Non-necrotizing autoimmune myopathy with pipestem type capillaries - a light and electron microscopic study.

We report on 7 cases of a nonnecrotizing type of autoimmune myopathy found in suspected connective tissue disease and related disorders. In muscle biopsy, these cases showed myopathic alterations characterized by irregular outlines of muscle fibers in cross and longitudinal section nearly or totally lacking inflammatory cell infiltrations or macrophages and no necroses of myofibers. They revealed deposition of immunoglobulins and of collagen IV antibodies attached to fiber surface. The number of capillaries per myofiber was enlarged. Capillaries of pipestem type with thickened wall were found at the electron microscopic level. This type of myopathy was seen especially in subacute cases presenting with muscle pain, weakness and increased unspecific inflammatory laboratory parameters. No evidence for polymyositis, dermatomyositis or inclusion body myositis could be found. In all cases, steroid therapy was of striking benefit.

Correlation between amplification of the gene for the epidermal growth factor receptor (EGFR), data from preoperative proton-MR-spectroscopy (1HMRS) and histomorphometric data of glioblastomas.

OBJECTIVE: To investigate a correlation between preoperative data from proton-MR-spectroscopy (1HMRS), genomic alterations (epidermal growth growth factor receptor [EGFR] gene amplification) and histomorphometric data from glioblastomas. STUDY DESIGN: In surgical specimens from 18 patients with glioblastomas, the degree of amplification of the gene for EGFR was determined in the region with the largest Ki-67 proliferation index by differential polymerase chain reaction. RESULTS: Correlation analysis showed significant positive correlation between degree of EGFR gene amplification and choline and total creatine (CHO/TCR) ratio, indicating increased membrane turnover. Cases with a high EGFR/interferon ratio showed a tendency toward a low lipid peak, whereas cases with a low EGFR/interferon ratio showed a large variation of the lipid peak. Differences were observed regarding quantitative histomorphologic data of tumor cell nuclei, especially nuclear size and shape. Together with the EGFR/interferon ratio, these morphometric data provided a good reclassification of cases with low and with high values for both spectroscopic variables by means of cross-validated linear discriminant analysis. CONCLUSION: The results provide further evidence for the biologic significance of metabolic data from preoperative 1HMRS, because these metabolic data showed a significant statistical relationship with histomorphology and a frequently occurring molecular biologic alteration (EGFR gene amplification) in glioblastomas.

The morphology of perinecrotic tumor cell nuclei in glioblastomas shows a significant relationship with survival time.

A deeper knowledge about histopathological criteria with a significant impact on the prognosis of patients with glioblastomas is worthwhile, since these patients may show a considerable difference in the time of survival. Investigation of the morphology of perinecrotic tumor cell nuclei is a promising approach, because the expression of specific molecules in these cells has been associated with a more aggressive behaviour of the tumors. In our series of patients with documented clinical course, 11 patients had a survival of at least 24 months and we compared this group with a group of 10 patients with maximum survival of 12 months. Digital microscopic image analysis was performed in paraffin sections from the primary surgical specimen. Three hundred perinecrotic tumor cell nuclei per case and 300 nuclei per case from tumor cells lying more distant from the tumor necroses ('distant zone' nuclei) were measured. The ratio for the numerical nuclear density between both types of nuclei was significantly larger in cases with short survival indicating a more pronounced nuclear density of perinecrotic nuclei in relation to the 'distant zone' nuclei in these tumors. In cases with long survival, on the contrary, perinecrotic tumor cells exhibited an only slightly larger numerical density compared with 'distant zone' nuclei. Mean values and standard deviations from parameters of nuclear shape (Fourier-amplitudes) had significantly smaller values in short-time survivors indicating a tendency towards a more circular nuclear shape with less pronounced intratumoral variation in that group. Based on the morphometric results, all cases could be correctly reclassified as short- or long-time survivors by means of cross-validated discriminant analysis. In conclusion, the results confirm significant morphological differences between tumors from patients with short and with long survival regarding morphology of both types of tumor cell nuclei. It seems likely, that histomorphometry of tumor cell nuclei could be a promising approach for the assessment of the individual prognosis of patients with glioblastomas.

Application of different methods for nuclear shape analysis with special reference to the differentiation of brain tumors.

OBJECTIVE: To study the discriminatory power of different methods designed for nuclear shape analysis with reference to the differentiation and grading of brain tumors and the differentiation between proliferating and nonproliferating nuclei. STUDY DESIGN: At least 300 tumor cell nuclei per case were measured by means of a digital image analysis system. Fourier amplitudes no. 1 to 15, moments no. 1 to 7 according to Hu, roundness factor, ellipse shape factor, concavity factor, Feret ratio, fractal dimension and bending energy were determined for each nucleus. The discriminatory power of these parameters was tested in three pairwise comparisons: (1) oligodendrogliomas WHO grade II (n = 13) vs. grade III (n = 11), (2) medulloblastomas WHO grade IV (n = 14) vs. anaplastic ependymomas WHO grade III (n = 12), (3) Ki-67-positive vs. Ki-67-negative tumor cell nuclei in the 14 medulloblastomas. RESULTS: When data from Fourier analysis were included in statistical analysis, cross-validated discriminant analysis led to a 100% correct reclassification for the first and for the second pairwise comparison and to a 75% correct reclassification when comparing Ki-67-positive and Ki-67-negative nucleifrom medulloblastomas. Different combinations of the other shape parameters led to a lower percentage of correctly reclassified cases for all three pairwise comparisons, especially when Fourier analysis was not included in the analysis. CONCLUSION: Fourier analysis provided an optimal statistical discrimination between different brain tumor entities and between data sets from proliferating and nonproliferating tumor cell nuclei. Since nuclear shape is an important criterion for the investigation of tumors, the application of Fourier analysis is therefore recommended for quantitative histologic investigations in neuro-oncology.

Ezrin immunoreactivity reveals specific astrocyte activation in cerebral HIV.

The actin-binding protein ezrin is associated with cellular shape changes, motility, tumor invasion, and lymphocyte activation. We have earlier shown that ezrin immunoreactivity (IR) is faintly present in normal astrocytes but increased in malignant human astrogliomas. We studied the role of ezrin in astrocyte activation, applying immunostaining on serial paraffin sections from human autopsied brain tissues (51 cases). Cerebral HIV infection was chosen as a model displaying consistent exemplary astrocyte activation. Semiquantitative ezrin-IR was compared with the common glial markers GFAP, ferritin, and HLA-DR in relation to clinical and morphologic criteria of HIV encephalopathy. In all cases with HIV infection, GFAP-, HLA-DR-, and ferritin-IR were elevated in comparison to normal brain tissues. In contrast, high ezrin-IR in HIV infection strictly correlated with additional HIV encephalopathy. HIV encephalopathy with particularly high ezrin-IR was correlated with neuronal apoptosis (TUNEL). Combined ezrin-IR and GFAP-IR thus reveals 2 distinct states of astrocytic activation. Normal ezrin-IR, when paralleled by upregulated GFAP, reflects astroglial activation not associated with neuronal apoptosis. High ezrin-IR indicates specific astrocyte stressors related to cellular damage within the central nervous system. Ezrin-IR might also provide a diagnostic tool for the classification of HIV encephalopathy.

Morphology of tumor cell nuclei is significantly related with survival time of patients with glioblastomas.

PURPOSE: To investigate whether histomorphology of tumor cell nuclei has a significant and independent relation to survival time of patients with glioblastomas. EXPERIMENTAL DESIGN: Seventy-two tumors from 72 patients were investigated by means of digital image analysis. Proliferating and nonproliferating nuclei were separately measured and parameters of nuclear size, shape, texture, and spatial relationships (topometric parameters) were detected. Survival analysis was done regarding morphometric data together with the patients' age, the amount of resection (total or subtotal), and the classification of the tumor as a "primary" (de novo) or "secondary" glioblastoma. RESULTS: The overall relation of all morphometric data to the time of survival was highly significant (Cox analysis, P < 0.0001). Apart from the extent of surgical resection, parameters of nuclear shape and topometric variables, such as the distance between two nuclei lying nearest to each other, showed an independent and significant relation to survival time. The patients' age had also a significant but comparably slight relation to survival time. CONCLUSIONS: The morphology of tumor cell nuclei, as represented by morphometric data, shows a significant relation to survival time of patients with glioblastomas. This relation is statistically independent from the amount of surgical resection, from the patients' age and from the classification of the glioblastoma as being primary or secondary. The results support the view that histomorphometry of tumor cell nuclei is a valuable prognostic marker for patients with glioblastomas. We believe that such a marker ought to be incorporated into the formation of individual therapeutic decisions.

Morphology of proliferating and non-proliferating tumor cell nuclei in glioblastomas correlates with preoperative data from proton-MR-spectroscopy.

In contrast to the growing interest in proton-MR-spectroscopy (1HMRS) for preoperative examination of patients with brain tumors, there is nearly no knowledge about a correlation between data from 1HMRS and histomorphology as confirmed by quantitative morphological methods. Whether a correlation can be confirmed between data from 1HMRS and quantitative histomorphology of glioblastomas representing the most frequent type of brain tumors was investigated in the present study. Furthermore, it was of interest, whether correlations between spectroscopic data and histomorphology can be confirmed for proliferating and non-proliferating tumor cell nuclei independently. Using stringent inclusion criteria for this study, 24 patients were investigated by means of preoperative 1HMRS and by means of digital image analysis of paraffin sections from the surgical specimen. Proliferating and non-proliferating tumor cell nuclei were investigated separately in the region with the highest proliferative activity in each tumor using immunohistological staining for the proliferation marker Ki67. Main results showed highly significant correlations between total creatine and variables of nuclear size, as well as correlations between choline and variables of nuclear shape. These results were confirmed for both proliferating and non-proliferating tumor cell nuclei. A significant correlation between N-acetyl-aspartate level and topometric variables (number of neighbors per nucleus, variables describing distances between tumor cell nuclei) was confirmed for proliferating tumor cell nuclei. Discriminant analysis provided a good separation of cases with high and with low values for these spectroscopic variables based on histomorphometric data. In conclusion, the results confirm a direct correlation between data from preoperative 1HMRS and histomorphological characteristics of glioblastomas supporting the biological relevance of spectroscopic data for the examination of brain tumor patients.

Regional heterogeneity of EGFR gene amplification and nuclear morphology in glioblastomas. An investigation using laser microdissection and pressure catapulting.

OBJECTIVE: To study the regional heterogeneity of epidermal growth factor receptor (EGFR) gene amplification (EGFR-GA) in glioblastomas, considering the relationship between this mutation and morphology of tumor cell nuclei. STUDY DESIGN: Tissue samples gained by laser microdissection and pressure catapulting were used for the performance of differential polymerase chain reaction in 32 morphologically different regions from 7 glioblastomas. Semiquantitative determination of EGFR expression and image analysis of tumor cell nuclei were performed in the same regions. RESULTS: Distinct regional differences concerning the degree of EGFR-GA were found in 2 tumor cases. When comparing regions with different degrees of gene amplification within these cases, morphologic differences in tumor cell nuclei were observed. The other tumor cases also showed distinct intratumoral heterogeneity concerning histomorphology but no regional heterogeneity in the degree of EGFR-GA. When comparing regions with a low densitometric EGFR/interferon (INF) band ratio (< 2.19, n = 18) and a high EGFR/IFN band ratio (> 4.39, n = 14), the latter type of region showed a significantly higher percentage of Ki-67--positive tumor cell nuclei and lower values for several shape variables (Fourier amplitudes), indicating a tendency toward a more regular nuclear shape in regions with distinct EGFR-GA. For the EGFR/IFN band ratio, a significant correlation was found with several morphometric variables, especially those of nuclear shape and distances between nuclei. CONCLUSION: In glioblastomas showing regional heterogeneity in the degree of EGFR-GA, morphology of tumor cell nuclei has been shown to be different when comparing regions with different degrees of EGFR-GA. Glioblastomas may also show distinct regional heterogeneity of histomorphology without evidence of regional heterogeneity of EGFR-GA. A significant statistical association has been confirmed between the degree of EGFR-GA and quantitative morphology of tumor cell nuclei.

Preoperative proton-MR spectroscopy of gliomas--correlation with quantitative nuclear morphology in surgical specimen.

A comparison between data from proton-MR spectroscopy (1HMRS) and quantitative histomorphology of tumor cell nuclei in gliomas has not been reported up to now. Therefore, the question must be answered, if there are any significant correlations between histomorphology of gliomas and quantitative data from 1HMRS concerning tissue metabolites. Surgical glioma specimen (glioblastomas, astrocytomas, oligodendrogliomas) from 46 patients with tumor grades II-IV according to WHO have been evaluated by means of a digital image analysis system using Ki-67-immunostained paraffin sections. Nuclear density, Ki-67-proliferation index, nuclear area and shape variables (roundness factor, Fourier-amplitudes) have been determined from 200 randomly selected tumor cell nuclei in each tumor specimen. These data have been correlated with preoperative data from 1HMRS. A positive correlation between Fourier-amplitudes, choline peak and lipide peak was observed, as well as a negative correlation between these variables and the nuclear roundness factor. This result indicates higher choline and lipide peaks with increasing irregularity of nuclear outlines. Proliferation index Ki-67 was positively correlated with the lipide peak, nuclear density showed a positive correlation with the choline peak. Glioblastomas (n = 29) showed an additional positive correlation between mean nuclear size and total creatine. Anaplastic gliomas (n = 12) showed a positive correlation between lactate peak and the standard deviation of the nuclear roundness factor. Further multivariate analyses have shown, that for the present collective of 46 cases, histometric variables have a higher significance than spectroscopic data for the differentiation of the different tumor grades. These results verify a significant correlation between preoperative data from 1HMRS and histomorphology of tumor cell nuclei in gliomas, supporting the biological significance of both histomorphometry and 1HMRS for the evaluation of these tumors.

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report.

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.

Correlation between preoperative magnetic resonance spectroscopic data on high grade gliomas and morphology of Ki-67-positive tumor cell nuclei.

OBJECTIVE: To investigate possible statistical correlations between metabolic data from preoperative proton magnetic resonance spectroscopy (1HMRS) and morphology of proliferating tumor cell nuclei in anaplastic gliomas and glioblastomas. STUDY DESIGN: Ki-67-positive tumor cell nuclei in paraffin sections of surgical specimens from 36 patients (7 anaplastic gliomas, World Health Organization grade 3; 29 glioblastomas, World Health Organization grade 4) were investigated by means of a digital image analysis system. Stringent inclusion criteria were formulated for all cases with respect to histologic quality and spectroscopic examination. As morphometric variables, nuclear area, shape variables (roundness factor, size-invariate Fourier amplitudes) and density of Ki-67-positive tumor cell nuclei per reference area were determined. RESULTS: Correlation analysis according to Spearman revealed a significant positive correlation between the total creatine (TCR) peak and nuclear area (P = .005). This correlation was also found within the glioblastoma group (P = .019). There was also a significant negative correlation of nuclear area with the ratio between choline and TCR in all cases (P = .014) and within the glioblastoma group (P = .046). No significant correlation of spectroscopic data was found with nuclear shape or density of Ki-67-positive tumor cell nuclei. CONCLUSION: The results demonstrate a correlation between spectroscopic data and morphology of proliferating tumor cell nuclei (nuclear size) in high grade gliomas. This study is part of a detailed investigation of the interrelationship between preoperative 1HMRS and quantitative histomorphology of gliomas.

Topometric analysis of diffuse astrocytomas.

OBJECTIVE: To investigate differences between astrocytomas of WHO grade 2 and anaplastic astrocytomas of WHO grade 3 in terms of topometric variables characterizing individual tumor cell nuclei. STUDY DESIGN: Paraffin sections from surgical specimens from 25 astrocytomas (grade 2, n = 11; grade 3, n = 14) were analyzed by means of an image analysis system. At least 300 tumor cell nuclei were measured in the region with the highest Ki-67 proliferation index. Three different kinds of topometric variables were determined for each tumor cell nucleus: (1) several distances; (2) the variable Angle 2/1, the angle between the straight lines representing the distance to the nearest and second-nearest nucleus; and (3) the number of neighbors according to our mathematical definition. RESULTS: Most topometric variables showed distinct differences between the 2 tumor grades (multivariate analysis of variance), with 88% cases correctly reclassified by means of cross-validated discriminant analysis. The variables with the highest discriminatory power were the SD of Angle 2/1 and the ratio between the distance to the second-nearest and nearest tumor cell nucleus, with lower values for these variables in anaplastic astrocytomas. Even variables concerning neighborhood relationships showed significant differences. CONCLUSION: The results of this pilot study show that this first set of topometric variables is sufficient to detect differences between topologic characteristics of tumor cell nuclei in astrocytomas grade 2 and grade 3. Topometry seems to be an important tool for grading astrocytomas.

Determination of histopathological tumor grade in neuroepithelial brain tumors by using spectral pattern analysis of in vivo spectroscopic data.

OBJECT: In this study, 1H magnetic resonance (MR) spectroscopy was prospectively tested as a reliable method for presurgical grading of neuroepithelial brain tumors. METHODS: Using a database of tumor spectra obtained in patients with histologically confirmed diagnoses, 94 consecutive untreated patients were studied using single-voxel 1H spectroscopy (point-resolved spectroscopy; TE 135 msec, TE 135 msec, TR 1500 msec). A total of 90 tumor spectra obtained in patients with diagnostic 1H MR spectroscopy examinations were analyzed using commercially available software (MRUI/VARPRO) and classified using linear discriminant analysis as World Health Organization (WHO) Grade I/II, WHO Grade III, or WHO Grade IV lesions. In all cases, the classification results were matched with histopathological diagnoses that were made according to the WHO classification criteria after serial stereotactic biopsy procedures or open surgery. Histopathological studies revealed 30 Grade I/II tumors, 29 Grade III tumors, and 31 Grade IV tumors. The reliability of the histological diagnoses was validated considering a minimum postsurgical follow-up period of 12 months (range 12-37 months). Classifications based on spectroscopic data yielded 31 tumors in Grade I/II, 32 in Grade III, and 27 in Grade IV. Incorrect classifications included two Grade II tumors, one of which was identified as Grade III and one as Grade IV; two Grade III tumors identified as Grade II; two Grade III lesions identified as Grade IV; and six Grade IV tumors identified as Grade III. Furthermore, one glioblastoma (WHO Grade IV) was classified as WHO Grade I/II. This represents an overall success rate of 86%, and a 95% success rate in differentiating low-grade from high-grade tumors. CONCLUSIONS: The authors conclude that in vivo 1H MR spectroscopy is a reliable technique for grading neuroepithelial brain tumors.

Isolation and molecular characterization of brain microvascular endothelial cells from human brain tumors.

Brain tumor formation and growth is accompanied by the proliferation and infiltration of blood capillaries. The phenotypes of endothelial cells that make up capillaries are known to differ not only in the tissues in which endothelial cells are located but also as a result of the microenvironment to which they are exposed. For this reason, primary cultures of brain endothelial cells were isolated from human brain tumors removed by surgery and compared with cells from normal tissue. The primary confluent monolayers that grew out of isolated capillary fragments consisted of closely associated, elongated, fusiform-shaped cells. But brain tumor-derived endothelial cells in culture exhibited significantly less expression of endothelial-specific Factor VIII-related antigen compared with cells isolated from normal tissue. Cultured cells that exhibited binding of Ulex europaeus lectin were shown to take up Dil-Ac-Ldl and formed continuous monolayers that were joined together by tight junctions. The cells also exhibited characteristics of the cells of the brain microvasculature in vitro as seen by the presence of large numbers of mitochondria and few pinocytotic vesicles and by the absence of Weibel-Palade bodies within the cells. The expression of vascular cell adhesion molecule-1, E-Selectin, and the tight junction associated protein ZO-1 but not intercellular adhesion molecule-1 was demonstrated by immunohistological staining or reverse transcriptase-polymerase chain reaction methodologies. Comparative studies of these endothelial cells with endothelial cells from normal tissue will be useful for determining and understanding how the blood-brain barrier differs and functions in tumor and healthy tissues and may lead to strategies for brain tumor therapeutic approaches.

HIV-related neuropathology, 1985 to 1999: rising prevalence of HIV encephalopathy in the era of highly active antiretroviral therapy.

Postmortem neuropathologic reports for a consecutive series of 436 HIV-seropositive patients who died between 1985 and 1999 were matched with clinical data for 371 of them. Cases were divided into four groups depending on the date of death. The chosen time periods reflected the type of antiretroviral therapy available: before 1987 (before zidovudine); 1987-1992, the period of monotherapy (nucleoside analog reverse transcriptase inhibitors [NRTIs]); 1993-1995, the era of the use of dual NRTI combinations; and 1996-1999, the era of highly active antiretroviral therapy (HAART) containing protease inhibitors. Fifty-seven percent of our cases in this group had been prescribed HAART. In our study population, accessibility to the latest antiretroviral therapy was widespread. The total number of HIV autopsies declined after the advent of combination therapy. The prevalence of opportunistic infections-cytomegalovirus, toxoplasmosis, cryptococcosis, and central nervous system lymphoma-decreased over time. Cerebral tuberculosis, aspergillosis, herpes, and progressive multifocal leukoencephalopathy showed a downward trend, but the numbers were too low for statistical analyses. The incidence of HIV encephalopathy increased over time (p =.014). The rising prevalence of HIV encephalopathy at time of death may reflect a longer survival time after initial HIV infection in the HAART era. Although combination therapies decrease overall mortality and prevalence of CNS opportunistic infections, these therapies may be less active in preventing direct HIV-1 effects on the brain.

Increased choline levels coincide with enhanced proliferative activity of human neuroepithelial brain tumors.

Proton MR spectroscopy ((1)H MRS)-visible total choline-containing compounds (tCho-compounds) are derivatives of membrane phospholipids and, in part, may act as a long-term second-messenger system for cellular proliferation. Experimental evidence suggests increasing concentrations of tCho-compounds during cellular proliferation. The present study was conducted in order to test the hypothesis that in vivo measurements of tCho-concentrations using (1)H MRS allow assessment of the proliferative activity of neuroepithelial brain tumors presurgically. Single-voxel (1)H MRS (PRESS, TR 1500 ms, TE 135 ms) was performed in 101 patients with neuroepithelial brain tumors prior to surgery and 19 healthy volunteers. Histological diagnoses were confirmed postsurgically according to the WHO classification. Measured tCho-compound signal intensities were corrected for coil loading, numbers of acquisitions and voxel size, and tCho concentrations calculated as institutional arbitrary units. They were matched with the mean immunohistochemical marker of cell proliferation, the Ki-67 (MIB.1) labeling index, using correlation analysis according to Spearman. Compared with low-grade tumors (i.e. WHO grade I/II) and normal white brain matter, high-grade tumors (i.e. WHO grade III/IV) revealed significantly (p < 0.05) elevated labeling indices paralleled by increasingly elevated tCho-concentrations. In contrast tCho-concentrations in low-grade tumor did not differ significantly from physiological values. A highly significant positive correlation (p < 0.0001, r(2) = 0.81) was found between the tCho-concentration and the labeling index. It was concluded that the determination of tCho-concentrations using in vivo (1)H MRS could provide a novel and noninvasive assessment of the proliferative activity of neuroepithelial brain tumors, pointing at (1)H MRS as a useful method for differentiating proliferating from non-proliferating tissues. Hence, potential indications for the clinical application of (1)H MRS are grading tumors presurgically, early detection of anaplastic transformation, and monitoring treatment.