Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1006

Bronchial Carcinoids

BY

DAN GRANBERG

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosopy (Faculty of Medicine) in Medicinepresented at Uppsala University in 2001

ABSTRACT

Granberg, D. Bronchial Carcinoids. Acta Universitatis Upsaliensis. ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Medicine 1006. 75 pp.Uppsala. ISBN 91-554-4957-3.

Bronchial carcinois are subdivided into typical and atypical. Atypical carcinoids are more malig-nant, but typical carcinoids may also influence survival. In the present study immunohistochemistrywas performed to identify prognostic markers in patients with typical bronchial carcinoids. Thediagnostic efficacy of octreoscan was evaluated, in comparison with CT and bone scan, and finallyour experience of treating patients with metastatic bronchial carcinoids is reported.

In an unselected material of 43 patients with typical bronchial carcinoids, metastatic disease wasfound in 12 patients (28%). Five patients (12%) developed distant metastases and died from theirdisease. High Ki-67 index, as well as positive staining for bcl-2 or p53 was associated with de-creased survival time. Positive staining for CD44s, v7-8 and v9, as well as positive nuclear stai-ning for nm23 correlated to decreased mortality. Staining for CD44 and Ki-67 should be performedroutinely for prognostic evaluation in these patients.

Octreoscan positive tumors were found in altogether 20/28 patients (71%). The primary tumorwas detectable in 81% and intrathoracic metastases in 78% of the patients on octreoscan; the corres-ponding figures for CT were 94% and 89% respectively. Liver metastases, as shown by CT, weredemonstable by octreoscan in 64% of patients. Octreoscan showed 70% and bone scan 90%sensitivity for identification of bone metastases.

Plasma chromogranin A was elevated in 28/30 patients (94 %) with metastatic bronchial carcinoidsand was the most sensitive tumor marker. Increased urinary 5’HIAA was found in 68%.

Biotherapy with α-interferon and Octreotide relieved carcinoid syndrome in 7/16 patients.However, only 4/27 patients showed stable disease during median 15 months, while 23 patientsprogressed. Treatment with cisplatinum + etoposide resulted in an objective response or stable dise-ase for 6–8 months in 3/8 patients with widespread tumors. Doxorubicin combined with strepto-zotocin or paclitaxel was associated with stable disease for 9 months in 2/2 patients each. All 7patients treated with streptozotocin+5-FU progressed.

Among the 43 unselected typical bronchial carcinoid patients, 5-year and 10-year survival was95% and 91%, respectively. The prognosis in patients with bronchial carcinoids showing distantmetastases was poor: 5-year survival was 70% from diagnosis and 22% from treatment start.

Key Words: Bronchial carcinoids, immunohistochemistry, prognostic markers, diagnosis,octreoscan, circulating hormones, treatment.

Dan Granberg, Department of Medicical Sciences, Uppsala University, SE-751 85Uppsala, Sweden

© Dan Granberg 2001

ISSN 0282-7476ISBN 91-554-4957-3

Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001

To my mother, who wanted me to make a discovery about cancerand

To my father, who has always wished to witness this momentand

To my beloved children

This thesis is based on the following papers which will be referred to in the text

by their roman numerals.

I. Granberg D, Wilander E, Öberg K, Skogseid B.

Prognostic markers in patients with typical bronchial carcinoid tumors.

Journal of Clinical Endocrinolology and Metabolism 2000;85(9):3425-3430

II. Granberg D, Wilander E, Öberg K, Skogseid B.

Decreased survival in patients with CD44-negative typical bronchial

carcinoid tumors.

International Journal of Cancer 1999;84:484–488.

III. Granberg D, Sundin A, Tiensuu Janson E, Öberg K, Skogseid B, Westlin J-E.

Octreoscan in patients with bronchial carcinoid tumors.

Submitted

IV. Granberg D, Eriksson B, Wilander E, Grimfjärd P, Fjällskog M-L, Öberg

K, Skogseid B.

Experience in treatment of metastatic bronchial carcinoid tumors.

Submitted

CONTENTS

Abbreviations 6

Introduction 7

Aims of the study 21

Patients 22

Methods 24

Results and comments 30

Clinical management 48

General summary 50

Acknowledgements 52

References 55

5

ABBREVIATIONS

ACTH Adrenocorticotropic hormonebax bcl-2-associated X proteinbcl-2 B-cell lymphoma/leukaemia-2 gene proteinCD44s Adhesion molecule CD44, standard (hematopoietic) formCD44v Adhesion molecule CD44, splice variantCEA Carcinoembryonic antigenc-erbB-2 c-erbB-2 = HER-2/neu oncoproteinCRF Corticotropin releasing factorCT Computerized tomography5-FU 5-FluorouracilGRP Gastrin releasing peptidehCGα Human chorionic gonadotropin subunit αhCGβ Human chorionic gonadotropin subunit β5’HIAA 5-hydroxy-indole-acetic acidKi-67 MIB-1 antibody against a nuclear antigen in proliferating cellsLCNC Large Cell Neuroendocrine CarcinomaLOH Loss of heterozygosityMeImAA Tele-methyl-imidazole-acetic acidMEN 1 Multiple endocrine neoplasia type 1MRI Magnetic resonance imagingnm23 Nonmetastatic protein 23/Nucleoside diphosphate kinaseNSE Neuron-specific enolasePP Pancreatic polypeptiderb Retinoblastoma proteinSCLC Small Cell Lung CarcinomaSPECT Single Photon Emission Tomography

6

INTRODUCTION

Neuroendocrine cells are widely distributed in various organs in the body, inclu-

ding the entire gastrointestinal tract and the lungs. The presence of neuro-

endocrine cells in the lungs was first described by Frölich in 1949 (Frölich, 1949)

and confirmed by Feyrter in 1954 (Feyrter, 1954). These cells, which are

argyrophilic (Tateishi, 1973), occur both as solitary cells and as clusters, termed

neuroepithelial bodies (Gould et al., 1983; Lauweryns and Peuskens, 1972).

Positive staining for NSE, serotonin, GRP/bombesin and calcitonin can be

detected both in solitary cells and neuroepithelial bodies, while leucine-

enkephaline is only found in solitary cells (Cutz et al., 1981; Gould et al., 1983;

Tsutsumi et al., 1983; Tsutsumi et al., 1983). In addition, hCGα-positive cells

have been demonstrated in normal bronchial epithelium (Fukayama et al., 1986)

and ACTH-immunoreactive cells in bronchiectasias (Tsutsumi et al., 1983).

Neuroendocrine cells are more frequent in fetal than in adult lungs, and may

proliferate in hypoxia and as response to irritants (Gould et al., 1983). The

number of neuroendocrine cells may increase in the airways of patients with

bronchiectasia (Gould et al., 1983; Memoli et al., 1983).

Neuroendocrine cells in the lungs are thought to be the origin of neuroendocrine

pulmonary neoplasias (Bensch et al., 1968; Bensch et al., 1965; Gould and

Linnoila, 1982; McDowell et al., 1976). Neuroendocrine lung tumors were

earlier subdivided into typical and atypical carcinoids (Arrigoni et al., 1972) and

small cell lung carcinomas, but several other classifications have later been

proposed (Capella et al., 1994; Paladugu et al., 1985; Travis et al., 1991; Warren

7

et al., 1985), and a fourth category, termed Large Cell Neuroendocrine

Carcinomas, has been defined (Travis et al., 1991). Between 81% and 88% of all

bronchial carcinoids are typical, while 12–19% are atypical (Ferguson et al.,

2000; Okike et al., 1976; Soga and Yakuwa, 1999).

Epidemiology

Bronchial carcinoids belong to the foregut neuroendocrine neoplasms (Williams

and Sandler, 1963), constituting 21–25% of all carcinoids (Modlin and Sandor,

1997; Soga and Yakuwa, 1999). They are rare tumors, accounting for 1–2% of

all lung tumors. Age-adjusted incidence is about 0.2:100 000. All ages are affec-

ted, even children, although the peak incidence is around 50 years. The disease is

slightly more common in women than in men (55% vs. 45%), and occurs more

often in whites than in blacks (Godwin, 1975). Although patients with multiple

endocrine neoplasia type 1 (MEN 1) have an increased risk, the etiology of

carcinoid is otherwise unknown. Smoking has not been recognized as a risk

factor.

Gross appearance

Bronchial carcinoids may be located centrally or peripherally. Central tumors are

usually cherry-red, vascular intrabronchial tumors, while peripheral tumors often

lack detectable origin from a bronchus. Both central and peripheral tumors fre-

quently infiltrate the surrounding lung parenchyma (Colby, 1994; Fraser et al.,

1989; Spencer, 1985). Between 5 and 20% of patients with typical carcinoids

develop metastases, while up to 70% of atypical carcinoid tumors metastasize

(Arrigoni et al., 1972; Colby, 1994; Okike et al., 1976). Metastases usually occur

8

to regional lymph nodes, but also distantly to the liver, bones, brain,

subcutaneous tissue or mammary glands. Metastases may be detected late,

several years or even decades after the initial diagnosis (Mokuno et al., 1999).

Microscopy

Bronchial carcinoids are epithelial in origin, reflected by their positivity for

cytokeratin (Bonato et al., 1992). Most tumors are argyrophilic; 100% of typical

and 86% of atypical carcinoids stain positive with Grimelius’ silver stain (Bonato

et al., 1992). Sevier-Munger’s silver stain may also be positive, but Masson’s

argentaffin stain is usually negative.

On light microscopy, bronchial carcinoids consist of small, polyhedral cells with

small, round or oval nuclei. The arrangement of the cells is regular and consists

of ribbons, nests, sheets, or spindling structures, separated by a fibrovascular

stroma. Typical carcinoids may contain amyloid material (Colby, 1994; Fraser et

al., 1989; Spencer, 1985). A spindle-cell variant exists (Ranchod and Levine,

1980). Necroses are only seen in atypical carcinoids (Arrigoni et al., 1972; Travis

et al., 1998). The proliferation rate is low. Staining with Ki-67 (MIB-1), which

recognizes a nuclear antigen in proliferating cells (Gerdes et al., 1983), yields

positive staining of 0.2–1.1% of the nuclei in typical and of 0.3–20.3% of the

nuclei in atypical carcinoids (Böhm et al., 1996). Electron microscopy of typical

carcinoids shows numerous neurosecretory granules varying in size from 90 to

450 nm, while atypical carcinoids contain fewer, diffusely distributed, secretory

granules between 100 and 200 nm in size (Travis et al., 1991; Warren et al.,

1984). Recently, the following criteria for atypical carcinoids (Travis et al., 1998)

9

were accepted by WHO: Carcinoid morphology in combination with either 2-10

mitoses per 2 mm2 (10 high power fields) or necroses. Typical carcinoids, on the

other hand, are characterized by carcinoid morphology, <2 mitoses per 2 mm2

(10 high power fields), and absence of necroses.

Figure 1. Typical bronchial carcinoid. Hematoxylin-eosin stain. Magnification,x200.

Immunohistochemistry

The vast majority of bronchial carcinoids stain positive for the general neuro-

endocrine markers chromogranin A (typical 100%, atypical 77–100%) , NSE

(typical 92–100%, atypical 83–100%), Leu 7 (typical 89%, atypical 100%) and

synaptophysin (typical 84–100%, atypical 80–92%) (Barbareschi et al., 1992;

Bonato et al., 1992; Travis et al., 1991; Warren et al., 1984) In most of the

10

tumors, positive staining is also found for various hormones, frequently two or

more, such as serotonin (30–84% of the tumors), GRP (14–79%), hCGα

(36–73%), leucine enkephalin (36–56%), vasoactive intestinal polypeptide

(18–60%), ACTH (12–67%), gastrin (0–60%), somatostatin (27–36%), PP

(35%), and calcitonin (5–40%). (Travis et al., 1991; Warren et al., 1985; Warren

et al., 1984; Warren et al., 1984; Wilander et al., 1985). GRP, which stains

positive in a considerable proportion of bronchial carcinoids, may act as a growth

factor for normal bronchial epithelial cells (Willey et al., 1984) and small cell

lung cancer (Cuttitta et al., 1985). High Ki-67 immunoreactivity is associated

with decreased survival in patients with bronchial neuroendocrine tumors (Böhm

et al., 1996) and endocrine pancreatic tumors (Pelosi et al., 1996).

Figure 2. Typical bronchial carcinoid. Chromogranin A stain. Magnification,

x200.

11

CD44The adhesion molecule CD44 is a glycoprotein existing in several isoforms. The

gene is localized on chromosome 11p13 close to a region causing suppression of

metastasis in prostatic cancer (Günthert, 1993; Ichikawa et al., 1992) and consists

of 20 exons (Screaton et al., 1993; Screaton et al., 1992). The 5 N-terminal exons

(exons 1-5) plus the 5 C-terminal exons (exons 16-20) encode the standard or

hematopoietic form (CD44s) which is a receptor for hyaluronan (Aruffo et al.,

1990) and is highly expressed on human lymphocytes. Several ”splice variants”

(v1–v10) exist, in which one or more epitopes encoded by the variant exons 6–15

are inserted into the extracellular portion close to the trans-membrane domain

(Günthert, 1993). CD44 is highly expressed in normal bronchial epithelium and

squamous cell lung cancers, as well as in a majority of bronchial carcinoid

tumors, while small cell lung cancers have only weak or absent expression

(Coppola et al., 1996; Givehchian et al., 1996; Mackay et al., 1994). Expression

of the standard or variant isoforms of CD44, especially variant 6 (v6) has been

coupled to more malignant tumor differentiation or worse prognosis in gastric

cancer (Mayer et al., 1993), colorectal cancer (Mulder et al., 1994; Wielenga et

al., 1993), renal cancer (Terpe et al., 1996) and non-Hodgkin lymphomas

(Stauder et al., 1995; Terpe et al., 1994), while CD44s is associated with good

prognosis in patients with neuroblastoma (Christiansen et al., 1995; Combaret et

al., 1995; Terpe et al., 1995). One study reported shorter survival in patients with

breast cancer positive for CD44v3, v 5 or v6 (Kaufmann et al., 1995), while

others have not been able to detect any correlation between CD44 and survival in

breast cancer (Friedrichs et al., 1995; Joensuu et al., 1993).

12

Oncogenes, Tumor Suppressor Genes

Bcl-2, which suppresses apoptosis, is counteracted by bax which hetero-

dimerizes with bcl-2, thereby accelerating programmed cell death (Oltvai et al.,

1993). Expression of bcl-2 correlates to neuroendocrine differentiation in lung

cancer and is found in 90–94% of small cell lung carcinomas (Jiang et al., 1996;

Jiang et al., 1995). Although bcl-2 suppresses apoptosis, non-small-cell lung

cancers positive for bcl-2 have a better prognosis than negative ones (Fontanini

et al., 1995). Positive staining for bcl-2 is rare among typical bronchial carcinoid

tumors, but is more frequent in atypical carcinoids (Coppola et al., 1996;

Lohmann et al., 1993). Decreased survival has been reported in bronchial

carcinoids with overexpression of bcl-2 and downregulation of bax (Brambilla et

al., 1996).

Mutations in the tumor suppressor gene p53 are found in many different cancer

types. Accumulation of mutated p53 protein, which can be detected by positive

immunostaining, is frequently found in highly malignant neuroendocrine lung

tumors and in atypical carcinoids, but is rare among typical bronchial carcinoids

(Coppola et al., 1996; Lohmann et al., 1993). Altered structure and expression of

the retinoblastoma gene (rb), another tumor suppressor gene, is common in small

cell lung cancer, and inactivation of rb may have pathogenetic implications for

the malignant phenotype and neuroendocrine differentiation in lung tumors

(Barbareschi et al., 1992; Gouyer et al., 1994; Hensel et al., 1990). Rb is usually

expressed in typical carcinoids, but only occasionally in atypical ones

(Barbareschi et al., 1992; Dosaka-Akita et al., 2000; Gouyer et al., 1998).

Amplification of the oncogene c-erbB-2 has been coupled to relapse and poor

13

survival in breast cancer (Wright et al., 1989), whereas c-erbB-2 has been

reported to stain negative in bronchial carcinoids (Roncalli et al., 1991).

nm23 was first identified in 1988 as a candidate metastasis suppressor gene

(Steeg et al., 1988). The protein product of nm23 is a heterodimeric enzyme

which acts as a nucleoside diphosphate kinase (Biggs et al., 1990; Gilles et al.,

1991). Two nm23 genes exist, nm23-H1 and nm23-H2 (Stahl et al., 1991), each

coding for one of the enzyme’s two chains (Gilles et al., 1991). Higher expres-

sion of nm23 has been associated with decreased risk of metastases in breast

cancer (Tokunaga et al., 1993), colorectal cancer (Ayhan et al., 1993; Yamaguchi

et al., 1993) and hepatocellular cancer (Yamaguchi et al., 1994). This antimetas-

tatic effect is usually coupled to the H1 form. In contrast, no correlation has been

found between nm23 expression and metastases in neuroendocrine lung tumors

(Gazzeri et al., 1996) or pulmonary adenocarcinoma (Higashiyama et al., 1992).

Genetics

Aneuploidy is found in 5–32% of typical carcinoids and in 17–79% of atypical

carcinoids (El-Naggar et al., 1991; Jones et al., 1988; Thunnissen et al., 1988;

Travis et al., 1991). Both typical and atypical carcinoids frequently show

deletions of 11q, including the MEN 1 locus. One study found homozygous

somatic inactivation of the MEN 1 gene in 36% of sporadic bronchial carcinoids

(Debelenko et al., 1997) and in a recent study 36% of bronchial carcinoids

showed deletions in 11q, but none had loss of 18p or 18q (Zhao et al., 2000). In

addition, atypical carcinoids frequently have deletions on 10q and 13q (Walch et

al., 1998). Other authors reported LOH at the rb locus to be infrequent in

14

carcinoids (around 20%) compared to LCNC (62%) and SCLC (71%). There was

also an increasing incidence of LOH at 5q21 from typical carcinoids (0%) to

atypical carcinoids (25%), LCNC (46%) and SCLC (86%). LOH at 5q21

correlated to shorter survival among carcinoid patients (Onuki et al., 1999).

Symptoms

A substantial proportion (13–51%) of patients with bronchial carcinoids are

asymptomatic, and the tumor is detected on routine chest X-ray. Presenting

symptoms include cough, hemoptysis, dyspnea, wheezing, chest pain and

recurrent pulmonary infections (Bertelsen et al., 1985; Harpole et al., 1992;

McCaughan et al., 1985; Mendonça et al., 1997; Mårtensson et al., 1987; Okike

et al., 1976). The carcinoid syndrome with flushing, diarrhea, wheezing and

elevated urinary 5’HIAA is infrequent, occuring in 2–12% of the patients

(Dusmet and McKneally, 1996; Harpole et al., 1992; Soga and Yakuwa, 1999).

Liver metastases are detected in most patients displaying the carcinoid synd-

rome (Davila et al., 1993; Dusmet and McKneally, 1996; Ricci et al., 1973). An

atypical carcinoid syndrome depending on histamine secretion may occasionally

be detected in patients with bronchial carcinoids. These patients suffer from

severe generalized flushing, swelling, lacrimation, asthma and diarrhea. Ectopic

Cushing’s syndrome, due to secretion of ACTH or CRF, is seen in 2–6% of

bronchial carcinoid patients (Dusmet and McKneally, 1996; Soga and Yakuwa,

1999). Acromegaly, caused by tumor production of growth hormone releasing

hormone, is exceedingly rare in patients with bronchial carcinoids (Ezzat et al.,

1994; Huber et al., 1991).

15

Figure 3. Bronchoscopy of a typical carcinoid in the right main bronchus

Diagnosis

More than 60% of the primary tumors are detectable on chest X-ray (Soga and

Yakuwa, 1999; Stefani et al., 1999). Peripheral tumors are visble as nodules,

while central tumors may be disclosed by a peripheral atelectasis or obstructive

pneumonia (Fraser et al., 1989). Computerized tomography or magnetic reso-

nance imaging is more sensitive, especially in detecting lymph node metatastases

(Ginsberg, 1994; Magid et al., 1989; Soga and Yakuwa, 1999). Up to 75% of the

central tumors are detectable by bronchoscopy (Ginsberg, 1994), Figure 3.

Despite a small risk for bleeding, it is usually safe to take biopsies (Chughtai et

al., 1997; Hurt and Bates, 1984). Plasma chromogranin A is a valuable tumor

marker in both midgut (O’Connor and Deftos, 1986) and gastric carcinoids

16

(Granberg et al., 1998), but its value in bronchial carcinoids has not been investi-

gated. Elevation of urinary 5HIAA or serotonin has been described in up to 25%

of patients with bronchial carcinoids (Harpole et al., 1992).

Treatment

The main treatment of bronchial carcinoids is surgery with removal of the

primary tumor and affected lymph nodes. Surgical procedures include pneumon-

ectomy, bilobectomy, lobectomy, segmentectomy, sleeve resection and wedge

resection. The aim is to remove the primary tumor and affected lymph nodes

radically, sparing as much lung parenchyma as possible (Chughtai et al., 1997;

Stamatis et al., 1990). Because of the risk of recurrence, extirpation by YAG

laser via bronchoscopy is not recommended except for patients with obstructive

symptoms unable to undergo radical surgery. Thoracic radiotherapy is some-

times administered when mediastinal lymph node metastaes are present; in

addition, radiotherapy is used to relieve pain in patients with bone metastases.

Chemotherapy with streptozotocin combined with cyclophosphamide or 5-FU

(Moertel and Hanley, 1979) or cisplatinum + etoposide (Moertel et al., 1991) has

been tried in patients with metastatic bronchial carcinoids, but the results are

poor. Octreotide may be of value for symptomatic relief of patients with the

carcinoid syndrome (Kvols, 1989). In case of carcinoid crisis, somatostatin

analogue treatment is life-saving .

17

Prognosis

The mortality rate varies between 0 and 8% for patients with typical carcinoids

(Padberg et al., 1996; Travis et al., 1991; Vadasz et al., 1993; Warren and Gould,

1990) and between 20 and 55% in patients with atypical carcinoids (Arrigoni et

al., 1972; Padberg et al., 1996; Valli et al., 1994). Patients with typical carcinoids

have 90-100% 5-year and 87-96% 10-year survival, while the 5- and 10-year

survival in patients with atypical carcinoids is only 40-72% and 35-60%� respec-

tively (Beasley et al., 2000; Chughtai et al., 1997; Ferguson et al., 2000; Garcia-

Yuste et al., 2000; Harpole et al., 1992; McCaughan et al., 1985; Rea et al., 1989;

Stamatis et al., 1990). Known adverse prognostic factors are atypical histology

(Arrigoni et al., 1972; McCaughan et al., 1985; Padberg et al., 1996; Travis et al.,

1998), lymph node metastases at diagnosis (El-Naggar et al., 1991; Jones et al.,

1988; McCaughan et al., 1985; Torre et al., 1989), presence of satellite lesions

(Chughtai et al., 1997), high proliferative activity assessed by staining with Ki-67

(Böhm et al., 1996), high mitotic rate (Beasley et al., 2000), high apoptotic index

(Laitinen et al., 2000) and elevation of urinary 5’HIAA or serum serotonin

(Harpole et al., 1992). The results of DNA aneuploidy and tumor size as

prognostic markers have been inconsistent (El-Naggar et al., 1991; Jones et al.,

1988; McCaughan et al., 1985; Padberg et al., 1996; Torre et al., 1989; Vadasz et

al., 1993). Positive immunostaining for CEA was a strong negative prognostic

factor in one study (Bishopric and Ordóñez, 1986).

18

111Indium labelled Octreotide

Somatostatin producing cells are widely distributed in the body. The peptide is

present in the central nervous system, in the pituitary gland, thyroid, adrenal

medulla, prostate, placenta, kidneys, in neuroendocrine cells in the gastro-

intestinal tract and in D-cells in the pancreas. Somatostatin has inhibitory effects

on the secretion of insulin and glucagon in the pancreatic islets (Koerker et al.,

1973), and on growth hormone secretion in the pituitary gland (Brazeau et al.,

1973). Somatostatin acts by specific G-protein coupled receptors, five of which

have so far been identified (Yamada et al., 1993; Yamada et al., 1992; Yamada et

al., 1992). For clinical use, more long-acting synthetic somatostatin analogues

are now available, such as octreotide and lanreotide.

Scintigraphy with 111Indium-labelled octreotide (octreoscan) is valuable for

diagnosis of neuroendocrine tumors. Octreoscan positive lesions can be found in

more than 90% of patients with midgut carcinoids (Kwekkeboom et al., 1993;

Kälkner et al., 1995; Shi et al., 1998), paragangliomas (Kwekkeboom et al.,

1993), and gastrinomas as well as 86% of pheochromocytomas are detectable on

octreoscan (Kwekkeboom and Krenning, 1997). Insulinomas are more rarely

(<50%) demonstrable by this method. Kälkner et.al. reported that 4/12 foregut

carcinoids could not be visualized on octreoscan (Kälkner et al., 1995) but in

another study 8/8 primary lung carcinoids were demonstrable (Musi et al., 1998).

The sensitivity for detection of liver metastases in neuroendocrine gastro-entero-

pancreatic tumors is about 90–100% (Chiti et al., 1998; Krausz et al., 1998;

Scherübl et al., 1993). In several case reports, octreoscan identified the primary

tumor in patients with occult ectopic Cushing’s syndrome (Carretta et al., 1997;

19

Mansi et al., 1997; Phlipponneau et al., 1994; Weiss et al., 1994). The value of

octreoscan in localizing small ACTH-producing bronchial carcinoids was

however questioned in two recent studies (Tabarin et al., 1999; Torpy et al.,

1999). In small cell lung cancer patients, octreoscan detects nearly all primary

tumors, but distant metastases are seen less frequently (Berenger et al., 1996;

O’Byrne et al., 1994; Reisinger et al., 1998). Other diseases that may be positive

on octreoscan include non-small-cell lung cancer (Lau et al., 2000), sarcoidosis

(Kwekkeboom et al., 1998), pneumonia (Castellani et al., 1999), malignant

lymphomas (Reubi et al., 1992) and Graves’ disease (Postema et al., 1994).

Octreoscan can be used to predict the response to treatment with somatostatin

analogues in patients with malignant midgut carcinoids (Tiensuu Janson et al.,

1994).

20

AIMS OF THE STUDY

The aims of the present study were to

• Find prognostic markers for patients with typical bronchial carcinoids

• Identify tumor markers in plasma or urine suitable for treatment monitoring

and early detection of recurrence

• Evaluate the effectiveness of octreotide scintigraphy for the diagnosis of

bronchial carcinoids and for identifying metastases

• Survey the treatment of patients with metastatic bronchial carcinoids at the

Department of Endocrine Oncology, University Hospital, Uppsala

21

PATIENTS

Paper I and II

All 43 patients admitted to our hospital between 1985 and 1995 whose pathology

specimens (paraffin blocks containing tumor tissue) fulfilled the criteria for

typical bronchial carcinoid (Figure 1) (Travis et al., 1998), were included. All

specimens showed less than 2 mitoses per 10 high power fields. Sixteen of the

patients were men and 27 were women. Their median age was 56 years (range

18-75). Median follow-up was 74 months (14–330) in paper I and 65 months

(14-325) in Paper II. The primary tumor was studied in 41 patients, while in one

patient only a brain metastasis and in another patient an intrathoracic lymph node

metastasis was available.

The carcinoid tumor diagnosis was made because of respiratory symptoms in 32

patients. Nine cases were found on routine chest X-ray and one patient was

operated on for a brain metastasis, that turned out to be derived from a typical

bronchial carcinoid. The last patient presented with ectopic Cushing’s syndrome.

Two patients had carcinoid syndrome at diagnosis, and two more patients

developed this syndrome when liver metastases were detectable 12 and 20 years

after primary surgery.

Altogether 12/43 patients (28%) displayed metastatic disease. Ten patients (23%)

had lymph node involvement at diagnosis (including one patient who recurred

locally 26 years after bronchoscopic extirpation of an intrabronchial carinoid),

and distant metastases have occurred in 5 patients (12%).

22

Surgical removal of the primary tumor and lymph node dissection was performed

in 42 patients, one of whom had undergone bronchoscopic extirpation of an

intrabronchial carcinoid 26 years earlier. In one patient surgery was denied

because of extensive intrathoracic tumor growth.

Paper III

All 28 patients referred for octreoscan at our hospital between 1992 and May

2000 who suffered from histologically verified bronchial carcinoids were

studied. One patient was subjected to octreoscan on 3 different occasions, while

the remaining 27 patients were examined once. Four patients had atypical and 24

patients had typical carcinoids (Travis et al., 1998). There were 13 men and 15

women. Their median age was 57 years (range 16–83). Carcinoid syndrome was

present in 9 patients and ectopic Cushings syndrome in 5; one of these patients

had both syndromes. The primary tumor had been surgically removed prior to the

octreoscan investigation in 12 patients. Altogether 22 patients harbored

metastatic disease: Intrathoracic recurrence or metastases (n=9), liver (n=14),

bones (n=10), breast, brain and abdominal lymph nodes (1 patient each). Five

patients were receiving treatment with octreotide.

Paper IV

All 31 patients (14 men and 17 women) treated at the Department of Endocrine

Oncology, University Hospital, Uppsala who harbored bronchial carcinoids with

distant metastases were included in this study. Four patients had atypical while

the remaining 27 had typical carcinoids (Travis et al., 1998). Their median age

23

was 60 years (15–84). Carcinoid syndrome was present in 16 patients, while 2

patients had ectopic Cushing’s syndrome. Metastases were present at the start of

treatment in 29 of the patients, while the remaining 2 developed metastases

during the observation period. Median follow-up from the start of treatment was

25 months (4–106). Surgery for the primary tumor had been performed in 22 of

the patients. In addition, 7 patients had been subjected to surgery of metastases in

the brain, ovaries, mammary glands or subcutaneous tissue.

METHODS

Immunohistochemistry

All specimens were stained with hematoxylin and eosin. Immunohistochemistry

was performed on all tumors in Papers I and II with the antibodies shown in

Table 1. In addition, 17 tumors of patients (15 with typical and 2 with atypical

carcinoids) in Paper IV were stained with bcl-2, CD44s, GRP, hCGα, Ki-67,

nm23, p53 and serotonin. The following procedure was used: Formalin-fixed and

paraffin-embedded blocks were cut in 4 µm sections. After deparaffinization and

rehydration of the sections, endogenous peroxidase was blocked with 0.3%

hydrogen peroxide for 30 min. For some of the antibodies, antigen unmasking

was performed by pretreatment according to Table 1. The slides were repeatedly

washed in phosphate saline buffer solution, pH 7.4 (PBS), whereafter horse or

goat serum diluted 1:5 in PBS was applied in order to avoid non-specific

secondary antibody binding. Primary antibody diluted in PBS was employed for

1.5 hours in room temperature or overnight at 4°C. Biotinylated horse anti-mouse

24

or goat anti-rabbit Ig, diluted 1:200, was used as secondary antibody and was

applied for 45 min. The reaction was visualized with an ’Elite’ kit (Vector

Laboratories, Burlingame, CA) with 0.006% hydrogen peroxide as substrate and

3-amino-9-ethyl-carbazole (Sigma Chemical Co., St Louis, MO) in dimethyl-

sulfoxide as chromogen. Finally, counterstaining of the slides was performed

with hematoxylin. A positive control was always included. All slides were

assessed by an experienced pathologist (E. Wilander) without knowledge about

the clinical data. The stainings of CD44 (standard form and splice variants) and

bax were graded as positive or negative. For Ki-67, the percentage of positive

cells was calculated. The remaining stainings were graded as follows: -, negative;

+, <10% positive cells; ++, 10-50% positive cells; +++, >50% positive cells. For

c-erbB-2 membranous staining, which correlates to gene amplification

(Gusterson et al., 1988) was re�quired for positivity.

Hormones

Plasma chromogranin A, plasma chromogranin B, serum PP, serum calcitonin,

serum hCGα and hCGβ were analyzed according to methods described earlier

(Almqvist et al., 1974; Hällgren et al., 1977; Stridsberg et al., 1993; Stridsberg et

al., 1995; Öberg and Wide, 1981). Urinary 5’HIAA was sampled on two conse-

cutive days and the mean value was calculated. Urinary MeImAA, the principal

metabolite of histamine, was collected for 24 hours and analyzed as described

earlier (Granerus et al., 1966).

25

TABLE 1. Antibodies used for immunohistochemistry

Antibody Type Dilution Pretreatment Company Pos controlACTH M 1/75 DAKO Pituitarybax P 1/100 Pepsin Santa Cruz Ileumbcl-2 M 1/80 Boiling DAKO LNCalcitonin M 1/200 DAKO MTCCD44s, clone DF1485* M 1/200 Boiling DAKO LNCD44v4, clone VFF-11 M 1/20 Bender UCECD44v5, clone VFF-8 M 1/80 Boiling Bender UCECD44v6, clone VFF-18 M 1/50 Bender UCECD44v7, clone VFF-9 M 1/10 Boiling Bender UCECD44v7-8, clone VFF-17 M 1/10 Boiling Bender UCECD44v9, clone 441v M 1/100 Seigagaku GastrinomaCD44v10, clone VFF-14 M 1/10 Boiling Bender UCEc-erbB-2 P 1/200 Boiling DAKO Breast cancerChromogranin A P 1/2000 B. Eriksson# NoneCytokeratin, clone MNF116 M 1/100 Protease DAKO NoneGastrin P 1/4000 MILAB GastrinomaGRP P 1/800 DAKO Fetal lunghCGα M 1/800 ICN PlacentahCGβ M 1/400 ICN PlacentaKi-67, clone MIB-1 M 1/100 Boiling Immunotech Ileumnm23 (H1 and H2) P 1/50 Boiling DAKO EPTp53 M 1/100 Boiling DAKO Pos tumorPP P 1/500 DAKO Pancreasrb M 3/100 Boiling Oncogene Sci ColonS-100 protein P 1/800 DAKO NoneSerotonin M 1/10 DAKO Duodenum

Abbreviations: M, mouse monoclonal; P, rabbit polyclonal. LN, lymph node; MTC,medul-lary thyroid carcinoma; EPT, endocrine pancreatic tumor; UCE, uterine cervixepithelium.

Santa Cruz, Santa Cruz Biotechnology, Santa Cruz, CA; ICN, ICN Biomedicals, CostaMesa, CA; DAKO, DAKO A/S, Glostrup, Denmark; Oncogene Sci, Oncogene Science,Cambridge, MA; Immunotech, Immunotech, Marseille, France; MILAB, MILAB, Malmö,Sweden; #, Dr. Barbro Eriksson, University Hospital, Uppsala, Sweden; Bender, BenderMedSystems, Vienna, Austria; Seikagaku, Seikagaku Corp, Tokyo, Japan.

Boiling, 15 min boiling in citrate buffer. Pepsin, pepsin (Sigma Chemical Co., St Louis,MO) was applied in a concentration of 0.1% in distilled water with 0.01 M HCl, pH 2.3 for6 min. Protease (Sigma Chemical Co., St Louis, MO) was applied in a concentration of0.05% in PBS for 10 min.

26

OctreoscanScintigraphy was performed with 111In-DTPA-D-Phe1-octreotide delivered by

Mallinckrodt Medical, Petten, the Netherlands. The labelling procedure (Bakker

et al., 1991) was briefly as follows: 244 MBq of 111In-chloride was added to 20

µg lyophilized (DTPA-D-Phe1)-octreotide and incubated for 30 min at room

temperature. The labelling yield, which was checked by chromatography using

SEP-PAK (Bakker et al., 1990), always exceeded 97%. Each patient received an

intravenous bolus injection of 220 MBq of the 111In-pentetreotide solution. Planar

antero-posterior and lateral images covering the whole body with the exception

of the extremities were obtained after 24 hours. In addition, a single photon

emission tomography (SPECT) study was performed over the abdomen after 24

hours, using a gamma scintillation SPECT-camera delivered by Nuclear

Diagnostics, Hägersten, Sweden. To collect the original SPECT data, a 64 step

rotation of 360° in a 64x64 word matrix was used. For the reconstruction of

SPECT images, a Wiener filter was applied (Westlin et al., 1992). The results

were compared to intravenously contrast enhanced computerized tomography

(CT) for detection of primary tumors and soft tissue metastases, and to

conventional bone scan and plain X-ray or magnetic resonance imaging (MRI)

using the standard protocl for bone metastases. Octreoscan and CT were assesed

by different observers without knowledge of the results of the other examination.

Treatment schedules

Our first line treatment in patients with metastatic bronchial carcinoids has been

α-interferon in a dose of 9–42 (median 15) million units/week administered in 3

or 5 subcutaneous injections. The dose had been adjusted according to leukocyte

27

count and side effects. In cases of disabling carcinoid syndrome, α-interferon has

been combined with octreotide. When this treatment has failed, we have changed

to a combination of streptozotocin + 5-FU. If the tumor has continued to grow,

we have used cisplatinum + etoposide as third line therapy. Other regimens used

as third or fourth line treatment are streptozotocin + doxorubicin, α-interferon +

5-FU, paclitaxel and paclitaxel + doxorubicin. The chemotherapy schedules are

shown in Table 2. Liver embolization with gel-foam has been used as second or

third line therapy when the liver contained the majority of the tumor burden. A

few patients with advanced disease have received targeted radiotherapy with111In-Octreotide or 131I-MIBG. An objective response was defined as a ≥50%

decrease in radiological size (product of two perpendicular measures), and a

biochemical response was defined as a ≥50% decrease of at least one hormone

marker. A ≥25% increase in tumor size or the occurrence of new metastases was

regarded as progression, and a ≥25% increase of at least one hormone marker

was considered as biochemical progression. Time to progression was defined as

the time from start of the treatment to the first occasion when progression was

observed. Duration of response was defined as time from start of treatment to the

last occasion when disease was considered stable. Our intention was to monitor

the patients every third month.

28

TABLE 2. Chemotherapy regimens used

Drugs, doses Interval

cisplatinum 45 mg/m2 day 2–3 + etoposide 100 mg/m2 day 1–3 4 weeks

streptozotocin 2000 mg day 1 + 5-FU 400 mg/m2 day 1 3 weeks

streptozotocin 2000 mg day 1 + doxorubicin 40 mg/m2 day 1 3 weeks

paclitaxel 150 mg/m2 day 1 + doxorubicin 40 mg/m2 day 1 3 weeks

paclitaxel 175 mg/m2 day 1 3 weeks

α-interferon 9–25 million units/week+ 5-FU 500 mg/m2 day 1–3 4 weeks

Statistics

The median and range were used as measures of central tendency and variation,

respectively. When calculating the statistics, comparison was made only between

positive and negative staining, except for Ki-67. Correlation between antibody

expression and metastasis or mortality was analyzed by the χ2 test or (for Ki-67)

the Mann-Whitney U test. Survival time was calculated by Kaplan-Meier

cumulative survival plot and Mantel-Cox logrank test except for Ki-67, where

Cox proportional Hazards was applied. p<0.05 was considered significant. A

patient who died from an unrelated cause (paper I and II) was omitted from the

survival calculations.

29

RESULTS AND COMMENTS

Proliferation

Figure 3. The results for Ki-67 are shown in tables 3, 4 and 5. Among the 43

patients in Paper I and II, Ki-67 did not correlate to the incidence of distant

metastases (p=0.2), or to metatstatic disease (p=0.4), although higher Ki-67 index

was associated with decreased survival time (p<0.01). If, however, the 15

additional immunostained typical carcinoids from Paper IV were included in the

analysis, Ki-67 correlated both to distant metastases (p<0.001), metastatic

disease (p<0.01), mortality and shorter survival time (p<0.01). This more

comprehensive patient material encompasses a higher number of metastatic

typical carcinoid tumors. Similar results were reported by Böhm et.al.: No

correlation was found between Ki-67 and survival for typical carcinoids, but

when patients with atypical carcinoids (well differentiated neuroendocrine

carcinomas) and SCLC were included, the difference was significant (Böhm et

al., 1996). Analysis of Ki-67 expression adds prognostic information in patients

with neuoendocrine lung tumors and should be performed routinely.

Immunohistochemistry

The results of the immunohistochemical stainings for the individual patients are

summarized in Tables 3 and 4 and the correlations are shown in Table 5. All 43

tumors in Paper I and II stained positive for chromogranin A (Figure 2). Cyto-

keratin was positive in 40 tumors, confirming their epithelial origin, and S-100

protein in 10. S-100 showed no correlation to metastases or survival. Positive

staining for mutiple hormones was common, although only a few patients

30

displayed endocrine symptoms (Figure 5). Correlation to lymph node metastases

was not found for any of the investigated antibodies.

Figure 4. A single Ki-67 positive cell (0.18% in total) in a patient with a typicalcarcinoid. Magnification, x400.

Figure 5. Positive ACTH staining in a patient without clinical evidence ofCushing’s syndrome. Magnification, x200.

31

Positive staining for the CD44 isoforms CD44s (Figure 6), v9 and v7-8 (Paper I

and II) correlated to decreased incidence of distant metastases and mortality

(p<0.001, p<0.01 and p=0.04 for the respective CD44 isoforms). This is consis-

tent with earlier observations that normal bronchial epithelium shows high

expression of CD44s and v5, v6, v9 while SCLC tumors have weaker or no

expression (Givehchian et al., 1996), and that CD44s and v6 are correlated to

more aggressive variants of neuroendocrine lung tumors (Coppola et al., 1996).

If the 15 immunostained typical carcinoids from Paper IV were included,

positive staining for CD44s still correlated to decreased risk for distant

metastases and death (p<0.0001). In addition, correlation was now reached to

metastatic disease (p<0.001). Thus, by increasing the number of observations

and the proportion of patients harboring distant metastases, the findings from

paper I and II were strengthened. CD44 expression did not correlate to lymph

node metastases, which is the predilective metastatic site. We conclude that

immunostaining with CD44s may give useful prognostic information in patients

with bronchial carcinoids and should be performed routinely. It is unclear if the

absence of CD44 expression has a causative role in the metastatic process or

merely accompanies poor differentiation. Several theories exist as to how CD44

can inhibit metastasis. One posiible mechanism is to promote adhesion between

tumor cells or between tumor cells and the basement membrane (Fujita et al.,

1994). Another theory is that CD44 mediates adhesion of tumor cells to hyalu-

ronic acid molecules in the intercellular matrix (Penno et al., 1994).

32

Figure 6. Strong positive staining for CD44s. Magnification, x400.

Figure 7. Positive staining for GRP. Magnification, x400.

33

TA

BL

E 3

. R

esul

ts o

f the

stai

ning

s, m

etas

tase

s and

surv

ival

(mon

ths)

of a

ll pa

tient

s in

Pape

r I a

nd I

IPa

t LN

D

MO

utco

me

mon

ths

Cyk

S-10

0K

i-67

%C

D44 s

CD

44v4

CD

44v5

CD

44v6

CD

44v7

CD

44v7

-8C

D44

v9A

CTH

Cal

GR

PhC

Gα

hCGβ

PP5-

HT

bax

bcl-2

c-er

bB-2

nm23

(nuc

l)nm

23(c

yt)

p53

rb

1N

oN

oA

, 25

-po

s0.

13po

spo

spo

s-

--

pos

--

++-

--

-po

s-

+++

++

--

2N

oN

oA

, 111

pos

-0.

13po

spo

spo

s-

pos

--

--

-+

--

+po

s-

+++

++

-+

3N

oN

oA

, 112

pos

-1.

00-

pos

pos

-po

s-

pos

--

--

-+

-po

s+

+++

++-

++4

No

No

A, 9

7po

spo

s0.

10po

spo

spo

spo

s-

pos

--

-++

+-

--

--

++++

++

-++

5N

oN

oA

, 70

pos

-0.

33po

spo

spo

s-

--

pos

--

--

--

-po

s-

-+

+-

-6

No

No

A, 6

7po

s-

0.10

pos

pos

pos

pos

-po

spo

s-

--

--

--

pos

--

+++

--

7N

oN

oA

, 170

--

0.46

pos

-po

s-

-po

spo

s+

++++

+++

+-

++-

--

+-

--

8N

oN

oA

, 120

pos

-0.

36po

spo

spo

spo

spo

spo

spo

s-

--

--

--

pos

--

+++

+-

+++

9N

oN

oA

, 87

pos

-2.

78po

spo

spo

spo

spo

spo

spo

s-

--

--

--

--

++++

+++

-++

10N

oN

oA

, 76

pos

-0.

78po

spo

spo

spo

spo

spo

spo

s-

--

+-

+++

pos

-++

++

-11

No

No

A, 5

8po

s-

0.10

--

pos

--

--

--

-+

-+

--

-++

+++

++

--

12N

oN

oA

, 101

pos

pos

1.11

pos

pos

pos

--

-po

s+

-+

+-

--

--

+++

++

+++

13N

oN

oA

, 43

pos

-0.

00-

pos

pos

-po

s-

--

--

--

--

pos

-++

++

+++

-+

14N

oN

oA

, 74

pos

-0.

10po

spo

spo

s-

pos

pos

pos

--

--

-+

-po

s-

++++

+-

+15

No

No

A, 1

62po

s-

1.17

pos

pos

pos

--

pos

pos

--

-+

-+

-po

s-

+++

+++

-+

16N

oN

oA

, 134

pos

pos

0.58

--

pos

--

--

--

-+

--

-po

s-

-++

+-

+17

No

No

A, 1

75po

s-

0.06

--

pos

--

--

--

-+

--

+po

s-

+++

+++

+-

-18

No

No

A, 6

5po

s-

0.50

pos

pos

pos

--

-po

s-

--

+-

--

pos

-+

+++

+-

-19

No

No

A, 1

18po

s-

0.33

pos

pos

pos

pos

pos

pos

pos

--

--

--

-po

s-

+++

++-

-20

No

No

A, 9

2po

s-

1.06

pos

-po

spo

spo

spo

spo

s-

--

++-

+-

pos

-+

+++

+-

-21

No

No

A, 9

3po

s-

1.40

-po

spo

s-

pos

--

--

-+

--

-po

s-

+++

+++

-+

22N

oN

oA

, 84

pos

-1.

00po

spo

spo

spo

spo

spo

spo

s-

--

+-

+-

pos

-++

+++

-+

23N

oN

oA

, 55

pos

pos

1.00

pos

-po

spo

spo

spo

spo

s-

--

+-

+-

pos

-+

+++

--

24N

oN

oA

, 32

pos

-0.

82po

spo

spo

s-

pos

pos

pos

--

-+

--

--

-+

+-

--

25N

oN

oA

, 37

pos

-0.

90-

--

-po

s-

--

--

--

-+

--

+++

++

-+

26N

oN

oA

, 34

pos

pos

0.73

pos

-po

s-

--

--

--

+-

--

--

++

+-

-27

No

No

A, 6

7po

spo

s0.

50po

s-

pos

-po

spo

spo

s+

--

+-

+-

pos

-+

+++

-+

28N

oN

o(†

), 45

pos

-0.

60-

--

-po

s-

--

--

+-

-++

pos

-++

++

++-

-29

No

No

A, 2

6po

s-

0.78

pos

pos

pos

-po

s-

pos

--

--

-++

--

-+

+++

--

30N

oN

oA

, 25

pos

-0.

91po

spo

spo

spo

spo

spo

spo

s-

--

++-

++++

pos

-++

-++

+-

++31

No

No

A, 2

1po

s-

0.30

pos

-po

s-

pos

-po

s-

--

++-

--

--

+++

+-

+32

Yes

No

A, 8

4po

spo

s0.

33po

spo

spo

spo

spo

spo

spo

s-

--

--

+-

pos

-+

++

--

33Y

esN

oA

, 134

pos

-0.

63po

spo

spo

spo

s-

pos

pos

--

--

-+

+-

-+

+++

-++

34Y

esN

oA

, 144

pos

pos

0.33

-po

spo

s-

pos

-po

s-

-++

+-

--

+++

pos

-+

+++

-++

35Y

esN

oA

, 70

--

0.27

pos

pos

pos

-po

s-

pos

+++

+++

+++

+-

++

--

+++

+++

-++

36Y

esN

oA

, 134

pos

-0.

18po

spo

spo

spo

spo

spo

spo

s-

--

--

--

pos

-+

+++

--

37Y

esN

oA

, 26

pos

-0.

10po

spo

s-

-po

s-

--

--

++-

+++

--

+++

-++

--

38Y

esN

oA

, 14

pos

-2.

78po

s-

pos

-po

s-

--

-+

+-

--

-+

+++

-++

-+

39N

oY

es†,

330

pos

-0.

10-

pos

--

pos

--

--

--

--

+po

s-

+++

-+

--

40Y

esY

es†,

60

pos

-10

.00

--

pos

--

--

+-

++-

--

++po

s++

++

-++

++++

41Y

esY

es†,

25

pos

pos

2.80

-po

spo

s-

--

--

--

--

--

--

-+

-+

++42

Yes

Yes

†, 3

8po

s-

0.33

--

pos

-po

s-

--

--

+++

--

--

-++

++

++-

-43

No

Yes

†, 1

74po

s-

1.40

--

pos

--

--

--

+++

--

--

pos

+++

-++

+-

+N

umbe

r of p

ositi

ve40

1029

2839

1327

1826

52

924

115

1327

437

3740

324

Cyk

, cyt

oker

atin

; Cal

, cal

cito

nin;

5-H

T, se

roto

nin;

nuc

l, nu

clea

r; cy

t, cy

topl

asm

ic. -

, neg

ativ

e; p

os, p

ositi

ve; +

, <1

0% p

ositi

ve c

ells

; ++,

10-

50%

pos

itive

cel

ls; +

++, >

50%

pos

itive

cells

. LN

, lym

ph n

ode

met

asta

ses a

t dia

gnos

is; D

M, d

ista

nt m

etas

tase

s; A

, aliv

e; †

, dea

d; (†

), de

ad o

f an

unre

late

d ca

use.

All

43 tu

mor

s sta

ined

pos

itive

for C

D44

v10.

34

TABLE 4. Paper IV. Results of the stainings, metastases and survival (months)of the 15 immunostained tumors of typical carcinoid patients harboring distantmetastases.

Pat Outcomemonths

Ki-67%

CD44s GRP hCGα 5-HT bcl-2 nm23(nucl)

nm23(cyt)

p53

1 †, 199 1.0 - +++ - ++ + + + -2 †, 55 2.1 - - +++ ++ +++ - +++ -3 †, 25 1.0 - - +++ - + +++ +++ -4 †, 202 4.3 - - - - - +++ +++ -5 †, 296 1.0 - ND - - ND - +++ -6 A, 114 1.6 pos - - - - - +++ -7 A, 105 4.8 - - +++ - +++ - +++ -8 †, 224 3.3 - - +++ - - - +++ -9 †, 87 0.5 - +++ +++ - - - +++ -10 †, 130 0.0 - ND +++ - - ++ +++ -11 †, 19 5.3 - - - - - +++ +++ -12 †, 172 2.0 - - - - - ++ +++ -13 †, 16 3.7 - - - - - +++ +++ -14 †, 164 2.0 - ++ ++ - - +++ +++ -15 A, 26 3.0 - +++ - - - - +++ -

Number of positive 1 4 7 2 4 8 15 0

5-HT, serotonin. -, negative; pos, positive; + , <10% positive cells; ++, 10-50% positivecells; +++, >50% positive cells. A, alive; †, dead; ND, not done; nucl, nuclear; cyt, cyto-plasmic.

None of the immunostained hormones listed in Table 3 correlated to lymph node

metastases, distant metastases or mortality. However, patients with GRP-positive

tumors (Figure 7) had increased risk of metastatic disease (p=0.04). This

correlation persisted if the 15 additional typical carcinoids from Paper IV were

included in the analysis. Whether this means that GRP has a stimulatory growth

effect on bronchial carcinoids as for SCLC (Willey et al., 1984), needs to be

further studied. Our results confirm earlier observations that hCGα� does not

35

correlate to malignancy in neuroendocrine lung tumors (Heitz et al., 1987;

Tsutsumi, 1989).

TABLE 5. Correlations of all the immunostained antibodies (Paper I and II) toclinical parameters. Figures in brackets denote values when the 15 immuno-stained typical carcinoids from Paper IV were included in the analysis.

Antibody Metastases Mortality Survival timeDistant All

ACTH NS NS NS NSbax NS NS NS NSbcl-2 ↑ (⇑) ↑ (↑) ↑ (↑) ↓ (↓)CD44s ⇓⇓ (⇓⇓) NS (⇓⇓) ⇓⇓ (⇓⇓) ND (ND)CD44v4 NS NS NS NSCD44v5 NS NS NS NSCD44v6 NS NS NS NDCD44v7 NS NS NS NSCD44v7-8 ↓ NS ↓ NDCD44v9 ⇓ NS ⇓ NDCD44v10 NS NS NS NSc-erbB-2 NS NS NS NSGRP NS (NS) ↑ (↑) NS (NS) NS (NS)hCGα NS (NS) NS (NS) NS (NS) NS (NS)Ki-67 NS (⇑⇑) NS (⇑) NS (⇑) ⇓ (⇓)nm23 (cytoplasmic) NS (NS) NS (NS) NS (NS) NS (NS)nm23 (nuclear) ⇓ (⇓⇓) ⇓ (⇓⇓) ⇓ (↓) NS (NS)p53 ⇑ (NS) NS (NS) ⇑ (NS) ⇓⇓ (⇓)PP NS NS NS NDrb NS NS NS NSSerotonin NS (NS) NS (NS) NS (NS) NS (NS)

NS, no significant correlation; ↑, positive correlation p<0.05; ⇑, positive correlationp<0.01; ⇑⇑, positive correlation p<0.001; ↓, negative correlation p<0.05; ⇓, negativecorrelation p<0.01; ⇓⇓, negative correlation p<0.001. ND, not possible to calculatebecause all patients who died stained negative.

36

Positive nuclear nm23 staining (Figure 8) correlated to decreased risk of distant

metastases and metastatic disease (p<0.01), a correlation that persisted if the

additional 15 patients with typical carcinoids from Paper IV were included. This

contrasts to an earlier study of bronchial carcinoids and other lung tumors

where nuclei stained negative and no corrlelation to clinical parameters was

found (Gazzeri et al., 1996). The antimetastatic effect of nm23 is usually attri-

buted to the H1 form. Although the antibody we used recognized both nm23-H1

and nm23-H2, our results are consistent with observations in other cancer types

(Tokunaga et al., 1993; Yamaguchi et al., 1993; Yamaguchi et al., 1994).

Regarding the relatively high frequency of positive nuclear staining for nm23

both in tumors of patients with distant metastases, 50%, compared with 92% in

patients without distant metastses, nm23 is however of limited value as prog-

nostic marker in patients with typical bronchial carcinoids (Paper I and II).

Figure 8. Strong positive nuclear staining for nm23. Magnification, x400

37

Positive staining for bcl-2 correlated to increased risk of distant metastases

(p=0.01) and metastatic disease (p<0.05) as well as decreased survival time

(p<0.05), indicating that apoptosis may have prognostic importance in these

tumors. This is consistent with earlier observations in patients with neuro-

endocrine lung tumors (Brambilla et al., 1996). Weak positive staining for p53

has earlier been found in bronchial carcinoids (Coppola et al., 1996). In our

material, 3 tumors were positive for p53, which correlated to increased risk of

distant metastases (p<0.01) as well as decreased survival time (p<0.001). If all 58

immunostained typical carcinoids from Papers I+II and IV were included, p53

still correlated to decreased survival time (p<0.01) but not to metastases or

mortality. Since positive staining is rare for both bcl-2 and p53 in typical

bronchial carcinoids (14% and 5% respectively in our material), they are not

suitable for routine use as prognostic markers (Paper I and II).

Octreoscan

Octreoscan positive tumors were found in altogether 20/28 patients (71%). The

sensitivity for detection of lesions in various locations is accounted for in Table

6. The primary tumor (Figure 9A and 9B) was detectable on octreoscan in 13/16

patients (81%) and on CT in 15/16 patients (94%). CT thus seems to be a more

sensitive method for diagnosis of primary bronchial carcinoids. Moreover, since

several non-endocrine diseases may be positive, octreoscan is not suitable for

differential diagnosis of an intrathoracic lesion found on chest X-ray or CT

(Castellani et al., 1999; Kwekkeboom et al., 1998; Lau et al., 2000). Only 40% of

the tumors in patients displaying ectopic Cushing’s syndrome were detectable on

octreoscan, although octreoscan was positive in the single patient presenting with

38

ectopic Cushing’s syndrome, whose tumor could not be identified on CT. This

supports the recent reports that octreoscan is of limited value in these patients

(Tabarin et al., 1999; Torpy et al., 1999). Intrathoracic metastases or recurrences

were localized on octreoscan in 7/9 patients (78%) and on CT in 8/9 (89%)

patients.

Figure 9A. Primary bronchial carcinoid demonstrated on octreoscan

Figure 9B. CT in the same patient as in Figure 1A.

39

Octreoscan managed to demonstrate liver metastases in 9/14 (64%) of the pati-

ents with liver metastases seen on CT, yielding 64% sensitivity for recognition of

liver involvement. This is slightly lower than earlier reported figures (89–93%)

in patients with neuroendocrine gastro-entero-pancreatic tumors (Chiti et al.,

1998; Krausz et al., 1998; Scherübl et al., 1993).

Bone metastases were visualized on octreoscan in 7/10 patients (70%) and on

conventional bone scan in 9/10 patients with bone metastases confirmed by plain

X-ray or MRI. Conventional bone scan or plain X-ray revealed a higher number

of metastases than octreoscan in 5 of the 7 patients with bone metastases seen on

octreoscan. Bone scan has thus a higher sensitivity for detection of bone

metastases than octreoscan. Octreoscan is nevertheless suitable for follow-up and

screening of relapse in patients with octreoscan positive bronchial carcinoids. In

order to select the appropriate patients to follow with octreoscan, it is crucial to

perform an examination before curative surgery. If this is not done, an alternative

is to immunostain the primary tumor for somatostatin receptor expression

(Tiensuu Janson et al., 1998) (Paper III).

The number of patients in this study is somewhat limited, and the recorded

differences of sensitivity for octreoscan and CT may reflect this circumstance. If

the sensitivity for octreoscan to detect lesions in various locations is calculated in

only those patients whose tumors are known to be octreoscan positive, the

figures will of course be slightly higher than in Table 6. The superiority of CT,

however, remains (Paper III).

40

TABLE 6. Sensitivity for octreoscan, CT and bone scan to

detect various lesions in patients with bronchial carcinoids.

Localization Octreoscan CT Bone scanPrimary tumor 81% 94% NA

Intrathoracic metastases 78% 89% NA

Liver metastases 64% 100% NA

Bone metastases 70% NA 90%

Tumor markers

The results are summarized in Table 7. Plasma chromogranin A was the most

frequently elevated hormone (94%) and thus appears to be the most sensitive

tumor marker in patients with metastatic bronchial carcinoids. Higher levels of

chromogranin A were found in patients with liver metastases than in patients

without, implying a correlation to tumor burden. The usefulness of chromo-

granin A as a marker in bronchial carcinoid patients without metastases needs to

be further evaluated. Moreover, the low specificity of circulating chromogranin

A (Granberg et al., 1999) makes it unsuitable for screening in patients with

respiratory symptoms or for differential diagnosis of a lesion found on chest X-

ray. The efficacy of chromogranin B, increased in 86% of the patients, needs to

be further investigated. Urinary 5’HIAA, which was elevated in 68%, may

however be used as a marker in patients with metastatic bronchial carcinoids. In

the absence of a clinically overt Cushing’s syndrome, measurement of plasma

ACTH or urinary cortisol is of limited clinical value (Paper IV).

41

TABLE 7. Hormone markers in patients with bronchial carcinoids

Hormone Measured(n)

Elevated(n)

Elevated(%)

P-Chromogranin A 30 28 93%

P-Chromogranin B 14 12 86%

U-5’HIAA 31 21 68%

S-hCGα 30 17 57%

U-MeImAA 21 6 29%

S-PP 30 8 27%

P-ACTH 25 2 8%

U-cortisol 25 2 8%

S-Calcitonin 24 1 4%

S-hCGβ 30 1 3%

Treatment

Biotherapy

In our material of bronchial carcinoids (Paper IV), decrease of tumor size was

not observed during treatment with α-interferon. Only 4/27 patients (15%), 3 of

whom received combination therapy with octreotide, showed stable disease

during median 15 months. The remaining 23 patients (85%), 12 of whom were

treated with single drug α-interferon, 10 α-interferon + octreotide and the last

patient α-interferon + γ-interferon, progressed. This implies less effect of α-

42

interferon on tumor growth in bronchial than in midgut carcinoids (Frank et al.,

1999). Symptomatic relief of carcinoid syndrome, which was achieved in 7/16 of

our patients, seems to be the principal effect of biotherapy with α-interferon and

octreotide in bronchial carcinoids.

Treatment with γ-interferon was used in altogether 14 patients without being

apparently beneficial. Intolerable side-effects necessitated withdrawal within 2

months in 4 patients (3 of whom received concomitant α-interferon). In another 7

patients (5 of these 7 were treated with α-interferon in combination with γ-

interferon) progressive disease was observed within 6 months. Only 3 patients (1

treated with α-interferon + γ-interferon and 2 receiving γ-interferon + octreo-

tide) showed stable disease during 10– 29 months. Progressive disease was

however later recorded in these cases as well. None of the patients treated with

octreotide (n=3) or lanreotide (n=1) as single drug therapy experienced an

objective response. This therapy is only indicated in patients with disabling

carcinoid syndrome. In addition, we have seen symptomatic improvement and

decrease of hormone levels during treatment with octreotide in bronchial carci-

noid patients displaying ectopic Cushing’s syndrome.

Chemotherapy

The response rates are summarized in Table 8. The combination of streptozoto-

cin + 5-FU, for which reponse rates of 45–63% have been reported in patients

with endocrine pancreatic tumors (Eriksson and Öberg, 1993; Moertel et al.,

1980; Moertel et al., 1992), was not effective in our patients (Paper IV). Six

patients progressed continously, while one patient, showing stable disease for 8

43

months, progressed after 12 months. Streptozotocin + 5-FU is thus not indicated

in bronchial carcinoid patients. On the other hand, doxorubicin combined with

either streptozotocin or paclitaxel resulted in stable disease for about 9 months in

2/2 patients each. Doxorubicin-containing regimens may be of value for patients

with bronchial carcinoids and should be further evaluated in controlled studies.

The combination of cisplatinum + etoposide, which is effective in small cell lung

cancer, was used in 8 of our patients (Paper IV), receiving 3–8 (median 6) cour-

ses. Two patients showed decrease in tumor size lasting 6 and 8 months respec-

tively and one had stablilization of the disease for 7 months. The remaining five

patients progressed after 3–4 months, although the hormones decreased and

symptomatic relief was obtained in a patient with ectopic Cushing’s syndrome

who received concomitant α-interferon. There was no correlation of response to

Ki-67 expression; both patients with objective responses had typical carcinoids

with 1% and 3% Ki-67-positive cells respectively. Our encouraging results

contrast to another study of patients with neuroendocrine tumors, where neither

of 2 patients harboring well differentiated lung carcinoids showed decreased

tumor size or reduction of urinary 5’HIAA during treatment with cisplatinum +

etoposide (Moertel et al., 1991). Although renal and neurologic side-effects limit

the number of tolerated courses, further studies of Cisplatinum + Etoposide are

justified in bronchial carcinoid patients.

44

TABLE 8. Response rates to medical treatment in patients with metastatic

bronchial carcinoids

Treatment Total Stable disease Duration* n n % months

α-interferon 27 4 15% 15

streptozotocin + 5-FU 7 1 14% 8

streptozotocin + doxorubicin 2 2 100% 9

cisplatinum + etoposide 8 3# 38% 7

paclitaxel + doxorubicin 2 2 100% 9

* Median duration in patients who showed stable disease or objective response.# Two of these patients displayed objective response.

Other treatments

Five patients underwent liver embolization with gel-foam (Paper IV). The proce-

dure was performed on one occasion in 1 patient, on two occasions in 2 patients

and on three occasions in 2 patients. Clinical improvement was achieved in 3

patients. Biochemical stabilization was observed in 2 patients, while 3 patients

experienced reduction of hormones. One patient showed decreased size of the

liver metastases lasting 11 months, while the remaing 4 patients showed radiolo-

gically stable disease for 8–23 months. As could be expeted from earlier data

regarding midgut carcinoids (Eriksson et al., 1998), liver embolization may as

well be of value for debulking of liver metastases in patients with bronchial

carcinoids, and may produce symptomatic relief and reduction of hormones. The

effect is, however, short-lived and most pronounced after the first embolization

in each lobe.

45

None of the 3 patients receiving targeted radiotherapy (111In-Octreotide, n=2; 131I-

MIBG, n=1) responded, but the limited number of cases and the fact that all

patients harbored advanced disease makes it difficult to settle the role for targe-

ted radiotherapy in patients with metastatic bronchial carcinoids.

Prognosis

All 5 patients with distant metastases in Paper I and II have died from their tumor

disease during the observation period; one of them, however, died after Paper II

was published. Moreover, one patient died from an unrelated cause, free of car-

cinoid recurrence. The relatively high mortality rate, 12%, contrasts to several

other reports (Padberg et al., 1996; Travis et al., 1991; Warren and Gould, 1990).

This may be due to differences in the patient populations. It is possible that, in

other studies reporting surgical materials, patients with widespread disease were

not referred to surgery and therefore not included. On the other hand, our inten-

tion was to identify all patients with typical carcinoids in the referral area of the

hospital. Confirming the results in previous studies (El-Naggar et al., 1991; Jones

et al., 1988; McCaughan et al., 1985; Torre et al., 1989), lymph node metastases

at diagnosis were associated with increased risk of distant metastases (p=0.04)

and shorter survival time (p<0.001). The 5- and 10-year survival in our unselec-

ted patient material of 43 typical bronchial carcinoids (Paper I and II) was 95%

and 91%, respectively, which is consistent with other reports (Chughtai et al.,

1997; Garcia-Yuste et al., 2000; Harpole et al., 1992; Okike et al., 1976). The

46

discrepancy between the relatively high mortality rate and the long survival time

reflects the slow growth of these tumors.

The prognosis in patients with bronchial carcinoids harboring distant metastases

was poor. Median survival from diagnosis among the patients treated at our unit

(Paper IV) was 76 months (16–325). Twenty-four patients (including all 4

patients with atypical and 20 with typical carcinoids) have died median 26

months (4–106) after treatment start, while 7 patients (all harboring typical

carcinoids) are alive with disease after median 23 months (18–71). Five- and 10-

year survival from the initial diagnosis was 70% and 54% respectively, while 5-

year survival from treatment start was only 22% (Figure 10). The survival rates

for all the patients with typical bronchial carcinoids displaying distant metastases

(Paper I and II + Paper IV) were identical; 5- and 10-year survival was 70% and

57% respectively from diagnosis. These results confirm the malignant potential

of bronchial carcinoids, including typical ones. Although these tumors are slowly

growing, the response to medical treatment, when distant metastases have

occurred, is dismal.

0 50 100 150 200 250 300 350

0

.2

.4

.6

.8

1

Cum.Survival

Months

Figure 10. Survival from start of treatment, , and from initial diagnosis, , inpatients (n=31) with metastasizing bronchial carcinoids. Kaplan-Meier plots.

47

CLINICAL MANAGEMENT

Figure 11. When a patient is found to have a lesion suspected to be a bronchial

carcinoid, either on chest X-ray or at bronchoscopy, histological diagnosis is

mandatory. If the tumor is not accessible by bronchoscopy, transthoracic needle

biopsy guided by CT should be considered. A CT scan of the thorax is warran-

ted for identification of intrathoracic lymph nodes, and CT or ultrasonography of

the abdomen is indicated to screen for liver metastases. An octreoscan ought to

be performed as well. Plasma chromogranin A should be analyzed; if the patient

presents with carcinoid syndrome, measurement of U-5’HIAA and U-MeImAA

is also indicated. Sampling of P-ACTH and U-cortisol is not necessary unless

Cushing’s syndrome is clinically overt. After these diagnostic measures, the

patient should be referred to surgery with curative intent. The aim of the surgical

procedure is to remove the tumor and all affected lymph nodes, sparing as much

lung parenchyma as possible. It is necessary to take intraoperative biopsies with

frozen section. For assessment of prognosis, the tumor should be classified as

typical or atypical and immunostained with CD44s and Ki-67. When radical

surgery of the primary tumor is not possible or distant metastases are present,

chemotherapy with doxorubicin combined with streptozotocin or paclitaxel may

be considered; another alternative is cisplatinum + etoposide. Thoracic

radiotherapy may be given to control local tumor growth. All patients with

bronchial carcinoids ought to be followed for more than a decade, since

metastases may occur late. The follow-up should include measurement of plasma

chromogranin A and regular screening for local recurrence and distant (liver and

bone) metastases.

48

Figure 11. Suggested management of patients with bronchial carcinoids.

Curative surgery Lymph node dissection Biopsies + frozen sections

CT thorax Bronchoscopy + biopsy P-chromogranin A CT/ultrasound liver Octreoscan

Clinically or radiologically sus-pected bronchial carcinoid

Distant metastases

No Yes

Stain tumor withKi-67 and CD44s

Consider chemotherapy: doxorubicin + streptozotocindoxorubicin + paclitaxel cisplatinum + etoposide

Follow-up >10 years

49

GENERAL SUMMARY

Typical bronchial carcinoids are potentially malignant tumors. In our unselected

material of 43 patients, altogehter 12 (28%) displayed metastatic disease. Distant

metastases were found in 5 patients (12%), who all died of their tumors. Lymph

node metastasis at diagnosis is associated with increased risk of distant

metastases and death. A high Ki-67 index correlates to increased risk of distant

metastases and mortality and to decreased survival time. Positive staining for

CD44s, v9 and v7-8, as well as positive nuclear nm23 staining is associated with

decreased risk of distant metastases and mortality. On the other hand, positive

staining for bcl-2 correlates to increased risk of distant metastases and death as

well as decreased survival time, while p53 only correlates to decreased survival

time. GRP merely shows correlation to metastatic disease. Immunostaining for

CD44s, CD44v7-8, CD44v9, nm23, Ki-67, bcl-2 and p53 might provide

additional information on the individual tumor biology and prognosis in patients

with typical bronchial carcinoids. For routine use as prognostic markers in these

patients, CD44s and Ki-67 are most suitable.

In patients with bronchial carcinoids, octreoscan is less sensitive for detection of

the primary tumor and liver metastases than CT. Conventional bone scan is more

effective than octreoscan for identification of bone metastases. Octreoscan may

nevertheless be useful in the follow-up of patients with somatostatin-receptor-

positive bronchial carcinoid tumors. An octreoscan should be performed in all

these patients prior to surgery with curative intent.

50

Plasma chromogranin A is the most sensitive tumor marker; in addition urinary

5’HIAA may be used as a marker in patients with metastatic bronchial carci-

noids. In the absence of clinically overt Cushing’s syndrome, measurement of

plasma ACTH or urinary cortisol seems to be of limited value.

Biotherapy with α-interferon and/or octreotide has mainly symptomatic effects in

patients with bronchial carcinoids, although a limited number of subjects may

experience stable disease. The combination of streptozotocin + 5-FU is not indi-

cated in these patients. On the other hand, doxorubicin combined with strepto-

zotocin or paclitaxel may result in stable disease and should be further studied.

Cisplatinum + etoposide resulted in tumor reduction or stable disease in 38% of

the patients. Although renal and neurologic side effects limit the number of

courses that can be administered, this combination seems to be useful in a subset

of patients with bronchial carcinoids. Liver embolization with gel-foam may for

limited periods result in symptomatic improvement in addition to reduction of

hormone markers and stabilization of tumor size.

The prognosis in bronchial carcinoid patients harboring distant metastases is

poor. Median survival was 76 months (16–325) from diagnosis, while 5- and 10-

year survival was 70% and 54% respectively. Five-year survival from treatment

start was only 22%. Although these tumors are slowly growing, the response to

medical treatment is discouraging. Radical surgery still offers the best hope for

cure. Since metastases may occur decades after the initial diagnosis, these

patients have to be followed for a long period of time.

51

ACKNOWLEDGEMENTS

This study was carried out at the Department of Endocrine Oncology, Division of

Medicine, University Hospital, Uppsala, Sweden. Financial support was received

from the Swedish Cancer Society, Lions Foundation for Cancer Research, the

Swedish Medical Council, the Swedish Society for Medical Research, the

Uppsala County Association against Heart and Lung Diseases and from Torsten

and Ragnar Söderbergs Foundation.

I wish to express my sincere gratitude to the following persons:

Britt Skogseid, my supervisor, for introducing me into the mysteries of research

Kjell Öberg, the former head of our department, for including me into his team

and providing me with the possibilities to perform research in the field of cancer,

and for his support, listening and interest

Erik Wilander, my co-supervisor, for fruitful collaboration and help with

interpretation of the immunohistochemistry

Barbro Eriksson, the head of our department, for her human attitude to

everybody

52

Anders Sundin and Jan-Erik Westlin, my co-authors in Paper IV, for their

interest, help and support and Ola Hessman for valuable comments about the

manuscript

Rajni Dyal, for teaching me the laboratory work, and Margareta Halin-

Lejonklou, for help with the immunohistochemical stainings

Lena Hultström, for excellent secretarial assistance and generous help with

practical problems of all kinds

Marie-Louise Fjällskog, Jarl Hellman, Eva Tiensuu Janson, Staffan Welin,

Håkan Örlefors and all other colleagues at our department for good cooperation

and interesting discussions

Peter Stålberg and Shu Wang for fruitful research cooperation

Gertrud Fredriksson, for generous help in finding patient records

Anita, Doris, Gunnel, Gunvi, Helena, Maj, Maud, Monica, Ragnvi and all

other staff at 30 B2 for their enthusiasm and warm care of the patients and

making our ward a nice place to work

Yesuf Taha, my friend, for interesting discussions and being a pleasant room-

mate

53

Gösta Tjernberg, my friend, for plenty of help in solving computer problems

Riselda, for love and tenderness

Kjell, my father, for his never-ending love, help with everything and support

Caroline and Niklas, my beloved children

54

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