Cutaneous T-cell lymphoma: molecular pathogenesis and
clinical behaviour.
Doorn, R. van
Citation
Doorn, R. van. (2005, October 26). Cutaneous T-cell lymphoma: molecular pathogenesis and clinical behaviour. Retrieved from
https://hdl.handle.net/1887/3630
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Cutaneous T-cell lymphoma:
Cutaneous T-cell lymphoma:
molecular pathogenesis and clinical behaviour
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magnificus dr. D.D. Breimer,
hoogleraar in de faculteit der Wiskunde en Natuurwetenschappen en die der Geneeskunde, volgens besluit van het College voor Promoties te verdedigen op woensdag 26 oktober 2005
te klokke 14.15 uur door
Remco van Doorn
Promotiecommissie
Promotoren: Prof. Dr. R. Willemze
Prof. Dr. Th. M. Starink, Vrije Universiteit, Amsterdam Co-promotor: Dr. C.P. Tensen
Referent: Prof. Dr. C.J.L.M. Meijer, Vrije Universiteit, Amsterdam Overige leden: Prof. Dr. C.J. Cornelisse
Contents
Chapter 1
General Introduction . . . 9
Chapter 2
Mycosis fungoides: disease evolution and prognosis of 309 Dutch patients . . . 31
Chapter 3
Follicular mycosis fungoides, a distinct disease entity with or without associated
follicular mucinosis: a clinicopathologic and follow-up study of 51 patients . . . 41
Chapter 4
CD8+ T cells in cutaneous T-cell lymphoma: expression of cytotoxic proteins, Fas
ligand, and killing inhibitory receptors and their relationship with clinical behaviour . . . . 51
Chapter 5
A novel splice variant of the Fas gene in patients with cutaneous T-cell lymphoma . . . 61
Chapter 6
Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor Twist in Sezary syndrome identified by gene expression analysis . . . 67
Chapter 7
Epigenetic profiling of cutaneous T-cell lymphoma: promoter hypermethylation of
multiple tumor suppressor genes including BL7a, PTPRG and p73 . . . 79
Chapter 8
Summary and Discussion . . . 93
Chapter 9
Chapter 1
11
Primary cutaneous T-cell lymphoma
The term primary cutaneous lymphoma refers to clonal proliferations of neoplastic lymphocytes presenting in the skin with no evidence of extra-cutaneous disease at time of diagnosis. Whereas most lymphomas develop in lymph nodes, a considerable proportion primarily involves extranodal sites. After the gastrointestinal tract, the skin is the second most common site of extranodal non-Hodgkin’s lymphoma with an estimated annual incidence of 1 per 100.000 persons.1 Primary cutaneous lymphomas often have a different clinical behavior and prognosis than nodal lymphomas of the same histological subtype. Lymphoma cells of nodal and primary cutaneous origin not only differ in the expres-sion of organ-specific homing receptors, but also with respect to the occurrence of genetic lesions such as specific chromosomal translocations.1,2 Based on the immunophenotypical character-istics of the neoplastic lymphocytes two major subgroups of primary cutaneous lymphomas are distinguished: primary cutaneous T-cell lym-phomas (CTCL) that account for approximately 75% of cases and primary cutaneous B-cell lym-phomas (CBCL) accounting for the remaining 25%. Clinical, histopathological, genetic and epigenetic aspects of CTCL are the subject of this thesis.
Classification of primary cutaneous lymphomas
A unique feature of cutaneous lymphomas is the fact that different clinical manifestations and disease stages are visible to the eye and easily accessible for histopathologic and molecular genetic study, thus allowing correlation between clinical appearance, disease behaviour, histopathology and genetic alterations. This approach has been taken to delineate distinct disease entities which have been included in the European Organization for Research and Treatment of Cancer (EORTC) for primary cutaneous lymphomas and the World Health Organization (WHO) classification.1,3 Accurate classification is significant from a clinical point of view since it dictates the management of
patients with cutaneous lymphoma. Recently a new classification system for cutaneous lymphomas was presented: the WHO-EORTC classification.4 Table 1 shows the WHO-EORTC classification of cutaneous lymphomas including the relative frequency and the actuarial 5-year survival rate of the different disease entities. The characteristics of CTCLs that have been the subject of studies included in this thesis are described in more detail below.
Mycosis Fungoides
Mycosis fungoides (MF) is a mature T-cell lym-phoma presenting in the skin with erythematous patches, plaques or tumors and is characterized histopathologically by epidermal and dermal infiltration of neoplastic T cells with cerebri-form nuclei. MF is the most common type of CTCL and accounts for approximately half of all primary cutaneous lymphomas (see Table 1).4 MF typically affects older adults and occurs more frequently in males than in females. The initial skin lesions are erythematous, slightly scaly patches and thin plaques that are com-monly located on the trunk. MF generally has an indolent disease course with subsequent evo-lution of limited patches to more widespread and infiltrated plaques over a period of years to decades. The disease may present with or progress towards more advanced forms, with development of skin tumors and dissemination of malignant T cells to lymph nodes and internal organs. Various cutaneous manifestations of MF are shown in Figure 1. Although MF behaves as a low-grade lymphoma with an apparent ten-dency to progress, actual disease progression with development of skin tumors and potentially lethal extracutaneous dissemination occurs in only a subset of patients. The clinical stage of MF is determined according to the nature and distribution of the skin lesions (limited or gen-eralized patches/plaques, skin tumors or eryth-roderma) and the presence of lymph node or visceral involvement.5-7
papil-12
Table 1. WHO-EORTC classification of cutaneous lymphomas
WHO-EORTC Classification Frequency(%)# Disease-specific 5-year survival
(%) CUTANEOUS T-CELL LYMPHOMA
Indolent clinical behaviour
Mycosis fungoides Folliculotropic MF Pagetoid reticulosis Granulomatous slack skin
Primary cutaneous anaplastic large cell lymphoma Lymphomatoid papulosis
Subcutaneous panniculitis-like T-cell lymphoma
Primary cutaneous CD4+ small/medium pleomorphic T-cell lymphoma*
Aggressive clinical behaviour
Sézary syndrome
Primary cutaneous NK/T-cell lymphoma, nasal-type Primary cutaneous aggressive CD8+ T-cell lymphoma * Primary cutaneous �/� T-cell lymphoma *
Primary cutaneous peripheral T-cell lymphoma, unspecified $
CUTANEOUS B-CELL LYMPHOMA
Indolent clinical behaviour
Primary cutaneous marginal zone B-cell lymphoma Primary cutaneous follicle center lymphoma
Intermediate clinical behaviour
Primary cutaneous diffuse large B-cell lymphoma, leg type Primary cutaneous diffuse large B-cell lymphoma, other Intravascular large B-cell lymphoma
44 4 <1 <1 8 12 1 2 3 <1 <1 <1 2 7 11 4 <1 <1 88 80 100 100 95 100 82 75 24 NR 18 NR 16 99 95 55 50 65 #Data are based on 1905 patients with a primary cutaneous lymphoma registered at the Dutch and Austrian Cutaneous Lymphoma Group between 1986 and 2002
13 lary dermis and the epidermis (epidermotro-pism).8 Pautrier’s microabscesses, collections of neoplastic T cells in the epidermis, are highly characteristic of MF. The neoplastic cells in MF have a mature CD3+, CD4+, CD8-, CD45RO+ memory T cell immunophenotype. Expression of the cell surface markers CD2, CD5 and CD7, present on benign mature T cells, may be lost on neoplastic T cells.9 Progression to tumor-stage MF is characterized by a loss of epidermotro-pism, an increase in the density of tumor cells in the dermis and is frequently associated with the acquisition by malignant T cells of a blast cell appearance. In Figure 1 characteristic histo-pathological features of MF are shown.
Patients presenting with localized patches or plaques are known to have an excellent prog-nosis and have a life expectancy that is similar to that of a matched control population.10 In patients presenting with more widespread plaques or skin tumors, the disease course is less favorable. The clinicopathological and biologi-cal determinants of the rate of disease progres-sion of patients with MF are largely unknown. MF confined to the skin is commonly treated with skin-directed therapies, including topical steroids, PUVA photochemotherapy, UV-B phototherapy, topical nitrogen mustard or total skin electron beam irradiation.11 Patients may also receive combination treatments consisting of PUVA photochemotherapy with systemic retinoids (acitretine, bexarotene) or interferon-alpha. Local radiotherapy is traditionally used to treat skin tumors and multi-agent chemotherapy is generally used only in case of extracutaneous involvement.12 Experimental and less studied interventions include autologous and allogeneic peripheral stem cell transplantation, recombi-nant fusion protein combining diphtheria toxin and interleukin-2, interleukin-12, imiquimod cream, pegylated liposomal doxorubicin, anti-CD4 and anti-CD52 monoclonal antibodies, the synthetic deoxynucleotide CpG7909 and the histone deacetylase inhibitors depsipeptide and suberoylanilide hydroxamic acid.13-17
Folliculotropic mycosis fungoides
Many clinical and histologic subtypes of MF
have been reported.18 In the WHO-EORTC classification, folliculotropic MF (also termed follicular MF) has been categorized as a sepa-rate entity.4 Folliculotropic MF is characterized histopathologically by the presence of follicu-lotropic infiltrates often with sparing of the epi-dermis, and clinically by preferential involve-ment of the head and neck area with pruritic follicular papules and nodules. Most cases show mucinous degeneration of the hair follicles (fol-licular mucinosis) and were designated as MF-associated follicular mucinosis in the EORTC classification.1,19 Similar cases, but without fol-licular mucinosis have been reported as follicu-locentric or pilotropic MF.20 In folliculotropic MF, malignant T cells are located around follic-ular structures, often sparing the epidermis. In contrast, the classic type of MF is characterized histopathologically by neoplastic T cells that are epidermotropic and located sub- or intra-epider-mally. Clinical experience has learned that fol-liculotropic MF is more refractory to treatment and has a worse prognosis than classic MF. This and the often typical clinicopathologic presen-tation have been the main reasons for its clas-sification as a separate disease entity. Treatment of folliculotropic MF usually consists of PUVA photochemotherapy, sometimes in combina-tion with retinoids or interferon-alpha, or total skin electron beam radiotherapy. Nodular skin lesions that are resistant to treatment are com-monly treated with local radiotherapy. Shown in Figure 1 are typical clinical and histological features of folliculotropic MF.
Sézary sydrome
14 a1 a2 b1 b2 c1 c2
Figure 1. Photographs of clinical and pathologic manifestations of various CTCLs.
a1) Plaque-stage MF, a2) Histopathological aspect of plaque-stage MF, b1) Tumor-stage MF, b2) Histopathological aspect of tumor-stage MF, c1) Folliculotropic MF, c2) Histopathological aspect of folliculotropic MF. Figure 1. Photographs of clinical and pathologic manifestations of various CTCLs.
15 d1 d2 e1 e2 f g Figure 1. continued
d1) Primary cutaneous anaplastic large cell lymphoma, d2) Histopathological aspect, e1) Lymphomatoid papulosis, e2) CD30-positive cells in a lymphomatoid papulosis lesion, f) Sézary syndrome, g) Primary cutaneous peripheral T-cell lymphoma unspecified. Figure 1 continued.
16 by systemic immunosuppression. As a conse-quence opportunistic infections often occur and are an important cause of death of patients with Sz. Although current treatments of Sz, including chemotherapy and extracorporal photophere-sis, have some efficacy, the prognosis generally remains poor.
Primary cutaneous anaplastic large cell lymphoma
Primary cutaneous anaplastic large cell lym-phoma (C-ALCL) is an indolent lymlym-phoma that generally presents with one solitary or few clus-tered skin tumors. Histopathologically C-ALCL is characterized by a dense dermal infiltrate com-posed of large cells with an anaplastic or pleo-morphic cytomorphology and expression of the CD30 antigen by the majority of tumor cells.24 In the EORTC classification this lymphoma was designated as primary cutaneous CD30+ large T-cell lymphoma (CD30+ LTCL), and this condi-tion will be referred to as such in some chapters of this thesis. C-ALCL and lymphomatoid papu-losis are considered as parts of a spectrum of primary cutaneous CD30-positive lymphopro-liferative disorders. Lymphomatoid papulosis is a chronic, recurrent, self-healing papulonecrotic or papulonodular skin disease with histologic features suggestive of C-ALCL. Also in patients with C-ALCL tumors may disappear spontane-ously. This CTCL rarely disseminates to extra-cutaneous sites and has an excellent prognosis with a disease-specific 5-year-survival of 96%.24 Whether signaling through the CD30 receptor itself contributes to the indolent behavior of this lymphoma is still a matter of debate.25 Radio-therapy or surgical excision is the first choice of treatment in patients presenting with a solitary or few localized tumors. In case of multifocal skin lesions patients may be treated with low dose methotrexate, whereas patients are usually only treated with multi-agent chemotherapy in case of extracutaneous dissemination.
Primary cutaneous peripheral T-cell lym-phoma unspecified
Primary cutaneous peripheral T-cell lymphoma (PTL) unspecified represents a heterogeneous
group that includes all T-cell neoplasms that do not fit into any of the other defined subtypes of CTCL. In the WHO-EORTC classification primary cutaneous CD4+ small/medium pleo-morphic T-cell lymphoma, primary cutaneous aggressive CD8+ T-cell lymphoma and primary cutaneous gamma/delta T-cell lymphoma are rec-ognized as provisional entities within the group of PTL unspecified (Table 1). In the EORTC-classification, the term primary cutaneous CD30-negative large T-cell lymphoma (CD30- LTCL) was used to designate most entities now referred to as PTL unspecified and in some chapters of this thesis the term CD30- LTCL is still used.1,26 These lymphomas usually present with generalized tumors and have an aggressive behavior. Rapid development of generalized skin lesions is more common than in C-ALCL. The estimated 5-year survival of patients with a CTCL categorized as PTL unspecified is approximately 16%.4
Pathogenetic aspects of cutaneous T-cell lymphoma
Phenotype of malignant T-cells in CTCL
17 profiles and effector functions.27 The generated antigen-specific T cells acquire the ability to home to the original site of inflammation, the skin. The interactions of skin-homing T cells with dermal capillary cells that facilitate extrav-asation and skin-homing capacity are conferred by expression on the cell membrane of CLA and the chemokine receptor CCR4, that interact with E-selectin and the chemokines CCL17 (TARC) and CCL22 (MDC) respectively on endothelial cells.28,29 The affinity of skin-homing T cells for the epidermis and superficial dermis is further-more mediated by expression of CXCR3, a che-mokine receptor that binds to CXCL10 (IP-10) and CXCL11 (IP-9/I-TAC) produced by basal keratinocytes.30
It has been assumed that malignant T cells in the early phase of MF proliferate in response to chronic stimulation by antigen present in the skin. This assumption is based on the observation that the malignant T cells have a CD45RO+ memory phenotype and are often found adjacent to antigen-presenting dendritic cells (sub)epidermally in the classic type of MF or infiltrating follicular epithelium in follicu-lotropic MF.31-33 Particularly in its early stages CTCL may therefore be considered as a group of diseases characterized by altered immune biology resulting from defective regulation of clonal T-cell expansion. During disease evo-lution the dependence of malignant T cells on antigenic stimulation is assumed to diminish and growth to become more autonomous. Similar to other malignancies the neoplastic T cells in CTCL are primarily characterized by dysregulation of proliferation. Under physi-ological circumstances T cells are programmed to undergo rapid clonal expansion upon recogni-tion of antigen, through activarecogni-tion of signaling cascades triggered by the T cell receptor and the interleukin-2 receptor. Because of the enormous potential for proliferation of a T cell upon activa-tion, the size of the expanding lymphocyte clone is regulated tightly.34,35 Homeostasis of clonally expanding T cells is mainly exerted through elimination of excessive T cells: once antigen-activated T cells have gone through several cell cycles, they become exquisitely susceptible to
apoptosis.36 If there is no further antigen stimu-lation, passive, lymphokine-withdrawal apop-tosis follows. In contrast, if cycling T cells are repeatedly stimulated by antigen, so called acti-vation-induced cell death occurs. At the conclu-sion of the contraction phase that follows clonal expansion, only a small number of memory T cells persist in secondary lymphoid organs and peripheral tissues. The cell surface receptor Fas (CD95) and Fas ligand are key regulators of mature T cell apoptosis and play a central role in the process of activation-induced cell death.37 CTCL is characterized by unrestricted expan-sion of a clone of skin-homing T cells. Given the importance of apoptosis in controlling the size of activated T-cell clones, it may be suspected that disruption of apoptosis signaling has an important role in the pathogenesis of CTCL. Genetic factors in the pathogenesis of CTCL Similar to other types of cancer, DNA lesions are primarily responsible for the development of CTCL. Alterations in the genome of cancer cells lead to increased activity of oncogenes and inac-tivation of tumor suppressor genes, which drives the neoplastic process mainly by stimulation of cellular proliferation and by inhibition of apop-tosis.38 Additionally, cancer cells often exhibit genomic instability and capacity to metastasize. Oncogene activation can result from chromo-somal translocation, amplification or activating mutations. Tumor suppressor genes are mostly inactivated through chromosomal deletions, inactivating mutations or promoter hypermeth-ylation.39 During evolution of the disease, also due to genomic instability, DNA lesions accu-mulate, cellular heterogeneity develops and more malignant subclones emerge.40
18 chromosomal translocations been identified. In addition to chromosomal instability, micro-satellite instability has been demonstrated in malignant T cells of a subset of patients with advanced MF.49,50 Genomic instability there-fore is a hallmark of CTCL, already occurring in early stages of the disease.41,48 The resulting acquisition of chromosomal alterations and mutations in the T cell genome is considered as an important intrinsic factor driving disease progression by allowing the emergence of more aggressive subclones.
Genes affected by chromosomal alteration, muta-tion or promoter hypermethylamuta-tion presumed to be implicated in the pathogenesis of CTCL are listed in Table 2. Among tumor suppressor genes affected in CTCL are the CDKN2A gene encod-ing the p16(INK4A) protein, TP53, Fas, MLH1, PTEN, TGF-beta receptor I and II genes. Inacti-vation of p16(INK4A), either through chromo-somal deletion or promoter hypermethylation,
occurs in a substantial percentage of CTCL cases and has been associated with tumor progression of MF.51-53 Point mutations in the TP53 gene were found at a low frequency in two studies and could not be detected in three other studies.53-57 More recently, point mutations in the Fas gene have been reported in MF, although at a low fre-quency.57,58 In tumor cells from a patient with progressed CTCL, a mutation in the TGF-beta receptor II gene has been demonstrated that dominantly inhibits the function of the wild-type receptor.59 In addition, an inactivating deletion in the TGF-beta receptor I gene was shown in a tumor cell line from a patient with C-ALCL.60 Gene amplification and overexpression of JUNB, a negative regulator of cellular proliferation, has been found in MF, Sz and C-ALCL.61,62 Para-doxically the BCL2 gene, that functions as an inhibitor of apoptosis and is often overexpressed in B-cell lymphomas, has been reported to be frequently deleted in CTCL.62
Table 2. Overview of genetic and epigenetic lesions reported in CTCL
Gene Mechanism Entity Frequency Effect Reference
BAX Point mutation MF 0/44 (0%) Diminished susceptibility
to apoptosis Dereure57 BCL2 Chromosomal deletion MF, Sz C-ALCL 17/42 (40%) 3/13 (23%) Disruption of intrinsic apoptosis pathway; altered susceptibility to apoptosis Mao62
BCR Amplification MF, Sz 4/7 (57%) Serine/threonine kinase
activity Mao61 CDKN2A, p16(INK4A) Chromosomal deletion Promoter hypermethylation Chromosomal deletion Promoter hypermethylation Promoter hypermethylation MF MF MF, Sz 4/13 (31%) 8/13 (62%) 9/49 (18%) 13/49 (27%) 19/66 (29%) Diminished CDK inhibitor activity, cell cycle deregulation Navas51 Navas53 Scarisbrick52 CDKN2A, p14(ARF)
Chromosomal deletion MF 2/50 (4%) Impaired DNA repair, apoptosis and senescence
Navas53 CDKN2B Promoter hypermethylation Chromosomal deletion Promoter hypermethylation MF MF, Sz 4/49 (8%) 1/49 (2%) 30/66 (45%) Diminished CDK inhibitor activity, cell cycle deregulation Navas53 Scarisbrick52 CTSB Amplification Amplification MF, Sz C-ALCL 5/7 (71%) 7/8 (88%) Increased invasive behaviour Mao61 Mao45
Fas Point mutation
Point mutation MF MF 6/44 (14%) 3/16 (19%) Disruption of extrinsic apoptosis pathway Dereure57 Nagasawa58
FGFR1 Amplification MF, Sz 4/7 (57%) Increased growth-factor
receptor signaling
19
HRAS Amplification MF, Sz 3/7 (43%) MAPK pathway,
growth-stimulation Mao61 JUNB Amplification MF Sz C-ALCL 4/14 (29%) 4/22 (18%) 7/10 (70%)
Component of the AP-1 transcription factor, regulating cell proliferation
Mao61
MLH1 Promoter hypermethylation MF 9/51 (18%) Diminished DNA
mismatch repair
Scarisbrick50
MYBL2 Amplification MF, Sz 3/7 (43%) Oncogenic transcription
factor
Mao61
MYCL1 Amplification MF, Sz 3/7 (43%) Oncogenic transcription
factor
Mao61
MYCN Amplification C-ALCL 4/8 (50%) Oncogenic transcription
factor
Mao45
PAK1 Amplification MF, Sz 5/7 (71%) Altered regulation of
cytoskeletal dynamics
Mao61
PIK3CA Amplification MF, Sz 3/7 (43%) Deregulation of the PI3K
pathway promoting survival
Mao61
PTEN Chromosomal deletion MF 10/44 (23%)
heterozygous 2/44 (5%) homozygous
Deregulation of the PI3K pathway, governing cell cycle and apoptosis
Scarisbrick49
PTPN1 Amplification MF, Sz 4/7 (57%) Tyrosine phosphatase
activity
Mao61
PTPN6 Promoter hypermethylation Cell line from
C-ALCL 3/3 (100%) Loss of negative regulation of cytokine receptor Jak-Stat pathways Zhang78 RAF1 Amplification Amplification MF, Sz C-ALCL 5/7 (71%) 7/8 (88%) Stimulation of growth-promoting MAPK pathway Mao61 Mao45
REL Amplification C-ALCL 6/8 (75%) NF-kB pathway,
anti-apoptotic
Mao45
TGFBR1 Deletion (intragenic) Cell line from
C-ALCL
1/1 (100%) Deregulation of SMAD pathway, escape from growth inhibition
Schiemann60
TGFBR2 Point mutation Cell line from
C-ALCL
1/1 (100%) Deregulation of SMAD pathway, escape from growth inhibition Knaus61 TP53 Point mutation Point mutation Point mutation Point mutation Point mutation CTCL MF MF MF MF 1/29 (3%) 6/29 (21%) 0/58 (0%) 0/9 (0%) 0/44 (0%)
Impaired DNA repair, apoptosis and senescence
Garatti54
McGregor55 Kapur56 Navas53
Dereure57
YES1 Amplification C-ALCL 4/8 (50%) Src-family kinase,
growth-stimulatory
Mao45
ZNF217 Amplification MF, Sz 3/7 (43%) Oncogenic transcription
factor
Mao61 Table 2. Overview of genetic and epigenetic lesions reported in CTCL
Gene Mechanism Entity Frequency Effect Reference
BAX Point mutation MF 0/44 (0%) Diminished susceptibility
to apoptosis Dereure57 BCL2 Chromosomal deletion MF, Sz C-ALCL 17/42 (40%) 3/13 (23%) Disruption of intrinsic apoptosis pathway; altered susceptibility to apoptosis Mao62
BCR Amplification MF, Sz 4/7 (57%) Serine/threonine kinase
activity Mao61 CDKN2A, p16(INK4A) Chromosomal deletion Promoter hypermethylation Chromosomal deletion Promoter hypermethylation Promoter hypermethylation MF MF MF, Sz 4/13 (31%) 8/13 (62%) 9/49 (18%) 13/49 (27%) 19/66 (29%) Diminished CDK inhibitor activity, cell cycle deregulation Navas51 Navas53 Scarisbrick52 CDKN2A, p14(ARF)
Chromosomal deletion MF 2/50 (4%) Impaired DNA repair, apoptosis and senescence
Navas53 CDKN2B Promoter hypermethylation Chromosomal deletion Promoter hypermethylation MF MF, Sz 4/49 (8%) 1/49 (2%) 30/66 (45%) Diminished CDK inhibitor activity, cell cycle deregulation Navas53 Scarisbrick52 CTSB Amplification Amplification MF, Sz C-ALCL 5/7 (71%) 7/8 (88%) Increased invasive behaviour Mao61 Mao45
Fas Point mutation
Point mutation MF MF 6/44 (14%) 3/16 (19%) Disruption of extrinsic apoptosis pathway Dereure57 Nagasawa58
FGFR1 Amplification MF, Sz 4/7 (57%) Increased growth-factor
receptor signaling
Mao61
20
Epigenetic factors in the pathogenesis of CTCL
In recent years it has increasingly been recog-nized that in addition to genetic lesions, also epi-genetic lesions are causally related to the devel-opment and progression of cancer. Epigenetic refers to heritable information related to gene function not encoded in the nucleotide sequence. The main epigenetic mechanism in humans is DNA methylation, which refers to addition of a methyl-group to the cytosine nucleotide, where located adjacent to a guanosine in a CpG dinu-cleotide. In normal human cells, CpG dinucleo-tides present in repetitive sequences are meth-ylated. CpG islands, sequences of hundreds of basepairs in length with a high density of CpG dinucleotides located in approximately half of all human gene promoters, are normally unmethyl-ated with the exception of genes on the silenced X-chromosome in females, imprinted genes and several genes with tissue-specific expression. In cancer cells this DNA methylation pattern is severely perturbed: normally unmethylated CpG islands located in gene promoters may demon-strate aberrant methylation, associated with silencing of these genes (promoter hypermeth-ylation).63,64 This epigenetic mechanism of gene silencing may contribute to malignant transfor-mation by inactivating tumor suppressor genes. Tumor types differ with respect to the frequency and patterns of promoter hypermethylation.65,66 In addition, normally methylated CpG dinucleo-tides present in repetitive sequences show loss of methylation (global hypomethylation), which is associated with chromosomal instability.67,68 A number of observations suggest that gene silencing associated with promoter hypermeth-ylation has an important role in the pathogenesis of CTCL. Firstly, lymphomas in general show promoter hypermethylation of tumor suppressor genes at a higher frequency when compared with other tumor types.66,69 Secondly, chronic inflam-mation is considered as a universal accelerator of DNA methylation, as indicated by studies in preneoplastic lesions of colon, oesophagus, liver and lung tissue.70-73 Tumors that develop in the setting of chronic inflammation, such as CTCL, are more likely to contain
hypermethyl-ated CpG islands.74 Thirdly, CTCL have been reported to respond favorably to treatment with histone deacetylase inhibitors (depsipeptide and suberanoylanilide hydroxamide).75 Aberrant transcriptional repression through histone modi-fication and promoter hypermethylation are linked processes and often coincide.76,77 Consis-tently, in the few studies performed thus far a number of genes relevant in the pathogenesis of CTCL have been demonstrated to be inactivated through promoter hypermethylation, including the CDKN2B, CDKN2A encoding p16(INK4A), MLH1 and PTPN6 (SHP-1) genes (see Table 2).49-53,78
Deregulation of signaling pathways in CTCL
Malignant transformation of cells is usually related to perturbation of signaling pathways that govern the regulation of proliferation, apop-tosis and differentiation, such as the p53, Rb, PI3K, receptor tyrosine kinase, NF-kB, SMAD, JAK-STAT and mitochondrial apoptosis signal-ing pathways.39 Deregulation of several signal-ing pathways involved in malignant transforma-tion due to altered expression or constitutive activity of their components has been identified in CTCL. In many cases however, no clear asso-ciation with specific mutations or chromosomal abnormalities has been identified. In particu-lar, defective apoptosis signaling is presumed to have an important role in the pathogenesis of CTCL, since apoptosis is the mechanism by which proliferating mature T cells are normally eliminated following stimulation with antigen. In addition, susceptibility to cellular immune responses directed towards malignant T cells depends on intact apoptotic pathways. As previ-ously mentioned, engagement of the Fas receptor by Fas ligand activates a signaling pathway with a central role in the regulation of T cell apop-tosis and homeostasis. In patients with tumor-stage MF and CD30- LTCL expression of the Fas receptor protein on malignant T cells is fre-quently lost.79 CTCL cells in early plaque-stage MF and C-ALCL however normally express the Fas receptor.
acti-21 vator of transcription (STAT) family of tran-scription factors in CTCL. Malignant T cells of patients with Sz and MF frequently demonstrate constitutive activity of STAT3.80,81 Activation of STAT3, observed in MF tumors but not in the early stages of MF, protects tumor cells from apoptosis in vitro.82 Promoter hypermethylation-associated silencing of PTPN6 has been shown to be induced by activated STAT3 in malignant T cells and may in turn contribute to the consti-tutive activation of this transcription factor.78,83 Furthermore predominant expression of a trun-cated isoform of STAT5 has been found in Sz. Expression of this STAT5 isoform has been related to suppression of the STAT5-dependent genes BCL2 and PIM1.84
Another signaling pathway reported to be dys-regulated is the TNF receptor pathway. Using microarray analysis Tracey and colleagues iden-tified 27 genes that were differentially expressed when comparing MF tumor biopsy samples with inflammatory dermatosis samples.85 Among the genes overexpressed in MF were TNFR-associ-atedfactor 1 (TRAF1) and TNFSF5 (CD40L), BIRC1 and BIRC3. The resultant deregulated signaling through TNFR1, TNFR2 and TNFR5 is hypothesized to have anti-apoptotic as well as T cell costimulatory effects. In other studies co-expression of TNFR5 (CD40) and TNFSF5 (CD40L, CD154), creating an autocrine loop, and increased expression of TNFSF10 (TRAIL) has been observed in CTCL.86,87 As previously mentioned, in C-ALCL and lymphomatoid papulosis signaling through the CD30 recep-tor (TNFR8), a TNF receprecep-tor family member present on activated T cells, may contribute to the malignant phenotype.25
Additionally, loss of sensitivity to TGF-beta has been reported in CTCL, either due to diminished surface expression of the TGF-beta receptor protein in Sz or due to deleterious mutations in the TGF-beta receptor genes.59,60,88,89 The result-ing disruption of the TGF-beta receptor signal-ing pathway is associated with loss of growth inhibition by TGF-beta, an anti-proliferative and pro-apoptotic cytokine for lymphoid cells.90
The immune environment in CTCL
The development and progression of CTCL appear to be determined in part by interactions with the immune micro-environment. Firstly, there is evidence to support that dendritic cells drive the proliferation of malignant T cells in CTCL, in particular during the early phases of the disease. In co-culture experiments it has been shown that immature dendritic cells stimulate the growth of CTCL cells.33 The exact nature of the co-dependency and stimulatory capacity of dendritic cells, including the question whether CTCL cells may recognize and be stimulated by antigen presented by dendritic cells, is as yet unclear.
con-22 taining cytotoxic proteins and interaction of Fas ligand expressed on the cytotoxic T cell and the Fas receptor on the target cell.99,100 Both of these stimuli activate an apoptotic signaling cascade in the neoplastic target cell (see Figure 2). The susceptibility of malignant T cells to cellular immune responses therefore depends on intact apoptotic pathways in these cells.
In advanced forms of CTCL and Sz in particu-lar, defective immune functioning occurs: the numbers of circulating CD8+ T cells, NK cells and dendritic cells are declined, the plasma
levels of interleukin12 and interferonalpha -cytokines that promote immune responses- are decreased, and the complexity of the T-cell repertoire is dramatically reduced.101-104 As a consequence, patients with CTCL are more sus-ceptible to opportunistic infection. Moreover, this immune dysregulation may contribute to disease progression by diminishing the anti-tumor immune response.
The immune defects in CTCL are in part directly attributable to activity of the malig-nant T-cell clone itself through the
produc-Fas Ligand Fas (CD95) FADD Procaspase 8 Caspase 8 Granzyme B Perforin Effector Caspase activation Apoptosis Cytotoxic T cell Target cell Figure 2
Figure 2. Apoptotic pathways activated by cytotoxic T cells.
23 tion of cytokines and expression of membrane molecules. Several studies suggest that CTCL cells originate from and display phenotypical characteristics of T helper 2 cells, including the production of cytokines such as interleukin-4, interleukin-5 and interleukin-10 compromising efficient immune responses.105-108 More recently it was reported that malignant T cells of CTCL, when cultured with dendritic cells, may produce significant levels of TGF-beta, interleukin-10 and express CTLA-4 and FoxP3, features of T regulatory cells, an immunosuppressive subset of T cells.109
Aims and outline of the thesis
General aims of this thesis have been to iden-tify clinicopathological determinants of disease progression of patients with CTCL and to gain more insight into the molecular pathogenesis of this group of T-cell malignancies.
In the first part of the thesis (chapter 2, 3 and 4) issues are addressed that relate to the disease course of CTCL and clinicopathological factors that may determine and predict disease progres-sion. This part of the study has been facilitated by the Dutch Cutaneous Lymphoma Group (DCLG). The DCLG consists of an expert panel of dermatologists and pathologists meeting on a regular basis to discuss and reach agreement on the diagnosis of Dutch patients presented with cutaneous lymphoma. Clinical characteristics and disease course data of patients are included in the registry of the DCLG. The fact that almost all Dutch patients with cutaneous lymphoma are registered by the DCLG allows unbiased clini-cal epidemiologiclini-cal studies. The department of Dermatology of the Leiden University Medical Center is the nation-wide referral center for CTCL patients and responsible for the registry of the DCLG. Biopsy specimens of many patients with cutaneous lymphoma are therefore availa-ble for studies of the pathological and molecular biological aspects of this group of lymphomas. In the first part of the thesis we addressed the
following issues:
- Although general survival figures for patients
with MF are available from epidemiological studies conducted in the United States, more specific information regarding the disease course stratified per clinical stage is limited. In chapter 2 we investigated the frequency of occurrence of disease progression from skin-limited to widespread disseminated disease as well as the survival of patients diagnosed with MF in The Netherlands. In addition, we evalu-ated which clinical features are associevalu-ated with an aggressive disease course and unfavorable prognosis.
- Results from the study presented in chapter 2 reinforced the clinical impression that fol-liculotropic MF has a more aggressive clini-cal behavior than the classic epidermotropic type of MF. In chapter 3 we report a detailed description of the clinical features, histopatho-logical alterations and prognosis of a large cohort of patients with folliculotropic MF. - There are many indications that the disease
24
Figure 3. Preparation of RNA en DNA for gene expression and DNA methylation profiling.
For gene expression analysis using oligonucleotide microarrays (Affymetrix) RNA is extracted from tumor tissue and reverse transcribed to yield complementary DNA. After second strand synthesis, the purified cDNA product is in vitro transcribed using T7 RNA polymerase, biotin-UTP and biotin-CTP to generate biotinylated aRNA. The fragmented aRNA is hybridized to an oligonucleotide microarray that contains short 25-mer oligonucleotide probes, that are photolithographically synthesized on the microarrays.
To detect methylated sequences, DNA isolated from tumor tissue is digested by MseI to obtain DNA fragments of a few hundred bp in length, to which linkers are ligated. These linker-ligated fragments are then sequentially digested by two methylation-sensitive restriction enzymes (BstUI and HpaII). Hypermethylated sequences, refractory to cleavage by the methylation sensitive enzymes, are then selectively amplified in a linker-PCR, fluorescently labeled and subsequently hybridized to the CpG island microarray. This microarray contains as probes a panel of 8640 CpG island tags, 200-2000 bp in length, prepared from a genomic library arrayed on a glass slide.
Figure 3
Total RNA Genomic DNA
AAA AAA
Reverse transcription using
T7 oligo(dT) primer Restriction digestion (MseI)Linker ligation double stranded cDNA
AAA AAA
TTT TTT
In vitro transcription using T7 RNA polymerase
Biotin labeling
Methylation-specific restriction digestion (BstUI, HpaII) biotin-labeled antisense cRNA
UUU
B BBB
UUU
B BBB
Fragmentation
Hybridization Fluorescence labeling (Cy5)PCR amplification Hybridization
oligonucleotide microarray
(Affymetrix) CpG island microarray
25 methylation.113,114 In microarray experiments, target cDNA, cRNA or genomic DNA is labeled with a fluorescent dye and hybridized to DNA probes arrayed on aplatform such as a glass slide (Figure 3).A scanner then measures fluo-rescence at the site of eachprobe corresponding to the abundance of the interrogated transcript or DNA sequence.
In the second part of the thesis we examined the following:
- Deregulated activity of the Fas signaling pathway has been suspected in CTCL, based on the central role of Fas-induced apoptosis in the homeostasis of mature T-cells and in con-ferring susceptibility to anti-tumor immune responses. Diminished Fas protein expression has been demonstrated in advanced stages of MF.79 The function of Fas may be disrupted in cancer cells through point mutations or alternative splicing of the gene encoding this apoptosis-inducing receptor.115-118 In chapter 5 we analyzed whether Fas gene alterations are present in neoplastic T cells of CTCL patients. - The gene expression pattern that distinguishes malignant T cells of CTCL patients from benign T cells promises to give insight into the signaling alterations relevant to the develop-ment and progression of CTCL. In chapter 6 we performed microarray-based gene expres-sion analysis on isolated T cells of patients with Sézary syndrome with the additional purpose of identifying tumor-associated markers for the molecular diagnosis and directed therapy of CTCL.
- Several observations suggest that promoter hypermethylation, associated with inactiva-tion of tumor suppressor genes, may be an important factor in the pathogenesis of CTCL. The purpose of the study presented in chapter 7 was to analyze the occurrence of promoter hypermethylation in CTCL on a genome-wide scale, focusing on pathogenetically significant epigenetic alterations.
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