Dépôt Institutionnel de l’Université libre de Bruxelles / Université libre de Bruxelles Institutional Repository
Thèse de doctorat/ PhD Thesis Citation APA:
El Hajj, P. (2015). New prognosis markers and new targets for therapy in high risk melanoma: evaluation of TYRP1 as a melanoma prognostic marker and its regulation by miRNA(s) (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté de Pharmacie, Bruxelles.
Disponible à / Available at permalink : https://dipot.ulb.ac.be/dspace/bitstream/2013/209064/7/a65b1280-0027-4b17-ba96-f2a0bc1ef482.txt
(English version below)
Cette thèse de doctorat a été numérisée par l’Université libre de Bruxelles. L’auteur qui s’opposerait à sa mise en ligne dans DI-fusion est invité à prendre contact avec l’Université (di-fusion@ulb.ac.be).
Dans le cas où une version électronique native de la thèse existe, l’Université ne peut garantir que la présente version numérisée soit identique à la version électronique native, ni qu’elle soit la version officielle définitive de la thèse.
DI-fusion, le Dépôt Institutionnel de l’Université libre de Bruxelles, recueille la production scientifique de l’Université, mise à disposition en libre accès autant que possible. Les œuvres accessibles dans DI-fusion sont protégées par la législation belge relative aux droits d'auteur et aux droits voisins. Toute personne peut, sans avoir à demander l’autorisation de l’auteur ou de l’ayant-droit, à des fins d’usage privé ou à des fins d’illustration de l’enseignement ou de recherche scientifique, dans la mesure justifiée par le but non lucratif poursuivi, lire, télécharger ou reproduire sur papier ou sur tout autre support, les articles ou des fragments d’autres œuvres, disponibles dans DI-fusion, pour autant que :
Le nom des auteurs, le titre et la référence bibliographique complète soient cités; L’identifiant unique attribué aux métadonnées dans DI-fusion (permalink) soit indiqué; Le contenu ne soit pas modifié.
L’œuvre ne peut être stockée dans une autre base de données dans le but d’y donner accès ; l’identifiant unique (permalink) indiqué ci-dessus doit toujours être utilisé pour donner accès à l’œuvre. Toute autre utilisation non mentionnée ci-dessus nécessite l’autorisation de l’auteur de l’œuvre ou de l’ayant droit.
--- English Version ---
This Ph.D. thesis has been digitized by Université libre de Bruxelles. The author who would disagree on its online availability in DI-fusion is invited to contact the University (di-fusion@ulb.ac.be).
If a native electronic version of the thesis exists, the University can guarantee neither that the present digitized version is identical to the native electronic version, nor that it is the definitive official version of the thesis.
DI-fusion is the Institutional Repository of Université libre de Bruxelles; it collects the research output of the University, available on open access as much as possible. The works included in DI-fusion are protected by the Belgian legislation relating to authors’ rights and neighbouring rights. Any user may, without prior permission from the authors or copyright owners, for private usage or for educational or scientific research purposes, to the extent justified by the non-profit activity, read, download or reproduce on paper or on any other media, the articles or fragments of other works, available in DI-fusion, provided:
The authors, title and full bibliographic details are credited in any copy;
The unique identifier (permalink) for the original metadata page in DI-fusion is indicated; The content is not changed in any way.
It is not permitted to store the work in another database in order to provide access to it; the unique identifier (permalink) indicated above must always be used to provide access to the work. Any other use not mentioned above requires the authors’ or copyright owners’ permission.
ULB
UNIVERSITé LIBRE DE BRUXELLES F a c u l t é d e P h a r m a c i e E c o l e D o c t o r a l e e n S c i e n c e s P h a r m a c e u t i q u e s Université Libanaise E c o l e D o c t o r a l e d e s S c i e n c e s et d e T e c h n o l o g i eNew prognosis markers and new targets for therapy in high risk
melanoma: Evaluation of TYRP1 as a melanoma prognostic marker
and its régulation by miRNA(s)
P e t r a EL HAJJ
Thèse en cotutelle présentée en vue de l'obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques- Université Libre de Bruxelles
Docteur en Sciences et Technologie- Université Libanaise
P r o m o t e u r s et c o - p r o m o t e u r :
Prof. Ghanem Elias G H A N E M (Laboratoire d'Oncologie et de Chirurgie Expérimentale, ULB) Prof. Bassam BADRAN (Service d'Immunologie, Faculté des Sciences, UL)
Prof. Fabrice JOURNE (Laboratoire d'Oncologie et de Chirurgie Expérimentale, ULB)
C o m p o s i t i o n du jury :
Prof. Jean-Michel KAUFFMANN- Président (Faculté de Pharmacie, ULB) Prof. Véronique MATHIEU- Secrétaire (Faculté de Pharmacie, ULB)
Prof. Wissam FAOUR - Rapporteur (School of Medicine, Lebanese American University) Prof. Nader HUSSEIN - Examinateur (Faculté de Sciences, UL)
Prof. Pierre DUEZ (Faculté de Médecine et de Pharmacie, UMONS)
Prof. J.C. GARCIA B O R R O N (School of Medicine, University of Murcia)
I wusla to tkiflkUe everyoi^^ wkio kieLpeci kvte coi^pLete tWis d'issertatiov^. WitV\out their cov^iwjutA efforts flkul support, ( wou.Lo( liflve ^wjt beei/v nbLe to bn.i^ kw-y worfe to n suûcessfutL ûompLetLoi/v.
Ft-rst fliA^ forentost \ offtr m,y sLkvcerest gratitude to lotu) sucpervisor. Prof. «^lofliA^Hc «^lifluvem., for tke dov^tiiM-ous sucpport of kw-y Plii> stucdy ai^ui Yts.ta.rcM, for Wis patLe^vce, vMtivatiovy^,
support, Mit to M^kvtt-okv kts iiM.vM\^t feiA^wLedge wliLLst providli^vg kvte wttM «kv exceLLeiAt
atkVLospl^erefordotiA/g reseflrcli. OIA^stm-pty couLd kw)twtsl^ f o r a betterorfrieiA^oILtersupervlsor.
ALSO, I W0U.L0I Lll^ to tkauvfe m.y super/usor. Prof. Bassam, B-adrai^ who uciA^ertoofe to act as kw-y supervisor despute Wis ^vui^vy otker flca(;<ei^tc a^ui frafe^wvuil cD\M.\Mitv\uvds. f-tts wLsdom.,
feiA/OwLetJlge l^as btet^ i\M/aluable OIA- aiA- aoadevw^ LeveL.
My sX\M,tYt thakv,les aLso go to i>r. Fmhrioejouriu wVo skiowed m.e tkie road avui kieLped to get kvte started. He was aLways avalLabLe for m.y c^M.estwkts a^vol gave gei^rousLy of lits tt-tue a^u^ vast fen^wLeotge. (-te aLways teiA.ew wliere to Loofe for tke avi^wirs. to obstacles wkit.Le Imdiv^ to the riglot source.
My ticcpest gratitude aLso goes to V>r. •Re^vato hAoravu^vd wVo heLped \M£ deveLop kvty baclegrout^d LIA- ceLL oulture, trakvsfîctlokv, bLottLi^^ aiA/d LiA-fonotattcs issues.
M y gratitude aLso goes to aLL mei/nbers of tliejury; prof, c^arcia. '%OYYOV^, prof. Pterre i>uez, prof.
jeaiA^MLûlieL \<JX\A.^WJX\M^, r>r. vérovûc^ue Matl^Leu, t^r. wlssa^M. Faour a^vd t^r. Nader f-tusseti^
wko, despLte tketr m.aiA,y dutLes, agreed tojudge thls tkiesLs.
Makvy tkauvfes to aLL SNOY^YS, LiA-tlie Laboratory crf oMoioQij avui experLkM.ekvtaL s,uYQeYij (LDCB),
juLes Bordet ikvstLtute, for slnowtkvg kvte a good kvuîkûpuLatLokv of tkie worte. Ttiey added truLy kvcekVLorflbLe experiewuie for ^vte. My researcl^ wouLd not kave beeiA, possible wttkiout tkieLr loelps.
with tl^eLr best wushes. FtuuiUy, i tkflu-le i^uj deflr kusbfliA^ c^eorQe^ wWo has. betv\, a source crf eM,DuraQeiMv\t, who aatlveLy supporte^ wte LIA, kw-y deter\Mivu^tU>\^ to flkvd n^u?! reaLvzt nty potekvti.aL, aiA^ to m,alee tWis oovdinhiàiiotA,. ht wos «Lwnys there ohetriv^ wjt w,p a^v/Ct SIIOWIIA^
S U M M A R Y
Clinical outcome of high-risk melanotna patients is not reliably predicted from histopathological analyses of primary tumor and is often adjusted during disease progression.
Our study aimed to extend our initial observations in skin métastases and to evaluate the prognostic value of tyrosinase-related protein 1 (TYRPl) in lymph node (LN) métastases of stages III and IV melanoma patients. TYRPl is a melanosomal enzyme that shares structural similitude with tyrosinase, a key enzyme regulating pigmentation.
TYRPl mRNA expression from 104 lymph node métastases was quantified by real-time PCR and normalized to S100 calcium binding protein B (SlOOB) m R N A expression to correct for tumor load. T Y R P l / S I 0 0 8 ratios were calculated and médian was used as cut-off value. TYRPl/SlOOB mRNA ratios were correlated to clinical follow-up and histopathological characteristics of the primary lésion.
A high TYRPl/SlOOB mRNA ratio significantly correlated with reduced disease-free and overall survival, increased Breslow thickness and présence of ulcération of the primaries. Moreover, high TYRPl/SlOOB was of better prognostic value for overall survival than Breslow thickness and ulcération of the primaries and it was well conserved during disease progression with respect to high/low TYRPl groups.
We found that high TYRPl/SlOOB mRNA expression in lymph node métastases from melanoma patients is associated with unfavorable clinical outcome. Its évaluation in lymph node métastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lésions and may allow différent management of the two groups of patients. Its conserved expression further supports its use as a target for therapy.
Second, by evaluating TYRPl protein expression by immunohistochemistry (IHC) in skin and LN métastases, we showed that TYRPl protein was not detected in half of tissues expressing mRNA and in contrast to mRNA, it was not associated with survival, suggesting a post-transcriptional régulation.
Récent data reported that the 3' untranslated région (3'-UTR) of T Y R P l mRNA contains three putative miR-155 binding sites, among which two are polymorphic (Single nucleotide
miRNA-mRNA interaction or not in the case of mismatched interaction. We aimed to examine if miR-155 may affect TYRPl mRNA and protein expression depending of thèse SNPs. First, we transfected two melanoma Unes harboring thèse SNPs with increasing concentrations of
pre-miR-155 and evaluated both miR-pre-miR-155 and TYRPl mRNA by Real-time PCR and TYRPl protein by Western blotting. We found that miR-155 overexpression induced a high TYRPl mRNA decay and disrupted its translation into protein in the line with the "match" génotype. Then, we examined TYRPl protein expression, TYRPl mRNA, miR-155 levels and SNPs in 192 melanoma métastases. We found that the group of samples with "match" génotype was significantly associated with a lower TYRPl protein level while no différence was found for TYRPl mRNA and miR-155 between both groups, confirming that SNPs may specifically affect protein expression. In addition, we showed that TYRPl mRNA inversely correlated with
R E S U M E
L'espérance de vie des patients atteints de mélanome à haut risque ne peut être prédite d'une façon fiable en se basant sur les analyses d'histopathologies de la lésion primitive et est souvent ajustée durant la progression de la maladie. Notre étude vise à élargir nos observations initiales au niveau des métastases cutanées et d'évaluer la valeur pronostique de tyrosinase relatedprotein 1 ( T Y R P l ) dans les métastases ganglionnaires des patients atteints de mélanome de stades 111 et IV. TYRPl est une enzyme mélanosomale qui partage des similitudes structurelles avec la tyrosinase, l'enzyme clé de la mélanogenèse.
L'expression de l'ARNm de TYRPl a été quantifiée dans 104 métastases ganglionnaires par PCR en temps réel et normalisée par rapport à l'expression de l'ARNm de SlOOB (marqueur reconnu du mélanome) pour corriger l'expression de TYRPl suivant la charge tumorale de l'échantillon. Le rapport TYRPl/SlOOB a été calculé et la médiane a été utilisée en tant que valeur seuil. Ensuite nous avons étudié la relation entre les valeurs de TYRPl/SlOOB, le suivi clinique et les caractéristiques histopathologiques de la tumeur primitive.
Un rapport élevé de l'ARNm TYRPl/SlOOB corrélait significativement avec une survie sans récidive et une survie globale plus courtes, avec une épaisseur de Breslow plus élevée et avec la présence d'une ulcération au niveau de la tumeur primitive. En outre, une expression élevée de TYRPl/SlOOB était de meilleure valeur pronostique pour la survie globale que l'épaisseur de Breslow et l'ulcération des primitifs. De plus, cette expression est bien conservée au cours de la progression de la maladie par rapport aux groupes de TYRPl bas/élevé.
Nous avons constaté qu'une expression élevée de TYRPl/SlOOB dans les métastases de patients atteints de mélanome est associée à un résultat clinique défavorable et une survie courte. Menée sur des patients atteints d'un mélanome à haut risque de récidive, cette première étude a suggéré que l'ARNm de TYRPl dans les métastases pourrait servir de biomarqueur pour affiner le pronostic initial des patients surtout ceux ayant des lésions primitives de localisation inconnues ou non évaluables et peut permettre une gestion différente des deux groupes de patients. Son expression conservée au cours de la progression de la maladie est en faveur de son utilisation comme cible thérapeutique.
moitié des tissus exprimant bel et bien l'ARNm correspondant et qu'elle, contrairement à l'ARNm, n'était pas associée à la survie.
A I M S
Cutaneous melanoma is the less common (4%) and the deadliest form of skin cancer; it causes the majority of deaths related to skin cancer (75%), with an increasing incidence in the last décades'. Melanoma is an extremely heterogeneous disease with a poor clinical outcome even in patients with in situ primary lésions or thin melanomas^.
Treatment options for advanced melanoma are limited and rarely curative. Early détection and complète surgical excision of melanoma are the best stratégies to reduce mortality. However, about 15% of patients diagnosed with primary melanoma develop distant métastases^. Although long-term survival for patients with advanced melanoma is low, it is highly variable'^. The variability in survival of patients with stage III (39-70% for 5-year survival) and stage IV melanoma (33-62% for 1-year survival)^ points to an insufficient understanding of the
heterogeneity of the disease and wams of diffîculties to select patients who could benefit from treatments.
The currently used staging system for melanoma^, based on histopathological and clinical criteria, such as Breslow tumor thickness, mitotic rate, lymph node status, and ulcération, is limited in its ability to provide a précise prognosis , upon first diagnosis, and is often adjusted during disease progression. Indeed, a large number of patients with similar or even identical clinical features have a variable outcome"*.
In this context, various groups identified gènes and proteins associated with patient outcomes^ or suggested the use of sérum and tissue markers in order to refme the prognosis of patients with high risk melanoma, to ensure adéquate follow-up, and to predict the possible benefit from a therapy. However, despite many attempts to identify complementary molecular markers
predicting clinical outcome in melanoma, most studies consist in small séries requiring additional évaluation in larger populations and thus, have not been shown to improve the current AJCC staging, preventing their translation into routine clinical assessment.
In our previous studies, we found, by gene expression profiling in melanoma skin métastases, that TYRPl expression ranked first and, alone, was prédictive of distant metastasis free survival (DMFS) and overall survival (OS) and correlated with Breslow thickness suggesting that T Y R P l can be of a prognostic value particularly useful when pathology parameters at the primary lésion are lacking^.
Our first aim was to investigate whether a prognostic relevance or any potential corrélation exist between TYRPl in lymph node (LN) métastases of stages III and IV melanoma patients and pathological features of the primaries. For that, the expression of TYRPl mRNA was measured by RT-qPCR in 104 LN melanoma métastases. Since LN biopsies may contain variable amounts of stroma and tumor tissue, we related TYRPl values to those of SlOOB mRNA. We calculated corrélations between TYRPl/SlOOB mRNA expression and disease-free survival (DFS), OS, and conventional histopathological parameters such as Breslow thickness, ulcération and lymph node involvement. Then, we compared the prognostic value of TYRPl/SlOOB to those of Breslow thickness and ulcération.
In addition, we checked for TYRPl protein expression by IHC in a panel of paraffin-embedded biopsies from skin and LN métastases and found a lack of association with patient survival. We also reported discrepancies with the TYRPl mRNA levels measured by real-time PCR in corresponding frozen tissues in about 50% of the cases where m R N A was expressed but not the protein, suggesting post-transcriptional regulation(s). In this context, Li et al. suggested that
miR-155 acts as a "rhéostat" to optimize TYRPl expression for local adaptation to differential UV radiation along the latitudes^. The authors showed that the 3'-UTR of TYRPl m R N A contains three putative miR-155 binding sites, among which two are polymorphic (SNPs rs683/rs910) inducing or disrupting régulation by miR-155.
TABLE OF CONTENTS
Acknowledgment Summary 4
Résume 6
Aim of the thesis 8 Table of contents 10 List of Abbreviations 15 Part L Introduction 18 Chapter 1. Melanogenesis 18
1. The human skin 18 1.1 Skin anatomy 18
1.2 Skin pigmentation 19 1.2.1 Melanocytes 19
1.2.1.1Melanocyte development: Melanocytic origin and embryonic development from neural crest through melanoblasts to melanocytes 19
1.2.2 Melanin: the pigment 20 1.2.2.1 Melanin types 20
1.2.2.2 Phenotypic diversity of pigmentation 21 1.2.3 Melanogenesis and Melanosome 21
1.2.3.1 Biogenesis and maturation of melanosomes 21 1.2.3.2 Transfer of melanosomes 21
1.2.4 Triggers of melanogenesis 22
1.2.5 Enzymes of melanogenesis and melanin synthesis 22
Chapter 2. Melanoma 24
1 Overview; Cutaneous melanoma 24 2 Stages of melanoma progression 24
2.1 Common Acquired/congenital melanocytic nevus 24 2.2 Dysplastic nevus 25
2.3 Radial Growth Phase 25 2.4 Vertical Growth Phase 26
2.5 Metastatic Melanoma 26 2.5.1 Sites of metastasis 26
3 Classification of cutaneous malignant melanoma 28 4 Risk factors for melanoma 29
4.1 Environmental risk factors 29 4.2 Phenotypic risk factors 30 4.3 Genetic risk factors 31 5 Epidemiology of melanoma 31 6 Pathology 33
6.1 Rôle of receptor tyrosine kinases 33
c-KIT. 34
AKT Pathway and PTEN in melanoma progression 34 Mitogen activating protein kinase (MAPK) pathway 34
6.2 Cell cycle 35
P53 36
6.3 MITF 36
6.4 Growth factor dependence in melanoma development 36 6.5 Cadherin expression in melanoma progression 37
7 Diagnosis 37
7.1 Diagnosis of primary melanoma 37 7.2 Sentinel lymph node biopsy 37 7.3 Imaging tests 38 8 Therapy 38 8.1 Surgery 38 8.2 Radiotherapy 39 8.3 Chemotherapy 39 8.4 Immuno-therapy 40 8.4.1 Interleukin-2 therapy 40 8.4.2 Interferon-a therapy 41 8.4.3 anti-CTLA-4 therapy 41 8.4.4 anti-PDl therapy 42 8.5 Combination therapy 42
8.6 Targeted thérapies for melanoma treatment 43
8.6.1 Targeting the RAS/RAF/MAPK Pathway in Melanoma 43 Résistance to BRAF inhibitors 44
8.6.2 Targeting the P13K/AKT Pathway in Melanoma 44 9 Prognostic factors in malignant melanoma 45
9.2 Progression markers 47
Prognosis for melanoma of unknown primaries 48
9.3 Blood markers and the need of new biomarkers in metastatic patients 49 9.4 Tissue biomarkers in metastatic patients 51
Chapter 3. Tyrosinase related proteinl, TYRPl 53
1 TYRP family 53 2 TYRPl gene 53
3 Synthesis and Transport of TYRPl 54 3.1 TYRPl cellular localization 54
3.2 Régulation of TYRPl transcription and rôle of MITF 54 4 Rôles of TYRPl in pigmentation 55
4.1 In mammals 55
4.1.1 TYRPl catalase acticity 55 4.2 In men 56
4.2.12 TYRPl régulation of tyrosinase activity 56 4.2.2 TYRPl and oxidative stress 57
5 TYRPl in melanoma 57
5.1 TYRPl gene variants and melanoma risk 57 5.2 TYRPl as a target for therapy 58
5.2.1 Preclinical studies 58 5.2.2 Clinical trials 59
5.3 TYRPl expression and melanoma progression 60
Chapter 4. MicroRNAs 62
1. Overview 62
1.1 miRNA Discover 62 1.2 miRNA Biogenesis 62
1.3 miRNA mechanisms of action 63
1.4 miRNA Nomenclature and Annotation 63 1.5 Target Prédiction and Algorithms 64 1.6 miRNAs in Cancer 64
2. miRNAs and melanoma 65
3. miR-155 in melanoma and its rôle in regulating TYRPl 66 4. mlRNAs and pigmentation 68
Part IL Material and methods 70
1. Patients and tissue collection 70
2. Melanoma cell Unes and cell culture conditions 70 3. Cell lysis and Western blot analysis 71
4. RNA extraction 71
4.1 Assessment of RNA integrityl 72 4.2 Reverse Transcriptase Reaction 73 5. Polymerase Chain Reaction (PCR) 73
5.1 m R N A Real-Time PCR 73 5.2 Primers choice for TYRPl 74
5.3 miRNA real time PCR 74 5.4 SNP Genotyping 75
5.5 PCR specificity and efficiency 76 5.6 Choice for endogenous contre! 76 5.6.1 SlOOB in melanoma tissues 76
5.6.2 18s in cell Unes 77
5.6.3 RNU44 control for microRNA(s) 77 6. Cell transfection 77
7 Stimulation of melanoma cell pigmentation 78
8 Taqman Low Density Array for miRNA profiling and MicroRNAs target prédiction 79 9. Immunohistochemistry 79
10. Statistical analyses 80
Part IIL Results and Discussion 81
1. Tyrosinase-related protein 1 mRNA expression in lymph node métastases predicts overall survival in high-risk melanoma patients 81
Summary 82
2. SNPs at miR-155 binding sites of TYRPl explain discrepancy between mRNA and protein and refme TYRPl prognostic value in melanoma 84
3. Differential expression profiles of microRNAs involved in malignant melanocyte pigmentation 88 Summary 89
4. General discussion 90
LIST OF ABBREVIATIONS
AJCC - American Joint Committee on Cancer ALM - Acral lentinigous melanoma
APC - antigen presenting cells
ARNm - acide ribonucleotide messager bp - base pair
BCC - Basai cell carcinoma Bcl-2 - B-cell lymplioma 2
bFGF - basic fibroblast growtti factor
BRAF - V-raf murine sarcoma viral oncogene homolog Bl cAMP - cyclic adenosine monophosphate
CDKN2A - Cyclin dépendent kinase inhibitor 2A cDNA - Complementary DNA
CREB - cAMP responsive-element-binding protein CT - Cycle threshold
CTL - cytotoxic T lymphocyte
CTLA-4 - Cytotoxic T-lymphocyte associated antigen 4 Da - Dalton
DCT - dopachrome tautomerase DFS - disease-free survival
DHICA - dihydroxyindole carboxylic acid DMFS - distant metastasis free survival DNA - deoxyribonucleic acid
dNTP - Deoxyribonucleotide triphosphate DTIC - dacarbazine
ER - endoplasmic reticulum
ERK - Extracellular signal-regulated kinases FDA - food and drug administration
FFPE - formalin-fixed paraffm embedded FGF - fibroblast growth factor
HGF - hepatocyte growth factor HR - Hazard Ratio
Ig - Immunoglobulin
IL - interleukin INF - interferon KDa - kilodalton
LDH - Lactate dehydrogenase
LMM - Lentigus malignant melanoma LN - Lymph node
mAb - monoclonal Antibody
MAPK - Mitogen activated protein kinase MC1R - Melanocortin-1 Receptor MEK - MAPK/ERK kinase
MIA - Melanoma inhibitory activity miRNA/miR - microRNA
MITF - Microphthalmia-associated transcription factor mRNA - messenger RNA
mlph - melanophilin
MSH - Melanocyte Stimulating Hormone/Melanotropin NM - Nodular melanoma
nt - nucleotide
0 C A 3 - Oculocutaneous Albinism type 3 OS - Overall survival
PCR - Polymerase chain reaction PD-1 - Programmed cell death 1 PDGF - Platelet-derived growth factor PDL-1- Programmed death-ligand 1 PFS - Progression-free survival PI3K - Phosphatidylinositide 3-kinase
PMEL - Premelanosome protein also known as silver locus protein homolog (SILV), P M E L I 7 a n d gplOO
Pre-miRNA - Precursor miRNA Pri-miRNA - Primary miRNA
PTEN - Phosphatase and tensin homolog RAS - Rat Sarcoma viral oncogene homolog RAF - Rapid Accelerated Fibrosarcoma RB - retinoblastoma protein
RGP - radial growth phase
RNA - Ribonucleic Acid RNase - Ribonuclease RT - reverse transcription
RT-qPCR - reverse transcription quantitative polymerase chain reaction RTK - Receptor tyrosine kinases
S100 - SI00 calcium binding protein s e c - Squamous cell carcinoma SCF - stem cell factor
SLN(B)- Sentinel lymph node (biopsy) snoRNA - small nucleolar RNA
SNP - single nucleotide polymorphisms
SRC - V-src sarcoma (Schmidt-Ruppin A-2) viral oncogene Homolog SSM - Superficial spreading Melanoma
TYRPl - Tyrosinase Related Protein 1 TYRP2 - Tyrosinase Related Protein 2 UTR - untranslated région
UVR - ultraviolet radiation
Part I. Introduction Chapter One: Melanogenesis
1. The human skin
The skin is the largest organ of the body, making up 16% of body weight, with a surface area of 1.5-2m . The skin is a dynamic organ as cells of the outer layers are continuously replaced by inner cells moving up to the surface^.
It constitutes an important physical barrier to the environment while providing protection against micro-organisms, ultraviolet radiation (UVR), toxic agents and mechanical insults. It is
associated with various afflictions, including developmental defects, autoimmune disorders, allergies and cancer.
1.1.Skin anatomy
The skin consists of three layers: the epidermis, the dermis and subcutis. Hair, nails, sebaceous, apocrine sweat glands are regarded as derivatives of skin'° (Figure 1).
The epidermis is the outer part of the skin; an avascular layer, mainly composed of keratinocytes
( S O ' / - ) constantly migrating firom the basai layer to the outer skin surface over a period of about 26
days, the keratinocytes provide cohésion to the epithelium, form a barrier from the exterior and protect against excessive light exposure". The epidermis is composed as well as smaller populations of pigment producing melanocytes containing melanosomes ( S - I O / - ) and
mechanosensory Merkel cells. The immune System is présent in the epidermis as the migratory Langerhans cells (4-8%) serve as antigen-presentation to lymphocytes and intraepidermal T-cells.
Beneath the epidermis is the dermoepidermal junction with the basai layer, and underlying this is the dermis, which mainly consists of supportive connective tissue with dermal fibroblasts and a complex network of vessels, nerves, eccrine glands and hair foUicles.
1.2 Skin pigmentation (Melanogenesis)
The epidermal units of the skin, composed of a melanocyte surrounded by keratinocytes (Figure 1) and regulated by a closed paracrine System, are responsible for melanin production and distribution, a process called melanogenesis.
1.2.1 Melanocytes
Melanocytes are dendritic cells found in the epidermis, in the inner ear, in the uveal tract, in the leptomeninges and in the hair folHcles. Melanocytes in the epidermis are dispersed along the basai layer at the dermoepidermal junction. They produce the pigment melanin within
melanosomes, organelles that are transferred to surrounding keratinocytes and hair follicle cells. Melanogenesis involves several steps: transcription of proteins required for melanogenesis; melanosome biogenesis; sorting of melanogenic proteins into melanosomes; transport of melanosomes to the tips of melanocyte dendrites, and transfer of melanosomes to keratinocytes. Disruptions in any of thèse events may resuit in hypopigmentation or hyperpigmentation. Melanin synthesis is largely influenced by UV radiation, as well as by signais from neighboring keratinocytes and to a lesser degree from dermal fibroblasts'^.
1.2.1.1 Melanocyte development: Melanocytic origin and embryonic development from neural crest through meianoblasts to melanocytes
Melanocytes originate in neural crest as a bipotential glial-melanocyte lineage progenitor that develops into an unpigmented precursor cell called the melanoblast. Meianoblasts migrate to différent destinations, including the basai layer of the epidermis and hair follicles. Their
migration, prolifération, and differentiation into melanocytes dépend on mediators produced by cells of the dorsal neural tube, ectoderm and keratinocytes, such as the family of glycoproteins WNT, endothelin 3 (EDN3), and stem cell factor (SCF) which binds the c-Kit receptor tyrosine kinase in melanocytes and meianoblasts .
A number of studies have identified gènes, such as MITF, c-Kit, and snail/slug that are important for melanocyte development: melanoblast survival is dépendent on MITF through its
In the epidermis and in response to UVR, keratinocytes secrète factors that regulate melanocyte survival, differentiation, prolifération and motility, stimulating melanocytes to produce melanin and resulting in the tanning response. Thereby, melanocytes have a key rôle in protecting our skin from the damaging effects of UVR, however, UVR and oxidative stress may create genetic mutations in the melanocytes capable of inducing malignant transformations.
1.2.2 Melanin: the pigment
Melanin is the primary déterminant of skin, hair, and eye color. It defines an important human phenotypic trait and has a critical rôle in photoprotection due to its ability to dissipate 99.9% of absorbed U V R ' ^
The melanin in the skin is produced by melanocytes and is transferred into the keratinocytes via a cellular vesicle, the melanosome. One melanocyte provides melanin to up to 36 keratinocytes in the surrounding area, named the epidermal melanin unit'^. The synthesis and transfer of melanin are regulated by several paracrine (mainly from the keratinocytes) and autocrine factors, in response to both endogenous and exogenous stimuli, such as UVR'^.
1.2.2.1 Melanin types
Two types of melanin are synthesized within melanosomes: eumelanin, a brown-black insoluble polymer and pheomelanin red-yellow soluble polymer formed by the conjugation of cysteine or glutathione (Figure 2).
Eumelanin is the major type in individuals with dark skin and hair and is more efficient in photoprotection. Eumelanin polymers comprise numerous cross-linked 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) polymers. The two types are recognized as black and brown.
1.2.2.2 Phenotypic diversity of pigmentation
Phenotypic diversity of pigmentation is not due to a variation in melanocyte number, which is relatively constant in différent ethnie groups, but to the size, number and distribution of melanosomes (defined below), the amount and type of melanin, and melanin transfer and distribution in keratinocytes. The melanosomes of dark-skinned individuals are larger, more numerous, and elongated, resulting in delayed dégradation in keratinocytes and consequently in mcreased visible pigmentation .
1.2.3 Melanogenesis and Melanosome
1.2.3.1 Biogenesis and maturation of melanosomes
Melanin synthesis occurs in melanosomes, lysosome-related organelles of melanocytes, in the présence of melanogenic enzymes (tyrosinase, TYRPl, TYRP2) and components of the fibrillar matrix that bind to melanin.
1 2
In thèse organelles, melanin is synthesized along four maturation stages (Figure 3).
Stage I melanosomes in eumelanogenesis and pheomelanogenesis are common and dérive from the late endosomes. In the stage II of maturation, premelanosomes are ellipsoïdal in
eumelanogenesis and contain well-organized lamellae/filaments riche in Pmell7, tyrosinase is not active yet. In stage II and III, Melanogenic enzymes from trans-Golgi are incorporated into
melanosomes which will then become electron-dense because of melanin déposition and the important activity of tyrosinase (stage III). In stage IV, melanosomes become amorphous'^. In contrast, pheomelanosomes are always spherical and contain only granular materials in ail four stages of melanosomal maturation. Stage IV melanosomes are ready to be transported to the surrounding keratinocytes.
1.2.3.2 Transfer of melanosomes
When melanin synthesis is completed, melanosomes move bi-directionally from the perinuclear area towards melanocyte dendrites, in a movement controlled by microtubule proteins (kinesin, dynein). This transport ends with melanosomes binding actin filaments through a complex formed by myosin Va, Rab27a, and melanophilin (mlph) (Figure 3).
différent mechanisms such as phagocytosis of naiced melanin, fusion of plasma membranes, and transfer by melanosome-containing vesicles are described'^.
1.2.4 Triggers of melanogenesis
The régulation of melanogenesis is achieved by keratinocytes that produce, in response to U V radiation, from the Pro-opiomelanocortin (POMC) the melanocyte stimulating hormone (a-MSH) and adrenocorticotropic hormone (ACTH) both agonists of melanocortin 1 receptor ( M C l - R ) a member of G-protein receptors that prédominâtes in melanocytes. Agonist-bound M C I R activâtes adenylyl cyclase, inducing cyclic AMP production, which leads to phosphorylation of cAMP responsive-element-binding protein (CREB) transcription factor family members by PKA. CREB, in tum, transcriptionally activâtes microphthalmia transcription factor (MITF) gene promoting eumelanogenesis, by binding to the CRE élément upstream of the human MITF transcription initiation site^". In addition to CREB, the expression of the MITF protein is
regulated by other transcription factors and mediators produced by keratinocytes and fibroblasts mcluding sex-determining région Y-box (S0X9) .
MITF is the transcription factor that régulâtes the expression of melanogenic enzymes; tyrosinase and tyrosinase related proteins. Moreover, it régulâtes the expression of the Rab27a protein and the melanosomal matrix protein Pmell7, which are important in melanosome transport, and régulâtes the anti-apoptotic protein (bcl-2) of melanocytes, which is often expressed in melanomas'^ (Figure 4).
1.2.5 Enzymes of melanogenesis and Melanin synthesis
Upon exposure of the skin to U V radiation, melanogenesis is enhanced by the activation of MITF and hence by the key enzyme of melanogenesis tyrosinase. Tyrosinase has an inner melanosomal domain with a catalytic région (approximately 90% of the protein), followed by a short
transmembrane domain and a cytoplasmic domain. Histidine residues présent in the catalytic portion bind copper ions and are required for tyrosinase activity.
Following the formation of dopaquinone, the eumelanin and pheomelanin pathways diverge. In the eumelanin pathway, dopaquinone is converted to leucodopachrome and dopachrome. Dopachrome is either spontaneously converted to 5,6-dihydroxyindole or is enzymatically converted to 5,6-dihydroxyindole-2-carboxylic acid via enzymatic conversion by dopachrome tautomerase (DCT also called TYRP2). DHICA can be converted to indole-5,6- carboxylic acid quinone by TYRPl (rôle not sure). The polymerization of indoles and quinones leads to
eumelanin formation (Figure 5).
There are two tyrosinase-related proteins, TYRPl and TYRP2, présent in the membrane of melanosomes. The rôle of TYRPl in eumelanin synthesis is not yet clarified; it is possible that it has a rôle in the activation and stabilization of tyrosinase'^.
The pheomelanin pathway branches from the melanin pathway at the L-dopaquinone step and is dépendent on the présence of cysteine or glutathione. Cysteine reacts with L-dopaquinone to form cysteinyl-dopa which is then converted to benzothiazin intermediates and polymerizes to
pheomelanin (figure 5). The type and ratio of melanin produced (eu/pheo) dépends on several
22
différent availability of cysteine .
While tyrosinase plays a major rôle in the biogenesis of both eumelanosomes and
pheomelanosomes, tyrosinase-related proteins are exclusively expressed by eumelanosomes. Eumelanin is photoprotective and acts as a scavenger to reactive oxygen species, unlike
23
Chapter Two: Melanoma
1 Overview; Cutaneous melanoma
Cutaneous melanoma is the most aggressive form of skin cancer. It arises from the melanocytes of the skin. Because malignant melanoma (MM) is a melanocyte-derived cancer; it can be found in ail organs that harbor melanocytes such as the ears, the eyes, the mucosal membranes (nose, oral cavity, anorectal mucosa and the genitourinary mucosa), the central nervous System (leptomeningeal melanoma) and in the gastrointestinal tract.
We will be focusing throughout this work on cutaneous melanoma that will be referred hereinafter as melanoma or malignant melanoma.
Cutaneous melanoma causes the majority of skin cancer related deaths; In fact, while it represents only around 4% of ail skin cancers, melanoma accounts for nearly 80% of skin cancer related deaths. Récent statistics are alarming, showing that the Worldwide incidence of melanoma is increasing at a rate of 3-7% per year. Cutaneous melanoma is among the most common types of cancer in young adults. The most common sites of Cutaneous melanoma are the trunk (43.5%), extremities (33.9%), acral sites (11.9%), and head and neck (10.7%)^"^
Cutaneous melanoma can arise de novo in the skin (about 70%) or have a common nevus or a clinically atypical nevus as a precursor lésion (in about 30%)^^. As long as the cutaneous
melanoma grows in the epidermis the tumor is characterized as in situ, but when it grows down in the dermis it is invasive with a potential to metastasize. Cutaneous melanoma can be classified according to location, stage and progression and are defined in 5 stages.
2 Stages of melanoma progression (Figure 6)
2.1 Common Acquired/congenital melanocytic nevus
The common acquired melanocytic nevus is a benign growth, it is the earliest hyperplastic melanocytic lésion and is common in humans. Thèse nevi are usually recognizable as brown areas on the skin of varying size and shape usually smaller than about 5 millimeters wide and are round or oval. This pigmented nevus, called a mole, develops as a prolifération of melanocytes
26
and the underlying dermis; they are flat and brown to black. They can be compound nevi, which are a mixture of junctional and intradermal prolifération which are slightly raised and brown to black (Beauty marks are usually compound nevi), also, they can be intradermal nevi which are in the dermal layer, most are raised and flesh-colored (not pigmented).
Melanocytic nevi can also be classified by âge of appearance. There are two classes: the
congénital nevi, which appear within the first six months after birth, and the acquired nevi, which appear when the patient is over 1 year of âge. The différence and clinical importance of this classification is that congénital nevi are thought to more likely become malignant in life^^. This can be related to the fact that congénital nevi, whilst clustered at the dermal-epidermal junction, go deeper into the dermis than acquired nevi and can invade blood vessels, nerves and erector pili muscles^^. Melanocytic nevi show higher relative risk for SSM and nodular melanoma.
2.2 Dysplastic nevus
The dysplastic nevus (DN) (atypical mole or atypical melanocytic nevus), usually a compound nevus with cellular and architectural dysplasia, has increased abnormal growth compared to the melanocytic nevi. D N may occur within a preexisting benign nevus or in a new location. It is often large usually more than 5 millimeters wide and flat and tends to have irregular borders and coloration. Dysplastic nevi are commonly found on the trunk. The risk of developing DN is estimated to be around 7-18 % in a lifetime .
People who have many dysplastic nevi (atypical mole syndrome) have a greater risk than others to develop melanoma. It is estimated that 22-36 % of malignant melanomas originate as DN.
28
However, most dysplastic nevi do not tum into melanoma .
2.3 Radial Growth Phase
The third stage, radial growth phase (RGP) called in situ melanoma, is the first recognizable malignant stage. During RGP the cells remain confined to the epidermis before invading the dermis passing through the basai lamina, however, some types of melanoma (nodular melanoma) arise directly in the dermis". Morphologically, RGP consist of atypical, rounded (epithelioid) or spindle (lentiginous) shaped cells, which initially aggregate in the epidermis and form nests above the basai membrane.
incapable of anchorage independent growth . At this stage, 5-year survival rates are high with complète surgical removal.
2.4 Vertical Growth Phase
The fourth stage is the vertical growth phase (VGP) in which the melanoma cells escape the control of keratinocytes and pass through the basai lamina to invade as an expanding mass the dermal layer and the basement membrane^^. They establish a close network with fibroblasts and are able to acquire growth factors and grow without anchorage with a high compétence for metastasis.
Not ail melanomas pass through each of thèse individual phases, RGP or VGP can both develop directly from isolated melanocytes or nevi, and both can progress directly to metastatic malignant melanoma^".
2.5 Metastatic Melanoma
Vertical growth phase can progress to the most aggressive form of cutaneous melanoma characterized by extensive vascularization and invasion; metastasis. As the tumor continues to expand deeper into the dermis and reaches the subcutaneous fat tissue, the risk of metastasis is high and systemic métastases are likely to occur. Cells that are shed from the primary lésion infiltrate the circulatory and lymphatic System, and migrate to new sites where they adhère to the walls of the capillary and invade new organs.
At the secondary site, micrometastases can survive for several years before they become proliferative, stimulate angiogenesis, and begin to form a metastatic tumor.
Advanced stage melanomas are described by the présence of régional or distant métastases.
2.5.1 Sites of metastasis:
Three routes of metastasis include in-transit and satellite métastases, régional lymph node métastases and distant métastases, including distant skin métastases.
When melanoma cells break away from the primary tumor, they enter the lymph vessels and spread along the lymph vessels, forming in-transit or satellite métastases to eventually spread to the régional lymph nodes. In-transit métastases are located within régional dermal and subdermal lymphatics prior to reaching the régional lymph nodes, in other terms it is any cutaneous
basin, whereas, satellite lésions are skin lésions within 2 cm of the primary tumor that are considered intralymphatic extensions of the primary mass^'.
In any area of skin where there is a penetrating melanoma, there will be a nearby sentinel node to which it drains. The sentinel lymph node is the very first lymph node to receive drainage from melanoma and is the one most likely to contain melanoma cells if any lymph nodes are involved.
Primary tumor location predicts most strongly where métastases would develop. As a whole, primary melanomas metastasize most frequently to the régional lymph nodes. Tumors located in the extremities and trunk had the strongest prédilection to develop satellite or in-transit
métastases. Tumors located in the head and neck areas tended to develop métastases via ail three routes^'. Of interest, tumor thickness was also related to certain pattems of metastasis
development. Tumors less than 0.75 mm and more than 1.5 mm thick developed preferentially satellite or in-transit métastases. Tumors of average thickness (0.75-1.5 mm) showed the highest rate of development of distant métastases. Time from initial présentation to the development of métastases was significantly longer for distant métastases, the most common sites of viscéral métastases, in order of frequency, are the lung, liver, brain, bone and intestine (Table 1).
2.5.2 Causing events of metastasis
The actual initiating event of metastasis and how tumor cells reach lymphatic vessels is not yet fully elucidated. Différent studies attempted to understand the initiating events: One concept suggests that the fusion of cancer cells with macrophages or other migratory bone marrow-derived cells underlies metastasis by activating pathways related to epithelial-mesenchymal transition, such as snail/slug .
Tumor cells that invade the extracellular matrix reach lymphatic vessels. Cells then flow within the lymphatics to reach the subcapsular sinus of the lymph nodes. Neoplastic cells may secrète cytokines that induce growth of lymphatics towards the tumor or within the tumor, for example, the binding of the melanoma secreted VEGF-C to VEGF-R3 expressed on lymphatic endothelial cells, induce the production of lymphatic vessels^'.
VAPG, that bind to receptors on melanoma: galectin-3, integrin aVpS and the elastin-binding protein which lead to the expression of more CXCR-4 that will be recognized by stromal cell-derived factor-1 (SDF-1) secreted by target organs, therefore melanoma cells become more aggressive^'.
Another study demonstrated that there is a différence in the sites of metastasis with melanomas expressing différent integrins; melanomas that express integrin avpS tend to develop lung métastases. On the other hand, melanomas expressing integrin a 4 p i develop lymph node métastases^'.
One study showed that of ail cancers, cutaneous melanoma is the most common type to metastasize to the submucosa of the small intestine; this migration is thought to be directed by CCL25, a cytokine produced by the epithelium of the small intestine that attracts CCR9-bearing melanoma cells^'.
In addition, melanoma cells develop mechanisms to escape from the immune System. In one example, melanoma cell prolifération is normally inhibited by cytokines, such as IL-2, TNF-a and IL-6. Metastatic melanomas can develop résistance to thèse cytokines through the
modification of the receptor of oncostatin M; an IL-6-related cytokines. This résistance is due to the histone hypoacetylation at the promoter rendered this receptor less responsive to oncostatin
3 Classification of cutaneous malignant melanoma
Primary melanoma lésions typically présent as any of four main subtypes: superficial spreading melanoma (SSM), nodular melanoma (NM), lentigo malignant melanoma (LMM) and acral lentiginous melanoma (ALM).
SSM is the most common primary cutaneous melanoma subtype (70%), it usually displays a prominent latéral spread throughout the epidermis and while most of the SSM arise de novo on chronically UV exposed skin especially at an early âge (predominantly on the trunk and lower extremities)^^, a quarter are associated with dysplastic nevi, indeed most nevus-associated primary melanomas are SSM^^. SSM initially displays a radial growth phase; it then progresses to the vertical phase of growth. SSM usually occurs over a period of 7 years.
mainly occurs in patients in their 4th or 5th décades and is equally prévalent in both men and women^^.
Nodular melanoma accounts for between 15-30 % of malignant melanoma cases. It is the most aggressive form of melanoma. It is characterized by its rapid growth, fréquent ulcération and lack of a radial growth phase. Nodular melanoma usually arises de novo; however it can also develop from a pre-existing nevus. Nodular melanomas are typically dome-shaped and lacking the ABCD properties (described below), making visual diagnosis more difficulté''. The tumors typically are blue-black nodules but may lack pigment in some circumstances, the nodule can extend down into the skin as far as subcutaneous tissues^^. NM affects men mainly in their 5* décade.
L M M is less common (10%) and the least aggressive melanoma type, it usually occurs in older patients, on chronically sun-exposed skin such as on the head and neck. This subtype is
histologically characterized by the confluent growth of atypical melanocytes along the dermal-epidermal junction with fréquent protrusions into the dermis^^.
ALM accounts for less than 5% of the total case numbers and is the most common type of melanoma in dark skinned people (35%). This type is most commonly found on the palms of the hands and soles of the feet or around the big toenail. Melanomas from acral lentiginous
frequently harbor activating mutations and/or increased copy number in the KIT tyrosine kinase receptor gene, which are very rare in the more common cutaneous tumors . Unlike other forms of melanoma, ALM is not associated with UV exposure.
In addition to thèse major subtypes, there are several rarer variants of malignant melanoma including desmoplastic, nevoid, verrucous, amelanotic melanoma.
Amelanocytic melanomas which do not contain any pigment, account for 2 % of ail malignant melanoma cases. They usually go undiagnosed due to their uncharacteristic appearance. For example desmoplastic melanoma, a rare amelanocytic melanoma variant, is usually not diagnosed due to it being mistaken as scar tissue^^.
4 Risk factors for melanoma 4.1 Environmental risk factors
UVR has damaging effects on the skin via direct and indirect mechanisms, such as the formation of cyclobutane pyrimidine dimers, gene mutations, immunosuppression and oxidative stress. Both UVA (}i=320-400nm) and UVB (?i=280-320nm) radiations damage skin and cause skin cancer; however, UVB rays are a more potent cause of melanoma.
Intermittent sun exposure is a risk factor for cutaneous melanoma development. Sunbum is a risk factor, in childhood sunbums cause the most damage^^'^*'^^ (Table 2). Sunbed users have an increased risk for cutaneous melanoma development^^. A dose dependency underlying this risk association has been also reported, expressed by the length, duration or number of treatment sessions. A meta-analysis by Boniol et al. showed an overall summary relative risk (RR) of 1.2 (95% CI 1,08- 1,34) of melanoma development in 'ever use' of sunbeds and a 1,8% increase of risk for each additional session of sunbed use per year. In a subgroup analysis of subjects who first used sunbeds at an âge below 35 years, the summary RR rose to 1.87 (95% CI 1,41-2,48) indicating a higher melanoma risk with an early onset of tanning bed exposure'*°. The
International Agency of Cancer Research recommends restricted access for minors and young adults to indoor tanning facilities.
4.2 Phenotypic risk factors
Red hair, fair skin, blue eyes, poor tanning ability and freckling are phenotypic features
associated with increased cutaneous melanoma risk. Thèse characteristics are strongly correlated with skin photosensitivity. Red hair carries the highest relative risk (RR = 3.64, 2.56-5.37) compared to dark hair. Patients with high-density freckling have double the risk compared with patients with little or no freckling'" (Table 2).
It is no longer accepted that a nevus is usually the precursor lésion. The présence of pre-existing nevi at the tumor site is unlikely, suggesting that the majority of lésions arises de novo (70^. The lifetime risk of any single nevus in a 20- year-old person transforming into melanoma by the âge of 80 years is 1/3000 for men and 1/11 000 for women^l
4.3 Genetic risk factors
Heritable factors play an important rôle in cutaneous melanoma prédisposition and a family history of melanoma is associated with a significant twofold increased risk of melanoma''^ (Table
2).
Several pigmentary gene polymorphisms favoring pale skin and red hair color phenotype have been associated with increased risk of melanoma. One example is the M C I R loss-of-function polymorphisms; M C I R loss causes a shift of melanogenesis from the photoprotective eumelanin to pheomelanin, resulting in a phenotypic spectrum of red hair color, pale skin and freckles'** and M C I R variants increase the risk of sporadic cutaneous melanoma in darker-pigmented
Caucasians**^.
In addition, a séries of génome wide association studies on pigmentary phenotypes and skin
• • 48 *
cancer risks have implicated several genetic risk loci affecting pigmentation . MITF variant was
-3 0
also associated with a higher nevus count and additional risk factors .
Approximately, 5-10% of melanoma occurs in families with hereditary melanoma prédisposition including multiple cases in family, multiple primary cancers in one individual, and melanoma diagnosis at a young âge (< 40 years). Inherited mutation of gènes with a critical rôle in cell cycle occurs in 3 0 - 4 0 % of familial melanomas. Examples of thèse gènes are cyclin dépendent kinase inhibitor 2A (CDKN2A) (autosomal dominant) and Phosphatase and Tensin homolog (PTEN). Defects in thèse gènes in familial melanoma principally are homozygous deletions"*^.
CDKN2A is located on chromosome 9p21. The mutation of this gene has been found in 25% to 40% of familial melanomas and affects two tumor-suppressor proteins, p l 6 r N K 4 A and p l 4 A R F both involved in régulation of cell cycle progression and induction of sénescence (Figure 11 ).
The altered signaling pathway related to thèse gènes causing carcinogenesis will be developed in the Pathology part.
Other risk factors include a personal history of non-melanoma skin cancer and immunosuppression related to organ transplantation, Xeroderma pigmentosum,
lymphoproliferative disease or human immunodeficiency virus infection/ AIDS. Exposure to heavy metals and insecticides has also been reported'*'*'''^.
5 Epidemiology of melanoma
populations, ranging from 3% to 7%, equal to a doubling of rates every 10-20 years . The calculated life-time risk for new-boms of developing melanoma being is 1:50.
Over the past twenty years, cutaneous melanoma has become a more prévalent cancer, indicating an influence of changing environmental risk factors^°; people are spending more time outside, in direct contact with the sun.
The incidence of cutaneous melanoma varies Worldwide with latitude and altitude, with generally higher incidence reported nearer to the equator and at higher altitude. Populations in Australia and New Zealand show the highest incidence rates (Figure 7) likely because they are subject to a combination of a high risk prédisposition (fair-skin) and environmental factors (intensive exposure to UV light)^*.
Some epidemiologic data showed:
In Belgium, 2008, the incidence of melanoma was 14.5/100.000 per year with a mortality rate of 3.1/100.000. No major différences in incidence rates were observed between the régions. Mean âge at diagnosis was 60 years in maies and 55 years in females (Belgian cancer registry for 2008). In 2012, melanoma constituted the 7"^ most fréquent tumor in maies (3.9%) and the S'*' most fréquent in females (6%). However, it was an uncommon cause of cancer death in maies (1.1%)) and females (1.3%) (Belgian cancer registry for 2012) (Figure 8).
In addition, in Lebanon, in 2013, melanoma constituted 0.8%) whereas other skin cancers constituted 8.6 % (10 times more) of total cancer registry (ministry of public health, Lebanon).
Cutaneous melanoma affects a younger patient population than many other malignancies. The médian âge at diagnosis is 62 years of âge and each death related to melanoma corresponds to about 19 years of life lost, one of the highest for any cancer.
6 Pathology
Three major events are required for melanoma progression, including clonal expansion of cells harboring initial mutation, acquisition of additional mutations allowing cells to overcome sénescence, and acquisition of mutations which resuit in suppression of apoptosis^'. A number of relevant oncogenes or tumor suppressor gènes have been found in association with melanoma development (Figure 9).
Initial mutations in melanocytes, such as activating mutations in BRAF, NRAS, or KIT would lead to hyperproliferation. Additional mutations, such as inactivation of retinoblastoma protein (Rb) pathways or activation of telomerase, are required to enable cells to overcome sénescence and to become proliferative and immortalized.
Lésions with changes described above, are early malignant melanocytic lésions in the RGP of melanoma (in situ) (Figure 9).
Additional changes are required to allow melanoma cells to escape keratinocyte dependence and invade deeper layers of the skin, a stage that characterizes the VGP of melanoma progression. This stage requires mutations that actively suppress apoptosis. Genetic altérations consistent with advanced melanomas include loss of phosphatase and tensin homolog (PTEN), RAS and RAF activating mutations and P-catenin activation^^.
Finally, vascularisation and melanoma growth, seen at later metastatic stages, are regulated by several factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF)^l
Bach factor will be detailed dépendent on the pathway it is involved in.
6.1 Rôle of receptor tyrosine kinases
Receptor tyrosine kinases (RTK) play a pivotai rôle in the normal régulation of ail basic cellular functions, including cell prolifération, differentiation, migration and survival. RTKs are trans-membrane polypeptides that contain both an extra-cellular ligand binding domain and a
c-Kit ^sum WêI0
c-kit is a receptor tyrosine kinase; it is stimulated by cytokine ligand stem cell factor (SCF). Until recently it was believed that C-Kit expression was lost with melanoma progression. However
récent studies have shown that C-Kit is overexpressed in a small percentage of melanoma
patients . Patients who have mutated c-Kit are generally not BRAF mutated. Thèse data suggest important différences between melanomas on a molecular level based on their site and
constitutional and genetic factors that are not yet entirely clear^"*.
AKT Pathway and PTEN in melanoma progression
Although PI3K itself is rarely mutated or overexpressed in melanoma, activation of downstream signaling components, e.g. AKT, have been implicated in melanoma progression. Increased
phosphorylated AKT was detected in primary and metastatic melanoma. The AKT pathway stimulâtes cell cycle progression by controUing Gl progression, cell prolifération and inhibition of apoptosis^^.
PTEN, a tumor suppressor gene, encodes phosphatase with dual speciflcity. The lipid phosphatase activity of PTEN can down-regulate the AKT pathway^^. Moreover, the protein
phosphatase activity of PTEN can inhibit MAPK signaling. Loss or mutation of PTEN is
associated with activation of the AKT pathway and has been found in approximately 30-50% of melanoma cell lines^'' (Figure 10).
Mitogen activating protein kinase (MAPK) pathway
Activation of the MAPK pathway is a fréquent and early event in melanoma. MAPK signaling is initiated at the cell membrane, either by RTKs binding ligand or integrin adhésion to extracellular
matrix, which transmits activation signais via the RAS GTPase, leading to cell prolifération, differentiation and survival through activation of various signaling pathways. In melanocytes, this
pathway is activated by growth factors such as SCF, FGF and HGF. Stimuli of the RAS family of proto-oncogenes cause the activation of the RAF family of serine/threonine kinases (e.g. BRAF,
CRAF and ARAF). RAF then phosphorylates the MAPK kinase MEK, which can lead to activation of the MAPKs, ERKl and ERK2^'*. ERKs relay proliferative or survival signais
through phosphorylation of a variety of cytoplasmic targets, such as prosurvival ribosomal S6 kinase (p90rsk) or proapoptotic bcl-2 interacting mediator of cell death (BIM); cytoskeletal
BRAF, a key player in the pathway, is mutated in 60-70 % of melanoma cases (somatic mutation, no germinal mutation described). Ail BRAF mutations are within the kinase domain, with a single substitution (V600E) accounting for 80%. This V600E mutant possesses 10.7 fold kinase activity versus wild-type BRAF^^. Other common BRAF mutations in melanoma, found in the same codon, are V600K (about 16% of mutations in melanoma) and V600D/R (3% of ail
mutations). Thèse less common variants are found at slightly higher rates in melanomas arising in older patients^^. Ali of thèse V600 mutations resuit in a mutant form of the BRAF protein that is constitutively active without the need for activation signais from growth factors through cell surface tyrosine kinase receptors. The resuit is uncontrolled prolifération, enhanced invasiveness, and résistance to apoptosis. BRAF mutations are early events in melanomagenesis: 80% of dysplastic nevi harbor the mutation^'.
V600E BRAF also contributes to neoangiogenesis by stimulating autocrine VEGF sécrétion . Thus, BRAF is implicated in several aspects of melanoma induction and progression.
Some groups have reported that despite the high prevalence of BRAF mutations in nevi, RGP has a low frequency of mutations that increases upon the transition to VGP^' and as melanoma metastasize^°. Also, studies have demonstrated that melanomas with the highest degree of BRAF mutations were those with intermittent sun exposure^'*.
RAS mutations, particularly NRAS, are also associated with melanoma. Studies have found that approximately 10-12 % of ail melanoma mutations are Ras mutations^"*. The most common mutation is the Q61L^'. Activating NRAS mutations have been correlated with chronic sun exposure . In the majority of cases, NRAS and BRAF mutations are mutually exclusive .
6.2 Cell cycle
Sénescence is a key cellular protection mechanism against cancer because it halts aberrant cell prolifération. P16rNK4a/Rb signaling is a key regulator of melanocyte sénescence and, consequently, to override sénescence melanoma cells must inactivate this pathway.
The cyclin-dependant kinases CDKS constitute a group of serine/threonine kinases that are responsible for driving progression of the cell cycle through a séries of sequential
phosphorylation events involving target proteins critical to nuclear transcription that become operative after their activation
damaged DNA. p l 6 mutations are found in both hereditary malignant melanoma and in sporadically developed melanomas^''. Somatic mutations in this gene can be point mutation, deletion, promoter methylation, for example C C ^ T T translocation; a hallmark of UVR-induced mutagenesis^^'^^.
p53:
CDKN2A gene also encodes the tumor suppressor pMARF protein that inhibits MDM2-mediated ubiquitination and subséquent dégradation of p53; thus, loss of ARE inactivates p53 which régulâtes growth arrest and apoptosis^^ (Figure 11). Despite the fact that p53 is one of the most commonly mutated gènes in cancer, p53 itself is rarely mutated in human melanoma (0% to 25%) or in melanoma cell lines^^.
6.3 MITF
MITF gene amplification is seen in approximately 10% of primary cutaneous melanomas and 20% of metastatic tumors, but not in benign nevi, and disruption of MITF is lethal to melanoma cells with thèse amplifications. In metastatic melanoma patients, amplification of MITF was associated with decreased 5-year survival rates and résistance to chemotherapy .
6.4 Growth factor dependence in melanoma development
Melanocytes are known to produce basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). When melanocytes undergo transformation to melanoma cells they show an increase in growth factor receptors and cytokine receptors^^. bFGF not only plays a rôle in the survival of melanoma cells but is also involved in regulating motility by up-regulating serine proteases and matrix metalloproteinases^*^.
HGF acts through its tyrosine kinase receptor (C-Met) présent on melanocytes, HGF stimulâtes prolifération and motility of melanoma cells through the disruption of adhésion between melanocytes and keratinocytes via down régulation of E-cadherin^^.
6.5 Cadherin expression in melanoma progression
The genetic and cellular différences that resuh in progression from RGP to VGP are not fully understood, however, one factor crucial to the progression to melanoma is a change in cadherin expression. Cadherins are a family of cell surface glycoproteins (type 1 transmembrane proteins) that promote calcium dépendent cell-cell adhésion. E-cadherin acts as a mediator between keratinocytes and normal melanocytes; it plays a key rôle in melanoma development and is lost during melanoma metastasis. Upon losing expression of E-cadherin, cells increase mobility and invasiveness and keratinocytes no longer control melanocytes^^.
7 Diagnosis
7.1 Diagnosis of primary melanoma
The clinical diagnosis of cutaneous melanoma is based on the morphological features of the skin lésion, according the 'ABCDE rule' that consists of asymmetry, border irregularity, color
variegation, diameter more than 6 mm and evolving lésion; a lésion that is différent in appearance compared to other moles or changing in size, shape, symptoms (eg, itching, tendemess), surface (eg, bleeding), or shades of color over time. Recognizing changes in moles, by following this ABCDE Chart, is helpful in detecting melanoma at its earlier, more curable stages^' (Figure 12). Not ail cutaneous melanoma have ail 4 ABCD features. It is the combination of features (eg, ABC, A+C,) that render cutaneous lésions most suspicious for early melanoma. Small-diameter melanomas represented less than 5% of ail invasive melanomas^'. Nodular melanomas, which frequently présent at more advanced stages, thus contributing greatly to melanoma mortality
71
rates, lack asymmetry, border irregularity, color variegation, and diameter greater than 6 mm . For clinical diagnosis, a dermatologist carefully examines moles and other suspicious spots. To get a better look, a dermatologist uses a dermatoscope that magnifies the skin and helps to see pigment in the skin. If doctor doubts of a melanoma, he performs a skin biopsy and the removed skin will be sent to a lab. Cutaneous melanoma cannot be diagnosed without a biopsy and spécial tests may be needed to confirm the diagnosis. Thèse tests are immunohistochemistry (IHC),
72
fluorescence in situ hybridization (FISH) .
7.2 Sentinel lymph node biopsy
first lymph node or group of nodes draining a cancer), the doctor injects a small amount of a radioactive substance or a blue dye into the area of the melanoma, then, a small incision is made. Thèse sentinel nodes are removed and looked at under a microscope. If the sentinel node does not contain melanoma cells, no more lymph node surgery is needed because it is very unlikely the melanoma would have spread beyond this point. If melanoma cells are found in the sentinel node, the remaining lymph nodes in this area are removed and looked at.
7.3 Imaging tests
Search for the distant métastases in patients includes physical examination and imaging test. Chest radiography, ultrasonography, computed tomography (CT) can show the détail in soft tissues such as internai organs. Magnetic résonance imaging (MRI) scans give detailed images of soft tissues in the body by using radio waves and strong magnets instead of x-rays. MRI scans are
31
very helpful in looking at the brain and spinal cord and to detect cérébral métastases . Positron émission tomography (PET) is most useful in patients with more advanced stages of melanoma; fluorodeoxyglucose used in PET marks malignant cells. Malignant cells accumulate glucose as a resuit of upregulation of glucose transport molécules on their surface. PET has a superior rôle in the détection of distant metastases^^.
8 Therapy
Treatment of cutaneous melanoma in its early stages is predominantly surgical and radical lymphadenectomy if the sentinel lymph nodes harbor metastasis. Patients with primary
melanoma thicker than 2 mm with ulcération, or thicker than 4 mm, patients with régional lymph node involvement, and with metastatic disease can be offered an adjuvant therapy, with the use of IFN-a2b or IL-2 (both approved by the FDA^''). Alternative treatment options include
dacarbazine, temozolomide and taxane chemotherapy. Other options are either in research, preclinical or clinical trial phases^'.
8.1 Surgery
