Heterogenous S-100B protein expression patterns in malignant melanoma and association with serum protein levels

OBJECTIVE: Serum S-100B is a reliable tumor marker of malignant melanoma, but efficient use is restricted to patients with metastatic disease. Therefore, the aim of our study was to assess serum S-100B levels at different stages of malignant melanoma and to compare these levels with the expression of the S-100B phenotype in primary tumors and lymph node metastases. METHODS: Fifty-nine patients were included in this study; serum S-100B protein was measured using an immunoluminometric assay while the expression pattern in the primary tumor was determined by immunohistochemistry using an anti-S-100B monoclonal antibody. RESULTS: Serum S-100B concentrations were significantly elevated in stage III (p = 0.01) patients, with normal levels in stage I-II. The most frequent S-100B protein expression pattern of the melanoma tissue was found to be diffuse staining observed in around half of the cases (52.5%) followed by heterogeneous (30.5%) and focal patterns (17%), being independent of the stage as well as the lymph node involvement. In stage I-II patients, the various staining patterns did not correlate with the serum concentration of the S-100B protein, while in stage III patients with heterogenous or diffuse S-100B staining patterns in tumor tissue, the serum marker concentration was significantly higher (p < 0.05) than in patients with focal staining. Furthermore, S-100B staining of the melanoma tissue also differed (low/negative, medium and strong staining), and serum marker concentrations corresponded to the pattern of the staining intensity. In stage I-II, only strong staining was associated with elevated serum S-100B concentrations while in stage III medium and strong staining was found to be associated with significantly higher serum marker concentrations compared to patients with tumors with low/negative staining (p < 0.05). CONCLUSIONS: In malignant melanoma characterized by focal and/or low S-100B staining in the tumor tissue determined by immunohistochemistry, S-100B monitoring in the serum may not suffice to detect disease progression.     

Macrophage-derived soluble factor enhances melanoma inhibitory activity expression by uveal melanoma cells in vitro

Melanoma inhibitory activity (MIA) is correlated with tumour progression and development of metastatic disease. Melanoma inhibitory activity, secreted by melanoma cells, is known to inhibit tumour cell attachment to the extracellular matrix enhancing their invasive potential. The regulatory pathways that lead to MIA expression have not yet been elucidated. It is well established that tumour cells and macrophages interact through soluble factors, preventing or enhancing tumour growth. The purpose of the present study was to determine whether soluble factor(s) derived from macrophages lead to the up-regulation of MIA production by human uveal melanoma cell lines (HUMCL) and whether MIA contributes to an increase in the invasive behaviour of HUMCL in vitro. Baseline MIA levels were measured by enzyme-linked immunosorbent assay in five HUMCL of known metastatic potential (92.1>SP6.5>OCM-1>MKT-BR>UW-1). Macrophage conditioned medium (MaCM) was placed on top of the HUMCL and MIA levels were measured at 6, 12, 24, and 36 h. The HUMCL were also seeded in a Matrigel chamber for 72 h and then cells invading the Matrigel were counted. The assay was repeated adding recombinant human MIA to the top layer of each well. All HUMCL expressed MIA at baseline (average of 31 ng/ml at 36 h). Following exposure to MaCM, MIA levels increased to an average of 45.2 ng/ml, with the 92.1 and SP6.5 cell lines expressing the highest MIA levels and UW-1 cell line expressing the lowest level. During the baseline invasion assay, the vast majority of cells (>95%) were found to adhere to the upper surface of the Matrigel. When MIA was added to the invasion chamber, no adhesion or invasion was observed. The results suggest, for the first time, that macrophages secrete a soluble factor(s) that may stimulate nearby melanoma cells to enhance their production of MIA in vitro. Furthermore, increased MIA production may, in turn, increase the invasive properties of the cells by modulating the attachment of HUMCL to the extracellular matrix.     

Prognostic value of serum S-100B in malignant melanoma

AIMS AND BACKGROUND: Although there is no established tumor marker of proven value for patients with melanoma, high serum levels of S-100B protein have been found in patients with melanoma and distant metastases. This study was performed to assess the prognostic value of this marker. METHODS AND STUDY DESIGN: Serum S-100B protein was measured by means of the LIA-mat System 300 (Sangtec S-100B LIA, AB Sangtec Medical, Bromma, Sweden) in 85 patients with melanoma. RESULTS: Mean serum S-100B protein was 0.075 microg/L (range, 0.001-0.470) in 66 patients with non-metastatic melanoma (stage I-III) versus 0.441 microg/L (range, 0.001-16.840) in 19 patients with metastatic melanoma (stage IV) (P <0.001, Mann Whitney U test). The median follow-up time was 329 days. Serum levels above 0.150 microg/L were found in 10 of patients with non-metastatic melanoma (15.2%) and in 17 of 19 patients with metastatic disease (89.4%). Median survival was 256 days for the 27 patients with serum S-100B levels above 0.150 microg/L versus 561 days for the 58 patients with normal values (P <0.3973). CONCLUSION: Serum S-100B is a useful tumor marker in melanoma.     

Serum S-100b protein as a prognostic marker in malignant cutaneous melanoma.

PURPOSE: To evaluate whether S-100B protein in serum is an independent prognostic marker in malignant melanoma. MATERIALS AND METHODS: S-100B protein in serum was analyzed in 1,007 consecutive patients with histologically verified cutaneous malignant melanoma. At the time of blood sampling, 876 patients were in clinical stage I, 35 were in stage II, and 96 were in stage III. The serum concentrations of S-100B protein were measured by a luminescence immunoassay (LIA). RESULTS: The mean serum concentration of S-100B protein was significantly related to clinical stage, with the lowest level in stage I and the highest in stage III. In a multivariate analysis, S-100B protein levels in serum showed the strongest prognostic impact of the factors analyzed with respect to disease-specific survival in clinical stages II to III, followed by clinical stage. Serum S-100B protein was not a significant independent prognostic factor in clinical stage I, where tumor thickness showed the strongest relation to melanoma-specific survival, followed by ulceration and satellites. CONCLUSION: This investigation contains the largest material of patients so far analyzed with the new LIA assay of S-100B protein in serum and confirms that S-100B protein in serum is correlated with clinical stage and is an independent prognostic marker in clinical stages II and III.     

The current status of S-100B as a biomarker in melanoma

Melanoma is the most malignant type of all skin cancer types. It causes over 75% of all skin cancer related mortality. In the Netherlands, the total number of new diagnosed melanoma patients is expected to increase from 2400 patients in 2000 to 4800 patients in 2015. After surgical treatment, 20-28% of melanoma patients present with loco-regional recurrence, 26-60% with regional recurrences, and 15-50% with distant metastases. Early detection of lymph node (micro) metastases by means of a sentinel lymph node biopsy (SLNB) is therefore of crucial importance since early lymph node dissection decrease treatment morbidity and improve overall survival. However when patients present with palpable nodes, given the heterogeneity in survival, the suspicion rises that numerous patients have a form of subclinical dissemination, which can remain undetected by current modern imaging methods. Biomarkers could illuminate on this matter, although there is very little understanding of their biological significance. It can be expected that the strongest biological markers are surrogates of key biological events. The protein S-100B seems to be the best analyzed biomarker in melanoma. It has the potential to identify high-risk stage III melanoma patients who may benefit from adjuvant systematic treatment. In the stratification of new adjuvant therapeutic trials in patients with loco-regional recurrences, we therefore recommend the use of S-100B in the stratification. Since an effective (adjuvant) therapy for loco-regional metastatic and disseminated melanoma is recently introduced, the use of S-100B seems to alter dramatically in the near future.     

The luminescence immunoassay S-100: a sensitive test to measure circulating S-100B: its prognostic value in malignant melanoma.

In this study we measured S-100B using a recently developed luminometric immunoassay with a detection limit of 0.02 microg l(-1). By measuring serum S-100B concentrations in 58 apparently healthy individuals a reference value of 0.16 microg l(-1) was found. To assess the sensitivity of the assay we measured levels of S-100B protein in the serum of 251 patients with cutaneous malignant melanoma before the start of treatment. Only one of 179 patients with limited disease had a serum concentration higher than the reference value, whereas elevated levels were seen in 79% of patients with metastasized disease. In the latter group the NSE serum concentration was elevated in 42%. Using a receiver operating characteristic (ROC) curve it is shown that S-100B is a significantly better parameter than neuron-specific enolase (NSE) for distinguishing patients with limited disease from those with extensive melanoma. Pretreatment S-100B values were highly predictive for the period of survival. Patients with limited disease have increased risk for early death with increasing levels of S-100B protein. Within the group of patients with positive lymph nodes and/or with distant metastases, elevated S-100B levels strongly identified high-risk patients. Our study indicates that the measurement of S-100B as a tumour marker in the management of patients with cutaneous malignant melanoma has clinical significance.     

S-100 protein serum levels in patients with benign and malignant diseases: false-positive results related to liver and renal function.

S-100 serum concentrations were analyzed in 39 healthy people, 130 patients with benign diseases and 304 patients with malignancies, including 49 patients with locoregional diseases and 255 with advanced diseases. S-100 was determined by a commercial immunoluminometric assay, and 0.20 ng/ml was considered to be the upper limit of normality. In none of the healthy people was S-100 higher than 0.2 ng/ml. Slightly high S-100 concentrations were found in 33 out of 130 patients (25%) with benign diseases (mean 0.21 +/- 0.45 ng/ml). Significantly higher S-100 serum levels were found in patients with liver cirrhosis (63%, 10/16) (p = 0.024) or renal failure (45%, 8/18) (p = 0.03) than in patients with other benign diseases or in healthy people. Abnormal S-100 serum levels were found in 68 of the patients (22.5%) with malignancy (mean 1.01 +/- 5.9 ng/ml). The highest S-100 concentrations were found in patients with malignant melanomas (p = 0.001). Excluding melanoma patients, the S-100 serum levels in patients with malignancies were not related to tumor origin or stage but were clearly related to the site of metastasis, with patients with liver metastases showing higher values than patients with metastases without liver involvement (p = 0.02). No statistical differences were found among patients with liver cirrhosis, primary liver cancer or liver metastases. In conclusion, S-100 is a useful marker for melanoma, but abnormal levels of this tumor marker may be found in benign and malignant diseases associated with liver or renal injury.     

Occult melanoma in lymph nodes detected by antiserum to S-100 protein

Sections of 1,273 nodes from 58 patients with melanoma were stained in an immunoperoxidase assay using an antiserum to S-100 protein and aminoethyl carbazole as indicator. By the hematoxylin and eosin (H. and E.) technique, 128 nodes were seen to contain melanoma (10%); by the anti-S-100 protein technique, 363 (29%) were found to be tumor-positive. The additional tumor-positive nodes contained single tumor cells or small groups of tumor cells and were adjacent to nodes that were partly or wholly replaced by melanoma. Penetration of nodes by single tumor cells was seldom seen in node groups containing no tumor that was visible on H. and E. staining. The anti-S-100 protein approach more accurately identified those patients who would die of their recurrent disease less than 5 years after lymphadectomy.     

Cadherin-7 interacts with melanoma inhibitory activity protein and negatively modulates melanoma cell migration

Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells, which strongly enhances melanoma cell migration and invasion. Detailed analyses performed by our group showed interaction of MIA with extracellular matrix proteins and integrin α4β1 and α5β1 leading to cellular detachment. In this study, we identified cadherin-7 as a new MIA-binding protein using surface-enhanced laser desorption/ionization-mass spectrometry technology and co-immunoprecipitation. Cadherin-7 is a classical cell–cell adhesion molecule which was shown to be upregulated in malignant melanoma. We demonstrated enhanced expression of cadherin-7 in primary tumor cells compared to metastatic cells. Upregulation of cadherin-7 expression in metastatic cell lines but also downregulation of expression in cells derived from primary melanomas resulted in reduced cell migration. In addition, we speculate that MIA/cadherin-7 interaction may regulate cell–cell adhesion of malignant melanoma cells influencing the migration of the cells. Interestingly, overexpression of cadherin-7 resulted in a decreased MIA mRNA expression. In addition, MIA effects on cell migration were abrogated in cell clones overexpressing cadherin-7. In conclusion, these findings suggest that cadherin-7 regulates the expression and activity of MIA and the migration of melanoma cells playing a role in tumor development of malignant melanoma. ( Cancer Sci 2009; 100: 261–268)     

Decitabine up-regulates S100A2 expression and synergizes with IFN-gamma to kill uveal melanoma cells.

PURPOSE: Metastatic uveal melanoma is resistant to conventional chemotherapy and immunotherapy. In this study, we investigated the responsiveness of uveal melanoma cell lines to IFNs and the hypomethylating agent decitabine. EXPERIMENTAL DESIGN: The uveal melanoma cell lines 92-1, UW-1, OCM-1, and MKT-BR were exposed to varying concentrations of IFN-alpha, IFN-gamma, and decitabine, alone and in combination. The effects of decitabine on gene expression were examined using DNA microarray analysis. RESULTS: We found that IFN-gamma and decitabine induced cell death in uveal melanoma. Whereas a high concentration of IFN-gamma (1,000 units/mL) was required to induce cell death, we observed a dose-related increase in cell death when decitabine was used at a range of 0.1 to 10 micromol/L. Strikingly, 1 micromol/L decitabine synergized with 10 to 1,000 units/mL IFN-gamma to induce massive cell death. In contrast, decitabine had no effect on three cutaneous melanoma cell lines and exhibited no synergy with either IFN. In uveal melanoma, decitabine up-regulated the expression of genes involved in growth control and apoptosis and down-regulated genes that have been implicated in the malignant phenotype of cutaneous melanoma. The gene up-regulated to the greatest degree by decitabine and whose expression showed a dose-effect across the three concentrations of decitabine was S100A2, a putative tumor suppressor. The genes modulated by decitabine in uveal melanoma were largely unaffected in cutaneous melanoma. CONCLUSIONS: These findings form a basis for testing the decitabine/IFN-gamma combination in metastatic uveal melanoma and for exploring the role of S100A2 in the susceptibility of uveal melanoma to IFN-mediated cell death.     

Molecular pathways mediating liver metastasis in patients with uveal melanoma

Uveal melanoma arises from melanocytes located in the uveal tract of the eye and is the most common primary intraocular tumor in adults. Metastatic liver disease is the overwhelming cause of death in uveal melanoma patients, with almost 50% of patients developing liver metastases up to 15 years after diagnosis. Most of these patients do not present with any evidence of overt metastasis at the time of initial diagnosis although it is assumed that they have undetectable micrometastases. Currently, there are no therapeutic modalities to prevent or efficiently treat the metastatic disease in uveal melanoma patients. Recent discoveries have shed light on the molecular pathways that may contribute to the progression of liver metastasis. The aim of this review is to describe new insights into the genetic and molecular pathways that may play a role in the development of liver metastases in uveal melanoma patients.     

Immunohistochemical evaluation of uveal melanocytic tumors. Expression of HMB-45, S-100 protein, and neuron-specific enolase

The authors compared the immunohistochemical reactivity of 13 uveal nevi and 20 uveal melanomas for HMB-45, S-100 protein, and neuron-specific enolase (NSE) in formalin-fixed, paraffin-embedded sections. All 33 of the lesions were positive for HMB-45. The false-negative rates for S-100 protein and NSE were 21% and 18%, respectively. If only strongly positive reactions were considered, more than 50% of the tumors would be interpreted as negative for S-100 protein and NSE. Nevi stained with less intensity than melanomas using all three antibodies. The expression of HMB-45 appeared to be greater in active nevi than in inactive nevi. There was a weak association between S-100 protein reactivity and the ability of the uveal melanomas to metastasize (P = 0.1); however, the standard deviation of nucleolar area was a much better predictor (P = 0.02). These results indicate that pathologists will find HMB-45 to be a useful tool in differentiating uveal melanoma from nonmelanocytic tumors.     

Comparison of prognostic significance of serum 5-S-Cysteinyldopa,LDH and S-100B protein in Stage III-IV malignant melanoma

5-S-cysteinyldopa is a precursor of pheomelanin. S-100B protein is a low molecular weight, acidic, calcium binding, cytoplasmatic protein. LDH was defined as the most important serum parameter in disseminated melanoma. The aim of the present study was to compare the prognostic values of serum 5-S-Cysteinyldopa, S-100B and LDH concentrations in Stage III-IV melanoma patients. Serum samples were taken from 179 Stage III-IV melanoma patients at diagnosis. Serum 5-S-CD concentrations were determined by HPLC, S-100B protein by immunoluminometric assay while LDH by UV kinetic method. The mean/median concentrations of LDH, S-100B protein and 5-S-CD in Stage III patients ranged around the normal level. In Stage IV, the markers ranked as S100B = 5-S-CD > LDH for sensitivity, S-100B > LDH > 5-S-CD for specificity and LDH = S100B = 5-S-CD for positive predictive value, respectively. Furthermore, mean marker concentrations of patients with progressive disease differed significantly from nonprogresssive cases (when staging categories have been disregarded). Survival analysis indicated, that the initially elevated LDH and S-100B level in Stage IV disease predicts comparably short survival. Results of our study suggest that these serum marker values correlate well with Stages and disease progression. In Stage IV melanoma, the markers had appropriate sensitivity, high specificity as well as important positive predictive value. Among the studied serum markers S-100B protein and LDH proved to be similarly reliable in respect to the clinical outcome.     

Use of serum 5-S-CD and S-100B protein levels to monitor the clinical course of malignant melanoma

5-S-Cysteinyldopa (5-S-CD) is a precursor of pheomelanin. S-100B protein is a low molecular weight, acidic, calcium binding, cytoplasmic protein. In this study, the concentration changes of serum 5-S-CD and S-100B protein in melanomas of all stages were examined in parallel and patients were monitored during and after treatment. Serum samples were taken from 478 melanoma patients on 1924 occasions. Of these, 180 cases were regularly monitored. Concentrations of 5-S-CD were determined by high performance liquid chromatography (HPLC), S-100B protein by immunoluminometric assay. The mean/median concentrations of 5-S-CD and S-100B protein in Stage I, II and III patients and in the control group ranged around the normal level. In Stage IV patients, 58.4/50.6% sensitivity, 100% specificity and 100/86.6% positive predictive values were obtained concerning S-100B protein and 5-S-CD, respectively. Recurrence was observed in 57/180 of the regularly monitored patients in Stages I, II and III. In 10/57 (17.5%) of these patients suffering from any type of disease progression increases in both marker levels preceded the detection of metastasis by conventional methods. We can confirm that changes in both marker concentrations correlated with the stages of the patient. The markers are most sensitive in Stage IV patients and also have a high specificity in these patients. In Stage IV melanoma patients, 5-S-CD and S-100B protein levels are independent significant prognostic factors. In almost one fifth of patients both marker levels increased before the detection of metastatic disease with other appropriate, routinely scheduled investigations. This study suggests that serial serum marker measurements in the management and follow-up of melanoma patients should be examined further.     

Significance of serum S-100B in melanoma patients before and after sentinel node biopsy

BACKGROUND AND OBJECTIVES: The clinical utility of the tumor marker serum S-100B has been described in determining prognosis, for early diagnosis of recurrence and for disease monitoring in melanoma patients. Sentinel node biopsy is increasingly used as staging procedure for patients with clinically localized melanoma. The aim of this study was to determine the value of serum S-100B in melanoma patients before and after sentinel lymph node biopsy. METHODS: S-100B values were measured prior to sentinel node biopsy in 89 patients and during follow-up (median 41 months; range 7-73 months) in 88 patients. The detection limit is < or =0.08 microg/L. In our laboratory levels of 0.16 microg/L and above are classified as increased. RESULTS: Twenty-four patients had tumor-positive sentinel nodes, 65 had tumor-free sentinel nodes. The median S-100B value prior to the operation was < or =0.08 microg/L for all patients. Sensitivity and specificity of S-100B to predict the tumor-status of the sentinel node were 13% and 98%, respectively. Eighteen patients developed a melanoma-related recurrence. Sensitivity for early diagnosis of recurrence was 55% and 33%, respectively for patients with a positive versus a negative sentinel node. Specificity was 100% in both patient groups. CONCLUSIONS: S-100B is not useful in predicting the tumor-status of the sentinel node, and questionable for early diagnosis of recurrence afterwards. Elevation of serum S-100B is highly specific for melanoma recurrence.     

Serum levels of S-100 protein and 5-S-cysteinyldopa as markers of melanoma progression

Serum S-100 protein is widely used as a marker of melanoma and since 5-S-cysteinyldopa (5-S-CD) is a precursor of melanin its serum and urinary levels can reflect melanoma progression. In this study we examined the concentration changes of serum S-100 protein and 5-S-CD in 252 melanoma patients of different clinical stages. Serum samples were taken from 252 melanoma patients at 860 times, from June 1996 to July 1998. The serum S-100 protein was measured by the immunoluminometric assay, levels of 5-S-CD was determined by HPLC. The value of S-100 protein in patients with primary melanoma (0.11m mg/l) and in patients without symptoms (0.15 m mg/l) ranged around the normal level (0.01 0.12 m mg/l). There was a significant difference between the values of patients with or without symptoms. There was a similarly significant difference between the S-100 values of clinical Stage I (0.11 mg/l) and Stage III (2.91 mg/l) as well as between those of clinical Stage II (0.47 mg/l) and Stage III (2.91 mg/l), respectively. Analyzing the values of patients with symptoms we observed significant difference between the S-100 protein values of patients with primary tumor and those with solitary or multiple distant metastases. In case of 5-S-CD significant difference was found between clinical Stage I and III as well as clinical Stage II and III. Furthermore, there was a significant difference between the mean marker values of patients with primary tumor, lymph node, lung metastasis and clinical stage III.     

Immune expression and inhibition of heat shock protein 90 in uveal melanoma.

PURPOSE: To examine the immunohistochemical profile of heat shock protein 90 (Hsp90) in uveal melanoma and the cytotoxicity of an Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in uveal melanoma cell lines. EXPERIMENTAL DESIGN: Hsp90 expression was determined by immunohistochemistry in 44 paraffin-embedded sections of primary human uveal melanoma and in five uveal melanoma cell lines (92.1, OCM-1, MKT-BR, SP6.5, and UW-1). Sulforhodamine B-based proliferation assay was used to compare uveal melanoma cell growth with a range of concentrations of 17-AAG. Changes in cell migration, invasion, cell cycle fractions, and apoptotic activity were also evaluated. Expression of intracellular proteins was determined by Western blot analysis after 17-AAG exposure. RESULTS: Immunohistochemical expression of Hsp90 was identified in 68% of the paraffin-embedded sections and significantly associated with largest tumor dimension (P = 0.03). 17-AAG significantly reduced the proliferation rates of uveal melanoma cell lines, with concentrations of 100 to 0.1 micromol/L. 17-AAG also significantly reduced the migratory and invasive capabilities of uveal melanoma cell lines. Cell cycle analysis showed that 17-AAG induced accumulations of cells in G(1). Caspase-3 protease activity analysis, a marker for apoptosis, showed a significant increase after drug exposure. The cytotoxic effect of 17-AAG was associated with decreased levels of phosphorylated Akt and cyclin-dependent kinase 4. CONCLUSIONS: The immunohistochemical expression of Hsp90 in uveal melanoma indicates worse prognosis. To the best of our knowledge, this is the first report showing the inhibitory effect on uveal melanoma cells using 17-AAG to target Hsp90. Therefore, Hsp90 may be used as a potential target for treatment of patients with uveal melanoma.     

S100B protein detection in serum is a significant prognostic factor in metastatic melanoma.

The serum detection of S100B, a new melanoma marker, has shown clinical significance in early studies. The aim of our study of 1, 339 serum samples from 412 different melanoma patients and 107 control patients was to prove the prognostic value of serum S100B levels in melanoma patients at different stages of disease and at follow-up (median: 30 months). Using a cutoff level of 0.2 microgram/l S100B, 5 of 286 patients (1.7%) with primary tumors (stage I/II), 14/73 (19.2%) patients with locoregional metastasis (stage III) and 57/84 (67.9%) patients with advanced disease (stage IV) were S100B positive (statistically significant differences for stage I/II vs. III, I/II vs. IV, and III vs. IV, p < 0.001). The estimated overall survival time was significantly longer (p < 0.001) for patients with S100B values below 0.2 microgram/l compared to patients with elevated S100B levels (>/=0.2 microgram/l), which was independent of the stage of disease (I-IV). Regarding prognosis, we were furthermore able to distinguish different subgroups among stage III and IV patients using S100B serum levels (p < 0.01). Patients with different cutaneous non-melanoma diseases served as S100B-negative controls. S100B serum evaluations using the Sangtec(R)100 IRMA are highly specific and sensitive for the detection of metastatic melanoma. S100B has been shown to be a relevant prognostic factor for survival in a study with a large sample size of melanoma patients including close follow-up evaluations.