Rapid polymer-based immunohistochemistry for intraoperative CNS tumor diagnosis: A validation study.

Tumors caused by failure of the DNA-mismatch repair system generally show microsatellite instability (MSI). High-frequency MSI cancers have been shown to be susceptible to immuno-oncology therapies. The aim of this study was to evaluate the clinical reliability of a rapid immunohistochemistry (IHC) technique for intraoperatively assessing molecular status through detection of tumoral deficiencies in the expression of mismatch repair proteins (dMMR; MLH1, MSH2, MSH6, and PMS2). The rapid IHC method uses non-contact alternating current (AC) mixing to achieve more rapid/stable staining within a minimum of 13min during surgery. Sixteen formalin-fixed paraffin-embedded (FFPE) tumor samples from 3 dMMR patients with Lynch syndrome and 6 FFPE samples from 6 dMMR-cancer patients were collected to establish an IHC protocol for MMR proteins. Next, 26 surgical patients treated and whose MSI status was determined using PCR-based tests were retrospectively analyzed. The concordance of dMMR diagnoses for thoracic tumors between the conventional (frozen section (FS)- and FFPE-IHCs) and rapid AC-mixing IHC with FSs were compared. A rapid IHC protocol using primary antibodies against four MMR proteins (mixed 5-10min) was established (entire process within 40min). The concordance rate for MMR-IHC between the conventional and rapid IHC was 100%. dMMR diagnoses including an MSI-high pulmonary sarcoma patient entirely matched between FS- and FFPE-IHC. Rapid MMR-IHC could potentially serve as a clinical tool for intraoperative determination of tumor MSI/dMMR status. AC-mixing technology will contribute to improving pathological diagnostic capability through the development of an original and innovative rapid IHC.

We read with great interest the recent article by Tanino et al. on the value of rapid immunohistochemistry (R-IHC) during brain tumor surgery. The authors developed a new immunohistochemistry method based on an alternating current electric field to facilitate the antigen–antibody reaction that could provide results for surgeons within 30 min [1]. The overall diagnostic accuracy of this method was 90.7 %. For the discrimination of lowand high-grade glioma, the authors evaluated supportive information of the Ki-67/MIB-1 index which could be completed within 16 min. Furthermore, in four cases of CNS lymphoma, CD20 and Ki-67/MIB-1 indices were successfully stained in all frozen samples using R-IHC. The authors concluded that R-IHC provides reliable results of IHC for CNS tumor diagnosis on frozen sections, and may contribute to an intraoperative rapid diagnosis [1]. Although flow cytometry has become a standard practice for the diagnosis, classification, staging and monitoring of patients with hematologic neoplasms, it has remained largely a research tool in solid tumors. Cell cycle analysis by flow cytometry can provide important information on tumor’s aggressiveness [2]. Furthermore, although flow cytometric analysis of DNA aneuploidy is not always associated with malignancy in several tumors, it is useful in brain tumors [3]. Recently, we have showed that based on G0/G1 and S-fraction we could differentiate lowfrom high-grade gliomas and benign from atypical/anaplastic meningiomas with high sensitivity and specificity (Fig. 1). In gliomas, a negative linear correlation between Ki-67 index and the G0/G1 fraction, and significant positive correlation between Ki-67 index and both S phase-fraction and mitoses were found. Apart from that, we have reported that cell cycle analysis has an additional prognostic significance. Glioma patients with G0/G1 value lower than 69 % and S phase value greater than 6 % were associated with worse survival [2]. The cell cycle analysis by flow cytometry usually requires 20 min. Recently, we developed a rapid cell cycle protocol that provides within 5 min intraoperative characterization of brain tumors and their margins [4]. The latter is of paramount importance to guide surgeon for tumor excision. Shioyama et al. also developed a rapid protocol for intraoperative characterization of resected gliomas by flow cytometry. By evaluating in 328 biopsy specimens the malignancy index, defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells, Shioyama et al. [5] found significant differences between neoplastic and G. Vartholomatos Haematology Laboratory-Unit of Molecular Biology, University Hospital of Ioannina, 45500 Ioannina, Greece

To evaluate the role of rapid immunohistochemistry (RIHC) based on ultrasonic thermal steam heating in improving diagnostic accuracy of intraoperative frozen section diagnosis and to recommend RIHC antibody panels for pathologic differential diagnosis. RIHC based on ultrasonic thermal steam heating was tested for intraoperative frozen diagnosis with difficulty in diagnosis, and all slides were reviewed and compared with the final diagnosis. Ninety-three cases of surgical specimens involving RIHC examination were studied. Discordance rates with paraffin immunohistochemistry were calculated. In 93 cases where RIHC was performed, 85 cases (91%) were proven to be helpful for the diagnosis. A total of 58 antibodies were used for RIHC 276 times, of which 19 antibodies were not effective 25 times. Fifteen RIHC antibody panels are recommended based on staining stability and utilization frequency. After improving the staining method, ultrasonic thermal steam heating RIHC is practical, convenient, and cost-effective, making it suitable for use in any pathology department with routine immunohistochemistry reagents. It plays an important auxiliary role in improving the accuracy of intraoperative rapid pathologic diagnosis.

Alterations in interstitial cells of Cajal (ICC) distribution and density may seriously influence gut motility. We and others have documented a disturbance of ICC and neurons in Hirschsprung's disease (HD), intestinal ischemia, and inflammation. The ability to remove intestine with permanent dysmotility improves significantly the prognosis. This may be important in the developing intestine especially in HD. More complete pathological information increases the accuracy of surgical decisions. Therefore we sought to develop a rapid, intraoperative immunohistochemical protocol for ICC and neurons in surgical specimens for routine diagnostic and therapeutic assessment of pediatric patients. To date, cKit is the only reliable immunohistochemistry for ICC identification. There are multiple antibodies in use for neuronal identification. A comparison of fixation methods and immunostaining using cKit and multiple intestinal neuronal antibodies was done. Fresh segments of surgically resected intestine were sectioned and stained using antibodies for ICC cell identification (anti-cKit) and for neuronal characterization. By carefully changing tissue fixation methods, different neuronal antibodies were tested to determine an optimal rapid protocol. Each suggested protocol was tested on normal and pathological intestinal tissue and compared to the previous overnight immunostaining of the same tissue. A new rapid tissue fixation and immunostaining protocol using cKit for ICC identification and NF 68 was developed. By employing this protocol, we could obtain ICC and neuro-immunohistochemistry in unfixed frozen sections within 1 h with tissue vibration to diminish the time for immunostaining. Without vibration the protocol takes 3 h. ICC and enteric neuronal changes could be readily observed and the quality of staining was comparable to standard immunohistochemistry. Each gut pathology displayed characteristic changes in ICC and neuronal density/distribution in the affected bowel. The time-scale of the 1-h immunoprocessing is still longer than the standard clinical pathological "quick" sections using H&E staining; however, the protocol duration is within the surgery timescale. Standard H&E stain used in combination with our rapid neuronal and ICC immunohistochemistry protocol enables a fast, comprehensive, and accurate assessment of the pathophysiology of signaling networks controlling gut motility while the patient is still in the operating room. We propose that the addition of this simple and rapid immunohistochemical assessment in the pathologist evaluation of surgical specimens would result in a more complete characterization for diagnosis and prognosis of the pediatric patient. Specifically, we propose that this test will differentiate good versus poor prognosis HD patients based on their neuron/ICC ratio and present a rapid, standardized method for use in general pathology.

Rapid immunohistochemistry (R-IHC) can contribute to the intraoperative diagnosis of central nervous system (CNS) tumors. We have recently developed a new IHC method based on an alternating current electric field to facilitate the antigen-antibody reaction. To ensure the requirement of R-IHC for intraoperative diagnosis, 183 cases of CNS tumors were reviewed regarding the accuracy rate of diagnosis without R-IHC. The diagnostic accuracy was 90.7 % (166/183 cases) [corrected] in which definitive diagnoses were not provided in 17 cases because of the failure of glioma grading and differential diagnosis of lymphoma and glioma. To establish the clinicopathological application, R-IHC for frozen specimens was compared with standard IHC for permanent specimens. 33 gliomas were analyzed, and the Ki-67/MIB-1 indices of frozen specimens by R-IHC were consistent with the grade and statistically correlated with those of permanent specimens. Thus, R-IHC provided supportive information to determine the grade of glioma. For discrimination between glioma and lymphoma, R-IHC was able to provide clear results of CD20 and Ki-67/MIB-1 in four frozen specimens of CNS lymphoma as well as standard IHC. We conclude that the R-IHC for frozen specimens can provide important information for intraoperative diagnosis of CNS tumors.

Rapid immunohistochemistry (R-IHC) can contribute to the intraoperative diagnosis of central nervous system (CNS) tumors. We have recently developed a new IHC method based on an alternating current electric field to facilitate the antigen-antibody reaction. To ensure the requirement of R-IHC for intraoperative diagnosis, 183 cases of CNS tumors were reviewed regarding the accuracy rate of diagnosis without R-IHC. The diagnostic accuracy was 90.7 % (166/183 cases) [corrected] in which definitive diagnoses were not provided in 17 cases because of the failure of glioma grading and differential diagnosis of lymphoma and glioma. To establish the clinicopathological application, R-IHC for frozen specimens was compared with standard IHC for permanent specimens. 33 gliomas were analyzed, and the Ki-67/MIB-1 indices of frozen specimens by R-IHC were consistent with the grade and statistically correlated with those of permanent specimens. Thus, R-IHC provided supportive information to determine the grade of glioma. For discrimination between glioma and lymphoma, R-IHC was able to provide clear results of CD20 and Ki-67/MIB-1 in four frozen specimens of CNS lymphoma as well as standard IHC. We conclude that the R-IHC for frozen specimens can provide important information for intraoperative diagnosis of CNS tumors.

Introduction: Determining a surgical strategy for early-stage lung cancer requires an accurate histologic diagnosis. Immunohistochemistry (IHC) enables reliable diagnosis of histological types but requires more time and more tumor tissue slides than hematoxylin and eosin staining. We aimed to assess the clinical validity of a new rapid multiplex IHC technique utilizing alternating current (AC) mixing for intraoperative lung cancer diagnosis. Methods: Forty-three patients who underwent radical resection of lung cancers were enrolled in a retrospective observational study. Frozen sections were prepared from lung tumor samples, and rapid IHC employing AC mixing was implemented alongside a multiplex IHC protocol targeting thyroid transcription factor-1 + cytokeratin 5, desmoglein 3 + Napsin A, and p63 + tripartite motif containing 29. We then evaluated the concordance between intraoperative diagnoses derived from rapid multiplex IHC and final pathology. Results: The concordance rate between the pathological diagnosis made with added rapid multiplex IHC and the final pathology was 93.0% (Cohen’s \U0001d705 coefficient = 0.860 and 95% CI: 0.727–0.993). When considering only adenocarcinoma and squamous cell carcinoma, the diagnoses were in agreement for all cases. Conclusions: We suggest rapid multiplex IHC as a promising tool for determining surgical strategies for lung tumors.

Intratumoral immune profiles have been related to prognosis and therapeutic efficacy; this could result in personalized treatment based on biomarkers. Although techniques for multifactorial evaluation of different tumor-immune microenvironments have been established, their practical implementation in clinical settings still requires further improvement, where the time required for testing needs to be reduced and the technology needs to be clinically validated. To develop a multiplex, quantitative, and rapid tissue evaluation method based on clinically established standard immunohistochemistry (IHC), we attempted to develop 6-marker rapid multiplex IHC based on our previously reported 14-marker multiplex IHC via the reduction of the number of labels and speeding up the staining procedure (Tsujikawa et al. Cell Reports, 2017). First, we confirmed whether immunological features that were found to be associated with prognosis in 14-marker multiplex IHC analyses could be identified with fewer labels. Quantitative results by selected six markers exhibited significant correlation with those by 14 markers in terms of immune classifications based on the balance of lymphoid and myeloid cells. Next, we developed a rapid staining protocol by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 hours and 49 minutes as opposed to several days required for the conventional multiplex IHC. By using six markers, validation in benign tonsil tissue and head and neck cancer tissue exhibited significant correlation between rapid and standard multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cells ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable a quantitative assessment of tumor-immune microenvironment on a clinically feasible time scale, which promote the development of tissue biomarker-guided therapeutic strategies. Citation Format: Alisa Kimura, Takahiro Tsujikawa, Hiroki Morimoto, Junichi Mitsuda, Sumiyo Saburi, Shigeyuki Mukudai, Hikaru Nagao, Nana Sakurai, Nanako Murakami, Aya Miyagawa-Hayashino, Hiroshi Ogi, Saya Shibata, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Development of a rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6879.

Objective To investigate the application of rapid immunohistochemical staining technique in intraoperative frozen section diagnosis of thyroid neoplasm. Methods MaxVision one-step rapid immunohistochemical staining technique was used to detect the expression of CK19,HBME-1,and Gal-3 in frozen section of papillary thyroid carcinoma (PTC) and thyroid benign lesions.MaxVision conventional immunohistochemistry of frozen remaining tissue was served as control.Results MaxVision one-step rapid immunohistochemical staining technique could be completed in 20 minutes.The positive localizations of three markers detected by rapid immunohistochemistry were similar to conventional immunohistochemistry, in general.The expression of CK19 was located in cytoplasm and cellular membrane.Gal-3 and HBME-1 were mainly detected in follicular luminal border and/or surface of papilla. The staining intensity in rapid immunohistochemistry was stronger than that in conventional immunohistochemistry. The positive rates of CK19,HBME-1,and Gal-3 by rapid immunohistochemistry in frozen sections were: 0 (0/28),10.7 ％ (3/28),0 (0/28),respectively,for benign lesions (nodular goiter,Hashimoto thyroiditis,thyroid adenoma); and 94.9 ％(37/39),92.3 ％ (36/39),92.3 ％ (36/39),respectively,for PTC.The expression of three markers between thyroid benign lesions and PTC had a significant difference (x2 =59.326,55.861,44.605,all P ＜ 0.001).In benign lesions,the rate of same case with two and more positive markers was 0,while in PTC it was 100 ％ and significantly different (x2 =67.000,P ＜ 0.05).Conclusion MaxVision one-step rapid immunohistochemical staining technique could be applied in intraoperative frozen section diagnosis.Detecting CK19,HBME-1,and Gal-3 expression in intraoperative frozen section has an auxiliary value for diagnosis of PTC. Key words: Thyroid neoplasms; Immunohistochemistry; Intraoperative pathology

Rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment

The role of intraoperative pathological diagnosis for central nervous system (CNS) tumors is crucial for neurosurgery when determining the surgical procedure. Especially, treatment of carmustine (BCNU) wafers requires a conclusive diagnosis of high-grade glioma proven by intraoperative diagnosis. Recently, we demonstrated the usefulness of rapid immunohistochemistry (R-IHC) that facilitates antigen-antibody reaction under alternative current (AC) electric field in the intraoperative diagnosis of CNS tumors; however, a higher proportion of water and lipid in the brain parenchyma sometimes leads to freezing artifacts, resulting in poor quality of frozen sections. On the other hand, squash smear preparation of CNS tumors for cytology does not affect the frozen artifacts, and the importance of smear preparation is now being re-recognized as being better than that of the tissue sections. In this study, we established the rapid immunocytochemistry (R-ICC) protocol for squash smears of CNS tumors using AC electric field that takes only 22min, and demonstrated its usefulness for semi-quantitative Ki-67/MIB-1 labeling index and CD 20 by R-ICC for intraoperative diagnosis. R-ICC by AC electric field may become a substantial tool for compensating R-IHC and will be applied for broad antibodies in the future.

BackgroundAlthough lobectomy is considered the standard surgery for any non‐small cell lung cancer (NSCLC), recent evidence indicates that for early NSCLCs segmentectomy may be equally effective. For segmentectomy to be oncologically safe, however, adequate intraoperative lymph node staging is essential. The aim of this study was to compare the results of a new rapid‐IHC system to the HE analysis for intraoperative nodal diagnosis in lung cancer patients considered for segmentectomy.MethodsThis retrospective study analyzed the pathological reports from NSCLC resections over a six‐year period between 2014 and 2020. Using a new device for rapid‐IHC, we applied a high‐voltage, low‐frequency alternating current (AC) field, which mixes the antipancytokeratin antibody as the voltage is switched on/off. Rapid‐IHC can provide a nodal diagnosis within 20 minutes.ResultsFrozen sections from 106 resected lymph nodes from 70 patients were intraoperatively evaluated for metastasis. Of those, five nodes were deemed positive based on both HE staining and rapid‐IHC. In addition, rapid‐IHC alone detected isolated tumor cells in one hilar lymph node. Three cStage IA patients with nodal metastasis detected with HE staining and rapid‐IHC received complete lobectomies. Five‐year relapse‐free survival and overall survival among patients receiving segmentectomy with rapid‐IHC were 88.77% and 88.79%, respectively.ConclusionsRapid‐IHC driven by AC mixing is simple, highly accurate, and preserves nodal tissue for subsequent tests. This system can be used effectively for intraoperative nodal diagnosis.Rapid immunohistochemistry based on alternating‐current field mixing (completed within 20 minutes) is simple and highly accurate. This system will assist clinicians when making intraoperative diagnoses of lymph node metastasis and deciding upon the appropriate surgical procedure in segmentectomy for lung cancer.Key pointsSignificant findings of the studyRapid immunohistochemistry driven by alternating‐current field mixing (completed within 20 minutes intraoperatively) is simple, highly accurate, and preserves lymph node tissue for subsequent pathological examination, including molecular assessments.What this study addsSegmentectomy for lung cancer is oncologically safe, but only when there is adequate intraoperative node staging. Rapid immunohistochemistry will assist clinicians when making intraoperative nodal diagnoses.

Knowledge of the histologic type and primary origin of pulmonary tumors is essential when preparing a surgical strategy. Intraoperative diagnosis of hematoxylin and eosin (H&E)‐stained frozen sections is the gold standard, but reliable pathology requires time‐consuming immunohistochemistry (IHC) to distinguish among histological types/organ origins and to analyze molecular status. The aim of this study was to evaluate the clinical reliability of a new rapid‐IHC technique for intraoperative diagnosis of pulmonary tumors. In total, 169 patients with undiagnosed pulmonary tumors were enrolled in a multicenter prospective observational study. At three institutes, pulmonary tumor samples were collected through core needle biopsy and/or surgery to determine surgical strategies. Using a new device for rapid IHC, we applied a high‐voltage, low‐frequency alternating current (AC) field, which mixes the available antibody as the voltage is switched on/off. Rapid IHC can provide tumor histologic type/origin diagnoses within 20 min, as opposed to the 3–6 h required for conventional IHC. No false diagnoses of malignancy were rendered in any of the cases when using simple H&E staining. With H&E staining alone, the overall definitive diagnosis rate, the rate of defined tumor origin, and the rate of determined histological type were 76.92%, 85.80%, and 90.53%, respectively. When rapid IHC was added, those rates were significantly improved to 88.76%, 94.67%, and 91.72%, respectively. By providing prompt and accurate intraoperative histological/molecular analysis, rapid IHC driven by AC mixing could serve as an effective clinical tool guiding the surgical strategy for undiagnosed pulmonary tumors.

The aim of this study was to evaluate whether the utility of cytokeratin (AE1/AE3) rapid immunohistochemistry (IHC) in combination with haematoxylin-eosin (H&E) staining improves the intraoperative diagnosis of spread through air spaces (STAS) on frozen sections (FS). This study included 153 patients. Three pathologists independently evaluated STAS on FS using either HE staining alone or in combination with rapid IHC, with postoperative paraffin sections serving as the gold standard. Sensitivity and specificity were compared, along with interobserver and intraobserver agreement (κ values), diagnostic time, and causes of misdiagnosis. Compared with HE alone, HE combined with rapid IHC staining increased mean diagnostic sensitivity from 73.7% to 87.8% and specificity from 85.5% to 89.9% across pathologists with varying levels of experience. Mean intraobserver agreement improved from κ = 0.644 (83.9%) to κ = 0.907 (95.9%), and mean interobserver agreement improved from κ = 0.485 (65.0%) to κ = 0.671 (77.2%). Average diagnostic time decreased from 2 min 53-23 s. Misdiagnoses were primarily attributable to a limited number of STAS clusters, confusion with macrophages, and artefacts. Intraoperative H&E combined with rapid IHC enhanced the sensitivity and specificity of STAS detection, improved interobserver/intraobserver agreement and reduced diagnostic time during FS evaluation without significantly extending the overall intraoperative FS processing time. Nevertheless, additional multicenter validation is required.

Rapid Immunohistochemistry With Thyroid Transcription Factor-1 for Pulmonary Adenocarcinoma