms and it has been proven to be superior to immunohistochemistry [1]. Although flow cytometry is able to provide objective and quantitative results, even on very small samples, and within a few hours, it has remained largely a research tool in solid tumors [1,2]. Flow cytometry provides a powerful tool to assess cells in G0/G1 phase versus S-phase, G2 or polyploidy [3,4]. We have recently reported that cell cycle analysis could differentiate low from high-grade gliomas and benign ⇑ Corresponding author. Address: P.O. Box 103, Neohoropoulo, Ioannina 455 00, Greece. Tel.: +30 26510 48795. E-mail address: [email protected] (G.A. Alexiou).

from atypical/anaplastic meningiomas. Furthermore, a prognostic significance was found in glioma patients [5]. Recently, by modifying the previously used cell cycle protocol we have developed a rapid protocol that allows cell cycle analysis within 6 minutes [6]. This could permit an intraoperative cell cycle analysis of the surgical specimen. Given that intraoperative pathologic analysis of frozen sections of tissue obtained during surgery is the gold standard for intraoperative diagnosis, we set out to evaluate the role of rapid cell cycle analysis for the intraoperative characterization of intracranial lesions and tumor margins. 2. Materials and methods Patients hospitalized in the Neurosurgical Department of our institution over a 2 year period who underwent surgery for an intracranial lesion suspicious for tumor on conventional

http://dx.doi.org/10.1016/j.jocn.2014.05.029 0967-5868/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Alexiou GA et al. Fast cell cycle analysis for intraoperative characterization of brain tumor margins and malignancy. J Clin Neurosci (2014), http://dx.doi.org/10.1016/j.jocn.2014.05.029

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G.A. Alexiou et al. / Journal of Clinical Neuroscience xxx (2014) xxx–xxx

radiological imaging (MRI/CT scan) were included in the study. A tumor sample was taken during surgery for flow cytometry analysis from at least two areas (tumor center and periphery), trying to avoid areas of necrosis. The preoperative CT scan/MRI findings (intra-axial or extra-axial tumor), and the intraoperative findings, were not disclosed to the investigator who performed cell cycle analysis. The histopathological examination of the tissue samples on permanent tissue sections was considered the gold standard. Diagnosed tumors were graded according to the World Health Organization (WHO) 2007 classification scheme. Regarding flow cytometry analysis, the tumors were classified as low-grade (WHO grade I and II) or high-grade (WHO grade III and IV). The study had the approval of our Institutional Review Board and was in accordance with the principles of the Declaration of Helsinki. 2.1. Modified DNA analysis protocol (Ioannina protocol) Immediately after tumor excision, tumor samples (0.5–2 mm3) are minced (Medimachine System, BD Bioscience, Franklin Lakes, NJ, USA) for 1 minute in phosphate buffered saline (Ca2+ and Mg2+ free, with 0.5 mg/ml RNase) and a cell suspension is obtained. The suspension is then filtered (Consult No. 10, Medicons, BD Bioscience) and cells are counted using an automated hematology analyzer to a final concentration of 1.0  106 cells/ml. Cells are then processed immediately for staining by adding propidium iodide (125 lg/ml) and after 3 minutes flow cytometric analysis is performed. All the stained samples are analyzed using a FACSCalibur flow cytometer, equipped with two lasers (488 nm, 635 nm) and six parameters (FSC, SSC, FL1–FL4) and using CellQuest software (both BD Bioscience). Chicken red blood cells and normal cells obtained from the peripheral blood mononuclear cells (Ficoll-Paque separation, GE Healthcare, Little Chalfont, Buckinghamshire, UK) are used as the standard to define the position of the diploid G0/ G1 peak in the DNA histograms. These cells can then be mixed with the sample in a second tube before staining and used as a reference to determine the degree of DNA content aberration. 2.2. Statistical analysis We used the Mann–Whitney U test to compare the G0/G1, S-phase and mitoses fraction of the low-grade versus high-grade tumors and tissue from tumor core and periphery. Receiver operating characteristic (ROC) analysis was used to define the threshold value most efficiently discriminating low-grade from high-grade tumors. Continuous data are expressed as mean ± standard deviation. The level of significance was defined as a probability value less than 0.05. 3. Results From June 2011 until June 2013, 31 patients (15 men, 16 women, mean age 57.3 years, range 19–86) met the inclusion criteria for the study. The histological diagnoses of the high-grade tumors were nine glioblastomas, one anaplastic astrocytoma, and one anaplastic meningioma; the low-grade tumors were one pilocytic astrocytoma and 15 benign meningiomas. There were also two metastases (from lung cancer), one primary central nervous system lymphoma and one case suspicious for tumor that proved to be intracerebral hemorrhage. High-grade gliomas had lower mean G0/G1, and higher S-phase and mitoses fraction than the one pilocytic astrocytoma (60.8 ± 17.9% versus 87.1%, 13.9 ± 15.4% versus 2%, and 18.6 ± 15.5% versus 4%, respectively). The one case of anaplastic meningioma had lower mean G0/G1 and higher S-phase and mitoses fraction than benign meningiomas (49.7%

versus 83.4 ± 14.6%, 13.1% versus 4.1 ± 4%, and 25.2% versus 5.9 ± 5.7%, respectively). When we analyzed the tumors as low-grade and high-grade there was a significant difference in G0/G1 phase between highgrade and low-grade tumors (median 62.4 versus 88.1%, respectively; p < 0.0001). ROC analysis indicated 75% of G0/G1 fraction as the optimal cutoff value thresholding the discrimination between low and high-grade tumors with 92.3% sensitivity and 91.7% specificity. There was a significant difference in S-phase between high-grade and low-grade tumors (median 9.5 versus 3.7, respectively; p = 0.0008). ROC analysis indicated 6% of S-phase as the optimal cutoff value thresholding the discrimination between these two groups, with 84.6% sensitivity and 91.7% specificity. There was a significant difference in mitoses between high-grade and low-grade tumors (median 16.5 versus 4.4, respectively; p = 0.0019). ROC analysis indicated 9.7% of mitoses as the optimal cutoff value thresholding the discrimination between these two groups, with 92.3% sensitivity and 66.7% specificity (Fig. 1). When we analyzed the G0/G1 phase between tumor core and perilesional tissue in the glioblastoma patients the difference was significant (median 58.3% versus 81.1%, p = 0.03). The difference was also significant for the median S-phase and mitoses fraction between tumor core and perilesional tissue (12.8 % versus 4.6%, p = 0.011 and 19.3% versus 6.8, p = 0.012) (Fig. 2). The cell cycle analysis of the intracerebral hemorrhage excluded the presence of a neoplasm as the G0/G1 phase was 96%, the S-phase was 1.8% and the mitosis fraction was 2.2% (Fig. 3). 4. Discussion The present study showed that low-grade tumors could be accurately differentiated from high-grade tumors by cell cycle analysis. Furthermore, glioma margins can be readily identified and the surgeon can be accordingly informed. The cell cycle analysis protocol reported here, to the best of our knowledge, is the fastest (6 minutes duration) that has been reported to date. Thus, rapid cell cycle analysis may be a novel intraoperative adjunct. Cell cycle analysis provides important information on tumor behavior and has been used in several malignancies [3]. We have previously reported that based on cell cycle analysis low-grade gliomas can be differentiated from high-grade gliomas and benign meningiomas from anaplastic gliomas. In gliomas, cell cycle analysis has an additional prognostic role [3]. Coons et al. found three groups of astrocytoma patients with significantly different survival based on S-phase fraction ranges of

Fast cell cycle analysis for intraoperative characterization of brain tumor margins and malignancy.

Flow cytometry, although indispensable for the characterization of hematologic malignancies, has not been extensively evaluated in solid tumors. To da...
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