Ludwig Suter

Infiltration of primary and metastatic melanomas with macrophages of the 25F9-positive phenotype

SummaryIn order to gain insight into the role of macrophages in human melanoma, we studied fresh-frozen material from 15 dysplastic nevi, 199 primary melanomas, 107 melanoma metastases, and paraffin sections from 98 primary melanomas with the monoclonal antibody 25F9 which recognizes an 86×103 dalton protein present on a subset of mature human macrophages. Considerable infiltration of tumors with 25F9-positive macrophages was observed in 2 dysplastic nevi (13%), 87 primary melanomas (44%), and 45 metastases (42%). The degree of intratumoral macrophage infiltration correlated with expression of class II HLA-DR antigens on tumor cells, in primary melanoma with a tumor thickness above 0.75 mm, and with the occurence of metastases within 2 years. In paraffin sections, intratumoral 25F9-positive macrophages also correlated with metastatic spread of primary tumors after longer follow-up. Metastases revealed a higher degree of macrophage infiltration following systemic or local immunotherapy, compared with untreated metastases, or metastases removed during chemotherapy. Of 38 patients who died within an observation period of 1 year, 19 (50%) had considerable infiltration of metastases with 25F9-positive macrophages, whereas this was found in only 4 of 12 patients (33%), who survived for longer than 2 years following metastases removal. A higher degree of 25F9-positive macrophages correlated with a shift towards the T8-positive subsets within the T cell compartment of the infiltrate. Our results suggest that accumulation of 25F9-positive macrophages in melanomas indicates more aggressive tumor properties.

Prognostic classification of malignant melanomas by combining clinical, histological, and immunohistochemical parameters

In a retrospective study the prognostic relevance of clinical, histopathological, immunohistochemical, and flow-cytometric parameters in primary malignant melanomas was evaluated using both the receiver operating characteristic ROC procedure and the logistic regression model. The proteolytic enzymes collagenase IV, cathepsin B, and cathepsin D proved to be significant prognostic factors. Combining the results obtained with these enzymes with gender, anatomic site, tumour thickness, Clark’s level, ulceration, pattern of invasive growth, and presence of large round cells resulted in greatly improved discrimination between metastasized and non-metastasized cases. It is anticipated that this method could allow for precise individual prognostic characterization and in particular for identification of high-risk patients for adjuvant therapy.

Increased Expression of the Tumor Suppressor PLZF Is a Continuous Predictor of Long-Term Survival in Malignant Melanoma Patients

Promyelocytic leukemia zinc finger (PLZF) is a transcriptional repressor and tumor suppressor inhibiting melanoma cell growth in vitro and in vivo in animal models. In this study, we analyzed the impact of in vivo primary tumor gene expression of PLZF on the long-term survival of malignant melanoma patients. PLZF expression was assessed by using DNA microarray and real-time polymerase chain reaction analysis of 41 primary malignant melanomas from patients with a defined histology and a close to 20-year clinical follow-up, of 29 melanoma metastases, and of 6 different melanoma cell lines. Kaplan-Meier survival analyses, log-rank statistics and Cox regression analysis were employed to identify the impact of PLZF expression on long-term survival. We detected PLZF expression in 92% of primary melanoma tumors in vivo but not in melanoma cell lines in vitro. By univariate analysis, we identified: (1) PLZF mRNA expression < or = 10,000 mRNA copies/mug total tumor RNA, (2) Breslow tumor thickness >4 mm, and (3) American Joint Committee on Cancer stages IIC, IIIB, IIIC, and IV as statistically significant pretreatment risk factors. We defined a continuous prognostic index (i.e., risk score) for primary melanoma patients based on the regression coefficient of PLZF mRNA expression. Applying a cutpoint to the prognostic index at - 1.65, patients were assigned to one of two risk groups: low-risk patients (n = 28) with a median overall survival of 79 months (5-year survival of 61%) and high-risk patients (n = 13) with a median overall survival of 32 months (5-year survival of 23%) (p < 0.05). This is the first time that PLZF mRNA expression has been linked to a prognostic model for primary malignant melanoma patients to derive prognostic groups for clinical purposes (e.g., improved melanoma immunotherapies).

p-Glycoprotein expression in malignant melanoma

SummaryIn some human malignancies resistance to chemotherapy is caused by an energy-dependent efflux system, responsible for the removal of chemotherapeutics out of the resistant tumor cells. A major component of this efflux system is the permeability glycoprotein (p-glycoprotein), which depends on the multidrug-resistance geneMDR1.We have tested p-glycoprotein in primary and metastatic human melanoma by use of the monoclonal antibody C219; a substantial expression was only observed in 1/37 primary melanomas and in 1/27 melanoma metastases. None of the patients with negative metastases responded to chemotherapy. Moreover a complete remission of metastasic growth was observed in the patient with the metastasis significantly expressing the p-glycoprotein. Sequential studies revealed no significant increase of p-glycoprotein-positive cells during and after chemotherapy. We conclude that drug resistance in human melanoma does not usually depend on the p-glycoprotein-related efflux system. Other mechanisms are obviously responsible for drug resistance in this human malignancy.

The expression of proteolytic enzymes at the dermal invading front of primary cutaneous melanoma predicts metastasis.

The expression of three proteinases Cathepsin B (Cath. B), Cathepsin D (Cath. D) and Collagenase IV (Coll. IV) has been retrospectively analyzed within an immunohistochemical study on routinely fixed, paraffin embedded tissues from 147 primary cutaneous melanomas belonging to the classes pT3 and pT4. The development of these melanomas has been followed for at least five years. We compared the expression of these proteolytic enzymes in tumors that metastized during the follow-up-period with that in tumors that did not metastize. The expression at the dermal invading front of the tumors showed higher prognostic values (Cath. B chi 2 = 40.03, p < 0.001; Cath. D chi 2 = 90.95, p < 0.001; Coll. IV chi 2 = 44.46, p < 0.001) than the overall expression of these enzymes (Cath. B chi 2 = 5.63, p = 0.018; Cath. D chi 2 = 6.21, p = 0.010; Coll. IV chi 2 = 6.57, p = 0.010). The distribution of protease-expression inside the tumor turned out to be important for the prognostic value, which might also lead to higher prognostic confidence when applied to other tumors.

Increased level of circulating U2 small nuclear RNA fragments indicates metastasis in melanoma patients.

Abstract Background: Melanoma is the most aggressive skin cancer and, despite recent advances in therapy, about 20% of the patients die of their disease. Early relapse detection and monitoring of therapy response are crucial for efficient treatment of advanced melanoma. Thus, there is a need for blood-based biomarkers in melanoma management. Serum-derived U2 small nuclear RNA fragments (RNU2-1f) were previously shown to be blood-based biomarkers for gastrointestinal and gynecologic malignancies. Here we examined whether RNU2-1f may also serve as diagnostic biomarker in advanced melanoma. Methods: Circulating RNU2-1f levels were quantified by comparative reverse transcription PCR in a training cohort of patients with metastatic melanoma (n=33, thereof regionally metastasized to skin and lymph nodes, n=23, and distantly metastasized, n=10) vs. patients with benign naevi (n=16) vs. healthy controls (n=39). Results were validated in an independent patient cohort with distant metastasis (n=16) vs. controls (n=18). Results: Circulating RNU2-1f levels in the training cohort were significantly increased in serum of regionally and distantly metastatic patients, compared with patients with benign naevi or healthy controls (p<0.0001) and allowed accurate detection of regional (AUC 0.80) as well as distant (AUC 0.84) metastasis. In the validation cohort, increased RNU2-1f levels were confirmed and enabled highly specific detection of distant metastasis (sensitivity 81%, specificity 100%, AUC 0.94). Conclusions: This is the first report to suggest a blood-based snRNA serving as a diagnostic biomarker for melanoma metastasis. Our data provide a rationale for further defining clinical utility of circulating RNU2-1f in metastasis detection in the management of melanoma patients at risk of relapse and/or with advanced disease.

The prognostic value of the expression of collagenase IV, cathepsin D and metallothionein in squamous cell carcinomas of the skin determined by immunohistochemistry.

Abstract  We analyzed 53 specimens from primary squamous cell carcinomas of the skin for the expression of collagenase IV, cathepsin D and metallothionein by means of immunohistochemistry along with histological data. We compared tumors that metastasized (30) with tumors that did not (23) during a follow-up period of at least 5 years. The expression of the two proteolytic enzymes cathepsin D and collagenase IV was also assessed at the invading front of the tumors. Statistical analyses revealed significant differences for the overall expression of cathepsin D ( P < 0.05), expression of cathepsin at the invading front ( P < 0.05) and the tumor thickness ( P < 0.01). The results showed that cathepsin D may be a prognostic factor for squamous cell carcinomas of the skin. We then combined the results of parameters with statistically significant differences by multiplication. The prognostic value of such a combined risk-factor score showed higher confidence than any of the single parameters alone: a sensitivity of 63.3%, a specificity of 87.0% and an efficiency of 73.6%. This kind of combined risk-factor score might be used to more accurately detect patients at high risk of metastasis.

Metastatic human melanoma

SummaryAccording to animal experiments, metastasis to a particular organ depends on the phenotype of the tumor cells. Widespread metastatic dissemination including internal organs would therefore, at least in part, depend on the capacity of tumor cells to modulate, resulting in increased phenotypic heterogeneity. We found evidence for this assumption in human melanoma by phenotyping metastases (mainly cutaneous/subcutaneous) from 59 patients by the use of six monoclonal antibodies. Interlesional antigenic heterogeneity was present in 22/33 (67%) patients with disseminated metastases including at least one internal organ, but only in 4/26 (15%) patients whose metastases were restricted to skin and/or skin-draining lymph nodes (P≤-0.01). Chemotherapy cannot be the main reason for interlesional phenotypic heterogeneity, as seen by comparison of treated and untreated patients. Aneuploid melanoma metastases, as an indication for instability on the chromosomal level, were found in the majority of patients (84%) regardless of their clinical situation. Widespread disease was significantly related to the loss of the cytoplasmatic antigen K-1-2 and to the expression of the 130-kDa membrane antigen A-1-43.

Cell-Surface Structure and State of Malignancy in Human Malignant Melanoma

Human malignant melanoma is a spontaneous tumor that progresses from the initial premalignant lesions to highly metastatic forms. Clinical and histological observations of this process and some recent knowledge gained on experimental tumor models suggest that tumor progression is a complex multistage process. In the course of this process, several distinct properties have to be aquired by the tumor cells either sequentially or in parallel (Poste and Fidler, 1980; Nicolson et al., 1977). Properties of prime importance are believed to be invasiveness, neovascularization, resistance to rejection mechanisms, and adaptation to and growth in a different organ or tissue. It has been shown in animal tumor models that the capacity to metastasize is a phenotypically expressed property of a tumor cell; moreover, even the target organ seems to be predetermined by the phenotype (Dexter et al., 1978; Hart and Fidler, 1980; Nicolson and Winkelhake, 1975; Nicolson et al.,1978; Nowell, 1976; Schirrmacher et al., 1979a; Susuki et al., 1978). The mechanisms by which primary tumors develop highly malignant variants have been circumscribed by the term retrodifferentiation and are unknown (Coggin and Anderson, 1974; Renselaer Potter, 1978; Uriel, 1979). That the process of retrodifferentiation might also be reversed is indicated in experiments wherein nonmetastasizing variants were isolated from metastases (Tao and Burger, 1977). It is logical to assume that phenotypic changes that are associated with the expression of different biochemical and biological properties are also associated with changes in the cell surface, which in fact has been demonstrated in several instances (Fogel et al., 1979; Killion and Kollmorgen, 1976; Nicolson and Winkelhake, 1975; Poste, 1977; Schirrmacher et al., 1979b; Sorg et al., 1978; Yogeeswaran et al., 1978, 1979).

Abstract 2861: Independent validation of a prognostic gene-signature based risk score in formalin-fixed paraffin-embedded melanomas

Current staging of melanoma, as defined in 2009 by the American Joint Committee on Cancer (AJCC), is based mainly on histopathological criteria but is limited in predicting outcome. Complementary molecular markers are not available for routine prognostic assessment. We have previously identified and validated a prognostic nine-gene signature expressed in fresh-frozen (FF) primary cutaneous melanomas (training cohort: n=91; validation cohort: n=44). A signature-based risk score predicts patient overall survival (OS) independently of AJCC staging (multivariate regression analysis: p = 0.0004; hazard ratio: 3.8). However, clinical application requires adaptation to formalin-fixed, paraffin-embedded (FFPE) melanomas. Therefore, we have transfered signature expression analysis onto FFPE melanomas. From FFPE melanomas matching the training and validation cohorts of the above FF melanoma study (n=125), RNA was prepared and transcribed into cDNA. Following cDNA pre-amplification, expression of the 9 signature genes, 2 additional candidate genes, and 3 housekeeping genes was quantified by real-time PCR. Correlation of gene expression with OS was evaluated using Cox regression analysis. Expression of a signature of 8 out of 11 genes (risk gene: KBTBD10; protective genes: DCD, GBP4, COL6A6, PIP, SCGB1D2, SCGB2A2, KRT9) was associated with OS in univariate regression and Kaplan Meier analysis. A signature-based risk score predicted OS independently of AJCC staging (multivariate analysis: p=0.0059, hazard ratio 3.09). The misclassification rates were 20% overall, 13.8% for low risk, and 5.7% for double low-risk (combined with AJCC staging). The risk score complemented and refined conventional AJCC staging. Thus, the FF melanoma risk score was successfully transfered onto FFPE melanomas. In order to validate the FFPE melanoma risk score, we analyzed signature expression in an independent cohort of 130 selected FFPE melanomas, which were particularly difficult to classify by AJCC staging (misclassification rate 40.8%), in order to stringently test the performance of the risk score. The misclassification rate of the FFPE melanoma risk score was comparable, even slightly better (39.2%) than that of AJCC staging, confirming its prognostic performance. The FFPE melanoma risk score was also externally validated in a Molecular Diagnostics Lab (Dermatologikum Hamburg). The concordance of melanoma classification exceeded 85%, demonstrating technical robustness of the risk score. We have established and independently as well as externally validated a quantitative, robust prognostic FFPE melanoma risk score that is complementary to AJCC staging in predicting outcome. This demonstrates clinical applicability and allows retrospective risk assessment of melanomas. The score identifies patients at low risk, not identified by AJCC staging, and defines high-risk patients in need of adjuvant therapy. Citation Format: Georg Brunner, Achim Heinecke, Ludwig Suter, Norbert Blodorn-Schlicht, Hans-Joachim Schulze, Jens Atzpodien. Independent validation of a prognostic gene-signature based risk score in formalin-fixed paraffin-embedded melanomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2014-2861