Malignancy Index Using Intraoperative Flow Cytometry is a Valuable Prognostic Factor for Glioblastoma Treated With Radiotherapy and Concomitant Temozolomide.

BACKGROUND: Intraoperative prediction of radiochemosensitivity is desirable for improving the clinical management of glioblastoma (GBM) patients. We have previously developed an original technique for intraoperative flow cytometry (iFC) and defined a malignancy index (MI). OBJECTIVE: To determine whether MI correlates with prognosis in GBM patients who underwent the standard treatment protocol of radiotherapy and temozolomide administration. METHODS: The current study included 102 patients with GBM that had been newly diagnosed from 2010 to 2015 who underwent our iFC analysis and received the standard treatment protocol. We evaluated MI values in each patient, then statistically analyzed the relationship between MI and prognosis using survival analysis that include other clinicopathological factors (age, sex, Karnofsky performance status [KPS], extent of resection, second-line bevacizumab, O6-methylguanine-DNA methyltransferase [MGMT] status, MIB-1 labeling index, and mutation of the isocitrate dehydrogenase 1 gene [IDH1]). RESULTS: Log-rank test revealed that age, KPS, extent of resection, MGMT status, IDH1 mutation, and high MI (≥26.3%) significantly correlated with overall survival. Multivariate analysis with Cox regression modeling identified MI as the most significant prognostic factor (hazard ratio = 2.246; 95% confidence interval = 1.347-3.800; P = .0019). MI showed strong correlation with IDH1 mutation status in chi-square test (P = .0023). In addition, log-rank test revealed that MI affects overall survival more strongly in patients with IDH1 wildtype than those with IDH1 mutant. CONCLUSION: MI from an iFC study may help predict the prognosis in patients with GBM who receive the standard treatment. Survival can be related to sensitivity to radio-chemotherapy.

A surgical strategy for lower grade gliomas using intraoperative molecular diagnosis.

Lower grade gliomas are both treated and diagnosed via surgical resection. Maximum tumor resection is currently the standard of care; however, this risks the loss of brain function. Glioma can be genetically subdivided into three different types, based on isocitrate dehydrogenase (IDH) mutation status and the presence of 1p/19q codeletion, which have radically different prognoses and responses to adjuvant therapies. Therefore, the means to identify the subtype and evaluate the surrounding tissues during surgery would be advantageous. In this study, we have developed a new surgical strategy for lower grade glioma based on the fourth edition of the World Health Organization Brain Tumor Classification, involving intraoperative molecular diagnosis. High-resolution melting analysis was used to evaluate IDH mutational status, while rapid immunohistochemistry of p53 and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) was used to evaluate the 1p/19q codeletion status, allowing genetic classification during surgery. In addition, intraoperative flow cytometry was used to evaluate the surgical cavity for additional tumor lesions, allowing maximal resection while mitigating the risk of functional losses. This strategy allows the rapid intraoperative diagnosis and mapping of lower grade gliomas, and its clinical use could dramatically improve its prognosis.

Intraoperative Flow Cytometry Enables the Differentiation of Primary Central Nervous System Lymphoma from Glioblastoma.

OBJECTIVE: Accurate preoperative and intraoperative differentiation between primary central nervous system lymphoma (PCNSL) and glioblastoma (GBM) is sometimes difficult. Distinguishing between these tumors during surgery is important because surgical treatment is different between the 2 tumors. In this study, we established a new method of intraoperative differentiation between PCNSL and GBM using intraoperative flow cytometry (iFC), and we retrospectively tested whether iFC was useful for the intraoperative diagnosis of PCNSL and GBM. METHODS: We analyzed the iFC data of 250 patients (28 with PCNSL and 222 with GBM) and then evaluated aneuploidy and S-phase population. RESULTS: Aneuploidy was detected in 54.5% of GBM cases but in only 7.14% of PCNSL cases. Aneuploidy indicated GBM, but it was difficult to distinguish PCNSL from GBM when a tumor had a diploid pattern. Thus, for tumors without aneuploidy, we evaluated the S-phase population: S2, the ratio of the average height of the S-phase to the height of the diploid peak. S2 was significantly higher in PCNSL than in GBM. Based on these results, we established an algorithm for differentiating between PCNSL and GBM using DNA aneuploidy and S2. Comparing this new iFC algorithm and the permanent pathologic diagnosis, the sensitivity was 89.3%, the specificity was 93.7%, and the accuracy was 93.2%. CONCLUSIONS: iFC is useful for the intraoperative differentiation between PCNSL and GBM and it aids in intraoperative decision making within a short time. The accuracy of intraoperative diagnosis of these tumors seems to be higher with the combination of iFC and intraoperative rapid pathologic diagnosis.

Intraoperative flow cytometry analysis of glioma tissue for rapid determination of tumor presence and its histopathological grade: clinical article.

OBJECT: Intraoperative histopathological investigation plays an important role during surgery for gliomas. To facilitate the rapid characterization of resected tissue, an original technique of intraoperative flow cytometry (iFC) was established. The objective in this study was evaluation of this technique's efficacy for rapidly determining tumor presence in the surgical biopsy sample and WHO histopathological grade of the neoplasm. METHODS: In total, 328 separate biopsy specimens obtained during the resection of 81 intracranial gliomas were analyzed with iFC. The evaluated malignancy index (MI) was defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells. The duration of iFC in all cases was approximately 10 minutes. Each sample was additionally investigated histopathologically on frozen and permanent formalin-fixed paraffin-embedded tissue sections. The latter process was used as a "gold standard" control for evaluation of the diagnostic efficacy of iFC analysis. RESULTS: The MI differed significantly between neoplastic and perilesional brain tissue (25.3% ± 22.0% vs 4.6% ± 2.6%, p < 0.01). Receiver operating characteristic curve analysis revealed a corresponding area under the curve value of 0.941. The optimal cutoff level of the MI for identification of tumor in the biopsy specimen was 6.8%, which provided 0.88 sensitivity, 0.88 specificity, 0.97 positive predictive value, 0.60 negative predictive value, and 0.88 diagnostic accuracy. Additionally, the MI showed a significant association with WHO histopathological grades of glioma (p < 0.01), but its values in Grade II, III, and IV tumors overlapped prominently and were on average 13.3% ± 11.0%, 35.0% ± 21.8%, and 46.6% ± 23.1%, respectively. CONCLUSIONS: Results of this study demonstrate that iFC with the determination of the MI may be feasible for rapidly determining glioma presence in a surgical biopsy sample.

New isolation system for collecting living cells from tissue.

A new semi-automatic living-cell isolation system was developed. The new system improves the quality of isolated cells, reduces cell isolation time, and isolates more cells with a higher cell viability compared to conventional manual methods. We successfully applied this system to isolate beating cardiomyocytes and fabricate electrical communicative cardiac tissue. In this study using the isolated cardiac cells we also fabricated a cardiac cell sheet that beat spontaneously and synchronously.