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Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Oncology
presented at Uppsala University in 2002.
ABSTRACT
Amini, R-M. 2002. Hodgkin lymphoma. Studies of advanced stages, relapses and the
relation to non-Hodgkin lymphomas. Acta Universitatis Upsaliensis. Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Medicine 1132. 64pp.
Uppsala. ISBN 91-554-5265-5.
The relationship between Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL)
is not entirely elucidated and a clonal relation may be present more often than
previously believed. Mechanisms of tumour progression and resistance to therapy are
poorly understood.
Between 1974 and 1994 all individuals in Sweden with both HL and NHL were
identified. Thirty-two cases were studied using clinical, histopathological and
immunohistochemical methods. The second lymphoma often appeared in an aggressive
clinical form and a significant correlation between the expression of p53 and LMP-1 in
the first and second lymphoma was demonstrated.
The treatment outcome for 307 patients with advanced stages of HL, in an unselected
population was in accordance with the treatment results of large centres world-wide.
Some patients were successfully selected for a shorter chemotherapy-regimen without
inferior treatment results.
In 124 patients with relapse, the survival of those primarily treated with radiotherapy
according to the National guidelines was in accordance with the survival of patients of
initially advanced stages. A worse outcome was found for those who received both
chemotherapy and radiotherapy initially, probably because of a higher frequency of
bulky disease in this group.
Immunohistochemical analysis of the tumour suppressor protein p53 and
retinoblastoma protein (Rb) of paired samples at diagnosis and at relapse in 81 patients
did not reveal any specific staining pattern affecting survival.
A novel B-cell line (U-2932) was established from a patient with a diffuse large B-cell
lymphoma previously treated for advanced stage and subsequent relapses of HL. An
identical rearranged IgH gene was demonstrated in tumour cells from the patient and in
U-2932. A p53 point mutation was detected and over-expression of the p53 protein was
found. A complex karyotype with high-level amplifications of the chromosomal regions
18q21 and 3q27, i.e. the loci for bcl-2 and bcl-6 were demonstrated.
Key words: Hodgkin lymphoma, advanced stages, relapse, p53, EBV, Rb, cell line.
Rose-Marie Amini, Department of Oncology, University Hospital, SE-751 85 Uppsala
Sweden
© Rose-Marie Amini 2002
ISSN 0282-7476
ISBN 91-554-5265-5
Printed in Sweden by Lindbergs Grafiska, Uppsala 2002
To Hashem, Jacob and Maria
LIST OF ORIGINAL PAPERS
This thesis is based on the following papers, which are referred to in the text by their
Roman numerals:
I
Patients suffering from both Hodgkin’s disease and non-Hodgkin’s lymphoma: A
clinico-pathological and immuno-histochemical population-based study of 32
patients.
R-M Amini, G Enblad, C Sundström, B Glimelius.
Int J Cancer 1997; 71: 510-516.
II
Treatment outcome in patients younger than 60 years with advanced stages (IIBIV) of Hodgkin’s disease: the Swedish National Health Care Programme
experience.
R-M Amini, G Enblad, A Gustavsson, T Ekman, M Erlanson, E Haapaniemi, B
Glimelius.
Eur J Haematol 2000; 65: 379-389.
III
A population-based study of the outcome for patients with first relapse of
Hodgkin lymphoma.
R-M Amini, B Glimelius, A Gustavsson, T Ekman, M Erlanson, E Haapaniemi,
G Enblad.
Accepted for publication in Eur J Haematol.
IV
Relapsed Hodgkin lymphoma: Immunostaining patterns in relation to survival.
R-M Amini, G Enblad, P Engström, B Christensson, B Glimelius, C Sundström.
Accepted for publication in Leuk Lymphoma.
V
A novel B-cell line (U-2932) established from a patient with a diffuse large B-cell
lymphoma following Hodgkin lymphoma.
R-M Amini, M Berglund, R Rosenquist, A v Heideman, S Lagercrantz, U
Thunberg, J Bergh, C Sundström, B Glimelius, G Enblad.
Accepted for publication in Leuk Lymphoma.
Reprints were made with permission of the publishers.
ABBREVIATIONS
ABVD
ALCL
ASCT
BEACOPP
cHL
CLL
CR
CS
DFS
DLBCL
EBER
EBV
EFS
EORTC
FL
GC
GELA
GHSG
HDCT
HLS
HRS
Ig
LDHL
LMP-1
LRCHL
MCHL
MIME
MOPP
NFκB
NHL
NLPHL
NSHL
PR
PS
PTL
Doxorubicin, bleomycin, vinblastine, dacarbazine
Anaplastic large cell lymphoma
Autologous stem-cell transplantation
Bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine,
procarbazine, prednisone
Classical Hodgkin lymphoma
Chronic lymphocytic leukaemia
Complete remission
Clinical stage
Disease-free survival
Diffuse large B-cell lymphoma
Epstein-Barr virus encoded messenger ribonucleic acid
Epstein-Barr virus
Event-free survival
European Organization for the Research and Treatment of Cancer
Follicular lymphoma
Germinal centre
Groupe d’Etudes des Lymphomes de l’Adulte
German Hodgkin Lymphoma Study Group
High-dose chemotherapy
Hodgkin lymphoma specific survival
Hodgkin and Reed-Sternberg cells
Immunoglobulin
Lymphocyte depleted Hodgkin lymphoma
Latent membrane protein-1
Lymphocyte rich classical Hodgkin lymphoma
Mixed cellularity Hodgkin lymphoma
Methyl-GAG, ifosfamide, methotrexate, etoposide
Mechloretamine, vincristine, procarbazine, prednisone
Nuclear Factor κB
Non-Hodgkin lymphoma
Nodular lymphocyte predominant Hodgkin lymphoma
Nodular sclerosis Hodgkin lymphoma
Partial remission
Pathological stage
Peripheral T-cell lymphoma
CONTENTS
ABSTRACT………………………………………………………………………. 2
LIST OF ORIGINAL PAPERS…………………………………………………. 4
ABBREVIATIONS………………………………………………………………. 5
BACKGROUND…………………………………………………………………. 9
Introduction………………………………………………………………………. 9
Epidemiology……………………………………………………………………... 10
Etiology…………………………………………………………………………… 10
Epstein-Barr virus……………………………………………………………… 10
Immunodeficiency……………………………………………………………....
Histopathology…………………………………………………………………….
The borderline between HL and NHL…………………………………………..
Tumour biology…………………………………………………………………...
Surrounding cells……………………………………………………………….
How do HRS cells escape from apoptosis?..........................................................
BCL-6…………………………………………………………………………...
Cytogenetics…………………………………………………………………….
Clinical presentation……………………………………………………………...
Clinical staging…………………………………………………………………
Treatment of early and intermediate stages…………………………………….
Treatment of advanced stages…………………………………………………..
Are eight courses of chemotherapy the golden standard?...................................
The International Prognostic Score…………………………………………….
Primary high-dose chemotherapy?......................................................................
What is the role of adjuvant radiotherapy?.........................................................
Prognostic markers of tumour-biological importance…………………………
Relapses and refractory disease………………………………………………..
Late complications……………………………………………………………...
Relationship between HL and NHL - tumour progression or therapyrelated?…………………………………………………………………………
11
11
13
15
15
16
18
18
19
19
20
21
22
22
22
23
23
24
25
26
AIMS OF THE STUDY………………………………………………………….. 29
PATIENTS, MATERIALS AND METHODS…………………………………. 30
Patients (I-IV)………………………………………………………………….. 30
Staging procedures (I-IV)……………………………………………………… 30
Treatment recommendations in the Health Care Programme (II, III, IV)……... 30
6
Histopathology and immunohistochemistry (I, IV, V)………………………….
Cytogenetic analysis……………………………………………………………
IgH gene rearrangement analysis………………………………………………
Sequence-based analysis of p53………………………………………………...
Statistical methods (I-IV)……………………………………………………….
31
32
32
32
33
RESULTS AND DISCUSSION…………………………………………………. 34
HL and NHL (I)………………………………………………………………....
Advanced stages (II)…………………………………………………………....
NHL misclassified as HL (II, III)……………………………………………….
Relapses (III)……………………………………………………………………
Immunostaining patterns of paired samples at diagnosis and relapse (IV)……
Cell line U-2932 (V)…………………………………………………………….
34
37
39
40
43
47
CONCLUSIONS…………………………………………………………………. 49
ACKNOWLEDGEMENTS…………………………………………………....... 50
REFERENCES…………………………………………………………………… 52
7
8
BACKGROUND
Introduction
Hodgkin lymphoma (HL) is a malignant disorder with an excellent outcome due to the
high cure rates achieved by modern polychemotherapy and/or radiotherapy. In Sweden,
the annual number of new cases is approximately 160, comprised mainly of young
adults. HL has a bimodal age curve with a first peak at 20-30 years of age and a second
peak in late life above the age of 60.
HL was first described by Thomas Hodgkin (1798-1866) in 1832, in a classical paper
entitled ”On some morbid appearances of the absorbant glands and spleen” (Hodgkin,
1832). In this article he described seven patients with tumours in lymph nodes and
spleen. The disease was later named after him as Hodgkin’s disease in 1865 by Sir
Samuel Wilks, who further described the disease (Wilks, 1865). Carl Sternberg
(Sternberg, 1898) and Dorothy Reed (Reed, 1902) independently described the tumour
cells of HL which are named hereafter as Reed-Sternberg (RS) cells. Both emphasised
the importance of making the proper histopathological examination. Not until recently
has it been proven that the disease is in fact a true lymphoma. The tumour cells in HL,
the Hodgkin and Reed-Sternberg (HRS) cells originate from germinal centre (GC)
derived B-cells (Küppers, 1994, Kanzler, 1996) although in some cases they can
possibly be of T-cell origin (Seitz, 2000, Müschen, 2000).
HL and non-Hodgkin lymphomas (NHL) have classically been considered as two
disease entities and the occurrence of both lymphomas in the same individual is
unusual. If the time interval is long, the second lymphoma has classically been regarded
as a therapy-induced, de novo lymphoma, but if the time interval is short other
mechanisms may be responsible for the development of a second lymphoma.
A clonal relation between HL and NHL has recently been demonstrated in a few cases
(Brauninger, 1999, Küppers, 2001). Thus, it is possible that in some of the cases where
HL and NHL appear in one individual, a clonal relation may be the underlying
mechanism.
Before the era of radiotherapy and combined chemotherapy, HL was an inevitably fatal
disease with a median survival of less than one year. Usage of radiotherapy as treatment
for lymphoma was introduced by Pusey in 1902, shortly after the discovery of roentgen,
and further developed by Gilbert in 1931 (Gilbert, 1931). Rapid advances in
radiotherapy changed the outlook for many patients (Peters, 1950, Kaplan, 1962).
Single agent chemotherapy resulted only in short remissions. In 1964, DeVita
introduced the MOPP (mechloretamine, vincristine, procarbazine, prednisone) regimen
and thereby revolutionised the treatment outcome giving an improved long term
survival (DeVita, 1967).
Today the majority of patients are cured by chemotherapy and radiotherapy, however,
relapses occur and the outcome for these patients is worse (Longo, 1992, Bonfante,
1997). The challenge in the treatment of HL patients today is not only to achieve cure,
but also to avoid long-term toxicity, the most serious being secondary malignancies.
Little is known about the mechanisms leading to tumour progression and resistance to
therapy. Further studies are needed to investigate the relationship between HL and
9
NHL, the mechanisms behind the development of secondary lymphomas, relapse
patterns and the optimal treatment for the younger patients with advanced stages of HL.
Epidemiology
The overall incidence of HL has decreased in parallell with a slight increase in young
adults (Merk, 1990, Hartge, 1994, Foss-Abrahamsen, 1997, Liu, 2000, Hjalgrim, 2001),
whereas the incidence of NHL has increased 3-4% per year during the past decades
world-wide (Hartge, 1994, Seow, 1996). Some of the lymphomas previously diagnosed
as HL are now classified as NHL and this shift is partially responsible for the declining
incidence of HL. HL occurrence varies by age, social class, geographic location, and
between different ethnic groups (Correa, 1971, Alexander, 1991, Glaser, 1996). HL is
particularly rare in Asians, but for Asians living in the United States the incidence is
doubled (Glaser, 2002). Thus, enviromental factors may influence the incidence of HL.
In addition, specific human leukocyte antigen (HLA) types are more often present in HL
cases, indicating a genetic susceptibility. Familial HL is estimated to represent
approximately 4-5% of all cases (Kerzin-Storrar, 1983, Ferraris, 1997). Shared
enviromental factors and genetic susceptibility have been proposed to explain familial
aggregation.
Etiology
Epstein-Barr virus
The etiology of HL is unknown. However, patients with a previous history of infectious
mononucleosis have an elevated risk of developing HL (Gutensohn, 1980, Hjalgrim,
2000). Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a
human herpes virus and more than 90% of the adult population world-wide is infected
by EBV (Niedobitek, 1996, Hjalgrim, 2000).
EBV has been postulated to play a role in the pathogenesis of classical HL (cHL) and
the prevalence varies according to the histological subtype and epidemiologic factors.
EBV can be detected in HRS cells in 30-50% of cHL (Pallesen, 1991, Herbst, 1992,
Glaser, 1997, Enblad, 1999, Stein, 2001). In mixed cellularity (MCHL) subtype, the
frequency of EBV is higher with approximately 50-75% of cases with EBV-positive
tumour cells compared to the nodular sclerosis (NSHL) subtype, where about 10-40%
are positive. EBV is monoclonal in HRS cells within the same lymph node (Boiocchi,
1993). Cases of cHL positive for EBV at diagnosis are also positive at relapse
(Nerurkar, 2000) with persistence of the same clonal EBV strain (Brousset, 1994).
EBV can transform resting human B-cells into permanently proliferating
lymphoblastoid cell lines. A set of latent viral gene products are expressed consisting of
EBV encoded mRNA (EBER), six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B,
3C, LP), and three latent membrane proteins (LMP-1, 2A, 2B). EBV-infected HRS cells
express LMP-1 and EBNA-1 without EBNA-2, a pattern which is characteristic of a
latent EBV type II infection (Rowe, 1987). LMP-1 can induce an up-regulation of
lymphocyte activation antigens such as CD30 in vitro (Wang, 1988) and has been
proposed to contribute to the malignant phenotype of HRS cells (Niedobitek, 1996).
LMP-1 also induces activation of Nuclear Factor κB (NFκB) in cHL by targeting IκBα
signaling pathways, probably by mimicking a constitutively activated receptor of the
10
tumour necrosis factor (TNF) receptor family (Kieff, 1995, Feuillard, 2000). LMP-1
might thus play a role for the HRS cells in evasion of apoptosis.
The causative role for EBV in HL is debated. A ”hit and run” mechanism hypothesis for
EBV negative cHL has been proposed (Nerurkar, 2000), where the EBV genome is
assumed to be lost after having initiated the neoplastic transformation. The search for
other infectious agents have so far been unsuccessful, although risk factor profiles
consistently suggest that delayed exposure to a common infectious agent may be
associated with HL in young adults, the so-called ”late-host-response” (Armstrong,
1998, Jarrett, 1999)
Immunodeficiency
Many HL patients suffer from an impaired immune system with a reduced number of
CD4+ lymphocytes in the peripheral blood (Grimfors, 1990). An immunological
impairment of lymphocytes in healthy family members of patients with HL has also
been observed (Merk, 1990).
It has been suggested that an LMP-1 specific immune defect may contribute to the
development of EBV-positive HL (Dolzetti, 1995). However, some patients with HL
have a normal immune system. It is therefore more likely that in EBV-associated cHL,
tumour specific factors in the microenvironment may elicit a localised suppression of
EBV-specific immunity and thereby contribute to the pathogenesis (Frisan, 1995).
This effect may partly be mediated by interleukin (IL)-10, a cytokine produced by HRS
cells with inhibitory effects on T-cell mediated immunity. Promising results from
treatment with EBV-specific cytotoxic T-cells (CTL) in relapsed patients with EBVpositive cHL (Rooney, 2001) support these data. Moreover, patients with elevated IL-10
in serum have a worse clinical outcome (Axdorph, 2000).
Histopathology
The diagnosis of HL is made from histological examination of biopsy material and is
based on observation of typical HL morphology. The neoplastic cells in HL- the
mononucleated Hodgkin (H) and multinucleated RS cells- constitute a minority of the
cells (0.1-10% of the cells, usually less than 3%) in affected lymph nodes. The HRS
cells are large, about 20-25 µm, have rounded nuclei with pale chromatin and prominent
eosinophilic nucleoli in two separate nuclear lobes in typical cases. The cytoplasm is
abundant and slightly basophilic.
HRS cells are seen in a typical background of reactive surrounding inflammatory and
accessory bystander cells; lymphocytes, plasma cells, eosinophils, neutrophils,
fibroblasts and histiocytes. Recently the importance of mast cell infiltration among
accessory cells was demonstrated (Molin, 2001).
Depending on the presence of fibrosis, amount of tumour cells and lymphocytes, HL
has been divided in four separate categories according to the Rye classification (Lukes,
1966). This has been reclassified in the Revised European-American classification of
lymphoid neoplasms (REAL) (Harris, 1994) and the present World Health Organization
(WHO) classification based on REAL (Jaffe, 2001).
11
In the WHO classification HL comprises of two disease entities, further divided in the
subtypes; NSHL, MCHL, lymphocyte depleted (LDHL) and lymphocyte rich (LRCHL),
and the nodular lymphocyte predominant HL (NLPHL).
The most common subtype is NSHL constituting about 70% of cHL. In this subtype, the
tumour growth is partially in a nodular pattern with fibrous bands separating the
nodules. The characteristic cell is the lacunar type RS cell, which may be numerous. In
MCHL, the second most common subtype comprising approximately 20-25% of cHL,
classical HRS cells are scattered in a mixed inflammatory background without nodular
sclerosing fibrosis. Histiocytes may show pronounced epitheloid differentiation, and
may form granuloma like clusters. LRCHL accounts for about 5% of cHL and HRS
cells are seen in a nodular or diffuse background characterised by an abundance of small
lymphocytes with few neutrophils and eosinophils. A proportion of the tumour cells
may resemble lymphocytic and histiocytic (L&H) cells and thus can easily be
mistakenly classified as NLPHL. In these cases, the diagnosis can be confirmed by
immunohistochemistry. The HRS cells of cHL are usually CD30+, CD15+/-, CD20-/+,
J-chain- (Stein, 2001).
The HRS cells in cHL originate predominantly from GC-derived B-cells (Küppers,
1994, Kanzler, 1996) and from T-cells in a few cases (Seitz, 2000, Müschen, 2000).
Single cell investigations of HRS cells in cHL (Marafioti, 2000) have revealed somatic
hypermutations of the immunoglobulin (Ig) gene and lack of Ig mRNA transcripts
regardless of absence or presence of crippling Ig gene mutations caused by an
inactivation of the Ig promotor. The reason for this appears to be due to a lack of the
octamer-dependent transcription factor Oct2 and/or its coactivator BOB.1 (Stein, 2001).
HRS cells are thus incapable of producing Igs and should therefore rapidly undergo
apoptosis, as do B-cells that have lost the capacity to express Ig (Lam, 1997).
NLPHL is a monoclonal B-cell neoplasm characterised by a nodular or a nodular and
diffuse proliferation consisting of small lymphocytes, histiocytes, epitheloid histiocytes
and intermingled neoplastic L&H cells. The L&H cells usually have one large nucleus
and scant cytoplasm and are also designated ”pop-corn cells” as the nucleus often is
folded and multilobated with vesicular chromatin, resembling the appearance of popcorn. Neutrophils and eosinophils are absent. L&H cells characteristically express
CD20, CD79a, BCL-6, and CD45 positivity in nearly all cases but are negative for
CD30 and CD15 (Kraus, 2000, Stein, 2001). The presence of CD57+ T-cells
surrounding L&H cells confirms the diagnosis of NLPHL (Hansmann, 1999, Kraus,
2000).
The tumour cells of NLPHL are derived from GC B-cells at the centroblastic stage of
differentiation and harbour identical monoclonally rearranged Ig genes (Marafioti,
1997). The Ig variable (V) heavy (H) chain genes carry a high load of somatic mutations
and also show signs of ongoing mutations (Brauninger, 1997, 1999, Marafioti, 1997,
2000).
12
The borderline between HL and NHL
A ”grey zone” exists between cHL, NLPHL, and NHL, despite improvements in
immunohistochemistry and molecular pathology. In many large materials a proportion
of cases not possible to classify as HL or NHL exists. Furthermore, reproducibility of
inter-and intraobservations are not 100%. This might partly be attributed to poor
technical quality, but true grey zone lymphomas likely exist (Bernhards, 1992, Georgii,
1993, Harris, 1994, Rüdiger, 1998).
DLBCL may in some cases be difficult to distinguish from both cHL and NLPHL,
especially the T-cell/histiocyte rich variant, in which case the majority of cells are nonneoplastic T-cells with or without histiocytes, and contain less than 10% of large
neoplastic, often atypical B-cells (Harris, 1994, Rüdiger, 1998, Gatter, 2001). With the
help of additional immunohistochemistry it is possible to distinguish cHL from T-cell
rich DLBCL, but it may still be difficult to distinguish NLPHL. In both NLPHL and Tcell rich DLBCL, the tumour cells express a B-cell phenotype in addition to CD30 and
CD15 negativity.
The histiocytes may in some T-cell rich DLBCL cases be epitheloid in appearance and
the neoplastic B-cells may also resemble both L&H cells as well as HRS cells.
In the anaplastic variant of DLBCL, the tumour cells may resemble HRS cells with
bizarre and pleomorphic nuclei, which also stain positively for CD30.
Mediastinal large B-cell lymphoma is a subtype of DLBCL of putative thymic B-cell
origin that arises in the mediastinum and must be recognised as it may exhibit features
overlapping with cHL (Banks, 2001). Sheets of large cells with abundant pale
cytoplasm are seen with variable fibrosis, interspersed small lymphocytes, and
eosinophils which may be difficult to distinguish from cHL. Diagnostic difficulties can
arise especially since biopsy samples are often sparse due to the mediastinal location.
Tumour cells are of B-cell phenotype and usually express markers such as CD19 or
CD20. CD30 expression is often present, although weak. CD45 is expressed in contrast
to HRS cells.
Another entity that might be misclassified as cHL is the anaplastic large cell lymphoma
(ALCL), where the tumour cells are usually large, pleomorphic and bizarre, sometimes
resembling HRS cells. In early stages, the growth pattern is sinusoidal and tumour cells
may colonise the paracortex and grow in cohesive sheets. In ALCL, the presence of the
t(2;5) translocation with the immunohistochemically detected expression of anaplastic
lymphoma kinase (ALK-1), may be a helpful tool in differentiating between cHL and
ALCL (Drexler, 2000, Stein, 2000, Delsol, 2001, Herling, 2001, Jaffe 2001).
The lymphoepitheloid cell variant (Lennert in the Kiel classification) among the
peripheral T-cell lymphomas (PTL) must also be differentially diagnosed from cHL. RS
cells are present in an inflammatory background with small lymphocytes, eosinophils,
plasma cells, and numerous small clusters of epitheloid histiocytes (Ralfkiaer, 2001).
Tumour cells express CD30 and a T-cell phenotype (CD4+, CD8-), which also may be
encountered, although rarely, in cHL. Morphological and immunohistochemical
charasteristics of cHL, NLPHL, T-cell/histiocyte rich and anaplastic variant of DLBCL,
ALCL and peripheral T-cell lymphoma (PTL) are summarised in table 1.
13
Table 1. Morphological and immunohistochemical characteristics of cHL, NLPHL, Tcell/histiocyte rich and anaplastic variant of DLBCL, ALCL and peripheral T-cell
lymphoma (PTL).
cHL
NLPHL
DLBCL
Anaplastic
variant
ALCL
PTL
(Lennert)
nodular,
nodular and
diffuse
DLBCL
T-cell/
histiocyte
rich variant
diffuse,
vague
nodularity
Growth
pattern
diffuse,
interfollicular
diffuse,
interfollicular,
sinusoidal,
cohesive
diffuse,
sinusoidal,
cohesive
pattern
diffuse
Background
lymphocytes
eosinophils
plasma cells
epitheloid
histiocytes
lymphocytes
histiocytes
plasma cells
lymphocytes
or
histiocytes
lymphocytes
lymphocytes
neutrophils
lymphocytes
eosinophils
plasma cells
epitheloid
histiocytes
Predominant
type of
background
lymphocytes
T
B
T
B
T
T
CD57+
lymphocytes
few
present
absent
absent
absent
absent
Tumour cells
HRS
L&H
B-cells
B-cells
Cytotoxic Tcells or nullcells
T-cells
Immunophenotype of
tumour cells
CD30
CD15
CD45
CD20
CD79a
CD3
Perforin/
Granzyme B
EMA
ALK
Oct2
BOB.1
BCL-6
Ig light chains
+
+/-/+
-/+1
-/+1
-/+1
-1
+
+
+
-
-/+
+
+
+
-
+
+
+
+
-
+
-1
+/+
+
+/+
+
-/+
-/+2
-/+
-/+3
+/+
+
+
+/-
-/+
+
+
+
-/+
-/+
+
+
+
-/+
+/+/na
na
+/-
na
na
-/+
-
+=all cases are positive, -=all cases are negative, +/-=majority of cases positive, -/+=majority of cases
negative, 1positive in rare cases, 2strong expression in less than 5% of the cases, 3reflects uptake of tumour
cells.
Abbreviations: cHL=classical Hodgkin lymphoma, NLPHL=nodular lymphocyte predominant Hodgkin
lymphoma, DLBCL=diffuse large B cell lymphoma, ALCL=anaplastic large cell lymphoma,
PTL=peripheral T cell lymphoma, HRS=Hodgkin and Reed-Sternberg cells, L&H=lymphocytic and
histiocytic cells, CD=cluster of differentiation, EMA=epithelial membrane antigen, ALK=anaplastic
lymphoma kinase, Ig=immunoglobulin, na=not analysed.
14
Tumour biology
Surrounding cells
In contrast to most forms of NHL and other tumour types, HL is characterised by a low
proportion of tumour cells. In spite of this, the proliferation, spread, and apoptotic
evasion of the tumour cells are the fundamental properties responsible for the
progression of the disease.
The abundant admixture of inflammatory cells in cHL consist of small T lymphocytes,
plasma cells, histiocytes, eosinophils, and mast cells. The interaction between the HRS
cells and surrounding cells is probably important for the pathogenesis of the disease.
The reason for this cellular admixture is due to the unique ability of the HRS cells to
produce a variety of cytokines and chemokines and/or their receptors (Gruss, 1997,
Kadin, 1999, Poppema, 2000). These are IL-1α, IL-2 receptor (R), IL-5 and IL-5R, IL-6
and IL-6R, IL-7, IL-8, IL-9 and IL-9R, IL-10 (Ohshima, 1995), IL-13 and IL-13 R
(Kapp, 1999), granulocyte-macrophage colony-stimulating factor (GM-CSF),
lymphotaxin-α, transforming growth factor-beta (TGF-β), tumour necrosis factor beta
(TNF- β) and CC chemokine thymus and activation related chemokine (TARC)
(Ohshima, 1995, Gruss, 1997, Poppema, 2000, Jaffe, 2001). The expression of CC
chemokine TARC, normally produced by antigen-presenting cells in the thymus and by
activated monocytes (van den Berg, 1999) has been demonstrated in cHL using serial
analysis of gene expression (SAGE). TARC produced by HRS cells may contribute to
the recruitment of T-cells with Th2-like phenotype in cHL. The influx of eosinophils
mediated by IL-5 and eotaxin and plasma cells recruited by IL-6 are examples of the
result of the production and induction of various paracrine loops.
One of the most prominent and important features of HRS cells is the expression of
CD30 (Hsu, 2000).RSHRS CD30 is a member of the TNF/nerve growth factor (NGF)
receptor superfamily and its ligand, CD30L stimulates the proliferation of HRS cells. In
vivo, CD30L is expressed by mast cells (Molin, 2001) and eosinophils (Pinto, 1996) in
the tumours. The CD30-CD30L interaction contributes to the proliferation of HRS cells
both by auto- and paracrine loops and is probably of importance for tumour progression
in HL. CD30 also mediates NFκB activation.
Primary and cultured HRS cells express not only CD30 but also CD40. The surface
receptor CD40 is virtually always expressed by HRS cells and its ligand, CD40L, is
expressed by T-cells surrounding the HRS cells (Carbone, 1995). The CD40-CD40L
interaction may be a critical element in the deregulated cytokine network. CD40 is the
first link in the pathway to NFκB activation, the central regulator of cytokine production
and apoptosis (Rothe, 1995, Annunziata, 2000).
The abnormal cytokine and chemokine expression produced by HRS and their
bystander cells in the tumour tissue infiltrated by cHL are probably necessary for the
growth and survival of the neoplastic cells. Thus, cytokines produced by the HRS cells
may induce the bystander cells to produce similar cytokines, leading to autocrine
cytokine loops in addition to paracrine loops. The differences between the histological
subtypes can probably be explained by these differences in cytokine and chemokine
production.
15
How do HRS cells escape from apoptosis?
Apoptosis, or programmed cell death, is an important mechanism for normal tissue
homeostasis in addition to tumour response to radiotherapy and chemotherapy.
However, the mechanisms by which HRS cells and their precursor cells escape
apoptosis are largely unknown.
One possible way of apoptotic evasion is the constant activation of NFκB found in HRS
cells. NFκB rescues cells from apoptosis probably by controlling the expression of a
number of growth-promoting cytokines (Bargou, 1996, 1997, Hinz, 2001). The
persistent activation of NFκB in HRS cells might be caused by mutations in IκB family
members, which are the natural inhibitors of NFκB (Bargou, 1997, Jungnickel, 2000) or
by aberrant activation of IκB kinase (Krappmann, 1999). NFκB activations may also be
caused by the TNF receptor associated factor TRAF 1 molecule, which is also
expressed in HRS cells (Dürkop, 1999). LMP-1, CD30 and CD40 can also induce
NFκB activity (Annunziata, 2000, Feuillard, 2000).
Apoptosis is also mediated via the Fas/apo-1/CD95 pathway by interaction between
CTLs expressing CD95 ligand and CD95 expressed by HRS cells (Schattner, 1995, Re,
2000). CD95 (Fas/apo-1) is a cell surface protein and member of the TNF receptor
family. Somatic mutation of the CD95 gene occurs in a fraction of cHL cases and may
favour the escape of the HRS precursor cells from apoptosis (Re, 2000, Müschen,
2000). In addition, CD40 and CD40L interaction also results in Fas-mediated apoptosis.
The anti-apoptopic gene bcl-2 is up-regulated in about 85% of follicular lymphomas
(FL) and approximately 20% of DLBCL mainly due to the t(14;18) translocation
(Korsmeyer, 1999, Skinnider, 1999, Nathwani, 2001, Gatter, 2001). Overexpression of
BCL-2 inhibits apoptosis and has been correlated with multi-drug resistance in in vitro
lymphoma and lymphocytic leukaemia cell lines (Reed, 1994). The presence of t(14;18)
has not been demonstrated in HRS cells but in bystander lymphocytes (Gravel, 1998),
whereas mRNA transcripts and over-expression of the protein have been detected
(LeBrun, 1994, Hell, 1995, Gravel, 1998) in about 50% of HL cases. An increased
expression of BCL-2 in HRS cells and a low expression in the bystander lymphocytes
have been associated with drug-resistance in HL (van Spronsen, 2000).
Proteins involved in cell cycle regulation are important in many types of cancer.
The tumour-suppressor gene p53 is involved in the induction of apoptosis, including
apoptosis induced by chemo- and radiotherapy (Hollstein, 1991, Giaccia, 1998,
Canman, 1998, Roth, 2001, Robles, 2001). p53 is also involved in cell cycle control and
regulation of angiogenesis (Giaccia, 1998). Overexpression of p53 protein and
mutations of the gene are frequently involved in human cancers.
In aggressive NHL, immunohistochemical overexpression of p53 implying high
intracellular levels of p53, but not necessarily presence of a mutated gene, predicts
treatment failure (Villuendas, 1993, Piris, 1994, Ichikawa, 1997, Navaratnam, 1998,
Nieder, 2001). Furthermore, in NHL, p53 mutations are associated with drug resistance
(Newcomb, 1995, Wilson, 1997). High levels of p53 expression have been detected in
the HRS cells at diagnosis with conflicting results on the relevance for clinical outcome
and prognosis (Xerri, 1994, Brink, 1998, Smolewski, 1998, Nieder, 2001).
16
Overexpression of p53 protein has been found in a high percentage of HL, but a
correlation between overexpression of p53 protein and the presence of p53 mutations
has not been demonstrated (Battifora, 1995, Chen, 1996, Elenitoba-Johnson, 1996,
Montesinos-Rongen, 1999, Lauritzen, 1999). It is not clarified why p53 frequently is
expressed in HRS cells (IV, Brink, 1998, Smolewski, 1998) and what the relevance is
for the clinical outcome and prognosis. Mutations in p53 have been demonstrated both
in EBV positive and in EBV negative cases of micro-manipulated HRS cells in a
frequency of about 15% (Poppema, 2000). These data suggest that p53 mutations in
addition to EBV infection of HRS cells might be early transforming events in HL cases.
The retinoblastoma tumour-suppressor protein (Rb) plays a critical role in cell cycle
control by determining the cell fate following DNA damage. Rb is inactivated in many
human malignancies, chiefly through phosphorylation. Rb is a ”conditional” tumour
suppressor, i.e in order for tumour cells to take advantage of the loss of Rb they must
also inactivate the apoptosis mechanisms that are inhibited by Rb (Kaelin, 1999,
Knudsen, 2000, Wang, 2001). The loss of Rb is almost always complemented by
mutations that inactivate apoptosis. The expression of Rb in NHL has not revealed any
prognostic information (Korkolopoulou, 2001). The expression of Rb in HL is not much
studied, however, loss of Rb expression at diagnosis is related to a worse prognosis in
HL (Morente, 1997, Montalban, 2000).
Recently, a novel putative tumour suppressor, ARF was identified that may provide an
important link between p53 and the p16INK4a-Rb pathway (Gronbaek, 2000). ARF and
p16INK4a are encoded by a single genetic locus, designated INK4a/ARF at chromosome
9p21. p16INK4a controls cell cycle progression by inhibiting phosphorylation of Rb,
while ARF prevents MDM2 degradation of p53. Furthermore, a concurrent disruption
of the p16INK4a and the ARF pathway predicts poor prognosis in NHL (Gronbaek,
2000). The importance of ARF in HL has not been studied.
In summary, there are many ways by which HRS precursor cells may escape apoptosis,
e.g. CD30, CD40, and LMP-1 mediated NFκB signaling, interaction between CTLs
expressing Fas ligand and the Fas/apo-1/CD95 pathway, BCL-2 expression, and
mechanisms involving p53 and Rb. However, it is not clarified which pathway is of
greatest importance in this intricate network and it is possible that the different
pathways also interact and thereby contribute to the resistance to apoptosis that is
observed in HRS cells. Furthermore, several signaling pathways might also,
contradictory, be activated at the same time, which might be the reason why HL is
curable by chemotherapy and radiotherapy since these treatments target and initiate
apoptosis in the HRS cells and their precursors. Mechanisms for therapy-resistance
needs further study, but probably the different apoptotic pathways are involved.
17
BCL-6
The HRS cells originate from GC derived B-cells in most cases.
The proto-oncogene bcl-6 is a nuclear zinc-finger transcription factor that is expressed
by virtually all GC B-cells and CD4+ T-cells within the GC (Cattoretti, 1995). BCL-6
acts by controlling the GC formation and represses Th2-mediated inflammatory
response. The bcl-6 gene is frequently involved in translocations affecting the
chromosomal band 3q27 and is rearranged in about 30% of DLBCL (Gatter, 2001). A
favourable clinical outcome for patients with DLBCL has been associated with BCL-6
protein expression, probably supporting the GC derived origin of DLBCL (Lossos,
2000, 2001). The presence of BCL-6 protein expression has been demonstrated in all
NLPHL cases investigated and in about 30% of cHL (Falini, 1996, Kraus, 2000). Tcells in NLPHL were strongly BCL-6+, but lacked CD40L, whereas in cHL the
surrounding T-cells were BCL-6- and CD40L+ (Seitz, 2001). CD40 signaling downregulates BCL-6 expression (Allman, 1996).
Cytogenetics
Cytogenetic analysis of HL is difficult since there are few HRS cells in the tumour and
they have a low mitotic rate. Conventional cytogenetics and fluorescence in situ
hybridisation (FISH) usually show polyploidy, which is consistent with the
multinucleated HRS cells. So far, no chromosome aberration specific for HL has been
found and most karyotypes described are hyperdiploid with several extra chromosomes,
frequently with complex chromosomal changes (Sarris, 1999). A number of nonrandom changes, including several that are also common in NHL, are frequently
present. Interestingly, the chromosomal bands 14q32 (the IgH locus), 11q13 (CCND1),
11q23 (IL10R, MLL, OBF1), 6q21 and 8q24 (c-myc), well-established breakpoints in
lymphoid malignancies are frequently involved in HL in earlier studies (Thangavelu,
1989, Sarris, 1999). In review of earlier studies, it must be taken into consideration that
some of the cases included might have been NHL instead of HL. Thus, these data must
be interpreted with caution. Using cell sorting techniques and microdissection, purified
samples may be obtained that allow investigation of small amounts of HRS cells.
In addition to triploidy and tetraploidy, deletions of 1p, 6q, 7q and loss of 4q25-27 are
commonly seen (Atkin, 1998, Falzetti, 1999). With comparative genomic hybridisation
(CGH), recurrent gains of the chromosomal sub-regions on chromosomal arms 2p, 9p,
12q and distinct high-level amplifications on chromosomal regions 4p16, 4q23-24 and
9p23-24 have been demonstrated (Joos, 2000). In another study, applying the CGH
technique the most commonly observed genetic aberrations were a loss on 16q11-21, a
gain on 1p13 and a gain on 7q35-36 (Ohshima, 1999).
Considerable variation from case to case has also been detected (Thangavelu, 1989,
Weber-Matthiesen, 1995, Atkin, 1998, Ohshima, 1999, Falzetti, 1999). A higher
number of chromosomal breakpoints have been detected in patients in more advanced
stages of HL (Falzetti, 1999) and karyotypes of HL cell lines are highly complex with
several non-random abnormalities (Drexler, 1992).
18
Clinical presentation
The typical patient with HL is a young adult who presents with enlarged lymph nodes in
the neck and chest X-ray often reveals an enlarged mediastinum. Also, abdominal
glands may be involved as well as extranodal sites, the most common being bonemarrow and lungs.
Clinical staging
The outcome in HL is strongly associated with the extent of the disease and thus
adequate staging procedures are of utmost importance. The staging definition generally
accepted worldwide is the Ann Arbor classification (Carbone, 1971) modified in
Cotswolds in 1989 (Lister, 1989).
The Ann Arbor classification differs between a clinical (CS) and a pathological stage
(PS) (table 2), the former being based upon non-invasive and clinical methods for
example X-rays and computerized tomography (CT) scan, in contrast to the latter,
which also includes histopathological examination after laparatomy with splenectomy.
Table 2. The Ann Arbor classification, modified in Cotswolds
Stage I
Stage II
Stage III
III:1
III:2
Stage IV
Involvement of a single lymph node region or lymphoid structure (e.g spleen,
thymus, Waldeyers ring) or a single extranodal site (IE).
Involvement of two or more lymph node regions on the same side of the
diaphragm or localised involvement of extranodal organ/tissue and one or
several lymph node regions on the same side of the diaphragm (IIE).
(the mediastinum is a single site)
Involvement of lymph node regions or structures on both side of the diaphragm.
With or without involvement of splenic, hilar, coeliac, or portal nodes.
With involvement of the paraaortic, iliac and mesenteric nodes.
Involvement of one or more extranodal site(s) in addition to a site for which the
designation ”E” has not been used.
A
B
No symptoms
Presence of any of the following symptoms:
1. Unexplained weight loss of >10% of the body weight in the preceeding
6 months.
2. Unexplained fever, with temperature above 38oC.
3. Night sweats.
E
Involvement of a single extranodal site, contiguous or proximal to known nodal
site
Bulky disease: A peripheral lymph node or region greater than 10 cm or a
mediastinal mass larger than 1/3 of the mediastinum at the level of the thoracic
verebral body Th5-6 on chest X-ray.
Clinical stage
Pathological stage (as determined by staging laparatomy and splenectomy)
X
CS
PS
19
Inclusion of staging laparatomy was introduced by Glatstein (Glatstein, 1970) but today
laparatomy with splenectomy has generally been abandoned (Foss Abrahamsen, 1996,
Carde, 1999). A staging laparatomy is associated with both acute and late morbidity
(Askergren, 1980, Carde, 1999). However, the major reason for the abandonment of this
invasive procedure is that the information provided is no longer needed. Limited
chemotherapy is, e.g used more and more also in early stage HL.
The diagnosis is based on a careful histopathologic examination complemented by
immunostainings if necessary. Staging procedures include a detailed medical history
especially regarding the presence of B-symptoms and a careful physical examination
with assessment of enlarged peripheral lymph nodes. Blood-sampling, chest X-ray, CT
scan of the thorax and the abdomen and bone-marrow biopsy, unless the disease is
localised, are also performed.
Treatment of early and intermediate stages
Patients in early stages (IA, IB, IIA) have an excellent prognosis with a long-term
survival of approximately 90%. These patients have traditionally been treated with
radiotherapy alone (Henry-Amar, 1990, Foss Abrahamsen, 1996, Brandt, 2001). The
number of recurrences decreased when larger radiation fields were used, which was first
observed by Gilbert in 1931 (Gilbert, 1931) when he introduced the principles of
treating uninvolved lymph node stations next to involved regions. Most patients with
supradiaphragmatic disease have thus been treated with extended-field radiotherapy, i.e.
mantle field, with or without the addition of a paraaortic field; subtotal nodal irradiation
(STNI). Some patients were treated with a total nodal irradiation (TNI). Patients with
infradiaphragmatic disease have been treated with inverted-Y fields.
An increased risk of fatal late complications such as secondary malignancies and
cardiac toxicity has been observed (Henry-Amar, 1992, Joensuu, 1993, Hancock, 1996,
Travis, 2002) and it is possible that the causes of deaths in the younger patients with
early stage HL is due to late complications to treatment, rather than deaths due to HL.
Evidence indicates that extended-field radiotherapy is responsible for at least some of
these late complications. It has been recognised that combination of radiotherapy and
chemotherapy would render less treatment failures. Since salvage chemotherapy is
usually very effective in relapsed patients, the overall long-term survival has not been
much improved (Specht, 1998, Brandt, 2001).
Today, the extended field radiotherapy has been abandoned and a combination of a
short course of chemotherapy and involved-field radiotherapy is given in order to
reduce toxicity (Andrieu 1980, 1985, Glimelius, 1994, Foss Abrahamsen, 1996, Sieber,
2002). The type of chemotherapy regimen and the radiotherapy dose is investigated in
many ongoing trials. In the Nordic countries, two to four courses of ABVD
(doxorubicin, bleomycin, vinblastine, dacarbazine) followed by involved-field
radiotherapy (30 Gy) is recommended. The number of courses is governed by the
presence or absence of risk factors; bulky disease, three or more involved sites, or
erythrocyte sedimentation rate (ESR) >50 mm. In Sweden, this strategy was already
introduced in 1984, and long-term follow-up shows a very favourable outcome with few
late complications (Molin, personal communication).
20
Treatment of advanced stages
Patients in advanced stages (IIB-IV) are treated with a full course of chemotherapy
(generally eight courses) often in combination with involved-field radiotherapy (if there
is presence of bulky disease at diagnosis or residual disease). However, despite
improved treatment results achieved in HL, these patients have a worse outcome and
only about 50-70% of the patients with advanced stages will reach long-term diseasefree survival (DFS) (DeVita, 1980, Longo, 1986, Oza, 1992, Urba, 1992, Viviani, 1996,
Glick, 1998, Wolf, 1998, Brandt, 2001, Diehl, 2001, Raemaekers, 2002). More than 8090% of the younger patients will, however, reach a first complete remission (CR).
The golden standard for treatment of advanced stages was for a long time MOPP, which
was introduced by DeVita in 1964 (DeVita, 1967, 1970). In the 1970s Bonadonna
introduced the ”non-cross resistant” regimen ABVD (Bonadonna, 1975) which induced
treatment responses in patients failing on MOPP. Results with ABVD were not inferior
to those of MOPP, when a comparison was made in a randomised study (Bonnadonna,
1975).
An advantage concerning both remission rates and DFS using alternating MOPP/ABVD
or ABVD treatment compared to MOPP alone was shown in a large study (Canellos,
1992). However, dose-reductions of MOPP were done, and a later study could not show
a survival disadvantage for MOPP compared to MOPP/ABVD (Somers, 1994). ABVD
is today considered the golden standard against which all new treatment protocols
should be compared. The reason for this is to avoid the unnecessarily high risk of
secondary leukaemias associated with MOPP. In order to further improve the results,
seven or eight drug regimens like MOPP/ABV has been introduced with apparently
superior results (Viviani, 1996, Duggan, 1997, Raemaekers, 2002, Canellos, 2002).
Other examples of new regimens are VAPEC-B (vincristine, doxorubicin, prednisolone,
etoposide, cyclophosphamide, bleomycin) (Radford, 1994), Stanford V
(mechloretamine, doxorubicin, vinblastine, vincristine, bleomycin, etoposide,
prednisone) (Horning, 1996, 2002) which gives promising results, CHOPE
(cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide) (Lester, 2001) and
MOPPEBVCAD (mechloretamine, lomustine, vindesine, melphalan, prednisone,
epidoxorubicin, vincristine, procarbazine, vinblastine, bleomycin) (Gobbi, 1998). The
German Hodgkin Study Group (GHSG) has recently completed a large clinical trial,
HD9, that addressed dose intensity. This study included 1180 evaluable patients with a
median follow-up of 40 months, when it was presented in Cologne, Germany, 2001
(Diehl, 1997, Tesch, 1998, Diehl, 1998, Diehl, 2001). Randomisation between standard
eight courses of COPP/ABVD (arm A), eight courses of BEACOPP (bleomycin,
etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone)
baseline (arm B) or eight courses of BEACOPP escalated (arm C) was made. Local
radiotherapy targeting initial bulky disease and/or residual disease followed
chemotherapy.
Very promising and apparently superior results were reported with failure-free survival
rates of 70%, 79% and 89% in arms A, B and C, respectively, at three years and overall
survival (OS) rates of 86%, 91% and 92%, respectively. Thus, intensification with doseescalated BEACOPP shows superior failure-free survival but did not, however show an
advantage in OS. A higher rate of acute myeloid leukaemias, and as expected, greater
haematologic toxicity was also shown to be associated with this regimen. Long-term
21
follow-up will shed light on whether a switch in therapy for the advanced stages should
be generally recommended.
Are eight courses of chemotherapy the golden standard?
Only one randomised study has addressed the question of the importance of the number
of courses for cure (Björkholm, 1995). In this study an early interim analysis showed a
progression-free survival disadvantage for those who received individual treatment
compared to the fixed-treatment arm and thus the study was closed. However, no
statistically significant OS disadvantage was observed after five years for the individual
treatment group. The statistical power of the study was restricted as a limited number of
patients were included. A fixed number of eight courses of chemotherapy has been
applied on all recent trials (Loeffler, 1998, Glick, 1998, Diehl, 1998, Raemaekers,
2002). In the Groupe d’Etudes des Lymphomes de l’Adulte (GELA) H89 trial, eight
courses of chemotherapy was concluded to be preferred when a CR was achieved after
six courses (Fermé, 2000). Doubling of the chemotherapy duration (12 months) did not
show any survival advantage compared to six months (Coleman, 1998).
The International Prognostic Score
Data from 25 centres and study-groups altogether including 5141 patients that were
treated with combination therapy for advanced stages, was used to create an
International Prognostic Score (IPS) (Hasenclever, 1998) and has gained wide
acceptance. Median five-year OS was 78% (range 56 to 90%) in the total material and
was based upon the number of adverse prognostic factors (male gender, age ≥ 45 years,
stage IV disease, serum albumin level ≤ 40g/L, haemoglobin level < 105 g/L,
leucocytosis ≥ 15x109/L and lymphocytopenia < 0.6x109/L or < 8% of white blood cell
count). Five-year freedom from progression rate ranged between 42% (five or more
factors present) to 84% (no adverse prognostic factor present).
Reports from the GHSG (Diehl, 2001) have shown an almost total eradication of the
need for the prognostic score after the introduction of the BEACOPP-regimen. This has
also been the case for other identified markers, like for instance the impact of bulky
disease, which almost is eradicted due to intensified treatment (Glimelius, 1994, II,
Hasenclever, 1998).
Primary high-dose chemotherapy?
The question has been raised of the possibility of identifying at diagnosis a high-risk
group of patients who should be primarily treated with high-dose chemotherapy
(HDCT) followed by autologous stem-cell transplantation (ASCT) (Armitage, 1994,
Fisher, 1996). Since the treatment results after conventional chemotherapy usually are
quite successful, it would be easy to study HDCT if one could define at diagnosis the
25-35% of patients who will fail to respond to the initial treatment. Unfortunately, in the
IPS, a distinct group of patients at very high risk could not be identified. A randomised
trial comparing HDCT followed by ASCT against conventional therapy for those
responding to four courses of ABVD or ABVD like regimens did not show any benefit
for those treated with HDCT (Federico, 2001). Several trials are ongoing that are testing
dose-intensification that also require stem cell support in high-risk patients.
22
What is the role of adjuvant radiotherapy?
The reason for the use of radiotherapy as an adjuvant after a full course of
chemotherapy includes the fact that relapse after chemotherapy occurs primarily in
initially involved sites. The additive effect of radiotherapy in advanced stages has been
much debated (Raemaekers, 1997, Canellos, 2002). A meta-analysis carried out showed
that the use of radiotherapy was associated with significantly poorer OS (Loeffler,
1998). In the German HD-3 trial, no survival difference was observed between those
treated with chemotherapy compared to radiotherapy as consolidation (Diehl, 1995).
In many studies, radiotherapy has been given with very varying extent, i.e. in the GHSG
HD-9 trial, 69% of the patients received radiotherapy. In the GELA H89 trial, there was
no difference in treatment outcome between patients in CR or patients in good partial
remission (PR) after six courses of chemotherapy treated with additional radiotherapy or
chemotherapy (Fermé, 2000).
In a recent European Organization for the Research and Treatment of Cancer (EORTC)
study (#20884) (Raemakers, 1997, 2001), no benefit from using involved-field
radiotherapy in patients with stage III-IV disease was seen in those who had reached a
CR. Patients in PR after six courses of MOPP/ABV were treated with involved-field
radiotherapy giving an apparently comparable outcome to those who reached a CR
(Raemaekers, 2002). In the ongoing HD12 trial by the GHSG, patients are randomised
between either eight courses of escalated BEACOPP or four courses of BEACOPP
escalated in addition to four courses of BEACOPP baseline. These patients are then
subsequently treated with radiotherapy to initial bulky disease and residual disease or
else given no further treatment (Diehl, 2000). No preliminary results have so far been
reported.
Prognostic markers of tumour-biological importance
The study of additional prognostic information in HL is of great importance. Although
the majority of patients will be cured, the goal must be to continously improve the
treatment results. A proportion of the patients will be over-treated and thereby at a
higher risk for secondary late complications (Björkholm, 1995). On the other hand,
some patients will need an intensified treatment from the start in order to control
refractory or progressive disease. Using markers of potential tumour-biological
importance, it will be possible to add prognostic information beyond that given by
clinico-pathological variables and thereby, hopefully, diminish the risk of treatment
failure. Such biological markers reported are, for example, infiltration of eosinophils
into tumour tissue correlating serum levels of eosinophil cationic protein (ECP)
(Enblad, 1993, Axdorph 2001, von Wasielewski, 2000, Molin, 2001), infiltration of
mast cells (Molin, 2001), S-IL10 (Axdorph, 2000), beta-2 microglobulin (Dimopoulos,
1993), S-IL2 R (Enblad, 1995), and S-CD30 (Enblad, 1997).
The difficulty is, however, proper validation of this information, since the markers often
correlate and only add limited information to the prognostic information of other
already known factors. Even if the study of new markers does not turn out to give new
clinical information, it can expand the knowledge of the tumour biology and the
behaviour of the tumour cells. This is even more important to understand in patients
with primarily therapy-resistant, refractory, relapsed or progressive disease.
23
Relapses and refractory disease
Patients who relapse after primary treatment with radiotherapy alone can successfully
be treated with conventional chemotherapy and thereby reach long-standing remissions
in about 60-70% of cases (DeVita, 1980, Henry-Amar, 1990, Hoppe, 1992, Healey,
1993, Horwich, 1997, Brandt, 2001). In some cases of relapse after primary
radiotherapy, a new CR can be achieved with radiotherapy if the relapse localisation is
limited and outside previously irradiated areas. Treatment outcome is worse for those
who relapse after primary chemotherapy (Longo, 1992, Brice, 1996, Bonfante, 1997,
Canellos, 1998, Josting, 2002). Therapeutic options for these patients include salvage
radiotherapy, salvage chemotherapy and HDCT followed by ASCT. Retreatment with
the same regimen that first induced a CR can be successful if the relapse occurs after
more than 12 months after completed primary treatment (Canellos, 1990, Fermé, 1995).
The choice of salvage chemotherapy will usually be non-cross-resistant regimens like
ABVD, MIME (methyl-GAG, ifosfamide, methotrexate, etoposide), DHAP
(dexametasone, cisplatin, araC), mini-BEAM (carmustine, etoposide, cytarabine,
melphalan) or CEP (CCNU, etoposide, prednimustine).
If the relapse occurs within one year after completed primary treatment, the outcome is
worse then if the relapse is diagnosed after more than 12 months (Salvagno, 1993, Lee,
1997, Horning, 1998, Garcia-Carbonero, 1998, Josting, 2002). Patients who fail to reach
a first CR or develop progressive disease (PD) during induction treatment have an
extremely poor prognosis (Lee, 1997). These patients with primary resistance or early
relapsing patients will constitute about 20-25% in larger patient materials and present
several treatment challenges. The results of ten studies have been summarised
concerning primary refractory and early relapsing HL (Armitage, 1994). An overall,
long term DFS may be achieved in 20-30% of the patients whereas there will be
treatment related deaths in about 10-15% of the patients. A randomised trial of
chemoresistant HL patients (non-responders, relapse within one year or relapse after
second chemotherapy regimen) was interrupted because of a significantly superior
progression-free survival (53% vs 10%) in the patients treated with HDCT compared to
patients not treated with HDCT, however, the OS rates did not differ (Linch, 1993).
Later studies of primary non-responders have, however, reported somewhat higher
survival rates of 35-40% five-year OS (Lazarus, 1999, Sweetenham, 1999, Schmitz,
1999, Lazarus, 2001, Fermé, 2002) with less treatment related deaths. The GHSG and
the European Bone Marrow Transplantation (EBMT) groups performed a randomised,
multi-center trial (HDR1) to determine the benefit of HDCT in relapsed HL. Patients
who relapsed early (3-12 months after initial treatment), late (>12 months) or patients
with multiple relapses were included. Treatment comprised of either four courses of
Dexa-BEAM (dexametasone, BCNU, etoposide, ara-C, melphalan) or two courses of
Dexa-BEAM followed by HDCT and ASCT. Only patients with chemosensitive disease
(CR or PR) after two courses of Dexa-BEAM were randomised. The interim analysis
reported in an abstract of 142 evaluable patients with a median follow-up of 33 months
described a significantly longer time until treatment failure for those treated with HDCT
and ASCT (53% vs 39%) (Schmitz, 1999). There is a wide agreement that patients who
fail to achieve CR or who relapse within 12 months should be treated with HDCT and
ASCT. However, most oncologists would also recommend this treatment to patients
relapsing after a longer initial remission. Even though the value of HDCT has not been
24
documented in large controlled trials with long follow-up (Brandt, 2001), most study
centres will use this therapy, since the results appear to be superior after HDCT.
Approximately 70-80% of chemotherapy-sensitive relapsers have apparently superior
five-year survival after HDCT treatment according to reports from the GELA H89 trial
(Fermé, 2002), the French Registry (Moreau, 1998) and the Autologous Blood and
Marrow Transplant Registry (Lazarus, 2001).
Allogenic bone-marrow transplantation has been performed in relapsed and refractory
HL patients in small series with unfavourable results due to high treatment-related
mortality and confers no benefit over ASCT (Milpied, 1996, Dann, 1997, Sureda,
2001). However, long-standing remissions have been reported in a few patients and the
advantages include haematopoietic rescue with tumour-free marrow, a potential
immune-mediated graft-versus-lymphoma effect and less treatment-related leukaemias.
Thus, allogenic transplantation may be considered in a subset of apparently chemoresistant patients.
Late complications
An increasing number of patients will have long-term survival due to the improved
treatment giving better outcome with excellent survival figures, especially for those in
early and intermediate stages. However, serious long-term side effects will arise in these
successfully treated patients. Above all, the most serious complications is the increased
risk of secondary malignancies, i.e. an increased cumulative incidence of acute nonlymphoblastic leukaemia (ANLL) often preceded by myelodysplastic syndrome (MDS)
of 3-10% over a period of ten years (Pedersen-Bjergaard, 1987, Kaldor, 1990, HenryAmar, 1996, van Leeuwen, 2000, Swerdlow, 2000). The risk is dependent on the type of
chemotherapy given, with a higher risk associated with MOPP and MOPP-like
regimens containing alkylating agents, the doses administered and the duration of the
chemotherapy treatment. The extent of radiation therapy might also be associated with
the risk (Henry-Amar, 1996). The cumulative risk reaches a plateau, ten to fifteen years
after completed primary treatment.
Secondary NHL was first described as possibly linked to HL treatment by Krikorian and
co-workers in 1979 (Krikorian, 1979). An increased risk of developing NHL has also
been confirmed in several other studies (Henry-Amar, 1992, van Leeuwen, 2000,
Swerdlow, 2000). Secondary NHL generally develops about five to fifteen years after
completed primary treatment with a cumulative incidence ranging from 1-5%.
An increased risk of solid tumours varies with a cumulative incidence of 10-15% over a
period of 15 years (Henry-Amar, 1996) and represents about two to three times the
incidence of ANLL and NHL. However, solid tumours are more common in the general
population and thus the increased relative risk is lower than for ANLL and NHL, which
are less frequent. The most common solid tumours are of lung, breast, stomach, thyroid,
bone and melanoma, with radiation therapy being the main risk factor (Swerdlow, 2000,
van Leeuwen, 2000). A recent collaborative study showed that the risk of lung cancer as
a result of radiotherapy was as great as the risk from chemotherapy (Travis, 2002).
Combinations of the two yielded additive effects. Virtually all patients with a secondary
lung cancer had smoked, underlining the cancer risk associated with smoking.
Other non-malignant complications to therapy are late infections due to splenectomy or
irradiation to the spleen, which can cause life-threatening pneumococcal septicemia.
25
Infertility can be caused both by radiotherapy (irradiation directly to the gonads) and
chemotherapy. Almost all men treated with more than two courses of MOPP will be
infertile after treatment, whereas ABVD causes reversible azoospermia in about half of
male patients. In women, amenorrhoea is dependent on the age at therapy, and over
60% of patients above 30 years of age will be amenorrhoic after a full course of
chemotherapy.
The most common therapy-related complication which must regularly be controlled is
hypothyreosis-caused by radiation included in mantle fields affecting the thyroid gland.
Pulmonary dysfunction with pulmonary fibrosis and radiation pneumonitis are
complications appearing one to three months and six to 18 months, respectively, after
radiotherapy. Chronic fatigue is also highly prevalent among long-term survivors
(Knobel, 2001) and has been associated with pulmonary dysfunction.
Cardiovascular dysfunctions such as pericarditis and, more seriously, myocardial
infarctions, are caused by previous treatment with irradiation of the mediastinum. These
complications will hopefully decrease in frequency due to the reduced doses and fields
of irradiation delivered with modern radiotherapy.
Relationship between HL and NHL - tumour progression or therapy-related?
HL and NHL have traditionally been considered as two disease entities. The
simultaneous occurrence of HL and NHL in one individual has been considered
coincidental. This view has, however, been reassessed as HL and NHL appear together
in the same individual, either simultaneously or sequentially, with a greater frequency
than would be expected by chance alone (Harris, 1992, Jaffe, 1994, Jaffe, 1999). Both
HL and NHL have also been diagnosed in the same anatomical place, so called
composite lymphoma (Gonzales, 1991, Jaffe, 1994).
There is an increased risk of NHL in patients successfully treated for HL (Bennett,
1991, Henry-Amar, 1992, Henry-Amar, 1996) and moreover, the risk of HL is also
elevated in NHL patients (Travis, 1992). If the time interval is long between the first
and second lymphoma, the second lymphoma has classically been considered as
therapy-induced. It the time interval is shorter, other mechanisms must be prevailing
and indeed, a clonal relationship has been proven in some cases (Brauninger, 1999,
Küppers, 2001).
In HL patients detection of chromosomal aberrations in normal peripheral blood
lymphocytes have been demonstrated (Barrios, 1988, M’kacher, 2001). This might pave
the way for the development of secondary lymphomas in patients successfully treated
for HL. Both radiotherapy and chemotherapy may add to the genetic instability and a de
novo lymphoma may arise in some of the cases. However, it is more likely that the
second lymphoma is a true progression of the first, where the treatment might aid in the
transformation process. The mechanisms leading to secondary lymphomas after an
initial diagnosis and treatment of another lymphoma are largely unknown, and in most
cases the clonality has not been investigated (Müller-Hermelink, 2001).
In the GHSG evaluation of a more intensive chemotherapy regimen, BEACOPP and
escalated BEACOPP, secondary lymphomas were much less frequent (Rüffer, 2001).
This could possibly mean that all tumour cells were eradicated by the intensive
treatment regimen delivered, or the alternative interpretation is that secondary
26
lymphomas rather are progression of the HL and are simply not induced by a large
amount of chemotherapy.
For the low grade B-cell lymphomas; chronic lymphocytic leukaemia (CLL) and FL,
progression to DLBCL is considered part of transformation of the original tumour clone
or in some cases an unrelated second lymphoma (Richter, 1928, Horning, 1984, Giles,
1998, Ohno, 1998, Matolcsy, 1999, Müller-Hermelink, 2001). This could possibly also
be the situation for HL.
27
28
AIMS OF THE PRESENT STUDY
The overall aim of the present study was to investigate the relationship between HL and
NHL to better understand the mechanisms involved in tumour progression. Further, it
was to analyse the outcome for younger patients with advanced stages of HL in a
population-based material and explore whether the number of chemotherapy courses
can be individualised based upon early response to treatment.
More specifically the aims were:
To investigate the occurrence of HL and NHL in the same individual in a defined
population over a period of 20 years, and to elucidate whether these patients differ in
clinical, histopathological and immunohistochemical behaviour from patients with
lymphomas not associated with a secondary lymphoma.
To analyse the outcome for younger patients relapsing with HL in an unselected
population-based material and evaluate response to salvage treatment in relation to
primary treatment.
To study the importance of the tumour suppressor genes p53 and Rb in paired samples
of HL at initial presentation and at relapse in order to better understand mechanisms of
tumour progression.
To characterise a novel B-cell line (U-2932) established from a patient that presented
initially with advanced stage of HL and subsequently treated for several relapses of HL
before the diagnosis of NHL, in order to try to elucidate mechanisms of tumour
progression that lead to secondary lymphomas.
29
PATIENTS, MATERIALS AND METHODS
Patients (I-IV)
In Sweden, a Health Care Programme providing clinical guidelines was initiated in
1985 that include all patients with HL in five of six health care regions (Glimelius,
1996). The programme includes all patients above the age of 16 years and is continously
cross-checked with the Regional Cancer Registries, to ensure that virtually all patients
diagnosed with HL are included. The programme is continously evaluated and renewed,
and contains information about adequate staging procedures, treatment and follow-up
data.
In papers I-IV, patients were recruited from this programme in addition to the Swedish
Cancer Registry. Patients included in paper II and III were younger than 60 years at
diagnosis.
Staging procedures (I-IV)
Initial staging included a clinical history, careful physical examination, biopsy of
affected site, laboratory investigations of blood, chest X-ray, CT scan of the abdomen
and if there was known mediastinal involvement CT-scan of the thorax,
ultrasonography of the abdomen, bone marrow aspirate and biopsy in advanced stages.
This information was prospectively recorded on special case record forms, except
results of blood analysis which was collected retrospectively. Stage at diagnosis is
defined according to the Ann Arbor classification (Carbone, 1971).
A CR was defined as disappearance of all disease and PR as reduction of > 50% of all
known tumour mass. Complete remission with residual disease of unknown significance
(CRu) was not clinically defined in the Health Care Programme when initial treatment
was given. Bulky disease was defined as a tumour mass exceeding a diameter of 10 cm
or a mediastinal mass constituting at least one third of the diameter of the thorax at the
level of thoracic vertebral body five to six on chest X-ray.
Treatment recommendations in the Health Care Programme (II, III, IV)
Patients in early and intermediate stages were recommended mantle irradiation (or
inverted-Y field) alone (mini-mantle to those with upper cervical presentation only) in
CS IA, PS IA and PS IIA and STNI in PS IIIA:1 if the disease was not bulky. All
patients in CS IA, PS IA, IIA and IIIA:1 with bulky disease and patients in PS IB, PS
IIB and CSIIA (limited disease, or less than three involved lymph node regions) were
recommended one or sometimes two cycles of MOPP/ABVD before radiotherapy. One
cycle consists of two monthly courses. In 1994 this regimen was replaced by two to four
courses of MOPP/ABV. For all other stages, principally stages IIB-IV, treatment
recommendations were three to four cycles of MOPP/ABVD (six to eight courses of
MOPP/ABV), the number of courses depending upon the response to treatment.
Treatment response had to be evaluated after every second course of chemotherapy with
both clinical and radiologic examination of primarily involved sites. If the patient
reached CR after two courses of chemotherapy, six courses were given in total, in all
other cases eight courses. Consolidating radiotherapy was given after chemotherapy to
patients with initial bulky disease (30 Gy to bulky area), if tumour regression was slow,
30
i.e. CR was not achieved until after six or more courses (30 Gy to all primarily involved
sites excluding bone-marrow), or if CR was not reached (40 Gy to residual disease and
15-30 Gy to involved sites). Staging laparatomy was recommended only in stage IIA
but could be performed in stages IB, IIB and IIIA in order to minimise the treatment; if
there was no HL involvement in the abdomen (PSI-IIB) two courses of chemotherapy
(one cycle of MOPP/ABVD or two courses of MOPP/ABV) were given followed by
radiotherapy. If staging laparatomy was not performed six to eight courses of
chemotherapy were recommended for stages IB, IIA-B and IIIA.
For patients initially treated with radiotherapy only, MOPP/ABVD was recommended
as salvage treatment, and for those treated with brief chemotherapy and radiotherapy,
re-treatment with the same regimen was recommended. Prior to 1988, the Care
Programme did not give any specific recommendations for salvage chemotherapy in
case of relapse after a full course of chemotherapy (6/8 courses). In 1988, a prospective
study with MIME as salvage regimen was started (Enblad, 1998). MIME was, however,
frequently used as second-line therapy for patients relapsing after combination
chemotherapy before 1988 (Enblad, 1990). Patients primarily treated with six to eight
courses of chemotherapy and younger than 60 years in PR or CR after salvage
chemotherapy were recommended HDCT with ASCT. Additional radiotherapy was
given to bulky sites at relapse if possible. For patients who were not candidates for
HDCT, the intention was to treat with MIME until CR and then to give two additional
courses, followed by radiotherapy as above. MIME was also used as palliation in
patients where a curative treatment was not considered possible.
Histopathology and immunohistochemistry (I, IV, V)
A re-examination of histopathological material was performed (I, IV, V) by an
experienced haematopathologist using the Rye-classification for HL (Lukes, 1966) and
for NHL the updated Kiel classification (Stansfeld, 1988) (I) and the REAL
classification (IV, V) (Harris, 1994).
Formalin-fixed, paraffin-embedded tissue material was sectioned at 3-4µm,
deparaffinised and rehydrated. Immunostainings were performed using the avidin-biotin
peroxidase complex (ABC) and AEC procedures (Wood, 1981). Antigen retrieval was
achieved by processing with citrate buffer in a microwave oven.
Tissue specimens were stained to detect the following markers (I, V); CD30 (Ber-H2
clone), CD15, CD45R, LMP-1, L26 (CD20), MB-2 (B-cell related antibody), 4KB5 (Bcell related antibody), CD3, CD45R0 (T-cell related antibody). In paper IV and V:
CD30, ALK-1, LMP-1, p53, Rb. Immunohistochemical staining with Rb was performed
with an automated immunostainer (Ventana 320 ES, Ventana medical systems, Tucson,
AZ).
In situ hybridisation for EBER was performed using kits from Dakopatts and according
to the manufacturers instructions. In short, paraffin sections were cut under RNAasefree conditions. After rehydration, the sections were pretreated with proteinase K for 30
minutes at room temperature. Hybridisation was performed at 55° C for 90 minutes,
using a FITC-conjugated RNA probe. A negative control probe was provided with the
kit. For visualisation alkaline phospatase-conjugated anti-FITC was incubated for 30
minutes at room temperature, followed by incubation with the chromogen substrate
31
NBT-BCIP with levamisole. Only large lymphoid cell nuclei with dark blue/black
labeling were considered positive for EBER.
Cytogenetic analysis
The composed karyotype (V) of U-2932 was based on results from G-banding, reversed
DAPI-banding, CGH and spectral karyotyping (SKY).
In CGH, tumour and reference DNA are labeled with green and red fluorochromes,
respectively, and cohybridised to normal metaphase chromosomes. The difference in
fluoroscence intensities is measured along the length of each chromosome and reflects
the relative copy number. Higher intensity of green fluoroscence reflects chromosomal
gains in tumour DNA, whereas higher intensity of red reflects chromosomal losses.
Twelve metaphase spreads were captured and analysed by digital image analysis. Green
to red ratios exceeding 1.2 were considered as gains whereas ratios below 0.8 were
scored as losses. The cut-off level for high-level amplification was set to 1.5.
SKY allows the simultaneous visualisation of all chromosomes in different colours.
Each chromosome is labeled with one to four different fluorochromes and in total five
different fluorochromes are used. The method combines Fourier spectroscopy
(interferometry), charge coupled device imaging and optical microscopy to measure the
emission spectrum in the visible and near infrared spectral range in all measure points.
This makes it possible to discriminate multiple and spectrally overlapping
fluorochromes.
IgH gene rearrangement analysis
High molecular weight DNA was prepared from ascites and from U-2932 using
standard protocols including proteinase K treatment. Polymerase chain reaction (PCR)
amplification was performed using six family specific VH primers and one JH primer.
PCR-single strand conformation polymorphism (SSCP) analysis was carried out to
compare the clonality of the IgH rearrangement of the tumour cells from the patient and
U-2932. This was performed by a GenePhor system electrophoresis and DNA was then
silver stained. The clonal PCR product amplified from U-2932 was ligated into the
pGEM-T vector and transformation into JM 109 competent bacteria. Plasmid DNA
from five colonies was extracted by plasmid miniprep technique and sequenced using
the BigDye Terminator Cycle Sequencing Reaction Kit. All sequence reactions were
analysed using an automated DNA sequencer. The sequences were aligned to IgH
sequences from the BLAST (National Center for Biotechnology Information, USA) and
the V-BASE databases (MRC Centre for Protein Engineering, Cambridge, UK). VH
gene sequences deviating more than 2% from the corresponding germline gene were
defined as mutated.
Sequence-based analysis of p53
Messenger RNA was prepared from the U-2932 sample, followed by reverse
transcription of the mRNA to complementary DNA (cDNA). p53 was PCR-amplified
from cDNA using four overlapping primer pairs covering the complete coding region of
the p53 gene. Biotin-labelled PCR products were generated by modifying one of the
primers in each primer pair with a biotin molecule facilitating solid-phase sequencing.
32
The sequenced products generated were analysed using an automated laser fluorescence
DNA sequencer, and sequence was compared against the sequence of wild-type p53.
Statistical methods (I-IV)
The survival curves were generated using Kaplan-Meier analysis.
Cause (HL)-specific survival was calculated from the time of diagnosis (relapse in paper
III-IV) to death from disease. Patients who died from causes other than HL were treated
as censored, provided they were in CR, whereas patients with known disease were
considered as having died from HL irrespective of the actual cause of death. DFS was
calculated from the time of diagnosis to the time of relapse. Deaths due to other causes
than HL were censored in the analysis. If no CR was obtained, DFS was zero. The OS
was calculated from the time of diagnosis to death of any cause. Event-free survival
(EFS) was calculated from the time of relapse to the time of second relapse, secondary
malignancy, or death from any cause.
Differences between survival curves were analysed by the log-rank test. Contingency
tables were analysed by the use of chi-square statistics. Uni- and multivariate analysis
were performed with the Cox proportional hazard model to assess differences between
curves while adjusting for other covariates. For analysis of the different stages and
histopathological subtypes, each stage/subtype was tested against the rest of the cohort.
Missing data were dealt with by carrying out ”complete case analysis”, excluding
patients for whom complete data for the respective analysis was not available.
Both the forward selection method in addition to the backwards elimination were
performed. For all the above mentioned analysis the Statistica 5.5 software StatSoft,
Tulsa, USA was used.
33
RESULTS AND DISCUSSION
HL and NHL (I)
Thirty-two patients with both HL and NHL in Sweden were identified during the time
period 1974-1994. It is likely that this number represents the majority of the patients
diagnosed with both lymphomas. During these 21 years, 4,188 patients were diagnosed
with HL and 19,559 patients with NHL. Thus, the simultaneous appearance of both
lymphomas in the same individual still remains unusual. The intention was, however,
not to study the relative risk of both HL and NHL; this question is better addressed by
other study designs.
HL was followed by NHL in 18/32 (56%) of the cases. Our cases differed from other
HL and NHL since many of the HL expressed a T-cell phenotype. In twelve (67%) of
these, the HL was of advanced stage at diagnosis. These patients received chemotherapy
and in a few cases complemented with additional radiotherapy. The time interval
between the diagnosis of HL and NHL ranged between four and 168 months with a
median of 54 months. NHL following HL were high-grade malignant (16/18) and often
of T-cell phenotype (7/18 cases).
Twelve (38%) patients had NHL followed by HL. NHL was often (10 patients) lowgrade malignant and untreated in four cases. HL following NHL was of advanced stage
in 10 (83%) patients. HL was preceded by B-CLL in seven cases and an interesting
finding was that there was no trace of CLL in six of seven cases when the HL was
diagnosed.
Two patients had HL and NHL diagnosed simultaneously. In one patient the NHL was a
high-grade malignant B-cell lymphoma and in the other ALCL.
In most cases, the second lymphoma, independent of whether it was HL or NHL,
appeared in an aggressive clinical form, disseminated, often with extranodal
involvement and with a fatal outcome (figure 1).
34
Lymphoma specific survival
Complete
Censored
5
7
100
90
80
Percent survival
70
60
50
40
30
20
10
0
1
2
3
4
6
8
9
10
11
12
13
Survival time (years)
Figure 1. Lymphoma specific survival in all patients with both HL and NHL (n= 32).
There are several plausible explanations for the development of HL and NHL in the
same patient; chance alone, therapy-induced, tumour biological transformation and
immunodeficiency-related.
If the secondary NHL had arisen independently of HL, distribution of NHL subtypes
similar to that in the general population, should have been expected. This was, however,
not the case.
One possible reason for the simultaneous occurrence of both HL and NHL is therapyinduced lymphomas arising from a non-related transformed lymphocyte, however, the
knowledge about therapy-induced lymphomas is somewhat sparse. The risk of
secondary lymphomas after treatment for other malignancies is also unknown.
Indications that the NHL might be therapy-induced include the rather long time interval
between diagnosis of the first and second lymphoma, and also the previous treatment
given to HL with advanced stages. However, in approximately one third of the cases,
the time interval was only one year and in these cases it is even more unlikely that the
secondary lymphoma is caused by therapy.
The NHL following HL appeared in an aggressive clinical form, with systemic
symptoms and extranodal involvement, which is more indicative of a progression of the
first lymphoma, rather than therapy-induction as the underlying mechanism. However,
for therapy-induced acute leukaemias, an aggressive clinical course is expected (Leone,
2001).
The development of HL and NHL in the same individual may be explained by an
alternative possibility- clonal progression. Support for a clonal relationship between HL
35
and NHL was indicated by statistically significant correlations between the expressions
of p53 and LMP-1 between the two lymphomas. One indication supporting the clonal
evolution theory is the finding in the GHSG studies of the chemotherapy regimen
BEACOPP standard and escalated (Diehl, 2000, Rüffer, 2001) after which a lower
incidence of secondary NHL was found. More effective eradication of all tumour cells
therefore seems to indicate a lower risk also of secondary NHL.
Since a large proportion of NHL following HL were of T cell phenotype, this should
imply that the corresponding HL cases also are of T cell phenotype. A T-cell phenotype
is expressed in a subset of HL cases (Stein, 2001). However, a T-cell phenotype of HL
does not necessarily imply a T-cell origin, but rather an aberrant antigenic expression
(Marafioti, 2000). T-cell lymphomas have previously been reported to be less often
associated with HL than B-cell lymphomas (Jaffe, 1994, Jaffe, 1999). The reasons for
this may be either that the coincidence is underreported due to difficulties in
distinguishing HL from PTL, or because B-cell NHL is indeed more common in HL
patients as HL often are of B-cell origin. In one case of HL associated with PTL
cytogenetic evidence of two distinct clones was demonstrated (Wlodarska, 1993).
For low-grade NHL followed by HL, in which one third of the patients were untreated,
it is even more likely that there is a clonal progression. This could be analogous to
tumour progression of FL (Horning, 1984), where previous treatment appears not to be
of major importance for progression. The phenomenon of CLL disappearing when HL
is diagnosed has been described earlier (Williams, 1991, Momose, 1992, Ohno, 1998)
and also the Richter-transformation of CLL might be part of this phenomenon (Ohno,
1998).
Immunological defects are known to be present in both HL and CLL patients, but the
role of immunodeficiency in the development of HL can only be speculated upon. In
addition, when a clonal relationship is absent, lymphomagenesis of HL and NHL may
be caused due to a genetic disruption of immunoregulatory pathways.
Misclassifications in a few cases are possible, however, this error has been minimised as
histopathological re-examination was performed by an experienced haematopathologist.
HRS cells are positive for CD30 in nearly all cases, but in this material some HL cases
were CD30 negative. This may be attributed to the fact that stainings were performed on
paraffin-embedded material and not fresh-frozen, and thus the epitope may have been
degraded or was blocked in some way. In addition, some of the cases were from the
1970-80s and during this time formalin used in the fixation process was not buffered.
NLPHL is particularly difficult to distinguish from the T-cell/histiocyte rich variant of
DLBCL. However, in this material only two of the included cases were lymphocyte
predominant HL; one had a simultaneous NHL and the other was followed by a highgrade T-cell lymphoma.
Re-examination with additional immunostainings was carried out on all cases for which
histopathological material was available. These cases were then subjected to an updated
reclassification in co-operation with Prof Hansmann in Frankfurt, Germany in order to
further investigate the clonal relationship with microdissection of single HRS cells.
Preliminary reports from this reclassification show that one of the CLL cases is mantle
cell lymphoma and one HL is actually a T-cell rich DLBCL, however, this case was
interestingly followed by a PTL.
36
Advanced stages (II)
A large and homogenously treated population-based patient material was studied. All
patients with advanced stages younger than 60 years were registered in five of six health
care regions in Sweden. Stage IIB was included among these advanced stages, even
though many study centres include only stage III-IV in the group of patients considered
to have advanced stage of the disease (Diehl, 1998, Raemakers, 2001, 2002, Fermé,
2000). The reason for including stage IIB was that these patients comprise an
unfavourable group of patients often having a worse outcome and high relapse rate,
especially if several supradiaphragmatic lymph node stations are involved at diagnosis
or if bulky disease is present (I Glimelius, 2002 in preparation). The decision to study
only the patients younger than 60 years at diagnosis was mainly because during these
years in Sweden, 60 years was the upper age limit for those considered to be in the
”elderly” unfavourable group of patients, as the outcome for patients above 60 years is
worse (Enblad, 1991).
In total, 307 patients were included in the study, and of these 267 (87%) reached a first
CR. The projected ten-year HLS was 80%, the OS was 76% (figure 2) and the DFS
67%. The overall high CR and survival rates are comparable with other groups of
patients reported from specialised treatment centres (Glick, 1998, Hasenclever, 1998,
Raemaekers, 1997).
Overall survival
Complete
Censored
5
7
100
90
80
Percent survival
70
60
50
40
30
20
10
1
1
2
3
4
6
8
9
10
11
12
13
Survival time (years)
Figure 2. Overall survival in all patients (n=307) with primarily advanced HL diagnosed
in Sweden between 1985-1992.
37
The recommendation to individualise treatment was based upon the idea that early
responding patients need less treatment for cure, and that more chemotherapy will
increase the risk for MDS/secondary leukaemias and possibly also NHL.
A full course of chemotherapy (six to eight courses) with or without additional
radiotherapy was initially planned for 270 patients. In order to specifically evaluate the
importance of the number of courses for treatment outcome, the 257 patients who
actually received at least five courses were studied separately. The patients were divided
into two groups; 92 patients who received six courses of chemotherapy and 165 patients
who received eight courses of chemotherapy.
The main difference between the two groups was a higher frequency of bulky disease in
those who received eight courses. There was no difference when applying the IPS
between the two groups (71% vs 68% had 0-2 risk factors in the six and eight courses
group, respectively). The CR rate of approximately 90% was the same in both groups
and furthermore there was no difference regarding survival (figure 3). Encouringly, the
method of selection of patients for the shorter regimen seems to have worked based on
the lack of difference in the treatment outcome. The decision to give two extra courses
of chemotherapy mainly seemed to be based upon the speed by which a tumour
response was reached. However, bias of selection may be present anyway, since the
treatment decision for each patient was made by their clinician.
It can, however, not be excluded that the group of early responding patients could have
had an even better survival if they had been treated with eight courses.
Hodgkin specific survival
Complete
Censored
100
90
80
Percent survival
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
8 CT
6 CT
Survival time (years)
Figure 3. Hodgkin-specific survival in patients with advanced HL treated with six
(n=92) or eight (n=165) courses of chemotherapy, p-value=0.6.
Abbreviations: CT=chemotherapy courses.
38
Additional radiotherapy did not appear to have a significant positive effect on survival
for those who reached CR after the first six courses of chemotherapy, and this is in
concordance with other studies where the value of adjuvant radiotherapy has not been
proven (Loeffler, 1998, Coleman, 1998, Brice, 2001, Raemaekers, 2001). The majority
of patients with bulky disease (87%) received additional radiotherapy in compliance
with the guidelines. This explains why the prognosis was not influenced by bulky
disease, a phenomenon also observed in the IPS study (Hasenclever, 1998) but
previously reported as an adverse prognostic factor (Specht, 1991, Mendenhall, 1997).
Patients treated with radiotherapy actually had a significantly worse (p=0.007) OS (fiveyear survival, 80 vs 90%) than those not treated with radiotherapy, but no significant
differences in HLS or DFS. This could be explained by the fact that radiotherapy was
given selectively to patient groups considered to have less favourable results after
chemotherapy alone. An alternative explanation is that some radiotherapy treated
patients died from late complications, which was also observed.
The late complications reported consisted of seven (2.3%) acute leukaemias, four
(1.3%) NHL, four (1.3%) cardiac toxicities (cardiomyopathia, angina pectoris, cardiac
failure, myocardial infarction), three (1%) solid tumours (colon, lung, sarcoma), 12
(4%) hypothyreosis and six (5%) persistent amenorrhoea. The majority of patients
received MOPP/ABVD treatment which is considered less leukaemogenic than MOPP
(Maurizi Enrizi, 1997). This perhaps offers an explanation for the low number of acute
leukaemias observed. Another explanation could be that relatively few courses of
chemotherapy were administered, however, the number of MOPP (LVPP) courses given
to those afflicted with leukaemia was also low and below six in all but one patient.
Causes of death were HL in 52/65 cases. Causes of death besides HL were all late
deaths: three acute leukaemias, two NHL, two infections (pneumococcal septicemia,
eight years after HL diagnosis in CR and toxoplasmosis encephalitis, one year after HL
diagnosis in CR), one colon carcinoma, one pneumonitis (two years after RT was
finished), one myocardial infarction, one aspiration, one adult respiratory distress
syndrome (ARDS) and one unknown.
NHL misclassified as HL (II, III)
The diagnosis of HL was the originally one made by the regional haematopathologists
or local pathologists, and was often made without immunohistochemical data. Reevaluation has not been performed (II, III), but the criteria for HL diagnosis have,
however, been sharpened since 1985 defining new disease entities, that may sometimes
require immunohistochemistry for differential diagnosis (Bernhards, 1992, Harris, 1994,
Jaffe, 2001, Stein, 2001). Thus it must be taken into consideration that some of the cases
might not be classified as HL, or may be grouped differently, if they were reclassified
today. The relapsed cases were reevaluated (II) and the original HL diagnosis was still
correct in 92% of cases.This is a good indication that the diagnosis of the non-relapsed
cases must also be correct as misclassification would be expected to occur at a higher
frequency in the relapsed cases. Five (8%) cases were identified at the re-evaluations of
all relapsed patients as being NHL instead of HL at the original diagnostic biopsy and
were included among the advanced stage patients in study II and seven (6%) were
included in study III.
39
The reason why these patients were not excluded from the studies was that not all the
cases were possible to re-evaluate, and therefore only a selected number of cases would
be excluded. In addition, comparisons regarding treatment outcome were made against
other patient materials that were also treated during the same time period and these
studies have not been subject to re-examination. It is also well established today that in
older patient series of HL about, 5-10% of the patients studied are in fact NHL
misclassified as HL.
Relapses (III)
Between 1985 and 1995, 124 patients with initial diagnosis of HL, younger than 60
years of age at the time of relapse, were registered up until January, 2001. This age limit
was chosen since 60 years of age was the upper limit for HDCT during that period of
time.
The median time to relapse from the date of completed primary treatment was 2.1 years
(range 0.1-10.5). Four patients had a remission duration of less than three months, a
time limit that has been applied in the GHSG trial (Josting, 2002), but not in the GELA
H89 trial (Fermé, 2002). Primary refractory patients were not included in the study, as
the aim was to evaluate the response to salvage treatment in relation to primary
treatment. The induction failures constitute a group of chemo-resistant patients who
need more effective first-line treatment (Josting 2000).
Fifty-eight patients relapsed after initial treatment with radiotherapy alone. Among
these 58 relapsers, 37 patients were initially treated according to national guidelines.
The survival for those treated according to the guidelines was not inferior to that of
patients with primarily advanced stages - five-year HLS 85% and 86% respectively (II).
However, 21 of the 58 patients were treated with primary radiotherapy alone even
though recommendations stated more extensive treatment due to either bulky disease
and/or B-symptoms. The survival after relapse was poorer in these 21 patients, although
not statistically significant.
Sixty-two patients relapsed after primary combination chemotherapy including 30 who
had received additional involved-field radiotherapy. The outcome was particularly poor
for these 30 patients (figure 4). When this was further analysed it emerged that the
majority of patients treated with a full course of chemotherapy and additional
radiotherapy had bulky disease at diagnosis.
40
Hodgkin specific survival
Complete
Censored
100
90
80
Percent survival
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
RT only
6/8 CT+RT
6/8 CT
Survival time (years)
Figure 4. Hodgkin specific survival in the different groups of patients with relapse
according to initial treatment. Abbreviations: RT=radiotherapy, CT=chemotherapy courses.
Multivariate analysis identified bulky disease as the strongest significant factor
affecting survival without additional information about whether radiotherapy was given
or not. Bulky disease is a well known prognostic factor for HL (Specht, 1991,
Mendenhall, 1997), however, it was not a negative prognostic factor in the total
Swedish material of advanced HL (II) or in the IPS study (Hasenclever, 1998). Thus,
bulky disease at presentation seems to predict poor prognosis at relapse, but doesn’t
predict the outcome of the primary treatment. It is possible that selectively given
radiotherapy in the primary treatment eradicates the relevance of tumour bulk for
prognosis. It is, however, difficult to elucidate the role of initial tumour bulk for the
poor outcome in the salvage situation.
Four patients relapsed after a short course of chemotherapy followed by extended-field
radiotherapy. This very small number of relapsing patients indicates successful selection
of patients for this treatment (during the time period, 95 patients were treated in this
manner and registered) (Glimelius, 1994, 1996). Similar excellent results have also been
reported by others (Andrieu, 1980, 1985, Foss-Abrahamsen, 1996, Sieber 2002),
favouring an international trend to treat all patients in early and intermediate stages
having unfavourable prognostic signs with this regimen. A short course of
chemotherapy followed by radiotherapy should also reduce late toxicity (Rüffer, 1999,
Brandt, 2001), although opposing opinions have been expressed (Gonzales-San
Segundo, 2001).
It is well-known that relapsed patients with a long initial remission have higher CR rates
and better survival than those with short initial remissions (Longo, 1992, Healey, 1993,
Salvagno, 1993, Bonfante, 1997, Garcia-Carbonero, 1998, Josting, 2002). In the present
41
study, early relapses were seen in about 40% and response to salvage treatment was
worse, although not statistically significant, compared to those who relapsed after one
year. Furthermore, there were no statistically significant survival differences, either in
the entire material, or in the different treatment groups (radiotherapy only, a full course
of chemotherapy with or without additional radiotherapy). There was also no survival
difference if the relapse was diagnosed within one year after completed therapy or not,
although it did appear that survival was compromised during the first years after relapse
(and statistically significant when using an alternative statistical test not initially used).
The time to relapse may thus still be of importance for the outcome, although less so
than previously seen. The greater armamentarium of treatments available today can
possibly be responsible for this shift. In earlier studies salvage therapy has usually
included retreatment with the same drugs used primarily, but in the present study,
salvage chemotherapy consisted of a non-cross resistant regimen in most patients.
In the present study less than one third of the patients received HDCT, which is
approximately the same proportion as in the GHSG material (Josting, 2002). The reason
for the relatively low number of patients treated with HDCT is probably because HDCT
was not an established therapy for relapsed HL in Sweden during the 1980s. For
patients who did not receive HDCT, but according to the criteria could have been
candidates for HDCT, EFS but not HLS or OS was statistically significantly lower.
However, firm conclusions can not be drawn, as few patients were treated, selection
mechanisms are unknown and data are non-randomised.
HDCT has become the standard treatment for chemosensitive relapsing HL (Jost, 1997,
Goldstone, 1998, Armitage, 2000, Brandt, 2001, Martin, 2001, Lazarus, 2001, Josting
2002). Superior progression-free survival has been shown in one randomised study
(Linch, 1993), and the interim analysis of a randomised GHSG/EBMT study has so far
shown superior freedom from treatment failure (Schmitz, 1999). Recently, the
promising reults from the GELA H89 trial demonstrated favourable survival (five-year
survival of 76%) of the relapsed patients (Fermé, 2002). Treatment related deaths and
risks of second malignancies associated with HDCT have been studied (André, 1998),
and unfortunately an increased risk of solid tumours must be taken into consideration.
A prognostic score has been presented for relapsed patients in which time to relapse,
clinical stage and anaemia at relapse were important (Josting, 2002). However, since
laboratory values at relapse were not recorded, this score can not be evaluated for this
patient material.
In total, 54 (43%) patients have died, of which 40 (32%) died of HL. The causes of
death besides HL were infections in five patients (all in CR), cardiovascular diseases
(2), disseminated intravasal coagulation (1), NHL (1), other malignancies (3) and two of
unknown causes (one in CR and one with no information of HL status).
We report again a rather low incidence of acute leukaemias and other secondary
malignancies. A secondary malignancy (NHL, acute leukaemia, lung cancer, pancreatic
cancer, mesothelioma of the pleura and disseminated adenocarcinoma with unknown
primary site) was diagnosed in six (5%) patients. This could be due to the tailored
treatment approach, thus limiting treatment for large groups of patients (Glimelius,
1996), as defined in the Health Care Programme.
42
Immunostaining patterns of paired samples at diagnosis and relapse (IV)
In 81 paired samples of HL, immunostainings were performed on biopsies from
diagnosis and relapse, and correlated to clinical data. This investigation was not
restricted to younger patients only, as the aim of the study was to better understand the
tumour biology of HL. Morphological re-examination was performed in 119 cases
where histopathological material was available both at diagnosis and at relapse. Six
cases were excluded from further analysis due to reclassification data; in five cases the
relapse of HL was instead a NHL (”HL followed by NHL”), and in one case the
diagnosis of HL was re-considered as NHL and the relapse was HL (”NHL followed by
HL”). In two patients, the relapse of HL was followed by NHL and these two patients
were included in the study. Histopathological blocks were available for about one third
of the relapsing cases during the time period, and thus a selection of cases was
observed. In some of the Swedish Health Care regions, the relapse diagnosis and
occasionally also the primary diagnosis were based on cytology only. However, a
geographical selection should not interfere with the possibilities of making general
conclusions on the tumour progression.
Clinically, these patients did not differ from other cases of relapsed HL, even though a
slight survival advantage was observed for cases included in this study (not statistically
significant), probably because in some of the patients a better performance status
permitted a biopsy sample to be taken prior to more intensive treatments, like HDCT.
A high proportion of cases were considered HL unclassified subtype at the time of
relapse. This could partly be due to relapse diagnosis being made based on needle
biopsy material, providing insufficient material for subtyping. Extranodal involvement
was also seen, making subtyping difficult. Surprisingly, a switch in HL subtype was
observed in 12 cases. Survival of these 12 patients was statistically significantly inferior
compared to the rest of the cohort (figure 5).
43
Hodgkin specific survival
Complete
Censored
100
90
80
Percent survival
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
No switch
Switch
Survival time (years)
Figure 5. Hodgkin-specific survival in patients where a switch in subtype (n=12) from
diagnosis to relapse was observed compared to those with no switch in subtype (n=44),
p=0.002.
Many possible explanations exist for a switch in subtype, e.g. tumour progression, de
novo HL, or even relapse of a different subtype which could have been present from the
initial diagnosis. The latter is less likely, however not yet investigated, since ”mapping”
of all involved sites is not routinely performed. In cases of tumour progression, it might
seem that NSHL should more likely be followed by MCHL, as this would imply a loss
of functions, i.e. mechanisms responsible for fibroblast activation and sclerosis.
However, most cases seen were MCHL that switched to NSHL, and in no case was a
switch to LDHL observed.
Immunohistochemical stainings with the monoclonal antibodies raised against CD30,
LMP-1, p53, Rb and ALK-1 were performed. In situ hybridisation for EBER was
performed if discrepancies existed between LMP-1 stainings at diagnosis and relapse, or
if LMP-1 staining was of poor technical quality.
Staining for CD30 was negative in a few cases, most probably due to poor quality of
archival paraffin-embedded material, and as stated previously, the tissue fixation
methods on the 1980s were not optimal. Bone-marrow biopsies are also subject to a
decalcifying process that might degrade antigenic epitopes, however, not all bonemarrow biopsies stained negatively. The NLPHL cases were all negative for CD30, as
expected.
The proportion of p53-stained tumour cells both increased and decreased in different
patients between diagnosis and relapse. There was no difference in p53 expression in
relation to histopathology subgroups or clinical characteristics. The prognosis was the
44
same whether p53 increased, decreased or remained the same, and was independent of
the primary treatment.
The overall p53 positivity was higher than found in one previous study of relapsed HL
that included 14 cases (Naresh, 1997). Different results concerning the prognostic
importance of p53 in primary HL have been reported (Brink, 1998, Smolewski, 1998,
Kanavaros, 2000, Montalban, 2000). An up-regulation of p53 has correlated to a
favourable prognosis, therefore assumed to be wild-type (Brink, 1998) but high levels
of p53 protein expression (probably mutant) (Smolewski, 1998) have negatively
influenced survival. It cannot be excluded that an explanation for these conflicting
results is that the antibodies, used by our group and others, detect both wild-type and
mutant p53 (Xerri, 1994, Battifora, 1995, Ferreira, 1999, Nieder, 2001). In addition,
inactivating p53 gene mutations may be rare in HL (Montesinos-Rongen, 1999,
Poppema, 2000).
Rb stainings showed a variation akin to that seen with p53. A change in Rb expression
did not influence prognosis and was not related to histopathology subgroups or clinical
characteristics. The high frequency of Rb protein expressed in this study was in
accordance with previous findings (Sanchez-Beato, 1996, Morente, 1997). Loss of Rb
has been correlated negatively with prognosis (Morente, 1997, Montalban, 2000), and a
loss of Rb expression was seen at relapse in some of our cases, but survival in this
group did not appear to be compromised.
High levels of the tumour suppressor proteins p53 and Rb detected in HRS cells, both at
diagnosis and relapse, could reflect an early transforming event leading to inactivation
and accumulation of p53 and Rb, possibly through unknown up-stream mechanism. In
our study, only relapsed cases of HL were analysed, and comparisons to non-relapsed
HL cases have not been performed. This hypothetical early transforming event might
cause a disruption of the p16INK4a and ARF-p53 pathway (Holstein, 1991, Kaelin,1999,
Knudsen, 2000, Gronbaek, 2000).
EBV is detectable in about 30-50% of of cHL cases and has been shown to be clonal in
HRS cells (Boiocchi, 1993, Brousset, 1994). A ”hit and run” mechanism hypothesis for
EBV negative HL has been proposed (Nerurkar, 2000) and the role of EBV in the
progression and relapse of HL is not entirely clarified. The number of positively stained
LMP-1 cases was comparable to that found in other series of HL (Herbst, 1992, Enblad,
1999, Stein, 2001) and all cases that were positive at diagnosis were also positive at
relapse. We thus assume that EBV is of minor importance in the pathogenesis of
relapsing HL, i.e it is either present from the time of diagnosis right throughout the
course of the disease, or else it is not present at all. We therefore did not find any
support for the ”hit and run” hypothesis for EBV in HL.
45
Antibody
/EBV
status
+/+
N (%)
5-year
HLS
(%)
-/N (%)
5-year
HLS
(%)
+/N (%)
5-year
HLS
(%)
-/+
N
(%)
5-year
HLS
(%)
CD30
60 (75)
68
6* (8)
100
10 (12)
58
4 (5)
67
EBV
35 (44)
65
45 (56)
68
-
-
-
-
p53
68 (84)
70
1 (1)
100
12 (15)
50
0
-
Rb
61 (84)
70
0
-
9 (12)
75
3 (4)
33
Table 3. Survival according to immunohistochemical expression of CD30, p53 and Rb
and EBV-status at diagnosis and at relapse. Staining for CD30 was performed in 80,
p53 in 81, Rb in 73, and EBV status in 80 cases.* 4 cases of NLPHL were included.
Abbreviations: HLS=Hodgkin lymphoma-specific survival, +/+=positive reaction both at diagnosis and at
relapse, -/-=negative reaction both at diagnosis and at relapse, +/-=positive reaction at diagnosis and
negative at relapse, -/+=negative reaction at diagnosis and positive at relapse.
A statistically significant correlation between the expression of p53 (p=0.02) and EBV
(p<0.0001) at diagnosis and relapse was observed in the paired samples, indicating a
possible clonal relationship. This is similar to the results in paper I. Consistent results of
biopsies at diagnosis and recurrence have also been demonstrated in a small series of
five patients (Gupta, 1992).
46
Cell line U-2932 (V)
A cell line was established from a patient with NHL (DLBCL) following cHL. U-2932
was established from ascites from the DLBCL. Sixteen years earlier, the patient was
diagnosed with cHL of advanced stage (IVB), relapsed four times with HL, and the fifth
”relapse”, was diagnosed as DLBCL. Extensive chemotherapy and radiotherapy were
administered both for cHL at primary diagnosis, at the subsequent relapses of cHL and
finally for DLBCL and relapse of DLBCL, before the patient died of progressive
DLBCL.
This novel cell line offers unique possibilities to study both the relationship between
cHL and NHL by means of a clonal relationship, in addition to the underlying
mechanisms of lymphoma progression and mechanisms of a possible therapy-induced
secondary lymphoma.
U-2932 is indeed a true B-cell lymphoma cell line with typical morphological features
and a B-cell phenotype. An identical Ig VH4 gene rearrangement was demonstrated in
ascites as in the cell line, by PCR-SSCP, thus confirming that U-2932 originated from
the tumour cells of ascites.
DLBCL has recently been divided into two different subgroups (Lossos, 2000,
Alizadeh, 2000), the GC-like and the activated lymphocyte-like, based on DNA
microarray analysis. Subsequent analysis of the IgH rearrangement in these groups
showed that the GC-like DLBCL had ongoing somatic hypermutations, whereas the
activated lymphocyte-like DLBCL lacked ongoing mutations (Lossos, 2000). DNA
sequencing of the VH4 rearrangement amplified from U-2932 revealed a VH4-39, D3-22
and JH4 gene usage. The VH4-39 gene was 92% homologous to the germ line sequence,
thus displaying 8% of somatic hypermutations. All five colonies sequenced showed
identical sequences and no confirmed ongoing mutations were found in five sequences
analysed. This finding may indicate a post-GC derived origin of the tumour cells.
However, more extensive sequencing has to be performed to clarify the issue of
ongoing mutations.
The composed karyotype, based on the results from the cytogenetic techniques used (Gbanding, reversed DAPI-banding, SKY and CGH) was as follows:
45,X,-X,der(1)del(1)(q23)dup(q12q23),del(2)(q11),der(3)ins(3;18)(q2?7;q21)?hsr(18)
(q21),der(6)t(6;18)(q16;q?),der(7)t(7;2;15)(q22;q24q34;q22),t(9;19)(q34;q13.1),der(10)
t(7;10)(q22;q2)der(11)t(1;11)(q12q23;q23)?hsr(11)(q21q23),del(13)(q12),
der(14)t(14;3;18;3)(q32;q13.2q2?7;q21;q2?7)?hsr(18)(q21),der(18)t(1;18)(q23;q12),
der(18)t(3;18)(q2?7;q23)?hsr(18)(q21),der(21)t(15;21)(q22;p11) [cp12]
with several structural and numerical aberrations including high-level amplifications of
the chromosomal regions 1q12-23, 3q27, 11q21-23 and 18q21.
Thus, high-level amplifications of the loci for bcl-2 and bcl-6 were seen in addition to
the immunohistochemical expression of both BCL-2 and BCL-6. Since both were overexpressed in the DLBCL biopsies and in U-2932, it is possible that the high-level
amplifications shown on 18q21 and 3q27 were responsible for the increased expression
of BCL-2 and BCL-6. Bcl-2 amplification is an alternative mechanism for BCL-2
overexpression in addition to t(14;18) (Monni, 1997).
47
In this situation, where HL might have transformed into a high-grade NHL, it is
possible that the HRS-precursor cells have gained the aberrant expression of the BCL-2
and BCL-6 proteins due to a transforming event. The BCL-2 and BCL-6 cooverexpression observed in this case might thus contribute to the development of a
secondary lymphoma.
A point mutation of the tumour suppressor gene p53 affecting amino acid position 176,
and immunohistochemical overexpression of the p53 protein was also demonstrated in
all samples of HL, DLBCL and in U-2932 cells.
The Rb was detected by immunohistochemistry in all samples of HL in addition to the
DLBCL and tumour cells of the cell line. The consistent finding of p53 and Rb
positivity in the consecutive tumour samples again supports the idea that the abnormal
overexpression of p53 and Rb is due to an early transforming event.
The p53 point mutation observed in the cell line was not investigated in HL samples due
to inability to extract enough material for the analysis. Even though
immunohistochemistry cannot prove clonality, a clonal relation may be implicated.
Based on the medical history of the patient, from whom U-2932 was established with a
late tumour response to primary HL treatment, an early relapse of HL, and subsequent
relapses of HL before the NHL developed it seems more likely that this case constitutes
a true lymphoma progression rather than a secondary lymphoma derived from a new
transformed lymphocyte. Furthermore, the mutagenic effects of chemo-and/or
radiotherapy appear not to play a major role in lymphoma progression (MüllerHermelink, 2001) as has been observed when studying the progression of follicular
lymphomas; untreated patients have similar frequencies of progression as those
previously treated (Horning, 1984).
48
CONCLUSIONS
I. The appearance of HL and NHL in one individual is uncommon.
The most common association between HL and NHL was HL preceding a high-grade
malignant NHL, whereas NHL preceding HL most often was of low-grade type.
The secondary lymphoma appeared in an aggressive clinical form, thus indicating
tumour progression. Further support for a clonal relationship between HL and NHL was
indicated by statistically significant correlations of expression of p53 and LMP-1
between the two lymphomas.
II.
Treatment outcome for younger patients with advanced stages of HL treated
according to a National Health Care Programme is in line with treatment results
reported from specialised centres. Outcome for patients treated with six courses of
chemotherapy as opposed to eight was not inferior, indicating that the number of
chemotherapy courses can be individualised. This is important to explore in future
trials. The overall results support the use of a Health Care Programme for such a rare
disease as HL. The results must be continously monitored and the guidelines repeatedly
revised in order to allow the Health Care Programmme to adapt with expanding
knowledge.
III. Patients in early stages of HL relapsing after primary treatment with radiotherapy
alone, and initially treated according to the recommendations in the National guidelines
have an outcome comparable to the outcome of patients of initially advanced stages.
The results also support the notion that the choice of primary treatment for a HL patient
is important. Even if salvage therapy is effective, it can not fully compensate for
inadequate choice of primary treatment.
Patients relapsing after combined chemotherapy and radiotherapy have a worse
outcome, and moreover, bulky disease at diagnosis was identified as an adverse
prognostic factor for outcome at relapse.
IV.
The tumour suppressor proteins p53 and Rb were frequently expressed both at
initial diagnosis and at relapse in paired samples of HL. No specific staining pattern of
p53 or Rb was detected that could be correlated to survival. However, a statistically
significant correlation was observed for the expression of p53 and EBV at diagnosis and
relapse, which may indicate a clonal relationship also in these cases. A switch in HL
subtype, observed in a subset of patients, appears to play a role in prognosis, although
no clear explanation can as yet be offered for this.
V.
In a novel B-cell line established from a patient with NHL who had been
previously treated for advanced stage and relapsed HL, a clonal relationship is likely
present. This lymphoma cell line might become a useful tool in further studies of the
relationship between HL and NHL, and lymphoma progression in contrast to therapyinduced mechanisms.
VI. The abnormal overexpression of p53 and Rb proteins in HRS cells might reflect
an early transforming event leading to the escape from apoptosis. The aberrant
expression of BCL-2 and BCL-6 can contribute to lymphoma progression.
49
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to:
Associate Professor Gunilla Enblad, my supervisor, for sharing not only her neverfailing enthusiasm and brilliant ideas in the field of Hodgkin lymphoma, but also the
experience of how to combine motherhood and research efforts.
Professor Bengt Glimelius, my co-supervisor, for patiently reading my manuscripts over
and over again, directing invaluable criticism in a skillful manner, never returning a
manuscript later than one week after receiving it (ususally within a few days) and
always being trusted as a ”living medline”.
Professor Christer Sundström, my co-supervisor, for sharing his deep knowledge on the
pathology of lymphomas both in the clinical and research field.
Professor Per Jacobsson and Professor Ingela Turesson, the former and present head of
the Dept. of Oncology, where this work was accomplished.
Professor Per Westermark, the head of the Dept. of Pathology, for encouraging
questions on the progress of my work, and the late Professor Jan Pontén, the former
head of the Dept. of Pathology, for providing excellent research conditions where part
of this work was performed.
My co-authors Mattias Berglund, Richard Rosenquist, Anita Gustafsson, Tor Ekman,
Martin Erlanson, Eva Haapaniemi, Peter Engström, Birger Christensson, Anne von
Heideman, Svetlana Lagercrantz, Ulf Thunberg and Jonas Bergh for fruitful scientific
discussions and constructive co-authorship.
Anneli Kraft, for her excellent technical assistance and guidance in the lab and for
having become such a dear friend.
Research secretary Inger Hjertström-Östh for her invaluable assistance whenever
needed and always knowing where to find Bengt.
Didde Simonsson-Westerström for help in every situation where a PhD student can be
trapped.
Margareta Hallin, Lila Shokohideh, Eva Pallin, Ellinor Berggren, Pernilla MalmrosEiderbrant, Lena Isacson and Ann Kristin Danielsson at the Haematopathology
laboratory for their excellent technical assistance when needed and interest in my work.
Dr Daniel Molin, my fellow PhD student for help with introducing the pc statistica
version and Carin Backlin for excellent technical assistance and sharing her experience
on how to survive as a PhD-student.
Sarah Walsh and Dr Nina Cavalli-Björkman for skillful linguistic revision.
50
Dr Ingrid Glimelius and Marie Rosén for help with data collection.
The other members in the ”Oncology lymphoma-group” in the Rudbeck laboratory:
Marie Fischer, Mia Thorsélius, Gerard Tobin, Jan Sällström and Ola Söderberg for
friendly support and encouragement.
All my former colleagues and the staff in the Dept. of Oncology, in particular the
members of the lymphoma division - Associate Professor Hans Hagberg, Associate
Professor Suzanne Rehn Ericsson, Associate Professor Mikael Kälkner, Dr Ulla
Martinsson, Dr Magdalena Adde and Dr Anna Johnson for taking such good care of the
Hodgkin patients.
All my colleagues and the staff in the Dept. of clinical Pathology, particularly ”STgänget” Dr Katharina Ericson, Associate Professor Alkwin Wanders, Dr Johan Botling,
Dr Anna Ybo, Dr Pia Waldenbäck and Dr Inga Wirström for their encouraging support
and friendship.
Professor Yngve Olsson for giving me great scientific advice and encouragement.
My fellow PhD students, present and former members of ”torsdagsfika-klubben” Anna
Asplund, Helena Bäckvall, Dr Jonas Isaksson and Dr Gao Ling for friendly support.
Professor Lore Zech and Eva Håkansson, and all the nice people at the Dept. of Clinical
Genetics for introducing me to their laboratory for chromosome G-banding, FISH and
the subsequent interpretation.
Associate Professor Nikos Papadogiannakis, my first supervisor at the Dept. of
Pathology in Huddinge, for advising me to start my medical studies, and awakening my
interest in medical research and clinical pathology.
My brothers, Peter, Michael, and Joachim for answering all my emergency phone-calls
and providing help whenever a computer problem arose and, Henric for being such a
great philosopher of life.
My parents Hjördis and Arne for all their love and support, without whom I would not
be here and for being such great grand-parents to Jacob and Maria.
Hashem, the love of my life, for his love, support and enormous belief in me and my
work, and for putting up with me not only during this time, but in general.
Our wonderful children, Jacob and Maria for making everything worthwhile and
without whose existence I never could have completed this thesis.
This work was financially supported by grants from the Swedish Cancer Society and
Lions Cancer Research Foundation.
51
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