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 (95 % 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 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.

Soluble adhesion molecules in human cancers: sources and fates.

Adhesion molecules endow tumor cells with the necessary cell-cell contacts and cell-matrix interactions. As such, adhesion molecules are involved in cell signalling, proliferation and tumor growth. Rearrangements in the adhesion repertoire allow tumor cells to migrate, invade and form metastases. Besides these membrane-bound adhesion molecules several soluble adhesion molecules are detected in the supernatant of tumor cell lines and patient body fluids. Truncated soluble adhesion molecules can be generated by several conventional mechanisms, including alternative splicing of mRNA transcripts, chromosomal translocation, and extracellular proteolytic ectodomain shedding. Secretion of vesicles (ectosomes and exosomes) is an alternative mechanism mediating the release of full-length adhesion molecules. Soluble adhesion molecules function as modulators of cell adhesion, induce proteolytic activity and facilitate cell signalling. Additionally, adhesion molecules present on secreted vesicles might be involved in the vesicle-target cell interaction. Based on currently available data, released soluble adhesion molecules contribute to cancer progression and therefore should not be regarded as unrelated and non-functional side products of tumor progression.

Attenuation of melanoma invasion by a secreted variant of activated leukocyte cell adhesion molecule.

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD), a marker of various cancers and mesenchymal stem cells, is involved in melanoma metastasis. We have exploited a secreted NH(2)-terminal fragment, sALCAM, to test the hypothesis that ALCAM coordinates tissue growth and cell migration. Overexpression of sALCAM in metastatic melanoma cells disturbed clustering of endogenous ALCAM and inhibited activation of matrix metalloproteinase-2 (MMP-2). Exposure of HT1080 fibrosarcoma cells to sALCAM similarly inhibited MMP-2, suggesting a broader effect on ALCAM-positive tumor cells. In contrast to the previously reported, promotive effects of an NH(2)-terminally truncated, transmembrane variant (DeltaN-ALCAM), sALCAM impaired the migratory capacity of transfected cells in vitro, reduced basement membrane penetration in reconstituted human skin equivalents, and diminished metastatic capacity in nude mice. Remarkably, L1 neuronal cell adhesion molecule (L1CAM/CD171), another progression marker of several cancers including melanoma, was suppressed upon sALCAM overexpression but was up-regulated by DeltaN-ALCAM. The partially overlapping and opposite effects induced by alternative strategies targeting ALCAM functions collectively attribute an integrative role to ALCAM in orchestrating cell adhesion, growth, invasion, and proteolysis in the tumor tissue microenvironment and disclose a therapeutic potential for sALCAM.

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.

Dietary fat and risk of colon and rectal cancer with aberrant MLH1 expression, APC or KRAS genes.

OBJECTIVE: To investigate baseline fat intake and the risk of colon and rectal tumors lacking MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) repair gene expression and harboring mutations in the APC (adenomatous polyposis coli) tumor suppressor gene and in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) oncogene. METHODS: After 7.3 years of follow-up of the Netherlands Cohort Study (n = 120,852), adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed, based on 401 colon and 130 rectal cancer patients. RESULTS: Total, saturated and monounsaturated fat were not associated with the risk of colon or rectal cancer, or different molecular subgroups. There was also no association between polyunsaturated fat and the risk of overall or subgroups of rectal cancer. Linoleic acid, the most abundant polyunsaturated fatty acid in the diet, was associated with increased risk of colon tumors with only a KRAS mutation and no additional truncating APC mutation or lack of MLH1 expression (RR = 1.41, 95% CI 1.18-1.69 for one standard deviation (i.e., 7.5 g/day) increase in intake, p-trend over the quartiles of intake <0.001). Linoleic acid intake was not associated with risk of colon tumors without any of the gene defects, or with tumors harboring aberrations in either MLH1 or APC. CONCLUSION: Linoleic acid intake is associated with colon tumors with an aberrant KRAS gene, but an intact APC gene and MLH1 expression, suggesting a unique etiology of tumors with specific genetic aberrations.

Environmental factors and colorectal tumor risk in individuals with hereditary nonpolyposis colorectal cancer.

BACKGROUND & AIMS: Individuals with hereditary nonpolyposis colorectal cancer (HNPCC) are at increased risk for colorectal cancer. Environmental factors might play a role in HNPCC-associated carcinogenesis. The aim of this study was to gain insight into the effects of environmental factors on colorectal tumor risk in individuals with HNPCC. METHODS: We examined associations between dietary factors, cigarette smoking, and HNPCC-associated colorectal tumors in a Dutch case-control study (145 cases, 103 tumor-free controls; all study participants were known or suspected carriers of a germline mutation in one of the DNA mismatch repair genes). We also assessed associations between the various environmental factors and occurrence of adenomatous polyposis coli (APC) mutations in HNPCC-associated polyps in a subset of the study population. RESULTS: Fruit consumption was inversely associated with ever developing HNPCC-associated colorectal tumors (odds ratio [95% confidence interval] for highest vs lowest tertile, 0.4 [0.2-0.9]; P(trend) = .03); a borderline significant inverse association was observed for dietary fiber intake (0.5 [0.2-1.0]; P(trend) = .06). Cigarette smoking seemed to increase the risk of HNPCC-associated colorectal tumors. Truncating APC mutations were detected in 30 (37.5%) of the 80 available HNPCC-associated polyps; frameshift mutations were most common (73.3%). None of the evaluated environmental factors was distinctively associated with a specific APC status of the polyps. CONCLUSIONS: Our data suggest that fruit consumption and dietary fiber intake might decrease the risk of colorectal tumors in individuals with HNPCC, whereas cigarette smoking might increase the risk of HNPCC-associated colorectal tumors. The observed associations support the hypothesis that HNPCC-associated outcomes might be modified by environmental factors.

Diet, lifestyle and risk of K-ras mutation-positive and -negative colorectal adenomas.

K-ras mutation-positive (K-ras+) and -negative (K-ras-) colorectal adenomas may differ clinically and pathologically. As environmental compounds may cause mutations in the growth-related K-ras oncogene or affect clonal selection depending on mutational status, we evaluated whether the aetiology of K-ras+ and K-ras- adenomas differs. K-ras mutations in codons 12 and 13 were assessed in colorectal adenoma tissue (K-ras+: n = 81, K-ras-: n = 453). Dietary and lifestyle data were collected through questionnaires that were also administered to 709 polyp-free controls. Multiple logistic regression analyses showed that intake of vitamin B2 and monounsaturated fat were differently associated with risk of K-ras+ and K-ras- adenomas; vitamin B2 was inversely associated with K-ras- (highest vs. lowest tertile: odds ratio (OR) = 0.70, 95% confidence interval (CI) = 0.50-0.97, p trend = 0.020), but not with K-ras+ adenomas, and a positive association with monounsaturated fat was confined to K-ras- adenomas (OR = 1.57, 95% CI = 1.06-2.34, p trend = 0.029). Besides, potential, not statistically significant, differences in risk arose because red meat was distinctly positively associated with K-ras+ adenomas (OR = 1.70, 95% CI = 0.94-3.09, p trend = 0.061); total dietary and polyunsaturated fat tended to be inversely associated with risk of K-ras+ but not of K-ras- adenomas; inverse associations with dairy products, calcium, protein and tea were confined to K-ras- adenomas, and smoking was more markedly positively associated with K-ras- adenomas. No differences in risk of K-ras+ and K-ras- adenomas could be detected for other factors. In conclusion, dietary and lifestyle factors may influence risk of K-ras+ and K-ras- adenomas differently. However, epidemiological literature on diet, lifestyle and colorectal K-ras mutations is inconsistent.

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study.

BACKGROUND: The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. METHODS: In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. RESULTS: Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CONCLUSION: CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD), a novel actor in invasive growth, controls matrix metalloproteinase activity.

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD) could function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in melanoma progression. We have tested the hypothesis that progressive cell clustering controls the proteolytic cascade for activation of gelatinase A/matrix metalloproteinase-2 (MMP-2), which involves formation of an intermediate ternary complex of membrane type 1 MMP (MT1-MMP/MMP-14), tissue inhibitor of metalloproteinase-2 (TIMP-2), and pro-MMP-2 at the cell surface. Surprisingly, truncation of ALCAM severely impaired MMP-2 activation in a nude mouse xenograft model, in which we previously observed diminished primary tumor growth and enhanced melanoma metastasis. Comparative studies of two-dimensional monolayer and three-dimensional collagen-gel cultures revealed that extensive cell-to-cell contacts, wild-type ALCAM, and cell-to-matrix interactions were all indispensable for efficient conversion of pro-MMP-2 to its active form in metastatic melanoma cells. Truncated, dominant-negative ALCAM diminished MMP-2 activation via reduced transcript levels and decreased processing of MT1-MMP. Failure of the proteolytic cascade after selective ALCAM depletion by RNA interference was mainly due to incomplete MT1-MMP processing, which was otherwise promoted by extensive cell-to-cell contacts. These data attribute a novel signaling role to ALCAM in regulation of proteolysis and support its previously postulated sensor function in invasive growth.

Fruits, vegetables, and hMLH1 protein-deficient and -proficient colon cancer: The Netherlands cohort study.

BACKGROUND: Clinical and pathologic differences exist between colon carcinomas deficient and proficient in the mismatch repair protein hMLH1. Animal and in vitro studies suggest that fruits, vegetables, folate, and antioxidants are associated with colonic expression of mismatch repair genes. METHODS: Associations between consumption of fruits and vegetables and hMLH1 protein-deficient and -proficient colon cancer were evaluated in the Netherlands Cohort Study on diet and cancer using a case-cohort approach. A self-administered food frequency questionnaire was completed, in 1986, by 120,852 individuals ages 55 to 69 years. Using immunohistochemistry, hMLH1 protein expression was assessed in colon cancer tissue obtained from 441 patients who were identified over 7.3 years of follow-up excluding the initial 2.3 years. Incidence rate ratios (RR) were estimated for hMLH1 protein-deficient and -proficient colon cancer. RESULTS: hMLH1 protein expression was absent in 54 tumors (12.2%) and present in 387 tumors. Fruit consumption was associated with hMLH1 protein-deficient colon cancer [highest versus lowest tertile, RR, 0.46; 95% confidence interval (95% CI), 0.23-0.90; P(trend) = 0.029] but not with hMLH1 protein-proficient tumors (highest versus lowest tertile, RR, 1.03; 95% CI, 0.78-1.35; P(trend) = 0.81). Total consumption of vegetables was not associated with either type of tumor (hMLH1 protein deficient: RR, 0.86; 95% CI, 0.45-1.65; P(trend) = 0.67; hMLH1 protein proficient: RR, 0.94; 95% CI, 0.72-1.23; P(trend) = 0.72). No associations were observed for folate, fiber, antioxidants, or subgroups of vegetables. CONCLUSION: These analyses indicate that an inverse association between consumption of fruits and colon cancer may be confined to the subgroup of tumors with a deficient mismatch repair system.

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.

Dietary factors and truncating APC mutations in sporadic colorectal adenomas.

Inactivating mutations in APC are thought to be early, initiating events in colorectal carcinogenesis. To gain insight into the relationship between diet and inactivating APC mutations, we evaluated associations between dietary factors and the occurrence of these mutations in a Dutch case-control study of sporadic colorectal adenomas (278 cases; 414 polyp-free controls). Direct-sequencing was used to screen adenomas for mutations in the mutation cluster region of APC; truncating mutations were detected in 161 (58%) of the adenomas. Red meat consumption was significantly differently related to polyps with truncating APC mutation (APC(+) polyps) compared to polyps without truncating APC mutation (APC(-) polyps) (highest vs. lowest tertile, odds ratio [OR] = 0.5, 95% confidence interval [CI] = 0.3-1.0). High intake of red meat and fat seemed to increase the risk of APC(-) polyps only (APC(+) vs. controls: red meat, OR = 1.0, 95% CI = 0.6-1.6; fat, OR = 1.1, 95% CI = 0.6-1.9; APC(-) vs. controls: red meat, OR = 1.8, 95% CI = 1.0-3.1; fat, OR = 1.9, 95% CI = 1.0-3.7). Intake of carbohydrates was inversely associated with both polyp groups, most noticeably with APC(-) polyps. Most other evaluated dietary factors were not distinctively associated with a specific APC status. None of the dietary factors was specifically associated with a particular type of truncating APC mutation. Our data suggest that red meat and fat may increase the risk of APC(-) polyps in particular, whereas carbohydrates may especially decrease the risk of APC(-) polyps. However, most examined dietary factors do not appear to be specifically associated with the occurrence of truncating APC mutations in colorectal adenomas but seem to affect both pathways equally.

The MMA1 gene family of cancer-testis antigens has multiple alternative splice variants: characterization of their expression profile, the genomic organization, and transcript properties.

Previously, we reported the identification of MMA1A by screening for differential gene expression in two human melanoma cell lines displaying diverse metastatic behavior after subcutaneous inoculation into nude mice. Splice variant MMA1B, which also was identified through database homology searches, showed a high degree of similarity with the MMA1A for exons 1, 2, and 4, but was missing exon 3. Through extensive expression profiling among normal and tumor samples, both MMA1A and -1B were found to belong to the family of cancer-testis antigens. In this study, we identified four additional alternatively spliced MMA1 variants, named MMA1C, MMA1D, MMA1E, and MMA1F. Generally, these novel MMA1 transcripts differ from MMA1A in that exon 2 or exon 3 is enlarged because of the use of alternative splice sites in intron 2 of the MMA1 gene. Moreover, MMA1E also lacks exon 3, as was previously seen in MMA1B. In screening for expression of the novel MMA1 transcripts in normal and tumor tissues, we demonstrated that MMA1C, -1D, and -1E also are members of the cancer-testis antigen family. MMA1F was found in only one melanoma metastasis sample and therefore is believed to have been expressed incidentally. Furthermore, we comprehensively elucidated the genomic structure of the MMA1 gene and the characteristic features of the alternatively spliced MMA1 transcripts.

Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1.

Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappé et al., Biochim. Biophys. Acta 1520, 1-6, 2001). Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues. Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR. Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining. We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens. To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins. TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9. Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells. The possible functional significance of this interaction is discussed.

Type I collagen synthesis parallels the conversion of keratinocytic intraepidermal neoplasia to cutaneous squamous cell carcinoma.

Neoplastic progression of solid tumours is often characterized by a simultaneous increase in matrix protein (eg collagen) synthesis and degradation, and results in the formation of a tumour stroma. At the tumour periphery, this process is believed to facilitate angiogenesis and invasive growth of tumour cells. In various types of carcinoma, differentiation of fibroblasts towards myofibroblasts is thought to play an important role in extracellular matrix remodelling as their emergence coincides with architectural changes in the tumour stroma. Here, distinct architectural changes in collagen fibres are reported in cutaneous squamous cell carcinomas (cSCC) with respect to normal skin and precursor lesions, ie keratinocytic intraepidermal neoplasia (KIN). Simultaneously, type I collagen mRNA was observed in fibroblasts in close proximity to cSCC lesions (19/19) but only in 2 of 10 KIN lesions tested. Interestingly, whereas emerging of myofibroblasts correlated with reduced differentiation of cSCCs, it was not a prerequisite for type I collagen synthesis. These data indicate that type I collagen synthesis by fibroblasts parallels the malignant transformation of human KIN to cSCC.

Detection of 2-O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n.

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of alpha-d-N-acetylglucosaminyl-2-O-sulfo-alpha-l-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V(H)3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.

Truncation of activated leukocyte cell adhesion molecule: a gateway to melanoma metastasis.

Progression of human cutaneous primary melanoma is, among others, accompanied by de novo expression of activated leukocyte cell adhesion molecule (ALCAM/CD166) and enhanced activity of proteolytic cascades in the invasive, vertical growth phase (VGP) of lesions. The homophilic cell adhesion function of wild-type ALCAM mediates homotypic clustering of melanoma cells and would, thus, antagonize cell release from the primary tumor, an early prerequisite for metastasis. Stable transfection of a transmembrane, amino-terminally truncated ALCAM (DeltaN-ALCAM) into metastatic cells diminished cell clustering mediated by wild-type ALCAM. We have addressed the biological effects of DeltaN-ALCAM on tumorigenicity and found that the relief of cell clustering constraints promoted motility in vitro and the transition from expansive tumor growth to tissue invasion in reconstructed skin in culture. In a transplant tumor model, the changes were reflected in reduced subcutaneous tumor growth and in accelerated, spontaneous lung metastasis. These data indicate that the intact cell adhesion function of ALCAM may both favor primary tumor growth and represent a rate-limiting step for tissue invasion from VGP melanoma. ALCAM induction could, thus, provide an attractive target for proteolysis as a part of a more complex cellular program that couples growth and migration and facilitates dissemination.

Human single-chain antibodies reactive with native chondroitin sulfate detect chondroitin sulfate alterations in melanoma and psoriasis.

Chondroitin sulfate (CS) belongs to the group of glycosaminoglycans (GAGs), which are linear polysaccharides, located in the extracellular matrix and on the cell surface. To study the structure and distribution of CS in human skin and skin disorders, we have selected antibodies using phage display technique against CS. Four unique human anti-CS single-chain antibodies were selected: IO3D9, IO3H10, IO3H12, and IO4C2. We determined their amino acid sequence and evaluated their CS reactivity using ELISA and immunohistochemistry. Antibodies were reactive with CS, but not with other GAGs except for IO4C2, which was also reactive with heparin. Antibody IO3D9 showed a strong reactivity with highly sulfated CS (CSE). All antibodies displayed a different staining pattern in rat kidney, indicating the recognition of unique CS epitopes. In normal skin, the papillary dermis but not the reticular dermis was strongly stained. Antibody IO3H12 also stained basal keratinocytes. We applied these antibodies to study CS expression and localization in melanoma and psoriasis. A strong immunoreactivity with the extracellular matrix of melanoma metastases could be observed for all four antibodies, while in atypical nevi a less extensive reactivity with only the papillary dermis was observed. In psoriatic lesions, CS could be observed in the papillary dermis and in the reticular dermis, whereas the specific location in the papillary dermis found in normal skin was completely lost. In conclusion, human phage-display-derived anti-CS antibodies have been selected, characterized, and applied to detect CS alterations in skin conditions. Altered CS composition was detected in melanoma and psoriasis.