In situ detection of antigen-specific T cells in cryo-sections using MHC class I tetramers after dendritic cell vaccination of melanoma patients.

Application of tetrameric MHC class I-peptide complexes has significantly improved the monitoring of antigen-specific T cell immune responses in mouse models as well as in clinical studies. Especially MHC class I tetramer analysis of tumor-specific T cells in suspension or on thick vibratome sections from viable tissue has been proven extremely useful. Using the well-characterized mouse tyrosinase-related-protein-2 specific cytotoxic T cell (CTL) clone LP9, we now developed a method that allows for specific identification of T cells with MHC class I tetramers in 8 mum thick, chemically fixed cryosections. The protocol was validated in a murine influenza virus-infection model. Moreover, analysis of delayed type hypersensitivity (DTH) skin biopsies from melanoma patients vaccinated with peptide-loaded mature dendritic cells, revealed the presence and location of anti-tumor CTLs. The specificity of the CTLs detected in situ correlated with both the DTH challenge specificity and reactivity of cell suspensions derived from the same biopsies. Collectively, our data demonstrate that in situ MHC class I tetramer staining provides a valuable tool to reveal the presence and anatomical location of specific CTLs in frozen tissue following immune-based treatment strategies in cancer patients.

Distinct phenotypic changes between the superficial and deep component of giant congenital melanocytic naevi: a rationale for curettage.

BACKGROUND: Giant congenital melanocytic naevi (GCMN) convey a 14-fold increased melanoma risk. In contrast, medium congenital melanocytic naevi (MCMN) are rarely associated with malignant transformation. Management of patients with GCMN is challenging and there is no consensus on the most appropriate strategy for treating these patients. OBJECTIVES: To provide a rationale for performing curettage of GCMN in the neonatal period in order to reduce the risk of malignant transformation to melanoma. METHODS: Twenty-six infants with GCMN who underwent biopsies before excisional surgery (n = 7) or curettage (n = 19) during the past 14 years (Academic Hospital, Vrije Universiteit Brussel) and 10 MCMN patients who underwent excision biopsies (Radboud University Nijmegen Medical Centre) were included in this study. Using these biopsies, we performed genetic and detailed immunohistochemical evaluations of changes that are associated with malignant transformation. Variables of interest included melanoma-associated BRAF mutations, proliferative activity, vascularity, cellular context and extracellular matrix architecture. RESULTS: GCMN and MCMN did not show oncogenic BRAF mutation and displayed similar features with respect to the amount of nonmelanocytic cells within the naevus and matrix architecture. Naevus cells in the superficial component of the GCMN, however, were more proliferative, and this component was more vascular compared with its deep component and with MCMN. In this study, none of the 19 newborn patients who underwent curettage developed a melanoma within a mean follow-up time of 7 years. CONCLUSIONS: The data presented here support the idea that curettage of GCMN in neonates has the potential for lowering the risk of developing cutaneous melanoma by not only obtaining an important numerical reduction of naevus cells but also removing the 'active' melanocytes.

Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays.

Melanoma is one of the most aggressive types of cancer and resection of the tumour prior to dissemination of tumour cells is still the most effective treatment. Therefore, early diagnosis of melanocytic lesions is important and identification of novel (molecular) markers would be helpful to improve diagnosis. Moreover, better understanding of molecular targets involved in melanocytic tumorigenesis could possibly lead to development of novel interventions. In this study, we used a custom made oligonucleotide array containing 298 genes that were previously found to be differentially expressed in human melanoma cell lines 1F6 (rarely metastasising) and Mel57 (frequently metastasising). We determined differential gene expression in human common nevocellular nevus and melanoma metastasis lesions. By performing nine dye-swap array experiments, using individual as well as pooled melanocytic lesions, a constant differential expression could be detected for 25 genes in eight out of nine or nine out of nine array analyses. For at least nine of these genes, namely THBD, FABP7, H2AFJ, RRAGD, MYADM, HR, CKS2, NCK2 and GDF15, the differential expression found by array analyses could be verified by semiquantitative and/or real-time quantitative RT-PCR. The genes that we identified to be differentially expressed during melanoma progression could be potent targets for diagnostic, prognostic and/or therapeutic interventions.

Differential patterns of insulin-like growth factor-I and -II mRNA expression in medulloblastoma.

Insulin-like growth factors (IGFs) play an important role in tumour growth and development. We hypothesized that this is also the case for medulloblastomas, which are highly malignant cerebellar brain tumours usually occurring in children. In these tumours the expression patterns of IGF-I and -II mRNA were studied. Tumour specimens obtained from 12 children and two adults at diagnosis were hybridized in situ with digoxigenin-labelled cRNA probes for hIGF-I and hIGF-II mRNAs. In all cases, tumour cells showed abundant expression of IGF-I mRNA. Nine of the 14 tumours showed variable but significant IGF-II expression. In these tumours, the hybridization signal almost exclusively colocalized with a subpopulation of Ki-M1P positive cells that were identified as ramified microglia (RM) cells. In the five tumours without IGF-II expression, microglia/brain macrophages with a more rounded amoeboid-like morphology predominated. RM cells in normal cerebellar tissues, residing abundantly in areas of the white and, to a less extent, in the grey matter, were IGF-II mRNA-negative. These RM cells showed a thinner and more extensively branched appearance and were more evenly distributed than those encountered in medulloblastoma. Probably, during the transformation from the resting ramified towards the amoeboid morphology (or vice versa) IGF-II mRNA expression is only temporarily induced. The physiological meaning of the induction of IGF-II mRNA expression by these cells in medulloblastoma remains unclear but any IGF-II peptide synthesized could exert unfavourable mitogenic and antiapoptotic effects on adjacent tumour cells. However, in this relatively small number of cases we could not find any indications for a relationship between clinical characteristics of the various cases and the extent of IGF-II mRNA expression.

Immunohistochemistry in melanocytic proliferative lesions.

Melanoma incidence is rising worldwide. Early diagnosis is very important, as the most effective treatment for melanoma still consists of excision of the tumour before onset of the metastatic growth phase. Immunohistochemistry is a valuable tool for (dermato)pathologists to aid establishing diagnosis. Melanoma markers can be classified into two main categories: melanocytic differentiation markers and melanoma progression markers. Melanocytic differentiation markers are mostly used to distinguish poorly differentiated melanomas from non-melanocytic tumours and for staging of melanocytic proliferative lesions. Melanoma progression markers are most suitable to determine the level of malignancy and/or aggressiveness of tumour cells. This review describes the classification of melanoma markers, including commonly used and recently identified antigens with potential marker function. We characterize their expression profile in melanocytic proliferative lesions and their potential usefulness for diagnosis, prognosis, microstaging, immunotherapeutic purposes and evaluation of therapies.

Detection of disseminated tumour cells in blood and bone marrow samples of patients undergoing hepatic resection for metastasis of colorectal cancer.

BACKGROUND: In 50-60 per cent of patients who undergo hepatic resection for metastasis of colorectal cancer the first site of tumour recurrence is extrahepatic, indicating the presence of more extensive disease at the time of resection. The aim of this study was to evaluate whether the presence of disseminated tumour cells in blood and bone marrow could predict extrahepatic tumour recurrence. METHODS: Cytokeratin 20 (CK20) reverse transcriptase-polymerase chain reaction was used to study the presence of tumour cells in preoperative peripheral blood and bone marrow samples from 41 patients with liver metastasis scheduled for surgical resection. RESULTS: CK20 expression was detected in six of 41 peripheral blood samples and in eight of 32 bone marrow samples. There was no correlation between CK20-positive samples and subsequent extrahepatic recurrence. Positive blood samples did, however, correlate with high serum carcinoembryonic antigen level and large tumour volume. None of the 14 patients previously treated with chemotherapy had CK20-positive samples, whereas six of 27 blood and eight of 20 bone marrow samples were positive in the chemotherapy-naive group. CONCLUSION: Although the number of patients in this study is limited, the presence of disseminated tumour cells did not predict subsequent extrahepatic recurrence. The results strongly suggest that the presence of circulating tumour cells in peripheral blood may reflect transient shedding of tumour cells related to large tumour volume.

Detection and clinical relevance of tumor cells in blood and bone marrow of patients with colorectal cancer.

In colorectal cancer, the predictive value of the currently used staging method is limited. Therefore, many parameters have been studied to improve the prediction of final clinical outcome, including several aspects of the primary tumor that are associated with its aggressiveness and the capacity of the host response. A more direct approach to predict the metastatic potential of a tumor may be the determination of limited disseminated disease at an early stage before it becomes clinically evident. Very sensitive techniques have been developed to detect single or very few tumor cells that have been disseminated into lymph nodes, blood, bone marrow and the peritoneal cavity. This review describes the advantages and disadvantages of the main detection techniques and discusses the current state of clinical relevance of disseminated tumor cells in patients with colorectal cancer.

Investigations for a multi-marker RT-PCR to improve sensitivity of disseminated tumor cell detection.

BACKGROUND: In order to develop a multi-marker RT-PCR, which as such may be more sensitive than a single marker assay for the detection of disseminated tumor cells, we evaluated six RT-PCR markers: cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), guanylyl cyclase C (GCC), epidermal growth factor receptor (EGFR), matrilysin (MMP-7) and HeLa metastatic gene (HLM). MATERIALS AND METHODS: The expression was studied in human colon tumor cell lines, in colon cancer tissues, and in blood and/or bone marrow samples of colorectal cancer patients and control subjects. RESULTS: The cell lines showed a differential expression pattern. The expression of all markers was detected in control blood samples with the lowest frequency for CK20 and EGFR. Semiquantitative analysis, which was performed to study threshold setting, demonstrated that GCC expression was elevated in patient compared to control samples. However, the reproducibility was questionable. CONCLUSION: The results presented in this study suggest an enhanced sensitivity for a combination of RT-PCR markers. Due to limited specificity however, the development of a multi-marker RT-PCR by using conventional PCR does not seem feasible. Future studies should focus on the potential of quantitative RT-PCR.

Cryosurgery of tumor tissue causes endotoxin tolerance through an inflammatory response.

INTRODUCTION: Earlier reports on animal studies showed inhibition of secondary tumor growth and metastases after cryoablation, probably mediated by an inflammatory response. In this study enhancement of this inflammatory response and its possible additive antitumor effect is evaluated in a mouse tumor model. MATERIALS AND METHODS: Mice received two subcutaneously implanted C--26B tumors on, respectively, day 0 (thigh) and day 7 (flank). The thigh tumor was treated by either cryoablation or resection. In addition the animals received a single dose of lipopolysaccharide (LPS) or anti-IL10 together with, or two days after, surgical treatment. The growth of the flank tumor was followed and plasma levels of IL-1 alpha and TNF-alpha were measured. RESULTS: Compared to excision of the primary tumor, cryosurgery clearly induced inhibition of secondary tumor growth while plasma levels of TNF (0.09) and IL-1 (0.06) were significantly elevated after cryosurgery when compared to excision (TNF 0.0, IL-1 0.03; p < 0.01). Administration of LPS two days after cryosurgery did not lead to extra inhibition of secondary tumor growth, even at high doses. Remarkably, dose--response studies with LPS administered two days after treatment showed a high mortality at a dose of 200 micrograms (75%) in the excision group while mortality in the cryo-treated group was 13% (p < 0.02). Mortality was directly related to cytokine levels that were significantly higher in the excision group (TNF 3.60, IL-1 0.30) when compared to the cryo-treated group (TNF 1.0, IL-10.15; p < 0.01). In contrast, when 25 micrograms LPS was given at the same time as treatment of the primary tumor either by cryosurgery or excision, mortality in the cryo-treated group (85%) was higher than in the excision group (14%, p < 0.05). Again mortality was related to post-treatment cytokine levels which now were significantly higher in the cryo-treated animals (TNF 1.30, IL-10.35) than in animals treated by excision (TNF 0.60, IL-10.10; p < 0.01). Administration of anti IL-10 did not lead to extra tumor growth inhibition. CONCLUSION: These experiments confirm the hypothesis that cryosurgery leads to a systemic inflammatory response. This reaction can lead to the inhibition of tumor growth. Administration of LPS after cryosurgery does not lead to an extra anti-tumor response; animals appear to become endotoxin tolerant. Adversely, when LPS is administered together with cryosurgery, the animals are extremely sensitive to LPS. These findings are in accordance with the clinical observation of cryoshock after cryoablation of liver metastases.

Relevance of disseminated tumour cells in blood and bone marrow of patients with solid epithelial tumours in perspective.

Currently-used systems to predict prognosis in patients with solid epithelial tumours after surgical resection of the tumour do not give any guarantees for the individual patient. In this respect the clinical relevance of the presence of disseminated tumour cells in blood and bone marrow has been frequently studied. Because of growing awareness that information on merely the presence of disseminated tumour cells is not sufficient for prognostic and therapeutic purposes, attention for characterization of disseminated tumour cells has increased. Numerous reviews have already been published on the detection and clinical relevance of disseminated tumour cells. Therefore, this paper will mainly focus on the biological significance of these cells and discusses the (in)efficiency of the metastatic process, the genotypic and phenotypic characteristics of disseminated tumour cells, and their structure of appearance. Despite the fact that information gained on the several individual aspects is substantial, it did not render any solid solutions for individual patient management yet. Hence, a combined approach of several aspects of disseminated tumour cells together with characteristics and behaviour of the primary tumour is needed to substantially improve our knowledge on the role of disseminated tumour cells in the complex process of tumour metastasis.

Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol.

Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.

Limitations of cytokeratin 20 RT-PCR to detect disseminated tumour cells in blood and bone marrow of patients with colorectal cancer: expression in controls and downregulation in tumour tissue.

AIMS: Despite informative staging of patients with colorectal cancer, some patients with localised disease at diagnosis will develop recurrence or metastasis. Attempts to improve staging include sensitive detection of disseminated tumour cells in blood and bone marrow by reverse transcriptase polymerase chain reaction (RT-PCR). The results of this study have been considered in relation to the controversial results in the literature to elucidate the usefulness of cytokeratin 20 (CK20) RT-PCR to detect disseminated tumour cells further. PATIENTS/METHODS: Blood and bone marrow samples from 30 patients with colorectal cancer were studied by CK20 RT-PCR. Specificity was evaluated in 47 blood and 15 bone marrow samples from non-cancer controls. In addition, the expression of CK20 mRNA and protein was studied in normal and tumour colon tissue samples. RESULTS: CK20 expression was detected in nine of 30 and nine of 19 of the blood and bone marrow samples from patients with colorectal cancer, respectively. In non-cancer control blood and bone marrow samples, CK20 expression was detected in 10 of 47 and four of 15, respectively. A difference between patient and control samples may be observed in terms of frequency of positive PCR tests. In tissue samples, CK20 mRNA expression was downregulated in tumour compared with normal colon tissue. CONCLUSIONS: CK20 expression was downregulated in tumour tissue compared with normal colon and a background expression of CK20 was seen in some control blood and bone marrow samples. Despite a lack of standardisation (which hampers comparison of studies), these results, together with other reports in the literature, suggest that CK20 may still be a suitable marker, but that background expression and threshold setting should be studied further.

Differentially expressed genes identified in human melanoma cell lines with different metastatic behaviour using high density oligonucleotide arrays.

The increasing incidence of melanoma and the lack of effective treatment, with the exception of tumour excision before the onset of the metastatic phase, make it important to identify genes that may function as new molecular markers for diagnosis and/or prognosis or as new targets for therapy. Recently, a new technique using high density oligonucleotide arrays has been developed to simultaneously screen for the expression of thousands of genes. We used this technique to compare the mRNA expression patterns of two human melanoma cell lines with different metastatic behaviour. Eight differentially expressed genes, namely apolipoprotein CII, tyrosinase-related protein 1, transforming growth factor-beta superfamily, subtilisin-like protein, elongation factor 1 alpha2, alpha2-macroglobulin, human cell division cycle 10 and serine/threonine protein kinase (DYRK1A), were selected to validate the array results by Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, a reliable correlation between differential expression of these genes in the melanoma cell lines and in fresh lesions of melanocytic tumour progression was demonstrated by RT-PCR analysis. Altogether, our data indicate that high density oligonucleotide arrays are a valuable and reliable tool to screen for differentially expressed genes, and that our study may be considered a basic step in the characterization of genes that are involved in the (malignant) progression of melanoma.