Interictal high-frequency oscillations, spikes, and connectivity profiles: A fingerprint of epileptogenic brain pathologies.

OBJECTIVE: Focal cortical dysplasia (FCD), hippocampal sclerosis (HS), nonspecific gliosis (NG), and normal tissue (NT) comprise the majority of histopathological results of surgically treated drug-resistant epilepsy patients. Epileptic spikes, high-frequency oscillations (HFOs), and connectivity measures are valuable biomarkers of epileptogenicity. The question remains whether they could also be utilized for preresective differentiation of the underlying brain pathology. This study explored spikes and HFOs together with functional connectivity in various epileptogenic pathologies. METHODS: Interictal awake stereoelectroencephalographic recordings of 33 patients with focal drug-resistant epilepsy with seizure-free postoperative outcomes were analyzed (15 FCD, 8 HS, 6 NT, and 4 NG). Interictal spikes and HFOs were automatically identified in the channels contained in the overlap of seizure onset zone and resected tissue. Functional connectivity measures (relative entropy, linear correlation, cross-correlation, and phase consistency) were computed for neighboring electrode pairs. RESULTS: Statistically significant differences were found between the individual pathologies in HFO rates, spikes, and their characteristics, together with functional connectivity measures, with the highest values in the case of HS and NG/NT. A model to predict brain pathology based on all interictal measures achieved up to 84.0% prediction accuracy. SIGNIFICANCE: The electrophysiological profile of the various epileptogenic lesions in epilepsy surgery patients was analyzed. Based on this profile, a predictive model was developed. This model offers excellent potential to identify the nature of the underlying lesion prior to resection. If validated, this model may be particularly valuable for counseling patients, as depending on the lesion type, different outcomes are achieved after epilepsy surgery.

Classification of brain lesions using a machine learning approach with cross-sectional ADC value dynamics.

Diffusion-weighted imaging (DWI) and its numerical expression via apparent diffusion coefficient (ADC) values are commonly utilized in non-invasive assessment of various brain pathologies. Although numerous studies have confirmed that ADC values could be pathognomic for various ring-enhancing lesions (RELs), their true potential is yet to be exploited in full. The article was designed to introduce an image analysis method allowing REL recognition independently of either absolute ADC values or specifically defined regions of interest within the evaluated image. For this purpose, the line of interest (LOI) was marked on each ADC map to cross all of the RELs' compartments. Using a machine learning approach, we analyzed the LOI between two representatives of the RELs, namely, brain abscess and glioblastoma (GBM). The diagnostic ability of the selected parameters as predictors for the machine learning algorithms was assessed using two models, the k-NN model and the SVM model with a Gaussian kernel. With the k-NN machine learning method, 80% of the abscesses and 100% of the GBM were classified correctly at high accuracy. Similar results were obtained via the SVM method. The proposed assessment of the LOI offers a new approach for evaluating ADC maps obtained from different RELs and contributing to the standardization of the ADC map assessment.

Twenty-five years of epilepsy surgery at a Central European comprehensive epilepsy center-Trends in intervention delay and outcomes.

OBJECTIVE: We analyzed trends in patients' characteristics, outcomes, and waiting times over the last 25 years at our epilepsy surgery center situated in Central Europe to highlight possible areas of improvement in our care for patients with drug-resistant epilepsy. METHODS: A total of 704 patients who underwent surgery at the Brno Epilepsy Center were included in the study, 71 of those were children. Patients were separated into three time periods, 1996-2000 (n = 95), 2001-2010 (n = 295) and 2011-2022 (n = 314) based on first evaluation at the center. RESULTS: The average duration of epilepsy before surgery in adults remained high over the last 25 years (20.1 years from 1996 to 2000, 21.3 from 2001 to 2010, and 21.3 from 2011 to 2020, P = 0.718). There has been a decrease in rate of surgeries for temporal lobe epilepsy in the most recent time period (67%-70%-52%, P < 0.001). Correspondingly, extratemporal resections have become more frequent with a significant increase in surgeries for focal cortical dysplasia (2%-8%-19%, P < 0.001). For resections, better outcomes (ILAE scores 1a-2) have been achieved in extratemporal lesional (0%-21%-61%, P = 0.01, at least 2-year follow-up) patients. In temporal lesional patients, outcomes remained unchanged (at least 77% success rate). A longer duration of epilepsy predicted a less favorable outcome for resective procedures (P = 0.024) in patients with disease duration of less than 25 years. SIGNIFICANCE: The spectrum of epilepsy surgery is shifting toward nonlesional and extratemporal cases. While success rates of extratemporal resections at our center are getting better, the average duration of epilepsy before surgical intervention is still very long and is not improving. This underscores the need for stronger collaboration between epileptologists and outpatient neurologists to ensure prompt and effective treatment for patients with drug-resistant epilepsy.

Safety assessment and redox status in rats after chronic exposure to cannabidiol and cannabigerol.

Cannabidiol (CBD) and cannabigerol (CBG) are the two main non-psychotropic phytocannabinoids with high application potential in drug development. Both substances are redox-active and are intensively investigated for their cytoprotective and antioxidant action in vitro. In this study, we focused on an in vivo safety evaluation and the effect of CBD and CBG on the redox status in rats in a 90-d experiment. The substances were administered orogastrically in a dose of 0.66 mg synthetic CBD or 0.66 mg/1.33 mg CBG/kg/day. CBD produced no changes in the red or white blood count or biochemical blood parameters in comparison to the control. No deviations in the morphology or histology of the gastrointestinal tract and liver were observed. After 90 d of CBD exposure, a significant improvement in redox status was found in the blood plasma and liver. The concentration of malondialdehyde and carbonylated proteins was reduced compared to the control. In contrast to CBD, total oxidative stress was significantly increased and this was accompanied by an elevated level of malondialdehyde and carbonylated proteins in CBG-treated animals. Hepatotoxic (regressive changes) manifestations, disruption in white cell count, and alterations in the ALT activity, level of creatinine and ionized calcium were also found in CBG-treated animals. Based on liquid chromatography-mass spectrometry analysis, CBD/CBG accumulated in rat tissues (in the liver, brain, muscle, heart, kidney and skin) at a low ng level per gram. Both CBD and CBG molecular structures include a resorcinol moiety. In CBG, there is an extra dimethyloctadienyl structural pattern, which is most likely responsible for the disruption to the redox status and hepatic environment. The results are valuable to further investigation of the effects of CBD on redox status and should contribute towards opening up critical discussion on the applicability of other non-psychotropic cannabinoids.

Spinal Metastasis in a Patient with Supratentorial Glioblastoma with Primitive Neuronal Component: A Case Report with Clinical and Molecular Evaluation.

Glioblastoma (GBM) is regarded as an aggressive brain tumor that rarely develops extracranial metastases. Despite well-investigated molecular alterations in GBM, there is a limited understanding of these associated with the metastatic potential. We herein present a case report of a 43-year-old woman with frontal GBM with primitive neuronal component who underwent gross total resection followed by chemoradiation. Five months after surgery, the patient was diagnosed with an intraspinal GBM metastasis. Next-generation sequencing analysis of both the primary and metastatic GBM tissues was performed using the Illumina TruSight Tumor 170 assay. The number of single nucleotide variants observed in the metastatic sample was more than two times higher. Mutations in TP53, PTEN, and RB1 found in the primary and metastatic tissue samples indicated the mesenchymal molecular GBM subtype. Among others, there were two inactivating mutations (Arg1026Ile, Trp1831Ter) detected in the NF1 gene, two novel NOTCH3 variants of unknown significance predicted to be damaging (Pro1505Thr, Cys1099Tyr), one novel ARID1A variant of unknown significance (Arg1046Ser), and one gene fusion of unknown significance, EIF2B5-KIF5B, in the metastatic sample. Based on the literature evidence, the alterations of NF1, NOTCH3, and ARID1A could explain, at least in part, the acquired invasiveness and metastatic potential in this particular GBM case.

Small RNA Sequencing Identifies a Six-MicroRNA Signature Enabling Classification of Brain Metastases According to their Origin.

BACKGROUND/AIM: Brain metastases (BMs) are the most frequent intracranial tumors in adults and one of the greatest challenges for modern oncology. Most are derived from lung, breast, renal cell, and colorectal carcinomas and melanomas. Up to 14% of patients are diagnosed with BMs of unknown primary, which are commonly characterized by an early and aggressive metastatic spread. It is important to discover novel biomarkers for early identification of BM origin, allowing better management of patients with this disease. Our study focused on microRNAs (miRNAs), which are very stable in frozen native and FFPE tissues and have been shown to be sensitive and specific diagnostic biomarkers of cancer. We aimed to identify miRNAs with significantly different expression in the five most frequent groups of BMs and develop a diagnostic classifier capable of sensitive and specific classification of BMs. MATERIALS AND METHODS: Total RNA enriched for miRNAs was isolated using the mirVana miRNA Isolation Kit from 71 fresh-frozen histopathologically confirmed BM tissues originating in 5 cancer types. Sequencing libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the NextSeq 500 platform. MiRNA expression was further validated by RT-qPCR. RESULTS: Differential analysis identified 373 miRNAs with significantly different expression between 5 BM groups (p<0.001). A classifier model was developed based on the expression of 6 miRNAs (hsa-miR-141-3p, hsa-miR-141-5p, hsa-miR-146a-5p, hsa-miR-194-5p, hsa-miR-200b-3p and hsa-miR-365b-5p) with the ability to correctly classify 91.5% of samples. Subsequent validation confirmed both significantly different expression of selected miRNAs in 5 BM groups as well as their diagnostic potential. CONCLUSION: To date, our study is the first to analyze miRNA expression in various types of BMs using small RNA sequencing to develop a diagnostic classifier and, thus, to help stratify BMs of unknown primary. The presented results confirm the importance of studying the dysregulated expression of miRNAs in BMs and the diagnostic potential of the validated 6-miRNA signature.

Disulfiram increases the efficacy of 5-fluorouracil in organotypic cultures of colorectal carcinoma.

Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.

Blood-Brain Barrier Alterations and Edema Formation in Different Brain Mass Lesions.

Differential diagnosis of brain lesion pathologies is complex, but it is nevertheless crucial for appropriate clinical management. Advanced imaging methods, including diffusion-weighted imaging and apparent diffusion coefficient, can help discriminate between brain mass lesions such as glioblastoma, brain metastasis, brain abscesses as well as brain lymphomas. These pathologies are characterized by blood-brain barrier alterations and have been extensively studied. However, the changes in the blood-brain barrier that are observed around brain pathologies and that contribute to the development of vasogenic brain edema are not well described. Some infiltrative brain pathologies such as glioblastoma are characterized by glioma cell infiltration in the brain tissue around the tumor mass and thus affect the nature of the vasogenic edema. Interestingly, a common feature of primary and secondary brain tumors or tumor-like brain lesions characterized by vasogenic brain edema is the formation of various molecules that lead to alterations of tight junctions and result in blood-brain barrier damage. The resulting vasogenic edema, especially blood-brain barrier disruption, can be visualized using advanced magnetic resonance imaging techniques, such as diffusion-weighted imaging and apparent diffusion coefficient. This review presents a comprehensive overview of blood-brain barrier changes contributing to the development of vasogenic brain edema around glioblastoma, brain metastases, lymphomas, and abscesses.

Metal Oxide Laser Ionization Mass Spectrometry Imaging of Fatty Acids and Their Double Bond Positional Isomers.

We present a novel combination of a metal oxide laser ionization mass spectrometry imaging (MOLI MSI) technique with off-line lipid derivatization by ozone for the detection of fatty acids (FA) and their carbon-carbon double bond (C═C) positional isomers in biological tissues. MOLI MSI experiments were realized with CeO2 and TiO2 nanopowders using a vacuum matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer in the negative mode. The catalytic properties of these metal oxides allow FA cleavage from phospholipids under UV laser irradiation. At the same time, fragile ozonides produced at the sites of unsaturation decomposed, yielding four diagnostic ions specific for the C═C positions. Advantageously, two MOLI MSI runs from a single tissue sprayed with the metal oxide suspension were performed. The first run prior to ozone derivatization revealed the distribution of FAs, while the second run after the reaction with ozone offered additional information about FA C═C isomers. The developed procedure was demonstrated on MSI of a normal mouse brain and human colorectal cancer tissues uncovering the differential distribution of FAs down to the isomer level. Compared to the histological analysis, MOLI MSI showed the distinct distribution of specific FAs in different functional parts of the brain and in healthy and cancer tissues pointing toward its biological relevance. The developed technique can be directly adopted by laboratories with MALDI TOF analyzers and help in the understanding of the local FA metabolism in tissues.

HLA-E and HLA-F Are Overexpressed in Glioblastoma and HLA-E Increased After Exposure to Ionizing Radiation.

BACKGROUND/AIM: Glioblastoma (GBM) is one of the deadliest human cancers responding very poorly to therapy. Although the central nervous system has been traditionally considered an immunologically privileged site with an enhanced immune response, GBM appears to benefit from this immunosuppressive milieu. Immunomodulatory molecules play an important role in immune tumor-host interactions. Non-classical human leukocyte antigens (HLA) class Ib molecules HLA-E, HLA-F, and HLA-G have been previously described to be involved in protecting semi-allogeneic fetal allografts from the maternal immune response and in transplant tolerance as well as tumoral immune escape. Unfortunately, their role in GBM remains poorly understood. Our study, therefore, aimed to characterize the relationship between the expression of these molecules in GBM on the transcriptional level and clinicopathological and molecular features of GBM as well as the effect of ionizing radiation. MATERIALS AND METHODS: We performed the analysis of HLA-E, HLA-F, and HLA-G mRNA expression in 69 GBM tissue samples and 21 non-tumor brain tissue samples (controls) by reverse transcription polymerase chain reaction. Furthermore, two primary GBM cell cultures had been irradiated to identify the effect of ionizing radiation on the expression of non-classical HLA molecules. RESULTS: Analyses revealed that both HLA-E and HLA-F are significantly up-regulated in GBM samples. Subsequent survival analysis showed a significant association between low expression of HLA-E and shorter survival of GBM patients. The dysregulated expression of both molecules was also observed between patients with methylated and unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Finally, we showed that ionizing radiation increased HLA-E expression level in GBM cells in vitro. CONCLUSION: HLA-E and HLA-F play an important role in GBM biology and could be used as diagnostic biomarkers, and in the case of HLA-E also as a prognostic biomarker.

Cytokine-chemokine profiles in the hippocampus of patients with mesial temporal lobe epilepsy and hippocampal sclerosis.

PURPOSE: Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is the most common drug-resistant epilepsy. Despite major advances in epilepsy research, the epileptogenesis of the MTLE-HS is not well understood. The altered neuroimmune response is one of the pathomechanisms linked to progressive epileptogenesis in MTLE-HS, and understanding its role may help design future cures for pharmaco-resistant MTLE-HS. Here, the neuroimmune function was evaluated by the assessment of cytokine-chemokine profiles in brain samples from the hippocampus of patients with MTLE-HS. METHODS: Brain samples from patients with MTLE-HS collected during epileptosurgical resection (n = 21) were compared to those obtained from autopsy controls (n = 13). The typing of HS was performed according to ILAE consensus classification, and patients were additionally sorted into subgroups based on the severity of neuronal depletion (Wyler grading system). Differences between patients with MTLE-HS with and without a history of febrile seizures were also assessed. RNA was isolated from native samples, and real-time gene expression analysis of cytokine-chemokine profiles, i.e., levels of IL-1ß, IL-6, IL-10, IL-18, CCL2, CCL3, CCL4, and STAT3, was carried out by qRT-PCR methodology. RESULTS: Upregulation of IL-1ß (p = 0.001), IL-18 (p = 0.0018), CCL2 (p = 0,0377), CCL3 (p < 0.001), and CCL4 (p < 0.001) in MTLE-HS patients was detected when compared to the post-mortem hippocampal samples collected from autopsy controls. The STAT3 expression was higher in more severe neuronal loss and glial scaring determined by different Wyler grades in HS patients. Furthermore, cytokine-chemokine profiles were not different in MTLE-HS patients with or without febrile seizures. CONCLUSION: The upregulation of specific cytokines and chemokines in MTLE-HS provides evidence that the neuroinflammatory process contributes to MTLE epileptogenesis. History of febrile seizures did not alter the immune profiles. Specific immune mediators and related immune pathways represent potential therapeutic targets for seizure control and pharmacoresistancy prevention in MTLE associated with hippocampal sclerosis.

Prolonged survival in patients with local chronic infection after high-grade glioma treatment: Two case reports.

High-grade gliomas are primary brain tumors with poor prognosis, despite surgical treatment followed by radiotherapy and concomitant chemotherapy. We present two cases of long-term survival in patients treated for high-grade glioma and concomitant prolonged bacterial wound infection. The first patient treated for glioblastoma IDH-wildtype had been without disease progression for 61 months from the first resected recurrence. Despite incomplete chemotherapy-induced myelosuppression in the second patient with anaplastic astrocytoma IDH-mutant, she died without disease relapse after 14 years from the diagnosis due to other comorbidities. We assume that the documented prolonged survival could be related to the bacterial infection.

Small RNA Sequencing Identifies PIWI-Interacting RNAs Deregulated in Glioblastoma-piR-9491 and piR-12488 Reduce Tumor Cell Colonies In Vitro.

Glioblastoma (GBM) is the most frequently occurring primary malignant brain tumor of astrocytic origin. To change poor prognosis, it is necessary to deeply understand the molecular mechanisms of gliomagenesis and identify new potential biomarkers and therapeutic targets. PIWI-interacting RNAs (piRNAs) help in maintaining genome stability, and their deregulation has already been observed in many tumors. Recent studies suggest that these molecules could also play an important role in the glioma biology. To determine GBM-associated piRNAs, we performed small RNA sequencing analysis in the discovery set of 19 GBM and 11 non-tumor brain samples followed by TaqMan qRT-PCR analyses in the independent set of 77 GBM and 23 non-tumor patients. Obtained data were subsequently bioinformatically analyzed. Small RNA sequencing revealed 58 significantly deregulated piRNA molecules in GBM samples in comparison with non-tumor brain tissues. Deregulation of piR-1849, piR-9491, piR-12487, and piR-12488 was successfully confirmed in the independent groups of patients and controls (all p < 0.0001), and piR-9491 and piR-12488 reduced GBM cells' ability to form colonies in vitro. In addition, piR-23231 was significantly associated with the overall survival of the GBM patients treated with Stupp regimen (p = 0.007). Our results suggest that piRNAs could be a novel promising diagnostic and prognostic biomarker in GBM potentially playing important roles in gliomagenesis.

Epilepsy miRNA Profile Depends on the Age of Onset in Humans and Rats.

Temporal lobe epilepsy (TLE) is a severe neurological disorder accompanied by recurrent spontaneous seizures. Although the knowledge of TLE onset is still incomplete, TLE pathogenesis most likely involves the aberrant expression of microRNAs (miRNAs). miRNAs play an essential role in organism homeostasis and are widely studied in TLE as potential therapeutics and biomarkers. However, many discrepancies in discovered miRNAs occur among TLE studies due to model-specific miRNA expression, different onset ages of epilepsy among patients, or technology-related bias. We employed a massive parallel sequencing approach to analyze brain tissues from 16 adult mesial TLE (mTLE)/hippocampal sclerosis (HS) patients, 8 controls and 20 rats with TLE-like syndrome, and 20 controls using the same workflow and categorized these subjects based on the age of epilepsy onset. All categories were compared to discover overlapping miRNAs with an aberrant expression, which could be involved in TLE. Our cross-comparative analyses showed distinct miRNA profiles across the age of epilepsy onset and found that the miRNA profile in rats with adult-onset TLE shows the closest resemblance to the profile in mTLE/HS patients. Additionally, this analysis revealed overlapping miRNAs between patients and the rat model, which should participate in epileptogenesis and ictogenesis. Among the overlapping miRNAs stand out miR-142-5p and miR-142-3p, which regulate immunomodulatory agents with pro-convulsive effects and suppress neuronal growth. Our cross-comparison study enhanced the insight into the effect of the age of epilepsy onset on miRNA expression and deepened the knowledge of epileptogenesis. We employed the same methodological workflow in both patients and the rat model, thus improving the reliability and accuracy of our results.

Pre-Radiotherapy Progression after Surgery of Newly Diagnosed Glioblastoma: Corroboration of New Prognostic Variable.

BACKGROUND: The aim of this retrospective study is to assess the incidence, localization, and potential predictors of rapid early progression (REP) prior to initiation of radiotherapy in newly diagnosed glioblastoma patients and to compare survival outcomes in cohorts with or without REP in relation to the treatment. METHODS: We assessed a consecutive cohort of 155 patients with histologically confirmed irradiated glioblastoma from 1/2014 to 12/2017. A total of 90 patients with preoperative, postoperative, and planning MRI were analyzed. RESULTS: Median age 59 years, 59% men, and 39 patients (43%) underwent gross total tumor resection. The Stupp regimen was indicated to 64 patients (71%); 26 patients (29%) underwent radiotherapy alone. REP on planning MRI performed shortly prior to radiotherapy was found in 46 (51%) patients, most often within the surgical cavity wall, and the main predictor for REP was non-radical surgery (p < 0.001). The presence of REP was confirmed as a strong negative prognostic factor; median overall survival (OS) in patients with REP was 10.7 vs. 18.7 months and 2-year survival was 15.6% vs. 37.7% (hazard ratio HR 0.53 for those without REP; p = 0.007). Interestingly, the REP occurrence effect on survival outcome was significantly different in younger patients (≤ 50 years) and older patients (> 50 years) for OS (p = 0.047) and non-significantly for PFS (p = 0.341). In younger patients, REP was a stronger negative prognostic factor, probably due to more aggressive behavior. Patients with REP who were indicated for the Stupp regimen had longer OS compared to radiotherapy alone (median OS 16.0 vs 7.5; HR = 0.5, p = 0.022; 2-year survival 22.3% vs. 5.6%). The interval between surgery and the initiation of radiotherapy were not prognostic in either the entire cohort or in patients with REP. CONCLUSION: Especially in the subgroup of patients without radical resection, one may recommend as early initiation of radiotherapy as possible. The phenomenon of REP should be recognized as an integral part of stratification factors in future prospective clinical trials enrolling patients before initiation of radiotherapy.

Real-World Evidence in Glioblastoma: Stupp's Regimen After a Decade.

The aim of this retrospective study is to provide real-world evidence in glioblastoma treatment and to compare overall survival after Stupp's regimen treatment today and a decade ago. A current consecutive cohort of histologically confirmed glioblastoma irradiated from 1/2014 to 12/2017 in our cancer center was compared with an already published historical control of patients treated in 1/2003-12/2009. A total of new 155 patients was analyzed, median age 60.9 years, 61% men, 58 patients (37%) underwent gross total tumor resection. Stupp's regimen was indicated in 90 patients (58%), 65 patients (42%) underwent radiotherapy alone. Median progression-free survival in Stupp's regimen cohort was 6.7 months, median OS 16.0 months, and 2-year OS 30.7%. OS was longer if patients were able to finish at least three cycles of adjuvant chemotherapy (median 23.3 months and 43.9% of patients lived at 2 years after surgery). Rapid early progression prior to radiotherapy was a negative prognostic factor with HR 1.87 (p = 0.007). The interval between surgery and the start of radiotherapy (median 6.7 weeks) was not prognostically significant (p = 0.825). The median OS in the current cohort was about 2 months longer than in the historical control group treated 10 years ago (16 vs. 13.8 months) using the same Stupp's regimen. Taking into account differences in patient's characteristics between current and historical cohorts, age, extent of resection, and ECOG patient performance status adjusted HR (Stupp's regimen vs. RT alone) for OS was determined as 0.45 (p = 0.002).

The tumor immune microenvironment and its implications for clinical outcome in patients with oropharyngeal squamous cell carcinoma.

BACKGROUND: We examined PD-L1 expression on tumor cells (TCs) and immune cells (ICs) and density of CD3+ and CD8+ tumor-infiltrating lymphocytes (TILs) in patients with oropharyngeal squamous cell carcinoma (OPSCC) and investigated their significance on clinicopathological characteristics and clinical outcomes. METHODS: In a cohort of 65 patients treated by definitive intensity-modulated radiotherapy (IMRT) with curative intent, immunohistochemical analysis of PD-L1 expression on TCs and ICs, and TIL subtyping was performed on primary biopsy tumor tissues, followed by prognostic evaluation of these immune response-related parameters including classification into four tumor immune microenvironment (TIM) types. To evaluate HPV status, p16 immunohistochemistry was performed. RESULTS: Densities of CD3+ and CD8+ TILs and PD-L1 expressions on TCs and ICs were significantly higher in p16+/HPV-mediated OPSCC. Patients with high densities of stromal CD8+ TILs displayed significantly better overall survival (OS) and progression-free survival (PFS). PD-L1 expression neither on tumor cells nor on immune cells affected survival outcomes. Distribution of TIM types based on the combination of PD-L1 expression on TCs and densities of CD8+ TILs is significantly different in p16+ compared with p16- OPSCC. In type III TIM (TC-PD-L1+/low CD8+ TIL density), significantly better OS was shown in p16+ group compared with p16- OPSCC. CONCLUSION: The prognostic and predictive role of tumor immune microenvironment was confirmed for patients with OPSCC. Combining HPV status with the evaluation of densities of CD8+ TILs and PD-L1 expression including TIM classification might be of high clinical interest and warrants further prospective evaluation.

Ozonization of Tissue Sections for MALDI MS Imaging of Carbon-Carbon Double Bond Positional Isomers of Phospholipids.

Visualizing the differential distribution of carbon-carbon double bond (C═C db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of C═C db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 µm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.

Composite Hemostatic Nonwoven Textiles Based on Hyaluronic Acid, Cellulose, and Etamsylate.

The achievement of rapid hemostasis represents a long-term trend in hemostatic research. Specifically, composite materials are now the focus of attention, based on the given issues and required properties. In urology, different materials are used to achieve fast and effective hemostasis. Additionally, it is desirable to exert a positive influence on local tissue reaction. In this study, three nonwoven textiles prepared by a wet spinning method and based on a combination of hyaluronic acid with either oxidized cellulose or carboxymethyl cellulose, along with the addition of etamsylate, were introduced and assessed in vivo using the rat partial nephrectomy model. A significantly shorter time to hemostasis in seconds (p < 0.05), was attributed to the effect of the carboxymethyl cellulose material. The addition of etamsylate did not noticeably contribute to further hemostasis, but its application strengthened the structure and therefore significantly improved the effect on local changes, while also facilitating any manipulation by the surgeons. Specifically, the hyaluronic acid supported the tissue healing and regeneration, and ensured the favorable results of the histological analysis. Moreover, the prepared textiles proved their bioresorbability after a three-day period. In brief, the fabrics yielded favorable hemostatic activity, bioresorbability, non-irritability, and had a beneficial effect on the tissue repair.

Prognostic impact of combined immunoprofiles in oropharyngeal squamous cell carcinoma patients with respect to AJCC 8th edition.

OBJECTIVES: To examine combined immunoprofiles of epidermal growth factor receptor (EGFR), CD44, and p16 in oropharyngeal squamous cell carcinoma (OPSCC) and to correlate them with radiotherapy treatment outcomes and clinicopathological parameters. Prognostic impact of the American Joint Committee on Cancer (AJCC) 8th edition staging system in comparison with 7th edition was analyzed. METHODS: The study included 77 OPSCC patients treated by definitive intensity-modulated radiotherapy (IMRT). Clinical staging was assessed according to the AJCC, both 7th and 8th edition. Immunohistochemical (IHC) analysis of CD44 and EGFR was performed on primary biopsy tumor tissues. To evaluate the HPV status, IHC detection of p16 was employed. RESULTS: The AJCC 8th edition staging system revealed correlations between overall survival (OS), progression-free survival (PFS), locoregional control (LRC), and clinical stage. EGFR and CD44 positivity (+) and p16 negativity (-) were associated with clinical stage IV of the disease. CD44+ and EGFR+ OPSCC displayed worse OS and LRC, and these cases also showed the worst 3-year OS and LRC. Combined analysis of protein expressions identified an association between p16- and EGFR+, p16- and CD44+, EGFR+, and CD44+. Combined immunoprofiles CD44+/p16-, EGFR+/p16-, and EGFR+/CD44+ were associated with worst OS and LRC. CONCLUSIONS: Combined immunoprofiles of p16, EGFR, and CD44 might provide valuable prognostic and predictive information for the individual OPSCC patients, especially in terms of response to IMRT and prediction of treatment outcomes. Application of the AJCC 8th edition staging for HPV+ OPSCC proved to improve hazard discrimination and prognostication of OPSCC.