Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool.

AIMS: Methylation profiling (MP) is increasingly incorporated in the diagnostic process of central nervous system (CNS) tumours at our centres in The Netherlands and Scandinavia. We aimed to identify the benefits and challenges of MP as a support tool for CNS tumour diagnostics. METHODS: About 502 CNS tumour samples were analysed using (850 k) MP. Profiles were matched with the DKFZ/Heidelberg CNS Tumour Classifier. For each case, the final pathological diagnosis was compared to the diagnosis before MP. RESULTS: In 54.4% (273/502) of all analysed cases, the suggested methylation class (calibrated score ≥0.9) corresponded with the initial pathological diagnosis. The diagnosis of 24.5% of these cases (67/273) was more refined after incorporation of the MP result. In 9.8% of cases (49/502), the MP result led to a new diagnosis, resulting in an altered WHO grade in 71.4% of these cases (35/49). In 1% of cases (5/502), the suggested class based on MP was initially disregarded/interpreted as misleading, but in retrospect, the MP result predicted the right diagnosis for three of these cases. In six cases, the suggested class was interpreted as 'discrepant but noncontributory'. The remaining 33.7% of cases (169/502) had a calibrated score <0.9, including 7.8% (39/502) for which no class indication was given at all (calibrated score <0.3). CONCLUSIONS: MP is a powerful tool to confirm and fine-tune the pathological diagnosis of CNS tumours, and to avoid misdiagnoses. However, it is crucial to interpret the results in the context of clinical, radiological, histopathological and other molecular information.

Surgical resection versus watchful waiting in low-grade gliomas.

BACKGROUND: Infiltrating low-grade gliomas (LGG; WHO grade 2) typically present with seizures in young adults. LGGs grow continuously and usually transform to higher grade of malignancy, eventually causing progressive disability and premature death. The effect of up-front surgery has been controversial and the impact of molecular biology on the effect of surgery is unknown. We now present long-term results of upfront surgical resection compared with watchful waiting in light of recently established molecular markers. MATERIALS AND METHODS: Population-based parallel cohorts were followed from two Norwegian university hospitals with different surgical treatment strategies and defined geographical catchment regions. In region A watchful waiting was favored while early resection was favored in region B. Thus, the treatment strategy in individual patients depended on their residential address. The inclusion criteria were histopathological diagnosis of supratentorial LGG from 1998 through 2009 in patients 18 years or older. Follow-up ended 1 January 2016. Making regional comparisons, the primary end-point was overall survival. RESULTS: A total of 153 patients (66 from region A, 87 from region B) were included. Early resection was carried out in 19 (29%) patients in region A compared with 75 (86%) patients in region B. Overall survival was 5.8 years (95% CI 4.5-7.2) in region A compared with 14.4 years (95% CI 10.4-18.5) in region B (P < 0.01). The effect of surgical strategy remained after adjustment for molecular markers (P = 0.001). CONCLUSION: In parallel population-based cohorts of LGGs, early surgical resection resulted in a clinical relevant survival benefit. The effect on survival persisted after adjustment for molecular markers.

Prognostic value of survivin and DNA topoisomerase IIα in diffuse and anaplastic astrocytomas.

Distinguishing WHO grade II astrocytomas from grade III is a difficult task. This study looks into the potential prognostic use of mitotic activity and the proliferation markers Ki67/MiB-1 (Ki67), survivin and DNA topoisomerase IIα (TIIα) in 59 WHO grade II diffuse astrocytomas (DA) and 33 WHO grade III anaplastic astrocytomas (AA), IDH1 R132H-mutated and not otherwise specified (NOS) by means of immunohistochemistry. All proliferation markers showed higher expression in AA compared with DA. Only Ki67 had significantly greater expression in astrocytomas, NOS vs. astrocytomas, IDH1-mutated. Uni-/multivariable COX-regression analyses showed that greater expression of both survivin and TIIα were associated with poorer survival when stratified for IDH1-mutation status and, additionally, achieved hazard rates surpassing clinically established prognostic factors such as age and WHO performance status. Ki67 achieved only statistical significance in univariable analyses, whereas mitoses did not reveal any relation to survival. IDH1-mutated astrocytomas had significantly better survival than astrocytomas, NOS. Among IDH1-mutated astrocytomas no significant difference in survival was shown between DA and AA. Our findings suggest a potential usefulness of proliferation markers in the prognostic setting of astrocytomas independent of IDH1-mutation status, and survivin and TIIα are potential candidates in that regard.

Primary Porcine Brain Endothelial Cells as In Vitro Model to Study Effects of Ultrasound and Microbubbles on Blood-Brain Barrier Function.

Focused ultrasound (FUS) in the presence of microbubbles transiently and reversibly opens the blood-brain barrier (BBB) in rodents and humans, thereby providing a time window for increased drug delivery into brain tissue. To get insight into the underlying mechanisms that govern ultrasound (US)-mediated opening of the BBB, in vitro models are a useful alternative. In this paper, we have utilized an in vitro BBB model that consists of primary porcine brain endothelial cells (PBECs). PBEC monolayers are grown on permeable membranes, which allow assessment of key features of BBB function as well as US treatment. This experimental model is characterized by low permeability for both small molecules and proteins, has a high transendothelial electrical resistance, and expresses tight junctions and efflux pumps. Here, we compare the effects of inertial and stable cavitation in the presence of SonoVue microbubbles on PBEC monolayers' electrical resistance and permeability properties. Our results point out the fragility of PBEC monolayers, which enhances results variability. In particular, we show that handling of the inserts, such as medium change and transfer to the US setup, modifies the cellular response, and immunostaining of the monolayers introduces damage and cell detachment within the US-exposed monolayers. Our results indicate that stable cavitation might have a more pronounced impact on cell permeability as compared with inertial cavitation in vitro. This paper might contribute to further development of experimental setups that are suitable to characterize the impact of FUS and microbubbles on BBB properties in vitro.

Mitosin and pHH3 predict poorer survival in astrocytomas WHO grades II and III.

AIMS: The limitations of the current WHO classification of astrocytomas call for a sustained effort to improve diagnostic and prognostic accuracy. The relationship between tumour growth and clinical outcome suggests that proliferative activity should be examined. The objective of this study was to evaluate the diagnostic and prognostic value of the proliferation markers mitosin and phosphohistone H3 (pHH3) in infiltrative astrocytomas WHO grades II and III and compare the findings with mitotic count and Ki-67/MiB-1 immunostaining. METHODS: Fifty-nine and thirty-three infiltrative astrocytomas WHO grades II and III, respectively, were immunostained with the proliferation markers mitosin and pHH3 using standard immunohistochemical procedures. The expression was quantified as a proliferative index (PI) and statistically evaluated with Spearman's rank correlation test, Wilcoxon-Mann-Whitney U test, and univariable and multivariable COX regression survival analyses. RESULTS: Significant positive correlations were found between these proliferation markers. The number of mitoses, pHH3 mitotic figures (MFs), the Ki-67/MiB-1 PI and the mitosin PI were greater in WHOgrade III anaplastic astrocytomas compared to WHO grade II diffuse astrocytomas, while pHH3 PI only showed a trend. All proliferation markers were associated with poorer prognosis, but mitotic count was not. Ki-67/MiB-1, mitosin and pHH3 MF achieved statistical significance in the univariable analyses of both time to relapse (TTR) and overall survival (OS). Only mitosin remained significant in both multivariable analyses. pHH3 was significant in the multivariable analysis of OS but not of TTR. Clinical factors including age, extent of surgical resection and WHO performance status were also significantly correlated with survival. CONCLUSIONS: In conclusion, mitosin and pHH3 immunostaining have prognostic and diagnostic value in the clinical assessment of patients with infiltrative astrocytomas. The inclusion of proliferation markers in a layered diagnosis should be considered in the upcoming revision of the WHO classification system.

Expression of Toll-like receptor-3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2.

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1ß and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.

Did survival improve after the implementation of intraoperative neuronavigation and 3D ultrasound in glioblastoma surgery? A retrospective analysis of 192 primary operations.

BACKGROUND: Numerous observational studies indicate that more aggressive resection may prolong survival in glioblastoma patients. In Trondheim, Norway, intraoperative 3D ultrasound has been in increasing use since November 1997. The aim of the present study was to examine if the introduction of 3D ultrasound and neuronavigation (i. e., the SonoWand® system) may have had an impact on overall survival. PATIENTS/MATERIAL AND METHODS: Patient data were obtained retrospectively for the 192 glio-blastoma patients who received surgery and postoperative radiotherapy between 1990 and 2005. Overall survival, before and after 1997, was compared using the log rank test. Possible confounders were adjusted for in a multivariate Cox regression analysis. RESULTS: We observed an increase in survival for patients in the last study period (9.6 vs. 11.9 months; HR = 0.7; p = 0.034). The significant improvement in the latest time period was sustained after adjusting for age, WHO performance status (≥2) and type of radiotherapy (normofractioned or hypofractioned), and chemotherapy (yes/no), p = 0.034. 10 out of 14 patients who survived more than 3 years received treatment after the implementation of 3D ultrasound. CONCLUSION: Our study demonstrates that survival has improved within the same period that intraoperative ultrasound and neuronavigation was introduced and established in our department. The demonstrated association is a necessity for causation, but given the nature of this study, one must be cautious to claim causality. The improvement was, however, significant after adjustment for known major prognostic factors.

Astroblastoma - a case report of a rare neuroepithelial tumor with complete remission after chemotherapy.

INTRODUCTION: Astroblastoma is a rare glial tumor of uncertain origin affecting mostly children, adolescents and young adults. Given the rarity and the definitional problems concerning this tumor entity, the prognosis and appropriate treatment are at this point unclear. CASE REPORT: A 50-yearold Caucasian female presented with a seizure. Radiological findings showed a welldefined circumscribed tumor located in the right cerebral frontal lobe. The patient underwent primary surgery followed by postoperative radiotherapy. After 6 months the tumor recurred with multiple small lesions not available for surgery. Chemotherapy was administered with complete radiological response. Seven years after surgery and more than 6 years after completed chemotherapy the patient is free of disease. Histopathology revealed a gliomatous tumor with gemistocyte-like tumor cells arranged in palisades or strings and areas with perivascular pseudorosettes, consistent with astroblastoma. Immunophenotype and ultrastructural findings confirmed the diagnosis and verified the neuroepithelial origin. CONCLUSION: Astroblastomas are rare brain tumors and pose a challenge in the diagnostic and clinical approach. In general, they have an unpredictable course with a tendency of recurrence. This and other case reports support a survival benefit of chemotherapy, suggesting this as an important treatment option for these patients.

Concurrent solitary fibrous tumor and low-grade fibrillary astrocytoma of the cerebellum.

OBJECTIVE: We report the clinicopathologic features of a solitary fibrous tumor (SFT) having undergone malignant transformation and being intimately associated with a WHO Grade II astrocytoma. CLINICAL PRESENTATION: A 7-month old patient presented with delayed motor development and hydrocephalus. INTERVENTION: Histologic and immunocytologic methods were applied in the study of the tumors. Resection was initially employed and the SIOP protocol employing vincristine and carboplatin was applied upon tumor recurrence. CONCLUSION: The biologic basis for the association of SFT and astrocytoma is unknown. The complex lesion differs substantially from WHO Grade IV gliosarcoma and from gliofibroma, lesions in which the disparate elements are linked by metaplasia. Indeed, it may represent a collision tumor. Lastly, induction of the glioma by the solitary fibrous tumor, a mechanism invoked to explain the poorly understood "sarcoglioma," deserves consideration.

Surgical resection of high-grade gliomas in eloquent regions guided by blood oxygenation level dependent functional magnetic resonance imaging, diffusion tensor tractography, and intraoperative navigated 3D ultrasound.

OBJECTIVE: The aims of this study of patients with high-grade gliomas in eloquent brain areas were 1) to assess the postoperative functional outcome, 2) to determine the extent of tumour resection in these difficult locations, 3) to evaluate the practical usefulness of navigated blood oxygenation level-dependent functional magnetic resonance imaging and diffusion tensor tractography. PATIENTS AND METHODS: 25 consecutive patients were included in the study. The patients' gross functional neurological status was determined using the 7-step modified Rankin scale. The extent of tumour resection was determined using pre- and postoperative T(1)-weighted or T(1)-weighted, contrast-enhanced MRI images. RESULTS: The average preoperative modified Rankin scale was 1.56+/-0.77, whereas the average postoperative modified Rankin scale was 1.08+/-1.29. There was a significant improvement in mean modified Rankin scale score after surgery. The mean percentage of residual tumour was calculated to 16+/-22% of the original tumour volume (median 8%). Blood oxygenation level-dependent functional magnetic resonance imaging and diffusion tensor tractography were performed in 23 and 18 patients, respectively. Blood oxygenation level-dependent functional magnetic resonance imaging and diffusion tensor tractography facilitated identification of probable functional regions in 91% and 94% of the respective investigations. CONCLUSION: We feel that the combination of blood oxygenation level-dependent functional magnetic resonance imaging, diffusion tensor tractography, and 3D ultrasound facilitated maximal tumour resection with minimal deficits. The method permits an image-based functional monitoring of the brain during surgery that may aid the preservation of motor and language function.

Coexpression of c-erbB 1-4 receptor proteins in human glioblastomas. An immunohistochemical study.

Studies support involvement of the erbB/HER (human epidermal growth factor receptor) family, comprising the c-erbB-1/2/3/4 receptor proteins, in the tumourigenesis of human gliomas, raising their potential role in diagnostic and therapeutic approaches to these tumours. Reliable detection systems for these molecules in glioma tissue are therefore needed. Formalin-fixed and paraffin-embedded sections from twenty-one human glioblastomas were investigated by standard immunohistochemical procedures for expression of c-erbB-1/2/3/4 receptor proteins using commercial antibodies. All the antibodies used worked satisfactorily on paraffin-sections. For EGFR (epidermal growth factor receptor) two antibodies reactive against the external and internal domain were used. The first revealed positive immunoreactivity in 13 of 21 tumours (62 %), whereas all were positive with the latter. All glioblastomas were negative for the mutated variant of EGFR (i.e. EGFRvIII). Nine of 21 tumours (43 %) were immunoreactive for c-erbB-2, 19 of 20 tumours (95 %) for c-erbB-3, and 21 of 21 for c-erbB-4. Kaplan-Meier plots as a function of growth factor receptor expression did not show any significant association with survival among the glioblastoma patients. In conclusion, immunohistochemistry is well suited for detection of erb receptor proteins in glioblastoma tissue and demonstrated abundant and simultaneous immunoreactivity of these receptors.

Progressive multifocal leukoencephalopathy in a lymphoma patient with complete remission after treatment with cytostatics and rituximab: case report and review of the literature.

A 44-year-old woman presented with dysarthria, visual disturbances, ataxia and cognitive impairment. There was a rapid progression of her neurological disease, and she died 8 months later. She was previously treated for a low-grade follicular B-cell lymphoma; complete remission was achieved by conventional radiotherapy and chemotherapy, including rituximab. Two years later, the neurological symptoms and signs started. MRI revealed a cerebral demyelinating process. Serology was negative. Autopsy disclosed areas in cerebral white matter with grey discoloration. Microscopy revealed demyelination, oligodendroglial viral inclusions and gliosis with bizarre astrocytes. Polymerase chain reaction (PCR) was positive for JC virus. These findings were consistent with progressive multifocal leukoencephalopathy (PML). This is one of recent reports on PML occurring in a patient treated with the anti-20 monoclonal antibody rituximab.

Ability of navigated 3D ultrasound to delineate gliomas and metastases--comparison of image interpretations with histopathology.

BACKGROUND: The objective of the study was to test the ability of a 3D ultrasound (US) based intraoperative imaging and navigation system to delineate gliomas and metastases in a clinical setting. The 3D US data is displayed as reformatted 2D image slices. The quality of the displayed 3D data is affected both by the resolution of the acquired data and the reformatting process. In order to investigate whether or not 3D US could be used for reliable guidance in tumour surgery, a study was initiated to compare interpretations of imaged biopsy sites with histopathology. The system also enabled concomitant comparison of navigated preoperative MR with histopathology. METHOD: Eighty-five biopsies were sampled between 2-7 mm from the tumour border visible in the ultrasound images. Biopsies were collected from 28 operations (7 low-grade astrocytomas, 8 anaplastic astrocytomas, 7 glioblastomas and 6 metastases). Corresponding cross-sections of preoperative MR T1, MR T2 and intraoperative US were concomitantly displayed, steered by the biopsy forceps equipped with a positioning sensor. The surgeons' interpretation of the images at the electronically indicated biopsy sites were compared with the histopathology of the samples. FINDINGS: The ultrasound findings were in agreement with histopathology in 74% (n = 31) for low-grade astrocytomas, 83% (n = 18) for anaplastic astrocytomas, 77% (n = 26) for glioblastomas and 100% (n = 10) for metastases. Excluding irradiated patients, the results for glioblastomas improved to 80% concurrence (n = 20). As expected tumour cells were found in biopsies outside the US visible tumour border, especially in low-grade gliomas. Navigated 3D US have a significantly better agreement with histopathology than navigated MR T1 for low-grade astrocytomas. CONCLUSION: Reformatted images from 3D US volumes give a good delineation of metastases and the solid part of gliomas before starting the resection. Navigated 3D US is at least as reliable as navigated 3D MR to delineate gliomas and metastases.

Prognostic significance of Ki-67/MIB-1 proliferation index in meningiomas.

Even though tumor grade, subtype, and extent of resection are strong prognostic factors in human meningiomas, the growth of this tumor is still unpredictable, and additional prognostic markers are needed. Thus, immunohistochemical determination of proliferative activity using the Ki-67 equivalent antibody MIB-1 has gained increased attention. However, the reported prognostic significance of this marker in meningiomas is not fully clarified. The aim of this study was to investigate the prognostic role of MIB-1 proliferation index (PI) in a series of meningiomas comprising 23 benign, 17 atypical, and 9 anaplastic tumors. MIB- 1 PI increased with increasing tumor grade and discriminated significantly benign from atypical and anaplastic meningiomas whereas no difference was found between the latter two grades. However, due to the considerable overlap of PI values between the various grades, one should be circumspect before using this criterion for tumor grading. Furthermore, MIB-1 PIs were significantly higher in recurrent tumors compared with non-recurrent and a reliable MIB-1 PI cut-off value of 10% was established. This value, however, cannot automatically be adapted by other laboratories and must be regarded just as a guideline. In conclusion, MIB-1 PI appears as an important prognostic factor and should be used in combination with traditional histological criteria for malignancy in order to identify meningiomas with increased risk of recurrence.

Immunohistochemical detection of epidermal growth factor receptor in human high-grade astrocytomas--a comparison between frozen- and paraffin sections.

The overexpression of epidermal growth factor receptor (EGFR) in human astrocytic tumours is associated with the oncogenesis of these tumours. Ongoing research on diagnostic, prognostic, and therapeutic aspects of this receptor is highly dependent on the development of reliable techniques for the detection of EGFR in tumour tissue. The aim of this study was to assess EGFR expression in human high-grade astrocytomas by means of immunohistochemistry on formalin-fixed, paraffin-embedded sections and to compare these findings with the results of our previous study on frozen sections from these tumours, in which we found about 60% EGFR positivity (10). Four anaplastic astrocytomas and 19 glioblastomas were included in this study. Two different antibodies were used, the monoclonal antibody E30 reactive against the extracellular domain of EGFR and the polyclonal antibody Ab-4 directed against its cytoplasmic domain. With E30, 3 out of 4 anaplastic astrocytomas (75%) and 12 out of 19 glioblastomas (63%) were found to express EGFR whereas Ab-4 demonstrated positive EGFR immunoreactivity in most of the tumours (18/19 glioblastomas and all the 4 anaplastic astrocytomas). In conclusion, immunohistochemistry represents a reliable and convenient technique for the detection of EGFR in tissue sections of human high-grade astrocytomas, and that EGFR immunoreactivity is comparable in frozen- and paraffin sections from these tumours.

Hypergastrinaemia induced by partial corpectomy results in development of enterochromaffin-like cell carcinoma in male Japanese cotton rats.

BACKGROUND: Among inbred female cotton rats (Sigmodon hispidus) 25%-50% of the animals develop spontaneous gastric carcinomas, whereas males have an incidence of less than 1%. The carcinomas are enterochromaffin-like (ECL)-cell derived. Animals with gastric carcinomas also have hypergastrinaemia and gastric hypoacidity, but the mechanism behind the hypoacidity is unknown. Carcinomas have been found in all female cotton rats with spontaneous hypergastrinaemia lasting more than 4 months, and a gastrin receptor antagonist prevents the development of carcinoma. The purpose of the present study was to investigate whether induced hypergastrinaemia in male cotton rats would also result in carcinomas. METHODS: Hypergastrinaemia was induced by partial corpectomy of male cotton rats, aiming at removal of 80%-90% of the corpus. A control group was sham-operated. RESULTS: All partially corpectomized animals developed persistent hypergastrinaemia. Six months after the operation, 9 out of 13 partially corpectomized animals developed gastric cancer. In the dysplastic mucosa surrounding the tumours there was an increase in chromogranin A immunoreactive cells, where numerous cells also were stained using the Sevier-Munger technique. Tumour tissue also contained cells that were chromogranin A positive and stained by Sevier-Munger. CONCLUSIONS: ECL-cell carcinomas can be induced in male cotton rats by partial corpectomy. This supports a previous statement that spontaneous carcinomas in female cotton rats are caused by gastric hypoacidity and hypergastrinaemia. In hypergastrinaemic animals, ECL-cell carcinomas develop independently of gender within a relatively short period of time, and cotton rats therefore represent an interesting model for studying gastric carcinogenesis.

Spontaneous enterochromaffin-like cell carcinomas in cotton rats (Sigmodon hispidus) are prevented by a somatostatin analogue.

Among inbred female cotton rats (Sigmodon hispidus) 25-50% of the animals develop spontaneous gastric carcinomas; the corresponding figure for male cotton rats is approximately 1%. Animals with carcinomas have hypergastrinaemia and gastric hypo-anacidity and the tumours are derived from enterochromaffin-like (ECL) cells. The mechanism behind the hypo-anacidity is unknown. Carcinomas are found in all female cotton rats with hypergastrinaemia lasting more than 4 months and this represents an excellent animal model for studying gastric carcinogenesis. In this study, the somatostatin analogue octreotide was given to female cotton rats to prevent carcinoma development caused by hypergastrinaemia. Twelve female cotton rats were given monthly injections of long-acting octreotide (5 mg i.m.) for 6 months. A control group of 20 animals was not given injections. Of the 20 control animals, 13 developed hypergastrinaemia and histologically invasive carcinomas or dysplasia. Of the 12 animals in the octreotide group, five developed hypergastrinaemia. None of these five animals developed histological cancer (P<0.05), whereas three had dysplasia. However, octreotide did not affect plasma gastrin concentration or antral gastrin mRNA abundance significantly. Dysplasia of the oxyntic mucosa in hypergastrinaemic animals was accompanied by a marked increase in chromogranin A-immunoreactive cells and cells positive for Sevier-Munger staining. The malignant tissue also contained groups of cells with Sevier-Munger staining. In conclusion, octreotide prevented ECL cell carcinomas in hypergastrinaemic cotton rats without lowering the gastrin concentration.

Determination of proliferation index in neoplasms using different Ki-67 equivalent antibodies.

Paraffin sections from 23 tumours were immunohistochemically stained with the following four Ki-67 equivalent antibodies: monoclonal MIB-1 (DAKO), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (DAKO). Ki-67 labelling indices were determined by counting in exactly the same area in each case. MIB-1 showed the highest labelling index in 21 of the 23 cases, and the mean MIB-1 index was approximately 30% higher than that of the other antibodies. The differences between MM1, NCL-Ki-67p and Rah Ki-67 were small and non-significant. There was a positive correlation between each of the four antibodies. As these findings may be of importance when the Ki-67 labelling index is used as a criterion for tumour grading or for clinical prognostication, this necessitate identification of the antibody used in every case.

Diagnostic and prognostic role of Ki67 immunostaining in human astrocytomas using four different antibodies.

OBJECTIVE: Astrocytomas have an inherent tendency to progress, and histopathological examination and grading do not always identify these subsets of tumors. The aim of this study was therefore to investigate whether different Ki67 antibodies could assist in the diagnostic and prognostic evaluation of these tumors. MATERIAL AND METHODS: Forty-one cerebral astrocytomas, graded according to the latest criteria of the World Health Organization, were included in the study: 22 diffuse fibrillary astrocytomas, 10 anaplastic astrocytomas and 9 glioblastomas. Standard immunohistochemical analyses were performed using 4 different commercially available Ki67 antibodies. The immunohistochemical data were correlated with the clinical course. RESULTS: There were positive correlations between the proliferation indices (PI) obtained with the different Ki67 antibodies. The Ki67 PIs increased significantly with increasing malignancy grade, and the antibodies discriminated well between low- (grade II) and high-grade tumors (grade III and IV). For the entire tumor material, the use of median values as cutoff divided the astrocytomas into 2 groups: those tumors with the higher Ki67 PIs had significantly poorer prognosis than those with lower indices. CONCLUSIONS: Ki67 immunostaining serves as an important supplementary tool in the diagnostic and prognostic evaluation of human astrocytomas.

Comparison of Ki-67 equivalent antibodies.

AIMS: To compare commercially available Ki-67 equivalent antibodies with regard to qualitative and quantitative immunohistochemical staining characteristics. METHODS: The following antibodies were used: monoclonal MIB-1 (Immunotech), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (Dako). All immunostainings were evaluated in squamous epithelium from formalin fixed and paraffin wax embedded pharyngeal tonsils. Labelling indices (LIs) were recorded twice to test their reproducibility. RESULTS: By application of all four antibodies the nuclear staining could be either diffuse, granular, or a combination of both (classified as granular in this study). The diffuse pattern generally showed a strong or moderate staining intensity, whereas the granular pattern displayed a continuum from strong to very weak, making it difficult to discriminate between positive and negative nuclei. The diffuse staining pattern was seen in approximately 59% of the nuclei with the MIB-1 antibody and in 35-45% when the other antibodies were used. The following mean LIs were recorded: MIB-1, 31%; NCL-Ki-67p, 21%; Rah Ki-67, 17%; and MM1, 14%. The reproducibility was excellent for all four antibodies, with the mean of differences between the two runs of counts ranging from 1.1% to 1.5%. CONCLUSIONS: The four tested Ki-67 equivalent antibodies revealed differences in qualitative and quantitative staining characteristics, which resulted in considerable variations in registered LIs. The MIB-1 antibody appears to have a higher sensitivity for detecting the Ki-67 antigen than the other three tested antibodies. These differences are important to consider when proliferative activity is determined by the Ki-67 LI.