Josef Zamecnik

Value of thyroid transcription factor-1 and surfactant apoprotein A in the differential diagnosis of pulmonary carcinomas: a study of 109 cases

Abstract Thyroid transcription factor-1 (TTF-1) and surfactant apoprotein A (SP-A) belong to tissue-specific markers expressed in the normal respiratory epithelium. Both proteins are expressed in some lung carcinomas, and they have potential diagnostic use. We performed an immunohistochemical study on 109 tumors to determine the usefulness of monoclonal SP-A (PE-10) and TTF-1 (8G7G3/1) antibodies in distinguishing primary and metastatic lung carcinomas (n=54) from a broad spectrum of nonpulmonary tumors (n=55). An immunoperoxidase method using a streptavidin—biotin kit was applied on paraffin sections. We found positive results for TTF-1 and SP-A in 75% and 46% of pulmonary adenocarcinomas and in 50% and 25% of pulmonary non-neuroendocrine large cell carcinomas (LCCs), respectively. Small cell lung carcinomas were TTF-1 positive in 89% of cases and completely negative for SP-A. Squamous cell carcinomas and carcinoid tumors were negative for both proteins. In the group of nonpulmonary tumors, TTF-1 was detected in 8 of 11 thyroid carcinomas and SP-A in 1 of 6 colorectal carcinomas. Other tumors, including seven cases of pleural mesothelioma, were negative for both TTF-1 and SP-A. The expression of both antibodies was independent of primary and metastatic sites of the tumor. We observed a significant decrease of SP-A immunoreactivity in poorly differentiated lung adenocarcinomas. The combination of anti-TTF-1 with anti-SP-A does not increase the diagnostic usefulness of TTF-1 alone. Because of its diagnostic utility TTF-1 should be added to a panel of antibodies used for assessing tumors of unknown origin.

Atypical Teratoid/Rhabdoid Tumors Are Comprised of Three Epigenetic Subgroups with Distinct Enhancer Landscapes

Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in infants. Although the prognosis of ATRT patients is poor, some patients respond favorably to current treatments, suggesting molecular inter-tumor heterogeneity. To investigate this further, we genetically and epigenetically analyzed 192 ATRTs. Three distinct molecular subgroups of ATRTs, associated with differences in demographics, tumor location, and type of SMARCB1 alterations, were identified. Whole-genome DNA and RNA sequencing found no recurrent mutations in addition to SMARCB1 that would explain the differences between subgroups. Whole-genome bisulfite sequencing and H3K27Ac chromatin-immunoprecipitation sequencing of primary tumors, however, revealed clear differences, leading to the identification of subgroup-specific regulatory networks and potential therapeutic targets.

Diffusion parameters of the extracellular space in human gliomas

Tumor cell migration through the extracellular space (ECS) might be affected by its pore size and extracellular matrix molecule content. ECS volume fraction α (α = ECS volume/total tissue volume), tortuosity λ (λ2 = free/apparent diffusion coefficient) and nonspecific uptake k′ were studied by the real‐time tetramethylammonium method in acute slices of human tissue. The diffusion parameters in temporal cortical tissue resected during surgical treatment of temporal lobe epilepsy (control) were compared with those in brain tumors. Subsequently, tumor slices were histopathologically classified according to the grading system of the World Health Organization (WHO), and proliferative activity was assessed. The average values of α, λ, and k′ in control cortex were 0.24, 1.55, and 3.66 × 10−3s−1, respectively. Values of α, λ, and k′ in oligodendrogliomas did not significantly differ from controls. In pilocytic astrogliomas (WHO grade I) as well as in ependymomas (WHO grade II), α was significantly higher, while λ and k′ were unchanged. Higher values of α as well as λ were found in low‐grade diffuse astrocytomas (WHO grade II). In cellular regions of high‐grade astrocytomas (WHO grade III and IV), α and λ were further increased, and k′ was significantly larger than in controls. Classic medulloblastomas (WHO grade IV) had an increased α, but not λ or k′, while in the desmoplastic type α and k′ remained unchanged, but λ was greatly increased. Tumor malignancy grade strongly corresponds to an increase in ECS volume, which is accompanied by a change in ECS structure manifested by an increase in diffusion barriers for small molecules. GLIA 42:77–88, 2003.

Expression of glutamate carboxypeptidase II in human brain.

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntingtons disease, Alzheimers disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.

The extracellular space and matrix of gliomas.

Changes in the properties of extracellular space (ECS) — its volume, shape, and composition — play an important role in influencing the biological behavior of brain tumors. Experimental methods allowing assessment of the volume and geometry of ECS by means of analyzing the diffusion of molecules within ECS have revealed a dramatic increase in the ECS volume of gliomas when compared with that of unaffected brain cortex, also correlating with increases in malignancy. However, the newly enlarged ECS of high-grade gliomas does not remain empty; ECS shape becomes more complicated than in normal brain tissue. In contrast to the low-grade tumors, where the diffusion of molecules is reduced mainly by the presence of a dense network of tumor cell processes, the increase of ECS barriers in high-grade gliomas is caused by the overproduction of certain components of the extracellular matrix (ECM), mainly of tenascin. These aberrantly or “overproduced” ECM glycoproteins not only stabilize the ECS volume, but also serve as a substrate for adhesion and subsequent migration of the tumor cells through the enlarged ECS. Interestingly, these same alterations in ECS structure may hinder the diffusion of neuroactive substances or even molecules of drugs into the neoplastic tissue. The presence of tenascin in the ECS of the neoplasm correlates significantly with increased malignancy and poor clinical outcome of the disease, which makes its immunohistochemical detection useful as a marker of an aggressive biological behavior of the tumors.

Pediatric Intracranial Ependymomas: Prognostic Relevance of Histological, Immunohistochemical, and Flow Cytometric Factors

The correlation between the histological features and clinical outcome remains poor in pediatric intracranial ependymomas. We performed a retrospective study of a group of 31 patients (diagnosed from 1985 to 1995) to assess prognostic implications of the current grading system, of histological and immunohistochemical features, and of ploidy status estimated by flow cytometry. Immunoexpression of a broad spectrum of antigens was evaluated, including MIB-1, topoisomerase-IIα, cyclin D1, glial and epithelial proteins (GFAP, EMA, cytokeratins), molecules involved in controlling apoptosis (bcl-2, caspase-3/CPP32), and p53 oncoprotein. Univariate and multivariate statistical analyses were performed to evaluate the influence of each variable on both the progression free survival (PFS) and the overall survival (OS) with at least 7-year follow up. Although we showed a significant correlation between histological grade and prognosis, the current grading system failed in predicting outcome in nearly one third of individual cases. Problems with interpathologist reproducibility were also demonstrated. The extent of surgical resection was the only clinical factor that was associated with survival. Both the PFS and the OS were significantly decreased for the following pathological variables: increased cellularity (>300 nuclei per HPF), mitotic activity of >7 per 10 HPF, increased MIB-1 labeling index (LI), topoisomerase-IIα LI, S-phase fraction, and p53 and bcl-2 positivity. Increased cyclin D1 LI was demonstrated to have only a marginally significant impact on PFS. A flow chart modeling was further performed to formulate a scheme for discriminating of prognostic subgroups. Based on that, p53 immunopositivity and/or MIB-1 LI of >5% (after subtotal resection) or MIB-1 LI of >15% (after complete resection) were the strongest indicators of the tumors aggressive behavior and of a poor prognosis of the disease. Foci of hypercellularity should be specifically looked for in ependymomas for assessing the immunohistochemical studies.

Human papillomavirus (HPV) profiles of vulvar lesions: possible implications for the classification of vulvar squamous cell carcinoma precursors and for the efficacy of prophylactic HPV vaccination.

The term vulvar intraepithelial neoplasia (VIN) introduced in 1986 incorporates 3 grades of usual VIN (u-VIN I-III) and the differentiated VIN (d-VIN). Although u-VIN is etiologically associated with the human papillomavirus (HPV) infection, d-VIN represents an alternative HPV negative pathway of vulvar carcinogenesis. In 2004, the u-VIN I category was abandoned and u-VIN II and III were merged. Further, an alternative Bethesda-like terminology scheme presenting the term vulvar intraepithelial lesion was proposed recently. To analyze the impact of HPV profiles of vulvar precancerous lesions for their classification and to assess the presumable efficacy of the prophylactic HPV vaccination, 269 vulvar excisions representing lichen sclerosus, lichen simplex chronicus, condylomata acuminata, d-VIN, all grades of u-VIN and squamous cell carcinomas were subjected to the HPV typing by use of GP5+/6+ polymerase chain reaction and reverse line blot hybridization. The results showed different HPV profiles, and also differing frequency of multiple-type HPV infection and the age structure in patients with u-VIN II and III. The biologic heterogeneity within the u-VIN II category was also demonstrated. u-VIN I was distinguished as a rare disorder associated with high-risk HPV infection. We conclude that the original VIN terminology proposed in 1986 seems to be appropriate for the classification of vulvar squamous dysplastic lesions. The spectrum of HPV types found in vulvar squamous cell carcinomas indicates that the efficacy of HPV vaccination in preventing vulvar cancer might be diminished in the studied population, because the recently developed prophylactic vaccines are targeted against a limited number of HPV types.

Good interobserver and intraobserver agreement in the evaluation of the new ILAE classification of focal cortical dysplasias

Purpose:  An International League Against Epilepsy (ILAE) consensus classification system for focal cortical dysplasias (FCDs) has been published in 2011 specifying clinicopathologic FCD variants. The aim of the present work was to microscopically assess interobserver agreement and intraobserver reproducibility for FCD categories among an international group of neuropathologists with different levels of experience and access to epilepsy surgery tissue.

Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate scaffolds in spinal cord injury in the rat

OBJECT Hydrogels are nontoxic, chemically inert synthetic polymers with a high water content and large surface area that provide mechanical support for cells and axons when implanted into spinal cord tissue. METHODS Macroporous hydrogels based on 2-hydroxyethyl methacrylate (HEMA) were prepared by radical copolymerization of monomers in the presence of fractionated NaCl particles. Male Wistar rats underwent complete spinal cord transection at the T-9 level. To bridge the lesion, positively charged HEMA hydrogels were implanted either immediately or 1 week after spinal cord transection; control animals were left untreated. Histological evaluation was performed 3 months after spinal cord transection to measure the volume of the pseudocyst cavities and the ingrowth of tissue elements into the hydrogels. RESULTS The hydrogel implants adhered well to the spinal cord tissue. Histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells into the hydrogels. Morphometric analysis of lesions showed a statistically significant reduction in pseudocyst volume in the treated animals compared with controls and in the delayed treatment group compared with the immediate treatment group (p < 0.001 and p < 0.05, respectively). CONCLUSIONS Positively charged HEMA hydrogels can bridge a posttraumatic spinal cord cavity and provide a scaffold for the ingrowth of regenerating axons. The results indicate that delayed implantation can be more effective than immediate reconstructive surgery.

Magnetic resonance tracking of human CD34+ progenitor cells separated by means of immunomagnetic selection and transplanted into injured rat brain.

Magnetic resonance imaging (MRI) provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied whether superparamagnetic nanoparticles (CD34 microbeads), used clinically for specific magnetic sorting, can be used as a magnetic cell label for in vivo cell visualization. Human cells from peripheral blood were selected by CliniMACS® CD34 Selection Technology (Miltenyi). Purified CD34+ cells were implanted into rats with a cortical photochemical lesion, contralaterally to the lesion. Twenty-four hours after grafting, the implanted cells were detected in the contralateral hemisphere as a hypointense spot on T2 weighted images; the hypointensity of the implant decreased during the first week. At the lesion site we observed a hypointensive signal 10 days after grafting that persisted for the next 3 weeks, until the end of the experiment. Prussian blue and anti-human nuclei staining confirmed the presence of magnetically labeled human cells in the corpus callosum and in the lesion 4 weeks after grafting. CD34+ cells were also found in the subventricular zone (SVZ). Human DNA (a human-specific 850 base pair fragment of α-satellite DNA from human chromosome 17) was detected in brain tissue sections from the lesion using PCR, confirming the presence of human cells. Our results show that CD34 microbeads superparamagnetic nanoparticles can be used as a magnetic cell label for in vivo cell visualization. The fact that microbeads coated with different commercially available antibodies can bind to specific cell types opens extensive possibilities for cell tracking in vivo.