Rob de Waal

Circulating tumour tissue fragments in patients with pulmonary metastasis of clear cell renal cell carcinoma.

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the ‘seed and soil’ hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF‐A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF‐A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fishers exact test). In patients with tumours that, in retrospect, were not of the VEGF‐A‐expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF‐A‐induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis. Copyright

Amyloid beta induces cellular relocalization and production of agrin and glypican-1

The major component of senile plaques and vascular amyloid in Alzheimers disease (AD) brains is the amyloid beta protein (Abeta). Besides Abeta, several other proteins have been identified in these lesions, in particular heparan sulfate proteoglycans (HSPG). However, it is still unclear, what causes the excessive accumulation of HSPG in AD brains. Therefore, we investigated if Abeta may influence production and expression of two major Abeta-associated HSPG species, agrin and glypican-1. When human brain pericytes (HBP) were cultured in the presence of Abeta, protein and mRNA expression of both agrin and glypican-1 were increased and more radioactive sulfate was incorporated in the glycosaminoglycan fraction of Abeta-treated HBP. Furthermore, after Abeta treatment, these HSPG were found in association with the amyloid fibrils attached to the cell membrane, in contrast to the intracellular agrin and glypican-1 staining observed in untreated cells. We conclude that Abeta can modulate the cellular expression of agrin and glypican-1, which may contribute to the accumulation of these HSPG in AD lesions.

Histological analysis of defective colonic healing as a result of angiostatin treatment.

Antiangiogenic therapy is a highly promising new strategy in the treatment of cancer. One of the first angiogenesis inhibitors described was angiostatin, a 38-kDa internal proteolytically generated fragment of plasminogen. In a previous study we found that angiostatin affected physiological angiogenesis as well as tumor angiogenesis. It impaired healing when administered during repair of experimental colonic anastomoses, as reflected by a decrease in mechanical strength. On histology, we observed a decrease in factor VIII-stained vessel amount and volume in angiostatin-treated colonic anastomoses. The exact working mechanism of angiostatin has not been elucidated. Based on the available studies on proposed working mechanisms of angiostatin, we have attempted to address histological differences in physiological angiogenesis between the tissues of colonic anastomoses of mice with impaired healing and control mice. After angiostatin treatment there was more inflammatory tissue as a result of impaired healing. Furthermore, we found fewer vessels in the granulation tissue after angiostatin treatment. However, especially with respect to extracellular matrix (ECM), endothelial cell apoptosis, proliferation, or neutrophil influx, no gross differences were discerned 1 week following surgery, using histology and immunohistochemistry techniques.

Neoadjuvant Sorafenib Treatment of Clear Cell Renal Cell Carcinoma and Release of Circulating Tumor Fragments

BACKGROUNDnClear cell renal cell carcinoma (ccRCC) is characterized by high constitutive vascular endothelial growth factor A (VEGF-A) production that induces a specific vascular phenotype. We previously reported that this phenotype may allow shedding of multicellular tumor fragments into the circulation, possibly contributing to the development of metastasis. Disruption of this phenotype through inhibition of VEGF signaling may therefore result in reduced shedding of tumor fragments and improved prognosis. To test this hypothesis, we investigated the effect of neoadjuvant sorafenib treatment on tumor cluster shedding.nnnPATIENTS AND METHODSnPatients with renal cancer (n = 10, of which 8 have ccRCC) received sorafenib for 4 weeks before tumor nephrectomy. The resection specimens were perfused, and the perfundate was examined for the presence of tumor clusters. Effects of the treatment on the tumor morphology and overall survival were investigated (follow-up of 2 years) and compared with a carefully matched control group.nnnRESULTSnNeoadjuvant sorafenib treatment induced extensive ischemic tumor necrosis and, as expected, destroyed the characteristic ccRCC vascular phenotype. In contrast to the expectation, vital groups of tumor cells with high proliferation indices were detected in postsurgical renal venous outflow in 75% of the cases. Overall survival of patients receiving neoadjuvant treatment was reduced compared to a control group, matched with regard to prognostic parameters.nnnCONCLUSIONSnThese results suggest that neoadjuvant sorafenib therapy for ccRCC does not prevent shedding of tumor fragments. Although this is a nonrandomized study with a small patient group, our results suggest that neoadjuvant treatment may worsen survival through as yet undefined mechanisms.

Better effect of sorafenib on the rhabdoid component of a clear cell renal cell carcinoma owing to its higher level of vascular endothelial growth factor‐A production

Sir: Antiangiogenic treatment of clear cell renal cell carcinoma (cc-RCC) targeting different steps of the tyrosine kinase (TK) cascade has brought a revolutionary change in the management of metastatic disease. These agents affect the blood flow around or to a tumour by blocking the effects of vascular endothelial growth factor (VEGF)-A in the TK cascade. The smallmolecule TK inhibitors sorafenib and sunitinib target VEGF receptor 2 and platelet-derived growth factor receptor and prevent downstream signalling. These agents are also used in experimental settings in adjuvant and neo-adjuvant therapy regimens to increase progression-free disease survival or downsize tumours in patients with locally extensive or metastatic cc-RCC. cc-RCC is the most common malignancy of the kidney. cc-RCC with rhabdoid features is an aggressive variant of ccRCC, and accounts for approximately 5% of adult RCC cases. The rhabdoid component is defined on the basis of morphological features, and its presence is related to high histological Fuhrman grade and advanced pathological stage. The rhabdoid and nonrhabdoid components in composite tumours have been shown to carry the same genetic aberrations, suggesting that these histologically distinct patterns arise from the same malignant precursor cell. Here, we present a patient in whom histological changes in a primary cc-RCC with rhabdoid features after 1 week of neo-adjuvant treatment with sorafenib were observed. The rhabdoid component of the tumour was more sensitive to sorafenib treatment. A 59-year-old woman with rheumatoid arthritis, hypertension and depression was diagnosed with a mass suspicious for cc-RCC in the upper pole of the left kidney. Clinical staging studies showed no metastases. The patient was referred to our university hospital for laparoscopic nephrectomy. At admission, she was enrolled in a clinical trial (http://www.clinicaltrials. gov/ct2/show/ NCT00602862) involving 1 week of neoadjuvant sorafenib (oral, 400 mg twice-daily) treatment. The time between cessation of medication and surgery was 60 h. The gross pathology specimen showed a centrally located nodular tumour of diameter 60 mm that extended into the perirenal fat tissue. The tumour was yellow–white with extensive necrosis and focal cystic changes. Histologically, the tumour was composed of a rhabdoid component with higher nuclear grade (grade 4) and a clear cell component (Figure 1A,B respectively). Treatment effects differed remarkably between the rhabdoid component and the non-rhabdoid cc-RCC component. In the rhabdoid component, extensive necrosis was observed, which we propose is secondary to prominent damage to the tumour vasculature. Loss of endothelial function was frequently observed, as shown by detachment of the endothelial cells, fibrinoid necrosis, thrombus formation, and obliteration of the vascular lumen. The perivascular tumour tissue showed ischaemic necrosis with mixed inflammatory cells (Figure 1C). The non-rhabdoid cc-RCC component sometimes showed perivascular hyalinization, which can be attributed to degeneration of the perivascular tumour tissue. The remaining perivascular vital tumour tissue showed clear cytoplasm, a lower nuclear grade, and shrinking of nuclei (Figure 1D). Immunohistochemical anti-VEGF-A staining showed stronger staining of the rhabdoid component than of the clear cell component (Figures 1E,F respectively). In a control group of five cc-RCCs with rhabdoid features, randomly chosen from our pathology archive, we could not detect blood vessel ⁄ endothelial damage. In 5 ⁄ 5 tumours, dispersed areas of necrosis could be observed in the rhabdoid components, which stands in contrast to the extensive necrosis observed in the current case. In 4 ⁄ 5 tumours, the rhabdoid component also showed stronger staining with anti-VEGF-A antibody than the ccRCC component (not shown). Our combined results suggest that the more aggressive rhabdoid component in ccRCC with rhabdoid features produces more VEGF-A and is therefore more susceptible to inhibition of VEGF-A effects by TK inhibitors. These findings indicate that the success of antiangiogenic therapy might be predicted on the basis of histological features of the tumour, but this should be verified in a larger patient population.

Explicit feedback to enhance the effect of an interim assessment: a cross-over study on learning effect and gender difference

In a previous study we demonstrated by a prospective controlled design that an interim assessment during an ongoing small group work (SGW) session resulted in a higher score in the course examination. As this reflects the so-called testing effect, which is supposed to be enhanced by feedback, we investigated whether feedback following an interim assessment would have an effect on the score of the course exam, and whether the effect is influenced by the gender of the student. During a General Pathology bachelor course all 386 (bio) medical students took an interim assessment on the topics cell damage (first week) and tumour pathology (fourth week). The intervention consisted of immediate detailed oral feedback on the content of the questions of the interim assessment by the tutor, including the rationale of the correct and incorrect answers. It concerned a prospective randomized study using a cross-over design. Outcome measures were: (1) the difference in the normalized scores (1–10) of the course examination multiple choice questions related to the two topics, (2) effect of gender, and (3) gender-specific scores on formal examination. The effect of feedback was estimated as half the difference in the outcome between the two conditions. Mixed-model analysis was used whereby the SGW group was taken as the study target. The scores of the questions on cell damage amounted to 7.70 (SD 1.59) in the group without and 7.78 (SD 1.39) in the group with feedback, and 6.73 (SD 1.51) and 6.77 (SD 1.60), respectively, for those on tumour pathology. No statistically significant effect of feedback was found: 0.02 on a scale of 1–10 (95xa0% CI: −0.20; 0.25). There were no significant interactions of feedback with gender. Female students scored 0.43 points higher on the formal examination in comparison with their male colleagues. No additional effect of immediate explicit feedback following an interim assessment during an SGW session in an ongoing bachelor course could be demonstrated in this prospective randomized controlled study. Gender analysis revealed a higher performance of female students on the formal examination, which could not be explained by the effect of feedback in the current study. In this particular learning environment, SGW, explicit feedback may have little added value to the interactive learning that includes implicit feedback.

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

BackgroundUnderutilization 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.MethodsProspective 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.ResultsThe mean score per student was 7.2 (intervention) and 6.9 (control; pu2009=u20090.22). Male students in the intervention group scored 0.5 point higher than controls (pu2009=u20090.05). In female students, this was only 0.1 point higher (pu2009=u20090.75).ConclusionsFormulating 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.

ABCA-1-mediated beta-amyloid uptake by cells of the blood-brain barrier

out animals. Results: Our data suggests that genetic knockdown of synaptojanin 1 (synj1) decreases Ab levels in cell culture, and reduces amyloid load in AD transgenic animals. The amounts of b CTF are increased. Further, there is a significantly increase of ApoE secretion in cortical neuron/astrocyte culture derived from synj1 -/animals if compared to controls. Conclusions: Together, our results suggest that one phosphoinositol modulator, synaptojanin 1, can regulate amyloid homeostasis, likely through its effects on Ab generation and clearance.

Small heat shock proteins associated with extracellular Aβ induce an inflammatory reaction in cultured human cerebrovascular cells

cent advances in molecular imaging and the use of different PET ligands for in vivo Ab imaging in AD has opened up a new dimension for further understanding of the complex disease processes occurring during the progression of disease. Longitudinal [C]-PIB PET imaging studies in subjects with mild cognitive impairment and AD have demonstrated that Ab accumulates in the brain early during life. It is suggested that this build-up of Ab in the AD brain triggers a cascade of neurodegenerative events, including inflammatory processes, oxidative stress, neurofibrillary tangles, neuronal network dysfunction with synaptic loss and neurotransmitter deficits leading to cognitive impairment. However, it is not yet clear how the amount and distribution of Aß in brain areas affected by AD is associated with the abovementioned neurodegenerative changes during disease progression. Methods: In the present study, we examined bymeans of quantitative in vitro autoradiography the laminar distributions of fibrillar Ab (H-PIB) and activated astrocytes (H)L-deprenyl) in relation to the distribution of cholinergic nicotinic receptors (H)-nicotine) in the autopsy brain hemisphere of a histopathological confirmed AD patient. Results: Different laminar distribution patterns of fibrillar Ab and activated astrocytes were observed in the various brain regions studied. Brain regions with high fibrillar Ab showed a reduced distribution of neuronal nicotinic receptors confirming a recent observation reported by our group (Kadir et al., 2011. Brain 134:301-317). Conclusions: The different laminar binding patterns for H-PIB andH-L-deprenyl as well as the reduced binding of [H]-nicotine in AD brain regions may reflect different pathological mechanisms during disease progression.

Enoxaparin improves cognition of APPswe/PS1dE9 and TgSwDI mice with minor effect on amyloid β levels

bapineuzumab reached maximum concentrations within one hour after each infusion in most subjects. At the four doses assessed, the average volume of distribution at steady state, total body clearance, and apparent terminal half-life of bapineuzumab ranged from 49-80 mL/kg, 0.07-0.09 mL/hr/kg, and 20-33 days, respectively. Exposure to serum bapineuzumab in terms of maximum concentration and area under the concentration-time curve (AUC) increased dose proportionally with the average AUC ratios between the third and first infusions ranging from 1.0-1.6. Plasma levels of Ab40 gradually reached maximum after approximately one day, and the exposure to plasma Ab40 increased less than dose proportionally. CSF bapineuzumab concentrations increased with dose (4.5 to 39.3 ng/mL), while the average ratios of CSF to serum bapineuzumab concentrations were consistent at 0.0020.003. No anti-bapineuzumab antibodies were detected in any subject. Conclusions: The pharmacokinetics of bapineuzumab can be characterized by small volume of distribution, slow clearance, and long terminal half-life. Exposure to bapineuzumab was dose proportional at the range observed with minimal-to-modest accumulation per current dosing interval. Plasma levels of Ab40 increased in response to bapineuzumab, reaching maximum approximately one day after each infusion. Consistent ratios of CSF to serum bapineuzumab concentrations indicated dose-independent flow from serum to CSF. The absence of anti-bapineuzumab antibodies indicated a low likelihood of immunogenicity. The observed pharmacokinetic profile of bapineuzumab supports the dose regimen currently tested in phase 3 clinical trials.