Fluorescein-stained confocal laser endomicroscopy versus conventional frozen section for intraoperative histopathological assessment of intracranial tumors.

BACKGROUND: The aim of this clinical trial was to compare Fluorescein-stained intraoperative confocal laser endomicroscopy (CLE) of intracranial lesions and evaluation by a neuropathologist with routine intraoperative frozen section (FS) assessment by neuropathology. METHODS: In this phase II noninferiority, prospective, multicenter, nonrandomized, off-label clinical trial (EudraCT: 2019-004512-58), patients above the age of 18 years with any intracranial lesion scheduled for elective resection were included. The diagnostic accuracies of both CLE and FS referenced with the final histopathological diagnosis were statistically compared in a noninferiority analysis, representing the primary endpoint. Secondary endpoints included the safety of the technique and time expedited for CLE and FS. RESULTS: A total of 210 patients were included by 3 participating sites between November 2020 and June 2022. Most common entities were high-grade gliomas (37.9%), metastases (24.1%), and meningiomas (22.7%). A total of 6 serious adverse events in 4 (2%) patients were recorded. For the primary endpoint, the diagnostic accuracy for CLE was inferior with 0.87 versus 0.91 for FS, resulting in a difference of 0.04 (95% confidence interval -0.10; 0.02; P = .367). The median time expedited until intraoperative diagnosis was 3 minutes for CLE and 27 minutes for FS, with a mean difference of 27.5 minutes (standard deviation 14.5; P < .001). CONCLUSIONS: CLE allowed for a safe and time-effective intraoperative histological diagnosis with a diagnostic accuracy of 87% across all intracranial entities included. The technique achieved histological assessments in real time with a 10-fold reduction of processing time compared to FS, which may invariably impact surgical strategy on the fly.

A balanced score to predict survival of elderly patients newly diagnosed with glioblastoma.

BACKGROUND: Over the past years, several treatment regimens have been recommended for elderly patients with glioblastoma (GBM), ranging from ultrahypofractionated radiotherapy (RT) over monochemotherapy (ChT) to combined radiochemotherapy (RChT). The current guidelines recommend active treatment in elderly patients in cases with a KPS of at least 60%. We established a score for selecting patients with a very poor prognosis from patients with a better prognosis. METHODS: One hundred eighty one patients ≥65 years old, histologically diagnosed with GBM, were retrospectively evaluated. Clinical characteristics were analysed for their impact on the overall survival (OS). Factors which were significant in univariate analysis (log-rank test, p < 0.05) were included in a multi-variate model (multi-variate Cox regression analysis, MVA). The 9-month OS for the significant factors after MVA (p < 0.05) was included in a prognostic score. Score sums with a median OS of < and > 6 months were summarized as Group A and B, respectively. RESULTS: Age, KPS, MGMT status, the extent of resection, aphasia after surgery and motor dysfunction after surgery were significantly associated with OS on univariate analysis (p < 0.05). On MVA age (p 0.002), MGMT promotor methylation (p 0.013) and Karnofsky performance status (p 0.005) remained significant and were included in the score. Patients were divided into two groups, group A (median OS of 2.7 months) and group B (median OS of 7.8 months). The score was of prognostic significance, independent of the adjuvant treatment regimen. CONCLUSIONS: The score distinguishes patients with a poor prognosis from patients with a better prognosis. Its inclusion in future retrospective or prospective trials could help enhance the comparability of results. Before its employment on a routine basis, external validation is recommended.

Absence of the mutated Trp2 allele but a common polymorphism of the COL9A2 collagen gene is associated with early recurrence after lumbar discectomy in a German population.

Genetic factors seem to play a role in symptomatic lumbar disc disease (LDD). It has been shown previously that a tryptophan mutation of the COL9A2 gene is a major risk factor for LDD in a Finish population. The impact of collagen gene variations on the relapse rate after lumbar discectomy, however, has not been studied so far. Here, we conducted a cross-sectional genotyping study of patients who underwent lumbar discectomy to determine the influence of a COL9A2 mutation on the recurrence rates. Biopsy samples from 288 patients suffering from LDD with and without relapse were analyzed by PCR restriction fragment analysis and direct sequencing. The mutated Trp2 allele was not detected in the patients' samples of the present study. However, nine patients with recurrent LDD, but only two without recurrence were homozygous for the Arg allele. Homozygosity for the Arg allele of Col9A2 seems to be more frequent in the patient group with early recurrence although the differences in the allele frequencies were statistically not significant. In contrast, the Trp2 mutation seems not to be a major susceptibility factor for LDD in a German population.

Diffusion tensor image features predict IDH genotype in newly diagnosed WHO grade II/III gliomas.

We hypothesized that machine learning analysis based on texture information from the preoperative MRI can predict IDH mutational status in newly diagnosed WHO grade II and III gliomas. This retrospective study included in total 79 consecutive patients with a newly diagnosed WHO grade II or III glioma. Local binary pattern texture features were generated from preoperative B0 and fractional anisotropy (FA) diffusion tensor imaging. Using a training set of 59 patients, a single hidden layer neural network was then trained on the texture features to predict IDH status. The model was validated based on the prediction accuracy calculated in a previously unseen set of 20 gliomas. Prediction accuracy of the generated model was 92% (54/59 cases; AUC = 0.921) in the training and 95% (19/20; AUC = 0.952) in the validation cohort. The ten most important features were comprised of tumor size and both B0 and FA texture information, underlining the joint contribution of imaging data to classification. Machine learning analysis of DTI texture information and tumor size reliably predicts IDH status in preoperative MRI of gliomas. Such information may increasingly support individualized surgical strategies, supplement pathological analysis and highlight the potential of radiogenomics.

Ultrasmall supraparamagnetic iron oxide-enhanced magnetic resonance imaging of antigen-induced arthritis: a comparative study between SHU 555 C, ferumoxtran-10, and ferumoxytol.

OBJECTIVES: We sought to compare the ability of 3 ultrasmall superparamagnetic iron oxides (USPIOs) to detect and characterize antigen-induced arthritis with MR imaging. MATERIALS AND METHODS: A monoarthritis was induced in the right knee of 18 rats. The left knee served as a normal control. Knees underwent magnetic resonance (MR) imaging before, up to 2 hours, and 24 hours after injection (p.i.) of 200 mumol Fe/kg SHU 555 C (n= 6), ferumoxtran-10 (n = 6), or ferumoxytol (n = 6), using T2-2D-SE 100/20,40,60,80/90 (TR/TE/flipangle), T2*-3D-spoiled gradient recalled (SPGR) 100/15/38, and T1-3D-SPGR 50/1,7/60 sequences. RESULTS: Quantitative signal to noise ratio and DeltaSI data of arthritic knees on T1- and T2*-weighted MR images showed no significant differences between the 3 USPIOs (P > 0.05). At 2 hours p.i., SNR and DeltaSI data were significantly increased from baseline on T1-weighted images and significantly decreased on T2*-weighted images (P < 0.001). At 24 hours p.i., the T1-enhancement returned to baseline, whereas the T2*-enhancement remained significantly elevated (P < 0.001). Immunostains demonstrated an USPIO compartmentalization in macrophages in the arthritic synovium. CONCLUSIONS: Based on the relatively small number of animals in our study group, inflammation in antigen-induced arthritis can be equally detected and characterized with any of the three USPIOs evaluated.

Prognostic Value of O-(2-[18F]-Fluoroethyl)-L-Tyrosine-Positron Emission Tomography Imaging for Histopathologic Characteristics and Progression-Free Survival in Patients with Low-Grade Glioma.

OBJECTIVE: O-(2-[18F]-fluoroethyl)-L-tyrosine positron emission tomography ((18)F-FET-PET) imaging is applied for tumor grading, prognostic stratification, and diagnosis of tumor recurrence, especially in high-grade gliomas. Experience with (18)F-FET-PET imaging in low-grade gliomas is limited. Therefore, the objective of the present study was to assess (18)F-FET-PET tracer uptake in low-grade gliomas and to investigate possible correlations with contrast enhancement in magnetic resonance imaging (MRI) and histopathology. METHODS: A total of 65 patients (29 female, 36 male, median age 38 years) with newly diagnosed or recurrent low-grade gliomas for whom preoperative MRI and (18)F-FET-PET imaging were available were included. Tumor entity, tumor location, as well as histopathology (isocitrate dehydrogenase [IDH] 1/2 mutation, Ki67, p53, oligodendroglial differentiation, 1p19q codeletion), and progression-free survival were assessed. (18)F-FET-PET images were acquired and fused to MRI (T2-weighted fluid-attenuated inversion recovery) and tumor volume was measured in areas with a tumor-to-background ratio >1.3, >1.6, and >2.0 and in MRI. RESULTS: PET tracer uptake was observed in 78.5% of all World Health Organization Grade I and II tumors. (18)F-FET uptake showed a high negative predictive value for oligodendroglial components and for 1p19q codeletion. No further significant correlation between histologic features, progression-free survival, or IDH1/2 mutation status and tracer uptake was observed. CONCLUSIONS: We found that 78.5% of low-grade gliomas do show elevated tracer uptake in (18)F-FET-PET imaging. Low-grade glioma without tracer uptake exclude oligodendroglial differentiation and 1p19q codeletion. Further differentiation between molecular subtypes is not possible with static (18)F-FET-PET. No correlation of progression-free survival to tracer uptake and IDH1/2-mutation status was observed.

In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways.

Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.

Epstein-Barr virus infection is strictly associated with the metastatic spread of sinonasal squamous-cell carcinomas.

BACKGROUND: Sinonasal squamous-cell carcinomas (SNSCC) are relatively rare. Thus, data regarding the rate of lymph node metastases are inconsistent in contrast with well-known high metastasis rates in squamous-cell carcinomas of the head and neck (HNSCC) (oral cavity, pharynx and larynx). Hence, the indication for elective neck dissection is difficult in SNSCC. The aim of this study was to assess common genetic alterations and EBV and HPV status as a function of metastasis in SNSCC and HNSCC. METHODS: We retrospectively analyzed 44 SNSCC and 65 HNSCC for TP53, EGFR, KRAS, PIK3CA and BRAF mutations using a high-resolution melting analysis followed by Sanger sequencing. EBV and HPV detection was performed using in situ hybridization for virus encoded RNA. Tumor-associated p16(INK4a) expression was visualized by immunohistochemistry and correlated with HPV infection. The mutation data, EBV and HPV status were statistically compared with the clinical data in SNSCC and HNSCC. RESULTS: TP53 mutations were exclusively associated with shorter survival in SNSCC (p=0.048). All the other markers had no effect on the metastasis rate and survival. In total, 20 of 44 SNSCC were EBV-positive. Only these EBV positive tumors developed lymph node or distant metastases (p=0.008). LMP1 was positive in 14/44 patients. When combining both methods significance for a correlation between EBV/LMP1 positive patients and metastases was even higher (p=0.001). CONCLUSION: In SNSCC, the presence of EBV is strictly associated with metastasis. We recommend an elective neck dissection in patients with EBV-positive SNSCC.

Mismatch repair protein hMSH2 in primary drug resistance in in vitro human malignant gliomas.

OBJECT: The mismatch repair (MMR) system has previously been implicated in acquired chemoresistance in malignant gliomas in humans. Its impact on the primary chemoresistance in glioblastoma multiforme (GBM) has not been determined in detail, however. METHODS: The authors investigated the expression of both the MMR genes (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6) at the transcriptional level through reverse transcription-polymerase chain reaction and the hMSH2 protein through Western blot and immunohistochemical analysis of tumor tissue and primary cell cultures of 25 in vitro human de novo GBMs without prior experimental treatment. Results of these analyses were compared with data on in vitro chemoresistance to nine drugs that are in general use in glioma therapy. All MMR genes were expressed in the GBMs, with no significant difference among the individual tumors except in one respect; that is, GBMs showed either relatively high levels or barely detectable levels of hMSH2 messenger (m)RNA and protein expression. All multiresistant tumors demonstrated high hMSH2 expression, and all but two of the sensitive tumors exhibited low hMSH2 mRNA levels. CONCLUSIONS: Analysis of the data indicates that functional alterations of the MMR system are involved in the primary drug resistance in in vitro human malignant gliomas. Analysis of hMSH2 expression might therefore predict therapeutic responses in humans with GBMs.

The role of growth factors in central nervous system tumours.

The role of growth factors in tumour growth and progression has increasingly been studied over the last few years. This review summarizes the available data and discusses their limitations as well as their potential influence on future therapeutic strategies. A large body of data suggests an important role of EGF, TGF-beta, PDGF and VEGF ligands and receptors in the vascularization of several brain tumour types, including gliomas and meningiomas. Recent experimental data indicate that inhibition of the signalling pathways may represent promising therapeutic strategies. Some inhibitory agents have now entered clinical trials, mainly for recurrent gliomas. Early results are presented.

Hypoxia upregulates aldehyde dehydrogenase isoform 1 (ALDH1) expression and induces functional stem cell characteristics in human glioblastoma cells.

Aldehyde dehydrogenase 1 (ALDH1) has been used to isolate tumorigenic stem-like cells in a large number of tumors, including glioblastoma multiforme (GBM). We recently showed that human glioblastoma cells with high ALDH1 (ALDH1(high)) activity contain stem-cell-like characteristics. In the study reported here, we isolated established and primary human glioblastoma cells based on their ALDH1 expression. When tested for asymmetric division, only cells with ALDH1(high) expression were able to restore heterogeneous populations after a few days, whereas cells with ALDH1(low) levels could not. Most interestingly, the capacity of cells with ALDH1(low) levels to divide asymmetrically into cells with either ALDH1(high) or ALDH1(low) expression could be restored after exposure to hypoxic culture conditions. Consequently, we found neurosphere formation reinstated in posthypoxic, formerly ALDH1(low), cells. The direct involvement of ALDH1 could be confirmed by ALDH1 small hairpin ribonucleic acid (shRNA) knockdown, suggesting ALDH1 as an intracellular marker for the identification and isolation of stem-like glioblastoma cells. In summary, we show that ALDH1 expression correlates well with asymmetric division capacity and tumor sphere formation. Furthermore, we demonstrated that hypoxic culture conditions induce and/or upregulate ALDH1 expression in established and primary GBM cell lines.

Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

MRI of arthritis: comparison of ultrasmall superparamagnetic iron oxide vs. Gd-DTPA.

PURPOSE: To compare the ability of the ultrasmall superparamagnetic iron oxide (USPIO) SHU555C vs. gadopentetate dimeglumine (Gd-DTPA) to detect antigen-induced monoarthritis with MRI. MATERIALS AND METHODS: Twelve seven-week-old female rats with an antigen-induced monoarthritis of the right knee were randomly assigned to two groups. Animals in group I (N = 6) underwent MRI using T1-weighted gradient-echo sequences before injection and at 2, 9, 17, 25, 33, 40, 47, 55, and 63 minutes postinjection (p.i.) of Gd-DTPA on day 1, and before injection and at 3, 23, 43, and 123 minutes p.i. of SHU555C on day 2. Animals in group II (N = 6) were imaged before injection and at 3, 23, 43, and 123 minutes p.i. using identical sequences. Signal-to-noise ratios (SNRs) and relative enhancement (DeltaSI%) of arthritic and normal synovium were determined from region-of-interest (ROI) measurements in consensus reading by two experienced radiologists. Data were tested for significant differences between the two agents and between the arthritic and normal knees using a mixed-effect model and F-tests (P < 0.05). Joints were processed for histopathology as the gold standard. RESULTS: USPIO and Gd-DTPA showed significant enhancement differences (P < 0.001). USPIO provided a progressive and persistent enhancement of arthritic joints while Gd-DTPA provided an early and rapidly declining enhancement. Maximal enhancement in synovitis was 400% at 40-120 minutes p.i. of USPIO vs. 300% at two minutes p.i. of Gd-DTPA. USPIO provided a significant higher difference in enhancement between the arthritic and normal synovium than Gd-DTPA (P < 0.001). Histopathology confirmed marked inflammatory synovial changes in all arthritis-induced right knee joints and normal synovium in all left knee joints. CONCLUSION: Both USPIO and Gd-DTPA detect arthritis by positive T1-enhancement. Compared to standard Gd-DTPA, the USPIO SHU555C provides a comparable maximal T1-enhancement (at two minutes p.i for Gd-DTPA and between 43 and 123 minutes p.i. for SHU555C), but in addition it provides a prolonged T1-enhancement of synovitis and a higher difference between the relative enhancement of arthritic and normal synovium.

Optical imaging of experimental arthritis using allogeneic leukocytes labeled with a near-infrared fluorescent probe.

PURPOSE: The purpose of this study was to assess the feasibility of inflammation detection in an antigen-induced arthritis model using fluorescent leukocytes and optical imaging. METHODS: Antigen-mediated monoarthritis was induced in the right knee of 12 Sprague-Dawley rats. Six rats remained untreated and six rats were treated with cortisone. All rats received ex vivo fluorescent-labeled rat leukocytes. Optical images of both knees were acquired before and at 5 min, 1 h, 4 h, and 24 h after cell injection. Images were evaluated qualitatively and quantitatively by calculating signal intensity ratios between the right arthritic (A) and contralateral normal (N) knee. A/N ratios were tested for significant differences between baseline values and values after cell injection using a paired t test as well as between the untreated and cortisone-treated group using an unpaired t test. Synovial specimens were processed and evaluated for labeled cells with fluorescence microscopy. RESULTS: At 4 h and 24 h p.i., the A/N ratios of untreated arthritic knees showed a significant signal increase compared with baseline values (p<0.05) and a significant difference compared with A/N ratios of cortisone-treated animals (p<0.05). Fluorescent microscopy confirmed the presence of labeled cells in the arthritic synovium. CONCLUSION: Inflammation in antigen-induced arthritis can be detected with ex vivo labeled allogenic leukocytes and optical imaging.

A missense mutation in the WD40 domain of murine Lyst is linked to severe progressive Purkinje cell degeneration.

Disturbance of intracellular trafficking plays a major role in several neurodegenerative disorders including Alzheimer or Parkinson's disease. The Chediak-Higashi syndrome (CHS), a life-threatening autosomal recessive disease with frequent mutations in the LYST gene, and its animal model, the beige mouse, are both characterized by lysosomal defects with accumulation of giant lysosomes. Clinically they manifest as hypopigmentation, abnormal bleeding and increased susceptibility to infection with various degrees of involvement of the nervous system. In the course of a recessive N-ethyl-N-nitrosurea (ENU) mutagenesis screen, we identified the first murine missense mutation in the lysosomal trafficking regulator gene (Lyst(Ing3618)) located at a highly conserved position in the WD40 protein domain. Nearly all described human Lyst alleles lead to protein truncation and fatal childhood CHS. Only four different missense mutations have been reported in patients with adolescent or adult forms of CHS involving the nervous system. Interestingly, the Lyst(Ing3618) model presents with a predominant neurodegenerative phenotype with progressive degeneration and loss of Purkinje cells and lacks severe impairment of the immune system. Therefore, the Lyst(Ing3618 )allele could represent a new model for adult CHS with neurological impairment. It could also provide an important tool to elucidate the role of neuronal lysosomal trafficking in the pathophysiology of neurodegeneration.

Expression of p53 and p21 in primary glioblastomas.

BACKGROUND AND PURPOSE: Primary glioblastomas (GBMs) are highly radioresistant, and in contrast to secondary GBMs, they bear wild-type (wt) p53 protein, which is stabilized in a proportion of these tumors. Therefore, it was investigated in vivo whether p53 expression has prognostic value in patients undergoing radiochemotherapy. Additionally, the authors tried to identify, in vitro, subgroups of primary GBM with different susceptibilities to irradiation, on the basis of their p53 and p21 responses to ionizing radiation. MATERIAL AND METHODS: Tumor tissue samples from 31 patients suffering from primary GBM undergoing a combined radiochemotherapy with topotecan were investigated. The percentage of cells expressing p53 protein was determined immunohistochemically. Additionally, primary cultures from eleven primary GBMs were established and investigated. p53 and p21 expressions were evaluated before irradiation with 10 Gy and at 2 and 8 h after irradiation. p53 protein expression was measured by Western analysis and p21 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The percentage of p53-positive cells within the tumor specimens obtained from the 31 patients ranged from 0% to 28%, the median value being 4.3%. No significant correlation with disease-free survival or overall survival was found. In vitro, p53 protein was detected in seven of eleven cultures from primary GBM. After irradiation a decrease in p53 protein expression was seen in six of the seven p53-positive cultures. Half of the cultures (two of four) without basal p53 expression showed an increase in p53 expression after irradiation. Basal overexpression of p21 was detected in six of the eleven cultures; in four out of six irradiation led to a decrease in p21 expression. In all cell lines (five of eleven) initially showing absent p21 expression, irradiation induced p21 expression. Despite these responses, G1 arrest was not detectable in any of the GBM cultures. CONCLUSION: p53 protein expression in vivo does not correlate with the outcome of patients with primary GBM. Therefore, p53 protein content per se does not appear to be a helpful prognostic factor for prognosis-adapted therapy in primary GBM. By contrast, primary GBM cells in vitro show different and independent responses in their p53 and p21 pathways to ionizing radiation. The failure of G1 arrest seems to be due to a functional defect in the p53 pathway, either because p21 was not induced or because of an unidentified defect downstream from p21.

Mutations in dynein link motor neuron degeneration to defects in retrograde transport.

Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.