Uncovering students' misconceptions by assessment of their written questions.

BACKGROUND: Misconceptions are ideas that are inconsistent with current scientific views. They are difficult to detect and refractory to change. Misconceptions can negatively influence how new concepts in science are learned, but are rarely measured in biomedical courses. Early identification of misconceptions is of critical relevance for effective teaching, but presents a difficult task for teachers as they tend to either over- or underestimate students' prior knowledge. A systematic appreciation of the existing misconceptions is desirable. This explorative study was performed to determine whether written questions generated by students can be used to uncover their misconceptions. METHODS: During a small-group work (SGW) session on Tumour Pathology in a (bio)medical bachelor course on General Pathology, students were asked to write down a question about the topic. This concerned a deepening question on disease mechanisms and not mere factual knowledge. Three independent expert pathologists determined whether the content of the questions was compatible with a misconception. Consensus was reached in all cases. Study outcomes were to determine whether misconceptions can be identified in students' written questions, and if so, to measure the frequency of misconceptions that can be encountered, and finally, to determine if the presence of such misconceptions is negatively associated with the students' course formal examination score. A subgroup analysis was performed according to gender and discipline. RESULTS: A total of 242 students participated in the SGW sessions, of whom 221 (91 %) formulated a question. Thirty-six questions did not meet the inclusion criteria. Of the 185 questions rated, 11 % (n = 20) was compatible with a misconception. Misconceptions were only found in medical students' questions, not in biomedical science students' questions. Formal examination score on Tumour Pathology was 5.0 (SD 2.0) in the group with misconceptions and 6.7 (SD 2.4) in the group without misconceptions (p = 0.003). CONCLUSIONS: This study demonstrates that misconceptions can be uncovered in students' written questions. The occurrence of these misconceptions was negatively associated with the formal examination score. Identification of misconceptions creates an opportunity to repair them during the remaining course sessions, in advance of the formal examination.

Diffusion tensor characteristics of gyrencephaly using high resolution diffusion MRI in vivo at 7T.

Gyrification of the human cerebral cortex allows for the surface expansion that accommodates many more cortical neurons in comparison to other mammals. For neuroimaging, however, it forms a feature that complicates analysis. For example, it has long been established that cortical layers do not occupy the same depth in gyri and sulci. Recently, in vivo diffusion imaging has provided insights into the fibre architecture of the cortex, usually showing radial tensor orientations. This makes it relevant to investigate whether cortical diffusion tensor metrics depend on the gyral pattern. High-resolution (1mm isotropic) diffusion weighted MRI of the medial wall of the hemispheres was performed at 7 T. Diffusion data were resampled to surfaces in the cortex and underlying white matter, where the cortical surfaces obeyed the equivolume principle for cortical laminae over the cortical curvature. Diffusion tensor metrics were averaged over bins of curvature to obtain maps of characteristic patterns in the gyrus. Diffusivity, anisotropy and radiality varied with curvature. Radiality was maximal in intermediate layers of the cortex next to the crown of the gyrus, not in white matter or on the crown. In the fundus, the deep cortical layers had tangential tensor orientations. In the white matter, tensor orientation changed from radial on the crown to tangential under the banks and fundus. White matter anisotropy gradually increased from the crown to the fundus. The characteristic pattern in the gyrus demonstrated here is in accordance with ex vivo diffusion MR microscopy and histological studies. The results indicate the necessity of taking into account the gyral pattern when cortical diffusion data is analysed. Additionally, the data suggest a confound for tractography approaches when reaching the gyrus, resulting in a possible bias towards the gyral crown. The implications for mechanisms that could drive cortical folding are discussed.

Challenging students to formulate written questions: a randomized controlled trial to assess learning effects.

BACKGROUND: Underutilization of dialogue among students during small-group work is a threat to active meaningful learning. To encourage small-group learning, we challenged students to generate written questions during a small-group work session. As gender differences have been shown to affect learning, these were also inventoried. METHODS: Prospective randomized study during a bachelor General Pathology course including 459 (bio) medical students, 315 females and 144 males. The intervention was to individually generate an extra written question on disease mechanisms, followed by a selection, by each student group, of the two questions considered to be most relevant. These selected questions were open for discussion during the subsequent interactive lecture. Outcome measure was the score on tumour pathology (range 1-10) on the course examination; the effect of gender was assessed. RESULTS: The mean score per student was 7.2 (intervention) and 6.9 (control; p = 0.22). Male students in the intervention group scored 0.5 point higher than controls (p = 0.05). In female students, this was only 0.1 point higher (p = 0.75). CONCLUSIONS: Formulating and prioritizing an extra written question during small-group work seems to exert a positive learning effect on male students. This is an interesting approach to improve learning in male students, as they generally tend to perform less well than their female colleagues.

Building on prior knowledge: schema-dependent encoding processes relate to academic performance.

The acquisition and retention of conceptual knowledge is more effective in well-structured curricula that provide an optimal conceptual framework for learning new material. However, the neural mechanisms by which preexisting conceptual schemas facilitate learning are not yet well understood despite their fundamental importance. A preexisting schema has been shown to enhance memory by influencing the balance between activity within the medial-temporal lobe and the medial pFC during mnemonic processes such as encoding, consolidation, and retrieval. Specifically, correctly encoding and retrieving information that is related to preexisting schemas appears rather related to medial prefrontal processing, whereas information unrelated or inconsistent with preexisting schemas rather relates to enhanced medial temporal processing and enhanced interaction between these structures. To further investigate interactions between these regions during conceptual encoding in a real-world university setting, we probed human brain activity and connectivity using fMRI during educationally relevant conceptual encoding carefully embedded within two course programs. Early second-year undergraduate biology and education students were scanned while encoding new facts that were either related or unrelated to the preexisting conceptual knowledge they had acquired during their first year of study. Subsequently, they were tested on their knowledge of these facts 24 hr later. Memory scores were better for course-related information, and this enhancement was associated with larger medial-prefrontal, but smaller medial-temporal subsequent memory effects. These activity differences went along with decreased functional interactions between these regions. Furthermore, schema-related medial-prefrontal subsequent memory effects measured during this experiment were found to be predictive of second-year course performance. These results, obtained in a real-world university setting, reveal brain mechanisms underlying acquisition of new knowledge that can be integrated into preexisting conceptual schemas and may indicate how relevant this process is for study success.

How to achieve synergy between medical education and cognitive neuroscience? An exercise on prior knowledge in understanding.

A major challenge in contemporary research is how to connect medical education and cognitive neuroscience and achieve synergy between these domains. Based on this starting point we discuss how this may result in a common language about learning, more educationally focused scientific inquiry, and multidisciplinary research projects. As the topic of prior knowledge in understanding plays a strategic role in both medical education and cognitive neuroscience it is used as a central element in our discussion. A critical condition for the acquisition of new knowledge is the existence of prior knowledge, which can be built in a mental model or schema. Formation of schemas is a central event in student-centered active learning, by which mental models are constructed and reconstructed. These theoretical considerations from cognitive psychology foster scientific discussions that may lead to salient issues and questions for research with cognitive neuroscience. Cognitive neuroscience attempts to understand how knowledge, insight and experience are established in the brain and to clarify their neural correlates. Recently, evidence has been obtained that new information processed by the hippocampus can be consolidated into a stable, neocortical network more rapidly if this new information fits readily into a schema. Opportunities for medical education and medical education research can be created in a fruitful dialogue within an educational multidisciplinary platform. In this synergetic setting many questions can be raised by educational scholars interested in evidence-based education that may be highly relevant for integrative research and the further development of medical education.

Challenging medical students with an interim assessment: a positive effect on formal examination score in a randomized controlled study.

Until now, positive effects of assessment at a medical curriculum level have not been demonstrated. This study was performed to determine whether an interim assessment, taken during a small group work session of an ongoing biomedical course, results in students' increased performance at the formal course examination. A randomized controlled trial was set up, with an interim assessment without explicit feedback as intervention. It was performed during a regular biomedical Bachelor course of 4 weeks on General Pathology at the Radboud University Nijmegen Medical Centre. Participants were 326 medical and 91 biomedical science students divided into three study arms: arm Intervention-1 (I-1) receiving one interim assessment; arm I-2 receiving two interim assessments, and control arm C, receiving no interim assessment. The study arms were stratified for gender and study discipline. The interim assessment consisted of seven multiple-choice questions on tumour pathology. Main outcome measures were overall score of the formal examination (scale 1-10), and the subscore of the questions on tumour pathology (scale 1-10). We found that students who underwent an interim assessment (arm I) had a 0.29-point (scale 1-10) higher score on the formal examination than the control group (p = 0.037). For the questions in the formal examination on tumour pathology the score amounted to 0.47 points higher (p = 0.007), whereas it was 0.17 points higher for the questions on topics related to the previous 3 weeks. No differences in formal examination score were found between arms I-1 and I-2 (p = 0.817). These findings suggest that an interim assessment during a small group work session in a randomized study setting stimulates students to increase their formal examination score.

Retrieval of associative information congruent with prior knowledge is related to increased medial prefrontal activity and connectivity.

We remember information that is congruent instead of incongruent with prior knowledge better, but the underlying neural mechanisms related to this enhancement are still relatively unknown. Recently, this memory enhancement due to a prior schema has been suggested to be based on rapid neocortical assimilation of new information, related to optimized encoding and consolidation processes. The medial prefrontal cortex (mPFC) is thought to be important in mediating this process, but its role in retrieval of schema-consistent information is still unclear. In this study, we regarded multisensory congruency with prior knowledge as a schema and used this factor to probe retrieval of consolidated memories either consistent or inconsistent with prior knowledge. We conducted a visuotactile learning paradigm in which participants studied visual motifs randomly associated with word-fabric combinations that were either congruent or incongruent with common knowledge. The next day, participants were scanned using functional magnetic resonance imaging while their memory was tested. Congruent associations were remembered better than incongruent ones. This behavioral finding was parallelized by stronger retrieval-related activity in and connectivity between medial prefrontal and left somatosensory cortex. Moreover, we found a positive across-subject correlation between the connectivity enhancement and the behavioral congruency effect. These results show that successful retrieval of congruent compared to incongruent visuotactile associations is related to enhanced processing in an mPFC-somatosensory network, and support the hypothesis that new information that fits a preexisting schema is more rapidly assimilated in neocortical networks, a process that may be mediated, at least in part, by the mPFC.

Molecular cytogenetics of cutaneous melanocytic lesions - diagnostic, prognostic and therapeutic aspects.

This review intends to update current knowledge regarding molecular cytogenetics in melanocytic tumours with a focus on cutaneous melanocytic lesions. Advantages and limitations of diverse, already established methods, such as (fluorescence) in situ hybridization and mutation analysis, to detect these cytogenetic alterations in melanocytic tumours are described. In addition, the potential value of more novel techniques such as multiplex ligation-dependent probe amplification is pointed out. This review demonstrates that at present cytogenetics has mainly increased our understanding of the pathogenesis of melanocytic tumours, with an important role for activation of the mitogen-activated protein kinase (MAPK) signalling pathway in the initiation of melanocytic tumours. Mutations in BRAF (in common naevocellular naevi), NRAS (congenital naevi), HRAS (Spitz naevi) and GNAQ (blue naevi) can all cause MAPK activation. All these mutations seem early events in the development of melanocytic tumours, but by themselves are insufficient to cause progression towards melanoma. Additional molecular alterations are implicated in progression towards melanoma, with different genetic alterations in melanomas at different sites and with varying levels of sun exposure. This genetic heterogeneity in distinct types of naevi and melanomas can be used for the development of molecular tests for diagnostic purposes. However, at the moment only few molecular tests have become of diagnostic value and are performed in daily routine practice. This is caused by lack of large prospective studies on the diagnostic value of molecular tests including follow-up, and by the low prevalence of certain molecular alterations. For the future we foresee an increasing role for cytogenetics in the treatment of melanoma patients with the increasing availability of targeted therapy. Potential targets for metastatic melanoma include genes involved in the MAPK pathway, such as BRAF and RAS. More recently, KIT has emerged as a potential target in melanoma patients. These targeted treatments all need careful evaluation, but might be a promising adjunct for treatment of metastatic melanoma patients, in which other therapies have not brought important survival advantages yet.

Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous melanoma.

Tumors are complex tissues composed of neoplastic cells, soluble and insoluble matrix components and stromal cells. Here we report that in melanoma, turn-over of type I collagen (Col(I)), the predominant matrix protein in dermal stroma affects melanoma progression. Fibroblasts juxtaposed to melanoma cell nests within the papillary dermis display high levels of Col(I) mRNA expression. These nests are enveloped by collagen fibers. In contrast, melanoma-associated fibroblasts within the reticular dermis express Col(I) mRNA at a level that is comparable to its expression in uninvolved dermis and reduced amount of collagen protein can be observed. To determine the significance of Col(I) expression in melanoma, we pharmacologically inhibited its transcription in a porcine cutaneous melanoma model by oral administration of halofuginone. When administered before melanoma development, it reduced melanoma incidence and diminished the transition from microinvasive toward deeply invasive growth by limiting the development of a tumor vasculature. Whereas invasive melanoma growth has been correlated with increased blood vessel density previously, our data for the first time demonstrate that the proangiogenic effect of Col(I) expression by fibroblasts and vascular cells precedes the development of invasive melanomas in a de novo tumor model.

Immunomonitoring tumor-specific T cells in delayed-type hypersensitivity skin biopsies after dendritic cell vaccination correlates with clinical outcome.

PURPOSE: Tumor-specific immunomonitoring is essential to evaluate the efficacy of vaccination against cancer. In this study, we investigated the predictive value of the presence or absence of antigen-specific T cells in biopsies from delayed-type hypersensitivity (DTH) sites. PATIENTS AND METHODS: In our ongoing clinical trials, HLA-A2.1+ melanoma patients were vaccinated with mature dendritic cells (DC) pulsed with melanoma-associated peptides (gp100 and tyrosinase) and keyhole limpet hemocyanin. RESULTS: After intradermal administration of a DTH challenge with gp100- and tyrosinase peptide-loaded DC, essentially all patients showed a positive induration. In clinically responding patients, T cells specific for the antigen preferentially accumulated in the DTH site, as visualized by in situ tetramer staining. Furthermore, significant numbers of functional gp100 and tyrosinase tetramer-positive T cells could be isolated from these DTH biopsies, in accordance with the applied antigen in the DTH challenge. We observed a direct correlation between the presence of DC vaccine-related T cells in the DTH biopsies of stage IV melanoma patients and a positive clinical outcome (P = .0012). CONCLUSION: These findings demonstrate the potency of this novel approach in the monitoring of vaccination studies in cancer patients.

Post-surgery adjuvant therapy with intermediate doses of interferon alfa 2b versus observation in patients with stage IIb/III melanoma (EORTC 18952): randomised controlled trial.

BACKGROUND: Individuals affected by melanoma with thick primary tumours or regional node involvement have a poor outlook, with only 30-50% alive at 5 years. High-dose and low-dose interferon alfa have been assessed for the treatment of these patients, with the former having considerable toxicity and a consistent effect on disease free survival, but not on overall survival, and the latter no consistent effect on either. Our aim was, therefore, to assess the effect of two regimens of interferon of intermediate dose versus observation alone on distant metastasis-free interval (DMFI) and overall survival in such patients. METHODS: We did a randomised controlled trial in 1388 patients who had had a thick primary tumour (thickness > or = 4 mm) resected (stage IIb) or regional lymph node metastases dissected (stage III) and had been assigned to 13-months (n=553) or 25 months (n=556) of treatment with subcutaneous interferon alfa 2b, or observation (n=279). Treatment comprised 4 weeks of 10 million units (MU) of interferon alfa (5 days per week) followed by either 10 MU three times a week for 1 year or 5 MU three times a week for 2 years, to a total dose of 1760 MU. Our primary endpoint was DMFI. Analyses were by intent to treat. FINDINGS: After a median follow-up of 4.65 years, we had recorded 760 distant metastases and 681 deaths. At 4.5 years, the 25-month interferon group showed a 7.2% increase in rate of DMFI (hazard ratio 0.83, 97.5% CI 0.66-1.03) and a 5.4% improvement in overall survival. The 13-month interferon group showed a 3.2% increase in rate of DMFI at 4.5 years (0.93, 0.75-1.16) and no extension of overall survival. Toxicity was acceptable, with 18% (195 of 1076) of patients going off study because of toxicity or as a result of refusal of treatment because of side-effects. INTERPRETATION: Interferon alfa as used in the regimens studied does not improve outcome for patients with stage IIb/III melanomas, and cannot be recommended. With respect to efficacy of the drug, duration of treatment seemed more important than dose, and should be assessed in future trials.

Induction of vascular endothelial growth factor receptor-3 expression on tumor microvasculature as a new progression marker in human cutaneous melanoma.

The anatomical relation between a malignant tumor and its vascular and lymphatic bed is an important factor influencing metastasis. Lack of specific markers for the lymphatic endothelium has long hampered a reliable detection of lymphatics. Here, we demonstrate that lymphatic endothelium can reliably be identified in a panel of different normal tissues and of benign and malignant tumors. Application of the previously described PAL-E/CD31 double staining protocol differentiates between blood capillaries and veins on one hand and lymphatic vessels on the other. Blood vessel marker CD34, absent from lymphatics, was used additionally to classify arteries. We found that the lymphatic vascular endothelial growth factor receptor-3 (VEGFR-3, also known as Flt-4) was present on both lymphatic and blood vessels in 76 of 113 malignant tumors [adenocarcinoma of kidney (n = 3), colon (n = 3) and liver (n = 3), breast (n = 9) and squamous cell carcinoma (n = 5), primary (n = 81), and metastatic (n = 9) melanoma]. No evident signs of tumor-induced lymphangiogenesis were observed. Evaluation of a series of 110 melanocytic skin lesions indicated that VEGFR-3 expression is confined to the lymphatic vasculature in benign lesions. However, its expression emerges on the blood neovasculature in malignant lesions as soon as metastatic potential develops. We conclude that induction of VEGFR-3 expression on tumor blood vessels may be a general phenomenon that would make VEGFR-3 a marker for tumor endothelium. In addition, we propose VEGFR-3 expression as a new microvascular progression marker in cutaneous melanoma.

Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the 121, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce endothelial cell proliferation, VEGF(121) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating endothelial cells accumulated in vessel lumina, giving these a microvascular, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neovascular bed. In VEGF(165)-expressing tumors, daughter endothelial cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different vascular phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(121)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.

Vascular endothelial growth factor-A(165) induces progression of melanoma brain metastases without induction of sprouting angiogenesis.

We investigated the mechanisms of vascularization in a brain metastases model of malignant melanoma. Parenchymal metastases expressing little vascular endothelial growth factor-A (VEGF-A) co-opted the preexistent brain vasculature, leading to an infiltrative phenotype. Metastases of the human melanoma cell line Mel57, engineered to express recombinant VEGF-A(165), showed accelerated growth in a combined expansive and infiltrative pattern with marked central necrosis. This difference in growth profile was accompanied by dilation of co-opted intra- and peritumoral vessels with concomitant induction of vascular permeability. Our data show that modulation of preexistent vasculature can contribute to malignant progression without induction of sprouting angiogenesis.

Stromal responses in human primary melanoma of the skin.

Tumour development and progression has long been considered as the consequence of an imbalance between apoptosis and proliferation of transformed cells. However, whereas genetic aberrations leading to the activation of oncogenes and/or loss of tumour suppressor genes are crucial for the transformation towards aberrant cell growth, progression towards a full blown malignancy requires a dynamic interaction between tumour cells and the environment in which they thrive. Over the recent years, it has become evident that the (early) inflammatory and angiogenic response, and remodelling of the extracellular proteins are key factors in creating a microenvironment that sustains tumour growth and metastasis. The host response towards cutaneous melanoma has received relatively little attention, most likely because the majority of these tumours develop without evoking a strong stromal response as can be observed in, e.g., carcinomas. This review discusses potential critical modulators of melanoma growth: turn-over of the most abundant extracellular matrix protein in skin (i.e. type I collagen), the early inflammatory response and angiogenesis.

Analysis of mutations in B-RAF, N-RAS, and H-RAS genes in the differential diagnosis of Spitz nevus and spitzoid melanoma.

A definite diagnosis cannot be established based on histologic features alone in a large number of Spitz nevi and spitzoid melanomas. In a vast majority of common benign and malignant melanocytic lesions, B-RAF and N-RAS mutations were described, but these were not detected in Spitz nevi. In contrast, H-RAS mutations were frequently encountered in Spitz nevi, but only rarely in melanomas. To date, B-RAF mutation analysis has not been reported in atypical Spitz nevi, and there are only a few reports of it in spitzoid melanomas. We analyzed 96 formalin-fixed, paraffin-embedded spitzoid melanocytic lesions for hotspot mutations in B-RAF, N-RAS, and H-RAS genes to test the assumption whether mutation analysis would assist a more accurate diagnosis of spitzoid melanocytic lesions, which are notoriously difficult to classify. B-RAF or N-RAS mutations were observed in 31 of 36 (86%) spitzoid melanomas, and in 6 of 7 (86%) spitzoid melanoma metastases. In contrast, none of the 14 Spitz nevi and none of the 16 atypical Spitz nevi had mutations in any of the three genes. A B-RAF or N-RAS mutation was found in 8 of 23 (35%) spitzoid lesions suspected for melanoma. H-RAS mutations were detected in 4 of 14 (29%) Spitz nevi, in 3 of 22 (14%) atypical Spitz nevi, in 1 of 15 (7%) spitzoid tumors suspected for melanoma, but in none of the spitzoid melanomas. These results strongly indicate that Spitz nevi and spitzoid melanomas are genetically unrelated entities. Furthermore, we can conclude that mutation analysis may be useful as an additional diagnostic tool to distinguish between benign and malignant spitzoid lesions.

INK4-ARF and p53 mutations in metastatic cutaneous squamous cell carcinoma: case report and archival study on the use of Ink4a-ARF and p53 mutation analysis in identification of the corresponding primary tumor.

So far, histopathologic, immunohistochemical and molecular properties of metastatic cutaneous squamous cell carcinomas (CSCCs) are relatively unexplored. In patients with multiple CSCCs, as for instance renal transplant recipients (RTRs), it might prove difficult to identify the primary tumor responsible for metastasis. We report a case of an RTR with multiple CSCCs, one of which metastasized. By using p53 and INK4a-ARF mutation analysis, we identified the responsible primary tumor due to an identical mutation in exon 2 of the INK4a-ARF locus. Archival study yielded 14 cases of metastatic CSCC (present case included). In only 8 of 14 metastases, DNA quality was sufficient to perform PCR reactions. In 7 of 8 metastases, either an INK4a-ARF (6 of 8 cases) and/or p53 (3 of 8 cases) mutation was present. In 6 of 7 cases, the corresponding primary could be identified by an identical mutation in p53 and/or INK4a-ARF. In conclusion, molecular analysis using a combination of p53 and INK4a-ARF mutation analysis can identify the corresponding primary skin tumor in case of CSCC metastases in the majority of cases. This is facilitated by the high frequency of these mutations in metastatic CSCC when compared with frequency spectra reported in the literature in primary CSCCs. The major limitation was formed by insufficient DNA quality in archival tissue.

Functional and morphologic analysis of the fluid-conducting meshwork in xenografted cutaneous and primary uveal melanoma.

PURPOSE: In primary uveal and cutaneous melanoma lesions, extracellular matrix (ECM) is often deposited in arcs, loops, and network patterns. Based on prognostic relevance, these patterns appear to play a significant role in facilitating metastasis. It has been demonstrated that these patterns were capable of transmitting fluid. The current study was undertaken to elucidate further the functional role of these patterns in tumor perfusion and to examine the composition of the patterns by immunohistochemistry. METHODS: To study the role of these patterns in perfusion, fluorochrome-labeled bovine serum albumin, bovine insulin, and dextrans of different molecular sizes were injected intravenously into nude mice bearing subcutaneous human cutaneous melanoma xenografts. Distribution of the human melanoma cells and murine host cells was analyzed by DNA in situ hybridization. To elucidate the composition of these patterns, human uveal melanoma tissues were analyzed for expression of ECM components by immunohistochemistry. RESULTS: Small molecules (Stokes' radius <4.4 nm) crossed the vessel wall and spread along the ECM patterns within 2 to 10 minutes, whereas larger molecules (Stokes' radius approximately 5.8 nm) required 30 to 45 minutes to enter. Murine host cells were found exclusively in the ECM pattern compartment. In primary uveal melanoma, different types of collagen, ECM-associated heparan sulfate proteoglycans, and different types of cells were present in the patterns. CONCLUSIONS: The data suggest that the ECM deposited as arcs, loops, and network patterns, accommodate the transport of plasma-derived molecules, (e.g., nutrients), to the tumor lesion, thus enhancing tumor growth and progression, and facilitating infiltration of tumor tissue by host-derived cells.

Decrease in circulating anti-angiogenic factors (angiostatin and endostatin) after surgical removal of primary colorectal carcinoma coincides with increased metabolic activity of liver metastases.

Removal of a primary colorectal tumor resulted in an increase in metabolic activity in its liver metastasis. Concomitantly, levels of angiostatin and endostatin in urine and plasma, respectively, dropped. This finding indicates that the primary tumor suppressed angiogenesis in its distant metastasis, and that removal of the primary lesion caused a flare-up in vessel neoformation and, thus, enhanced metabolic activity in its liver metastasis.

Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.

Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.