Volker Waldmann

Analysis of the T cell response to tumor and viral peptide antigens by an IFNγ-ELISPOT assay

We have established a sensitive ELISPOT assay measuring interferon γ (IFN γ) release on a single‐cell basis to detect influenza peptide‐specific CD8+ T cells in uncultured peripheral blood mononuclear cells (PBMC). Using this method, we studied the T cell response to HLA‐A1 and HLA‐A2.1 binding peptide epitopes derived from the MAGE‐1 and MAGE‐3 proteins, from the melanoma‐associated antigens tyrosinase, Melan‐A/MART‐1 and gp100, and from influenza proteins in stage IV melanoma patients and healthy controls. In 18 of 24 HLA‐A2‐positive donors (75%), but only in 9 of 25 HLA‐A2‐positive melanoma patients (36%) T cells reactive with the influenza matrix peptide were demonstrated (p = 0.007). T cells responding to one or several of the melanoma‐associated peptides were detected in 5 of 25 HLA‐A2‐positive patients with metastatic melanoma. Four of these 5 patients had been treated with interleukin‐2‐ and IFNα‐containing therapy. Two of the 24 healthy donors had T cells reactive with the MART‐1 27‐35 peptide. No reactivity with the HLA‐A1‐binding peptides from MAGE‐1 or MAGE‐3 was detected in any of the HLA‐A1‐positive healthy controls or melanoma patients. These results show that the IFNγ‐ELISPOT assay is suitable to determine quantitatively T cells reactive with melanoma‐associated and influenza peptide epitopes in uncultured PBMC. The failure to detect T cells responding to influenza in many melanoma patients with progressive disease may indicate an impairment of their T cell function. Int. J. Cancer 71: 932‐936, 1997.

S-100β-Protein im Serum als Tumormarker beim malignen Melanom Aktueller Kenntnisstand und klinische Erfahrungen

ZusammenfassungS-100 ist ein saures, kalziumbindendes Protein, das als Heterodimer aus 2 isomeren Untereinheiten α und β besteht und erstmalig in Zellen neuroendokrinen Ursprungs beschrieben wurde. Es spielt eine Rolle bei verschiedenen zellulären Prozessen, wie z.B. der Zelldifferenzierung und der Proliferation und interagiert mit dem Tumorsuppressorprotein p53. S-100 ist ebenfalls in Melanomzellen vorhanden, und sein immunhistochemischer Nachweis ist bei der histopathologischen Diagnostik des malignen Melanoms weit verbreitet. Nachdem S-100β im Serum von Patienten mit malignem Melanom nachgewiesen wurde, folgten zahlreiche klinische Studien zur Etablierung dieses Proteins als Tumormarker in verschiedenen Stadien der Erkrankung. Die Resultate zeigen, daß S-100β-Protein im Serum von Patienten mit malignem Melanom ein unabhängiger prognostischer Marker und ein ergänzender klinischer Parameter für die Progression der metastasierten Erkrankung sowie für das Monitoring der Patienten während einer systemischen Therapie sein kann. Bei Lymphknoten- oder Fernmetastasierung gibt es jedoch auch Patienten mit negativen S-100-β-Werten, bei denen eine Korrelation mit dem Krankheitsverlauf nicht hergestellt werden kann. Eigene Ergebnisse bestätigen diese Grundaussage für Patienten im Stadium III/IV. Werden aber wiederholt positive S-100β-Serumwerte bei der statistischen Auswertung nur einmalig berücksichtigt, zeigt sich ein deutlich geringerer Anteil von Patienten im Stadium III/IV mit positiven S-100-Werten, als in der Literatur angegeben. Für das Monitoring im Stadium I und II scheint S-100β nicht geeignet.SummaryS100 is an acidic-calcium-binding protein, composed as a heterodimer of two isomeric subunits α and β and was first described in cells of neuroendocrine origin. It plays an important role in various cellular processes such as cell differentiation and proliferation and interacts with the tumour suppressor gene p53. S100 is also present in melanoma cells and its immunhistochemical detection is widely used in the histopathological diagnosis of malignant melanoma. S100 has been detected in the serum of patients with malignant melanoma and many clinical studies have been performed to establish this protein as a tumor marker in different stages of the disease. The data suggest that S-100β-protein in serum of patients with malignant melanoma could be an independent prognostic marker and an additional clinical parameter for progression of metastatic disease and serological monitoring during systemic therapy. However there are patients in stage of lymph node- or systemic metastasis with negative S-100β-serum levels and no correlation to the course of disease. Our results confirm the findings for patients in stage III/IV. However, the percentage of S-100β-positive patients in stage III/IV is lower than reported in the literature, if repeatedly positive samples are excluded from statistical analysis. For monitoring in stage I and II it seems to be not helpful.

Therapie des metastasierten malignen Uveamelanoms

ZusammenfassungDie Uvea ist nach der Haut der zweithäufigste Entstehungsort maligner Melanome, und das Uveamelanom der häufigste primäre intraokuläre Tumor des Erwachsenenalters. Aufgrund seiner Lokalisation, Biologie, Histologie, Genetik und seiner von kutanen Melanomen differierenden Prognose wird diese maligne Neoplasie zunehmend als eigenständige Entität in der Gruppe der malignen Melanome angesehen. Während Primärtumoren von Ophthalmoonkologen therapiert werden, findet die Behandlung von Patienten im Stadium der Metastasierung häufig in dermatoonkologischen Zentren statt. Die hämatogene Tumoraussaat betrifft häufig primär und überwiegend die Leber und bleibt oftmals über einen lüngeren Zeitraum auf dieses Organ beschränkt. Fernmetastasierte Uveamelanome zeigen eine weitgehende Resistenz gegenüber Zytostatika, die für das kutane maligne Melanom etabliert sind. Neue Therapieansätze mit einer lokalen intraarteriellen Chemotherapie über die A. hepatica, ggf. in Kombination mit einer Embolisation der die Lebermetastasen versorgenden Gefäße, konnten zu einer Prognoseverbesserung beitragen. Bei der lokal-hepatischen und systemischen Chemotherapie scheint das Nitrosoureaderivat Fotemustin anderen Zytostatika überlegen und ist als Therapeutikum der 1. Wahl anzusehen. In dieser Arbeit werden nach einer Charakterisierung des okulären Tumors Uveamelanom die verfügbaren Daten über bisherige Behandlungsergebnisse im Stadium der Metastasierung zusammengefasst und ein Überblick über neue und zukünftige Behandlungsstrategien gegeben.AbstractThe uvea is the most common site for extracutaneous melanoma and uveal melanoma is the most frequent primary intraocular tumour in adults. Because its different location, biology, histology, genetic features and prognosis in comparision to cutaneous melanoma, this tumour is considered as a distinct entity in the group of malignant melanoma. While primary uveal melanoma is usually treated by ophthalmologic oncologists, metastatic diseases is often managed by dermatologic oncologists. Hematogenous spread predominantly involves the liver and is often restricted to this organ for a long period. Metastatic uveal melanoma is usually resistant to chemotherapeutic regimens established for the therapy of cutaneous melanoma. Newer therapeutic modalities, such as local intra-arterial chemotherapy into the hepatic artery, perhaps combined with embolisation of feeder blood vessels of liver metastases, improves the prognosis of metastatic uveal melanoma. Currently the nitrosourea derivate fotemustine is the drug of choice in the local hepatic and systemic treatment and seems to be superior to other chemotherapeutic agents. Following the characterisation of primary uveal melanoma, we summarize the results of different treatment protocols for metastatic disease and give an overview of new strategies.

Prognosis of metastatic melanoma: no correlation of tyrosinase mRNA in bone marrow and survival time.

Recent publications suggest that tyrosinase mRNA in blood as well as in bone marrow is detectable only in a subgroup of patients with metastatic melanoma. This would imply that tyrosinase mRNA is of limited value as a tumor marker. We addressed the question of whether patients with metastatic melanoma and RT-PCR-detectable tyrosinase mRNA in blood or bone marrow have a different prognosis than tyrosinase mRNA-negative patients. Twenty melanoma patients with widespread clinical metastases were enrolled; the survival time after first diagnosis of visceral metastases was correlated to tyrosinase mRNA presence in blood and bone marrow samples. The time of survival of eight patients with metastatic melanoma and detectable tyrosinase mRNA in either blood or bone marrow was not different from the prognosis of 12 patients without detectable tyrosinase mRNA in either blood or bone marrow. Detection of tyrosinase mRNA in blood or bone marrow samples of melanoma patients with advanced disease seems to have no substantial relevance for survival time and outcome of disease. In this constellation, detection of tyrosinase mRNA by RT-PCR is not a valid tumor marker. Nevertheless, tyrosinase positivity in bone marrow in earlier tumor stages might indicate increased risk for the development of distant metastases. This should be addressed in further studies.

Pathogenese des malignen Melanoms Molekularbiologische Aspekte

ZusammenfassungDas maligne Melanom ist der bösartigste Tumor der Haut mit weltweit steigender Inzidenz. Über die genetischen Veränderungen, welche die Melanominitiation und -progression auslösen und in Gang halten, ist wenig bekannt. Mutationen von p16 (CDKN2), p53, ras, Neurofibromatose Typ I (NF-1), bcl 2 und dem Retinoblastom-Gen (Rb) wurden beschrieben, liegen jedoch sämtlich nur in relativ geringen Prävalenzen bezogen auf alle Melanomerkrankungen vor. Familiäre genetische Alterationen von potentiellen Tumorsuppressorgenen wie dem p16, aber auch interpersonelle Unterschiede in der Reparaturkapazität von UV- und anders bedingten DNA-Schädigungen tragen zum individuellen Melanomrisiko bei. Wichtigstes Karzinogen scheint die UV-Exposition zu sein, deren mutagener Effekt bis in die molekulare Analyse detektierter Punktmutationen entscheidender Gene verfolgt werden kann. Aktivieren UV-induzierte DNA-Schädigungen Protoonkogene oder inaktivieren sie Tumorsuppressorgene, ist die molekularbiologische Kausalkette zwischen UV-Exposition, DNA-Schädigung, Mutation und Tumorinitiation bzw. -progression geschlossen. Ein zum molekularen Mehrstufenkarzinogenesemodell für kolorektale Karzinome analoges stadienabhängiges Modell der Melanomprogression konnte bislang trotz der Existenz von morphologisch und histopathologisch gut abgrenzbaren Melanomvorläuferläsionen der Haut nicht etabliert werden.SummaryThe incidence of melanoma, the most aggressive tumor of the skin, is increasing worldwide. The genetic mechanisms responsible for the initiation and progression of melanoma are poorly understood. Mutations of p16 (CDKN2), p53, ras, neurofibromatosis type I gene (NF-1), bcl 2 and the retinoblastoma gene have been described, but none are common. Suggesting heterogeneous mechanisms of carcinogenesis. Both familial inheritance of potential tumor suppressor genes, e.g. p16, and differences in DNA-repair capacity contribute to the individual risk for melanoma. The most important carcinogen for melanoma seems to be UV exposition whose mutagenic effects can be demonstrated by molecular analysis of detected point mutations in relevant genes. The UV-induced DNA damage generates mutations which are capable of activating proto-oncogenes or inactivating tumor suppressor genes, demonstrating the molecular link between UV exposition, DNA damage, mutations and tumor initiation and/or progression. A stage-dependent model of melanoma carcinogenesis analogous to colorectal cancer remains to be established, despite the existence of morphologically and histopathologically well defined melanoma precursor lesions in the skin.

Disseminierte Melanomzellen in Blut und Knochenmark Bedeutung und Nachweis durch potenzielle Tumormarker

ZusammenfassungDie weit überwiegende Mehrzahl aller primären Melanome werden in sano exzidiert. Somit hängt die Prognose einer Melanomerkrankung davon ab, ob zum Zeitpunkt der Exzision eine Tumorzellaussaat stattgefunden hat bzw. ob eine solche sich in der Folge etablieren kann und zum Auftreten klinisch apparenter Metastasen führt. Ein valider, prospektiver Nachweis einer solchen “minimal residual disease” des malignen Melanoms ist bis heute nicht möglich. Die wichtigsten, gegenwärtig bekannten sog. Marker einer Melanomerkrankung, Tyrosinase, S100 und MIA zeigen zwar alle einen, mit dem vorliegenden Stadium der Melanomerkrankung korrelierten Anstieg positiver Patienten, eine valide Prognosebestimmung konnte aber bisher nur für S100 für Patienten mit bereits metastasierten Melanomen statistisch nachgewiesen werden. Es muss daher weiterhin in prospektiven Studien abgeklärt werden, ob eine routinemäßige Bestimmung der genannten Marker im klinischen Alltag einen signifikanten Informationsgewinn darstellen kann.AbstractAs the majority of primary malignant melanomas can be cured by surgical excision, the prognosis of melanomas is dependent on whether tumor cells have disseminated orare capable of doing so at the time of surgery. A prospective and valid detection of this minimal residual disease is not currently possible. The most important known so-called markers of melanoma disease, tyrosinase, S100 and MIA, all are more likely to be present in patients with more advanced disease.A valid prognostic effect has only been shown for S100 in patients with already identified metastatic disease. Further prospective studies are required to determine the potential gain of information by routine determination of these markers in melanoma patients.

Mutations of the PH Domain of Protein Kinase B (PKB/AKT) Are Absentin Human Epidermal Skin Tumors

Background: While for most human solid tumors genetic alterations of few distinct genetic regions have been found, studies on basal cell carcinomas (BCC) have shown the prevalence of several abnormalities including alterations of the three ras genes, GAP (GTPase activating protein), p53, PTCH (the human homologue of Drosophila patched) and SMOH (the human homologue of Drosophila smoothened). On the other hand, during the last decade, a new oncogene, protein kinase B (PKB/AKT), has been characterized and found to be overexpressed in certain human tumors. In vivo activation of PKB/AKT necessitates its recruitment to the cell membrane mediated by the N-terminal pleckstrin homology (PH) domain. Objective: We investigated whether mutations of this mandatory domain are present in a subset of human epidermal skin tumors. Methods: RNA of 19 human skin tumors including 13 BCC, 4 squamous cell carcinomas (SCC; including 1 keratoacanthoma) and 2 neurofibromas of different size and tumor stage were used for reverse transcription and subsequent PCR amplification of the PH domain of PKB/AKT. Results: Cycle sequencing of the purified PCR products did not reveal any mutation of the PH domain of PKB/AKT. Conclusion: In human BCC and SCC, mutations of the PH domain of PKT/AKT do not play a major role during the carcinogenesis of these tumors.

No Correlation of Tyrosinase mRNA in Bone Marrow with Prognosis of Metastatic Melanoma

Background: Recent publications suggest that tyrosinase mRNA in blood as well as in bone marrow is detectable only in a subgroup of patients with metastatic melanoma. Objective: We addressed the question, whether patients with metastatic melanoma and with RT-PCR-detectable tyrosinase mRNA in blood or bone marrow have a different prognosis compared to tyrosinase mRNA-negative patients. Methods: 20 melanoma patients with widespread clinical metastases were enrolled and the survival time after first diagnosis of visceral metastases was correlated to tyrosinase mRNA presence in blood and bone marrow samples. Results: The time of survival of 8 patients with metastatic melanoma and detectable tyrosinase mRNA in either blood or bone marrow was not different from the prognosis of 12 patients without detectable tyrosinase mRNA in either blood or bone marrow. Conclusion: Although based on a limited number of patients our results suggest that detection of tyrosinase mRNA in blood or bone marrow samples of melanoma patients with advanced disease seems to have no substantial relevance for survival time and outcome of disease. For this purpose, detection of tyrosinase mRNA by RT-PCR is not a valid tumor marker. Nevertheless, tyrosinase positivity in bone marrow in earlier tumor stages might indicate increased risk for the development of distant metastases. This should be addressed in further studies.

Book Review / Announcement

The objective of this book is to provide profound knowledge of the impact of vitamins, electrolytes, trace elements, fatty acids and amino acids in the prevention and treatment of diseases caused by malnutrition and malabsorption. The book is divided into four parts covering the basics of micronutrients, the physiological and therapeutic impact of individual micronutrients and their link to civilization diseases such as diabetes, cancer and arteriosclerosis and possible therapeutic interventions. The last part of the book displays recommended daily reference doses in tables and contains a detailed glossary. The book is very well structured with short and precise sentences and well documented with good reference material. A very useful reference book for any kind of questions about micronutrients. P. Pedrazzetti