Touch Imprint Intraoperative Flow Cytometry as a Complementary Tool for Detailed Assessment of Resection Margins and Tumor Biology in Liver Surgery for Primary and Metastatic Liver Neoplasms

Abstract

Liver resection is the main treatment for primary and metastatic liver tumors in order to achieve long-term survival with good quality of life. The ultimate goal of surgical oncology is to achieve complete tumor removal with adequate clear surgical margins. Flow cytometry is a powerful analytical technique with applications such as phenotypic analysis and quantification of DNA content. Intraoperative flow cytometry (iFC) is the application of flow cytometry for DNA content/ploidy and cell cycle distribution analysis during surgery for tumor cell analysis and margin evaluation. It has been used for cell analysis of intracranial tumors and recently of head and neck carcinomas and breast carcinomas, as well as for tumor margin evaluation. Herein, we present a novel touch imprint iFC protocol for the detailed assessment of tumor margins during excision of malignant hepatic lesions. The protocol aims to offer information on surgical margins after removal of malignant liver tumors based on DNA content of cancer cells and to corroborate the results of iFC with that of histopathological analysis. Based on the established role of iFC in other types of malignancies, our specialized protocol has the potential, through characterization of cells in liver transection surface post hepatectomy, to offer significant information on the type of resection and tumor biology. This information can be used to effectively guide intra- and postoperative patient management.

Intraoperative Flow Cytometry for the Characterization of Gynecological Malignancies

“…This method offers numerous advantages, and can be successfully used for tumor diagnosis, as clonal expansion of cancer cells can be monitored as subpopulations with distinct genetic characteristics. It contributes towards precise intraoperative identification of tumor margins, offering the potential of complete removal, and is currently applied in brain [ 21 , 22 ], head-and-neck [ 23 , 24 ], breast [ 25 , 26 , 27 ], liver [ 28 ], pancreatic [ 29 ], and colorectal neoplasms [ 30 ]. To the best of our knowledge, the present study is the first application of iFC in the detection and characterization of cancer cells in gynecological malignancies.…”

“…An average of 5000 events (cell nuclei) counted in each sample were evaluated. A gating strategy based on area/width analysis of propidium iodide fluorescence was utilized to rule out cell doublets from DNA content quantification, as previously described [ 28 ]. The total analysis time per sample was 5–6 min.…”

“…Intraoperative flow cytometry (iFC) is a breakthrough technique enabling the cytometric analysis of DNA content/ploidy and cell-cycle distribution of cells acquired during surgical resection of tumors to characterize cancer cells and to evaluate the limits of resection, bridging the aforementioned gap between evaluation and surgery. This procedure has been applied by our team during central nervous tumor surgeries, and its significance has since been confirmed in several additional cancer types, including head-and-neck malignancies, breast cancer, hepatocellular cancer, pancreatic cancer, and colorectal cancer [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. Intraoperative flow cytometry offers good diagnostic potential with high sensitivity and specificity, while a prognostic role is also suggested.…”

“…Intraoperative flow cytometry offers good diagnostic potential with high sensitivity and specificity, while a prognostic role is also suggested. With an analysis time of less than 10 min, iFC is considered to be an accurate next-generation cancer-cell evaluation technique that can be applied intraoperatively [ 24 , 27 , 28 , 31 , 32 ]. The diagnostic and prognostic potential of iFC has been independently verified for central nervous system malignancies by a research team in Tokyo, Japan [ 33 , 34 , 35 , 36 , 37 ].…”

Accurate Characterization of Bladder Cancer Cells with Intraoperative Flow Cytometry

“…Our team has utilized intraoperative flow cytometry, originally for brain malignancies [ 20 , 21 , 22 , 23 ]. Based on the high diagnostic potential of iFC, the methodology has further been standardized and applied in several types of malignancy, including head and neck [ 24 , 25 ], breast [ 26 , 27 , 28 ], liver [ 29 ], pancreatic [ 30 ] colorectal [ 31 ], as well as gynecological neoplasms [ 32 ]. Since iFC facilitates near real time detection of aneuploidy and calculation of S- plus G2/M-phase fraction in surgical samples, it offers an accurate measurement of cancer hallmarks in 3–5 min.…”