Das Basalzellkarzinom (BZK) der Haut gilt als einer der häufigsten semimalignen Tumoren. Trotz der niedrigen Metastasierungsrate von < 0,1 % können BZK schwerwiegende Infiltrationen und Destruktionen knorpeliger sowie knöcherner Strukturen verursachen. Für die Tumorentstehung ist vor allem die kumulative UVB-Dosis in Kindheit und Adoleszenz bedeutsam. Aber auch die Embryonalentwicklung scheint eine Rolle in der Pathogenese von Tumoren zu spielen. Die so genannte embryonale Fusionszone (eFZ) entsteht zwischen der 5-10 Entwicklungswoche durch die Verschmelzung der fünf Gesichtswülste. Es konnte gezeigt werden, dass BZK innerhalb dieses Kompartiments nicht nur gehäuft auftreten sondern auch durch ein ausgeprägtes Tiefenwachstum charakterisiert sind. Als mögliche Ursache für das verstärkt invasive Wachstum von BZK innerhalb der eFZ wurden Änderungen im Hyaluronsäure (HA)-Stoffwechsel der Stromazellen angenommen. Neben der Bedeutung in der Embryonalentwicklung und bei der Geweberegeneration zeigten verschiedene Studien zudem die essentielle Rolle von HA im Rahmen von malignen Zelltransformationen. Vermehrte HA-Ablagerungen in der Tumorumgebung oder erhöhte HA-Serumkonzentrationen wurden bei einer Vielzahl von Tumoren als Zeichen einer fortschreitenden Tumorprogression beschrieben. Um den HA-Stoffwechsel von Stromafibroblasten um BZK gezielt zu untersuchen, wurden der HA-Gehalt, die HA-Größe und die exprimierten Enzyme des HA-Stoffwechsels in Abhängigkeit von ihrer Lokalisationen miteinander verglichen. Dabei zeigte sich, dass sowohl innerhalb als auch außerhalb der eFZ vergleichbare Mengen und Polymergrößen von HA sezerniert werden. Molekularbiologische Untersuchungen an expandierten Fibroblasten aus Biopsien verschiedener Lokalisationen zeigten ebenfalls keine Unterschiede in der Expression von mRNA der HA Stoffwechselenzyme nach Herkunft der Fibroblasten. Somit wird geschlussfolgert, dass HA zwar auch im Stroma von BZK gebildet wird, der HA-Stoffwechsel von Stromafibroblasten jedoch kein Merkmal ist, das mit dem vermehrten Auftreten und invasiven Wachstum von BZK im Bereich der embryonalen Fusionszone des Mittelgesichts korreliert.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Το βασικοκυτταρικό καρκίνωμα του δέρματος αποτελεί τον συχνότερο τύπο καρκίνου του δέρματος. Από τους διάφορους ιστολογικούς υποτύπους του βασικοκυτταρικού καρκινώματος, ο διηθητικός ιστολογικός υπότυπος συνοδεύεται από αυξημένη διηθητική ικανότητα, υψηλότερο κίνδυνο τοπικής υποτροπής και πιο επιθετική βιολογική συμπεριφορά. Σε προηγούμενη μελέτη μας στο ίδιο υλικό βασικοκυτταρικού καρκινώματος, δείξαμε ότι η υπερέκφραση της Integrin-linked kinase (ILK), της ακτίνης των λείων μυικών κυττάρων (α-SMA), της πυρηνικής β-κατενίνης, του μεταγραφικού παράγοντα Snail, καθώς και η μείωση της έκφρασης της Ε-καντχερίνης που συντελούν δείκτες επιθηλιομεσεγχυματικής μετατροπής σχετίζονται με αυξημένη ικανότητα διήθησης και επιθετική βιολογική συμπεριφορά. Η επιθηλιομεσεγχυματική μετατροπή (Epithelial mesencymal transition-EMT) είναι μια βιολογική διαδικασία που επιτρέπει στα επιθηλιακά κύτταρα να αποκτήσουν έναν μεσεγχυματικό κυτταρικό φαινότυπο με ενισχυμένη μεταναστευτική ικανότητα, διεισδυτικότητα, υψηλή αντίσταση στην απόπτωση και ικανότητα παραγωγής συστατικών της εξωκυττάριας θεμέλιας ουσίας. Κύρια χαρακτηριστικά της ΕΜΤ είναι η απώλεια των ζωνών πρόσφυσης, καθώς και η ανοδιοργάνωση του κυτταροσκελετού. Ο κυτταροσκελετός της ακτίνης, που αποτελείται από νημάτια ακτίνης και πρωτεΐνες που αλληλεπιδρούν και ρυθμίζουν την δυναμική του κυτταροσκελετού συμμετέχει σε πολλές σημαντικές κυτταρικές λειτουργίες όπως η κυτταρική κίνηση, η κυτταρική διαίρεση κ.α. και εμπλέκεται σημαντικά στην καρκινογένεση. Ο ανεξέλεγκτος πολλαπλασιασμός των νεοπλασματικών κυττάρων, καθώς και η ικανότητα διήθησης και μετάστασης σχετίζονται με μεταβολές του κυτταροσκελετού της ακτίνης. Οι πρωτεΐνες CTEN (COOH-Terminal tensin-like protein) και η παξιλλίνη εντοπίζονται στις περιοχές πρόσδεσης των κυττάρων μέσω ιντεγκρινών στην εξωκυττάρια ουσία όπου αλληλεπιδρούν με τον κυτταροκελετό ακτίνης και συμμετέχουν στην ρύθμιση οδών μεταγωγής σημάτων που ελέγχουν λειτουργίες όπως η κυτταρική κίνηση, η επιθήλιο μεσεγχυματική μετάβαση, η διήθηση και μετάσταση. Η εζρίνη, μια κυτταροπλασματική πρωτεΐνη συνδέει μόρια της πλασματικής μεμβράνης με τον κυτταροσκελετό της ακτίνης και εμπλέκεται και αυτή σημαντικά σε διεργασίες κυτταρικής μετανάστευσης καθώς και στην διήθηση και μετάσταση των καρκινικών κυττάρων. Προηγούμενες μελέτες υποστηρίζουν την συμμετοχή των πρωτείνων CTEN, εζρίνης και παξιλλίνης στην καρκινογένεση στον άνθρωπο. Ωστόσο λίγα είναι γνωστά για το ρόλο τους στο βασικοκυτταρικό καρκίνωμα του δέρματος. Σκοπός της παρούσας εργασίας ήταν η μελέτη της έκφρασης και του ρόλου των πρωτεϊνών CTEN, εζρίνης και παξιλλίνης στο βασικοκυτταρικό καρκίνωμα του δέρματος. Για το σκοπό αυτό μελετήσαμε ανοσοϊστοχημικά την έκφραση αυτών των πρωτεϊνών που σχετίζονται με τον κυτταροσκελετό της ακτίνης σε 76 περιστατικά βασικοκυτταρικού καρκινώματος και εξετάσαμε την πιθανή συσχέτιση της έκφρασής τους με παραμέτρους που έχουν προγνωστική σημασία, όπως ο ιστολογικός υπότυπος, ο βαθμός κινδύνου και το επίπεδο διήθησης των όγκων. Ακόμη ελέγχθηκε η πιθανή συσχέτιση με την έκφραση των ILK, α-SMA, β-κατενίνης, Ε-καντχερίνης και Snail τα οποία εμπλέκονται σημαντικά στην διεργασία της ΕΜΤ και τα οποία είχαν μελετηθεί στο ίδιο υλικό βασικοκυτταρικού καρκινώματος σε προηγούμενη μελέτη. Η πρωτεΐνη CTEN εμφάνισε ασθενώς θετική και εστιακή ανοσοχρώση στο 33% των βασικοκυτταρικών καρκινωμάτων, χωρίς στατιστικά σημαντική συσχέτιση με τις κλινικοπαθολογοανατομικές παραμέτρους που εξετάσθηκαν όπως ο ιστολογικός υπότυπος και το βάθος διήθησης. Αντιθέτως η εζρίνη υπερεκφράστηκε στο κυτταρόπλασμα των κυττάρων του όγκου στο 98,6% των περιστατικών βασικοκυτταρικού καρκινώματος. Η έκφρασή της ήταν σημαντικά μεγαλύτερη στα οζώδη και επιφανειακά (χαμηλού κινδύνου) βασικοκυτταρικά καρκινώματα σε σχέση με τα διηθητικά και μικροοζώδη (υψηλού κινδύνου) βασικοκυτταρικά καρκινώματα. Δεν υπήρξε στατιστικώς σημαντική συσχέτιση με το επίπεδο διήθησης των όγκων, ούτε με την έκφραση των δεικτών ΕΜΤ, ILK, α-SMA, β-κατενίνης, Ε-καντχερίνης και Snail. Όσον αφορά στην παξιλλίνη, εκφράστηκε στο κυτταρόπλασμα των κυττάρων του όγκου στο 90,4% των περιστατικών βασικοκυτταρικού καρκινώματος, σε αντίθεση με το παρακείμενο μη νεοπλασματικό δέρμα, όπου η ανοσοχρώση ήταν αρνητική ή ασθενής. Σε σχέση με τον ιστολογικό υπότυπο, παρατηρήθηκε σημαντική απώλεια της έκφρασής της στα διηθητικά και μικροοζώδη βασικοκυτταρικά καρκινώματα, σε σύγκριση με τα οζώδη και επιφανειακά. Ωστόσο δε βρέθηκε καμία συσχέτιση στατιστικώς σημαντική με το βάθος διήθησης των όγκων και με την έκφραση των δεικτών ΕΜΤ. Επίσης υπήρξε στατιστικώς σημαντική συσχέτιση της ανοσοϊστοχημικής έκφρασης της παξιλλίνης με την έκφραση της εζρίνης. Συμπερασματικά, τα ευρήματα αυτά υποστηρίζουν ότι η πρωτεΐνη CTEN πιθανώς δεν έχει σημαντικό ρόλο στην παθογένεια του βασικοκυτταρικού καρκινώματος του δέρματος. Αντιθέτως η εζρίνη και η παξιλλίνη φαίνεται να εμπλέκονται στο βασικοκυτταρικό καρκίνωμα του δέρματος στον ανθρώπο και μάλιστα τα μειωμένα επίπεδα έκφρασης τους σχετίζονται με ιστολογικούς υποτύπους υψηλής επικινδυνότητας και πιο επιθετικής βιολογικής συμπεριφοράς. Τα χαρακτηριστικά μειωμένα επίπεδα έκφρασης της εζρίνης και παξιλλίνης στα βασικοκυτταρικά καρκινώματα διηθητικού ιστολογικού υποτύπου σε συνδυασμό με τα ευρήματα της προηγούμενης μελέτης ότι αυτός ο ιστολογικός υπότυπος βασικοκυτταρικού καρκινώματος εκφράζει δείκτες επιθηλιομεσεγχυματικής μετάβασης υποστηρίζουν επίσης την άποψη ότι τα διηθητικά βασικοκυτταρικά καρκινώματα αποτελούν σε μοριακό επίπεδο μία διακριτή οντότητα. Η υπόθεση αυτή συμφωνεί με το γεγονός ότι τα διηθητικά βασικοκυτταρικά καρκινώματα έχουν διακριτά μορφολογικά χαρακτηριστικά ( μικρές ομάδες κυττάρων με οξύαιχμα άκρα που εντόνως διηθούν το άθφονο εξωκυττάριο στρώμα) και πιο επιθετική συμπεριφορά συγκριτικά με τα οζώδη βασικοκυτταρικά καρκινώματα. Τελος, η μελέτη της εζρίνης και παξιλλίνης σε υλικό βιοψίας βασικοκυτταρικών καρκινωμάτων δερμάτος μπορεί μελλοντικά να συντελέσει στην ταυτοποίηση εκείνων των περιστατικών που ενδέχεται να εμφανίζουν μία επιθετικότερη βιολογική συμπεριφορά. / Basal Cell Carcinoma (BCC) of the skin is the most common type of skin cancer. Among the various histological subtypes of basal cell carcinoma, infiltrative histological subtype is accompanied by increased invasiveness, higher risk of local recurrence and more aggressive biological behavior. In our previous study, in the same material of BCC, we showed that overexpression of Integrin-linked kinase (ILK), actin of smooth muscle cells (α-SMA), nuclear beta-catenin, the transcription factor Snail and the reduction of E-cadherin’s expression, which constitute markers of epithelial-mesenchymal transition, are associated with increased invasiveness and aggressive biological behavior. The epithelial-mesenchymal transition (EMT) is a biological process, through which the cells acquire enhanced migratory capacity, invasiveness, high resistance to apoptosis and increased production of extracellular matrix components. Main features of EMT are the loss of adherence junctions and the reorganization of the cytoskeleton. The actin cytoskeleton which is composed of actin filaments and proteins that interact with and regulate the cytoskeleton’s dynamics is involved in many important cellular functions such as cell movement, cell division and it is critically implicated in carcinogenesis significantly. The uncontrolled proliferation of tumor cells, increased invasiveness and metastasis are all related to cytoskeleton changes. Proteins CTEN (COOH-Terminal tensin-like protein) and paxillin are located in cell- extracellular matrix contact sites (focal adhesions) where they interact with actin cytoskeleton and transduse signals that regulate actin cytoskeletal dynamics, cell motility, epithelial mesenchymal transition, invasion and metastasis. Ezrin is a cytoplasmic protein which links molecules of the plasma membrane to the actin cytoskeleton. Ezrin is involved significantly in processes of cell migration, invasion and metastasis of cancer cells. Previous studies support the involvement of proteins CTEN, ezrin and paxillin in human tumorigenesis. However, little is known about their role in basal cell carcinoma of the skin. The purpose of this study was to investigate the protein expression and respective role of CTEN, ezrin and paxillin in basal cell carcinoma of the skin. For this reason, we studied immunohistochemically the protein expression of CTEN, ezrin and paxillin in 76 cases of BCC and we examined the possible association of their expression with parameters of prognostic significance, such as histological subtype, low-high risk groups and tumor invasion. Further we tested potential correlations with the expression of ILK, α-SMA, beta-catenin, E-cadherin and Snail, which are involved significantly in the process of EMT and have been studied in the same material of BCC in a previous study. CTEN showed weakly positive and focal immunostaining in 33% of BCC, without statistically significant correlation with clinicopathological parameters, such as histological subtype and depth of invasion. Ezrin and paxillin were overexpressed in the cytoplasm in 98,6% and 90,4% of cases respectively with significantly higher expression levels in low risk variants (nodular and superficial BCC), compared to the high risk variants (micronodular and infilrative BCC). No correlation was found between the expression of ezrin and paxillin and depth of tumor invasion nor with the expression of EMT markers, ILK, α-SMA, beta-catenin, E-cadherin and Snail. However, there was a statistically significant correlation between the immunohistochemical expression of paxillin and ezrin. In conclusion, these findings argue that CTEN probably has no significant role in the pathogenesis of basal cell carcinoma of the skin. Conversely, ezrin and paxillin seem to be involved in human BCC and their decreased expression levels are associated with high risk histological subtypes and a more aggressive tunor behavior. The decreased expression levels of ezrin and paxillin in infiltrative BCC in conjuction with the findings of the previous study that this histological subtype of BCC expresses markers of epithelial-mesenchymal transition, also support the view that infiltrative BCC is a distinct entity at the molecular level. This hypothesis is consistent with the fact that infiltrative BCC have distinct morphological characteristics (small groups of cells with sharp ends that strongly infiltrate the extracellular matix) and more aggressive behavior compared to nodular basal cell carcinoma. Finally, the study of ezrin and paxillin in biopsy material of BCC may, in the future, help to identify those cases that may exhibit a more aggressive behavior.

Das Basalzellkarzinom ist nicht nur die häufigste Neoplasie der Haut generell, es stellt auch die häufigste maligne Entität im Bereich der Augenlider dar. Es handelt sich um eine Erkrankung vornehmlich des höheren Lebensalters, jedoch sind auch zunehmend jüngere Patienten betroffen. Trotz geringer Metastasierungstendenz kann es bei Tiefeninfiltration zu schweren Krankheitsverläufen kommen. Aufgrund wachsender Inzidenz und damit stetig steigender Kosten im Gesundheitswesen sollte die Behandlungsstrategie kontinuierlich überprüft und optimiert werden. Ziel der vorliegenden Arbeit ist es, ein ausgewähltes Patientenkollektiv im Zeitraum von 2003 bis 2006 hinsichtlich epidemiologischer, klinischer und therapeutischer Aspekte zu analysieren und mit der Literatur zu vergleichen. Therapie der Wahl ist die chirurgische Exzision. Es wurden 216 Fälle von 204 Patienten auf Grundlage der Krankenakte detailliert untersucht und ausgewertet. Zwar konnten keine signifikanten Prädiktoren zur Vorhersage des Behandlungsverlaufes und der Rezidiventwicklung gefunden werden, anhand der 216 klinischen Fälle können die aktuelle Datenlage aber unterstützt und Empfehlungen zur Therapie und Nachbehandlung erweitert werden.

Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

Doctor of Philosophy(PhD) / BCC is the commonest cancer in European-derived populations and Australia has the highest recorded incidence in the world, creating enormous individual and societal cost in management of this disease. The incidence of this cancer has been increasing internationally, with evidence of a 1 to 2% rise in incidence in Australia per year over the last two decades. The main four epidemiological risk factors for the development of BCC are ultraviolet radiation (UVR) exposure, increasing age, male sex, and inability to tan. The pattern and timing of UVR exposure is important to BCC risk, with childhood and intermittent UVR exposure both associated with an increased risk. The complex of inherited characteristics making up an individual’s ‘sun sensitivity’ is also important in determining BCC risk. Very little is known about population genetic susceptibility to BCC outside of the rare genodermatosis Gorlin syndrome. Mutations in the tumour suppressor gene patched (PTCH) are responsible for this BCC predisposition syndrome and the molecular pathway and target genes of this highly conserved pathway are well described. Derangments in this pathway occur in sporadic BCC development, and the PTCH gene is an obvious candidate to contribute to non-syndromic susceptibility to BCC. The melanocortin 1 receptor (MC1R) locus is known to be involved in pigmentary traits and the cutaneous response to UVR, and variants have been associated with skin cancer risk. Many other genes have been considered with respect to population BCC risk and include p53, HPV, GSTs, and HLAs. There is preliminary evidence for specific familial aggregation of BCC, but very little known about the causes. 56 individuals who developed BCC under the age of 40 in the year 2000 were recruited from the Skin and Cancer Foundation of Australia’s database. This represents the youngest 7 – 8% of Australians with BCC from a database that captures approximately 10% of Sydney’s BCCs. 212 of their first degree relatives were also recruited, including 89 parents and 123 siblings of these 56 probands. All subjects were interviewed with respect to their cancer history and all reports of cancer verified with histopathological reports where possible. The oldest unaffected sibling for each proband (where available) was designated as an intra-family control. All cases and control siblings filled out a questionnaire regarding their pigmentary and sun sensitivity factors and underwent a skin examination by a trained examiner. Peripheral blood was collected from these cases and controls for genotyping of PTCH. All the exons of PTCH for which mutations have been documented in Gorlin patients were amplified using PCR. PCR products were screened for mutations using dHPLC, and all detectable variants sequenced. Prevalence of BCC and SCC for the Australian population was estimated from incidence data using a novel statistical approach. Familial aggregation of BCC, SCC and MM occurred within the 56 families studied here. The majority of families with aggregation of skin cancer had a combination of SCC and BCC, however nearly one fifth of families in this study had aggregation of BCC to the exclusion of SCC or MM, suggesting that BCCspecific risk factors are also likely to be at work. Skin cancer risks for first-degree relatives of people with early onset BCC were calculated: sisters and mothers of people with early-onset BCC had a 2-fold increased risk of BCC; brothers had a 5-fold increased risk of BCC; and sisters and fathers of people with early-onset BCC had over four times the prevalence of SCC than that expected. For melanoma, the increased risk was significant for male relatives only, with a 10-fold increased risk for brothers of people with early-onset BCC and 3-fold for fathers. On skin examination of cases and controls, several phenotypic factors were significantly associated with BCC risk. These included increasing risk of BCC with having fair, easyburning skin (ie decreasing skin phototype), and with having signs of cumulative sun damage to the skin in the form of actinic keratoses. Signs reflecting the combination of pigmentary characteristics and sun exposure - in the form of arm freckling and solar lentigines - also gave subjects a significantly increased risk BCC. Constitutive red-green reflectance of the skin was associated with decreased risk of BCC, as measured by spectrophotometery. Other non-significant trends were seen that may become significant in larger studies including associations of BCC with propensity to burn, moderate tanning ability and an inability to tan. No convincing trend for risk of BCC was seen with the pigmentary variables of hair or eye colour, and a non-significant reduced risk of BCC was associated with increasing numbers of seborrhoeic keratoses. Twenty PTCH exons (exons 2, 3, 5 to 18, and 20 to 23) were screened, accounting for 97% of the coding regions with published mutations in PTCH. Nine of these 20 exons were found to harbour single nucleotide polymorphisms (SNPs), seen on dHPLC as variant melting curves and confirmed on direct sequencing. SNPs frequencies were not significantly different to published population frequencies, or to Australian general population frequencies where SNP database population data was unavailable. Assuming a Poisson distribution, and having observed no mutations in a sample of 56, we can be 97.5% confident that if there are any PTCH mutations contributing to early-onset BCC in the Australian population, then their prevalence is less than 5.1%. Overall, this study provides evidence that familial aggregation of BCC is occurring, that first-degree relatives are at increased risk of all three types of skin cancer, and that a combination of environmental and genetic risk factors are likely to be responsible. The PTCH gene is excluded as a major cause of this increased susceptibility to BCC in particular and skin cancer in general. The weaknesses of the study design are explored, the possible clinical relevance of the data is examined, and future directions for research into the genetics of basal cell carcinoma are discussed.

Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

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