Serum and tumour biomarkers in non-small cell lung cancer
Hanne Astrid Eide, MD
Department of Cancer Genetics Institute for Cancer Research The Norwegian Radium Hospital
Division of Cancer Medicine Oslo University Hospital
Faculty of Medicine University of Oslo
© Hanne Astrid Eide, 2020
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-570-9
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
3 Table of contents
Acknowledgements ... 5
Abbreviations ... 7
Aims ... 9
List of papers ... 10
Introduction ... 11
Background ... 12
Lung cancer epidemiology ... 12
Aetiology ... 15
Diagnosis ... 16
Classification and staging of non-small cell lung cancer ... 18
Treatment and survival in patients with non-small cell lung cancer ... 22
Surgery ... 23
Radiotherapy ... 24
Chemotherapy ... 25
Targeted therapy ... 27
Immunotherapy ... 28
The development of cancer - tumour biology ... 30
A genetic and epigenetic disease ... 30
The tumour microenvironment ... 33
The immune system ... 35
Cytokines and metalloproteinases ... 38
Tumour biology and radiotherapy ... 41
Methods ... 43
Patient material... 43
The lung cancer biobank... 43
The ThoRaT-study ... 44
COPD control group ... 45
Radiotherapy cohort ... 46
Laboratory methods ... 47
RNA isolation and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for gene expression. ... 47
MicroRNA expression microarray ... 48
Immunohistochemistry ... 49
Blood sampling ... 51
Multiplex bioassays ... 51
FDG PET/CT ... 54
Statistical methods ... 55
Differences between variables ... 55
Correlations and hierarchical clustering ... 56
Survival analyses ... 56
Multiple testing. ... 57
4
Summary of individual papers ... 58
Paper I... 58
Paper II... 59
Paper III ... 59
Paper IV ... 60
Discussion ... 62
Methodological considerations ... 62
Patient material ... 62
Investigations... 64
Statistical considerations ... 68
Discussion of main results in papers ... 70
Tumour characteristics as biomarkers in NSCLC ... 71
Circulating proteins as biomarkers in NSCLC ... 75
Conclusions and future aspects ... 81
References ... 83
5 Acknowledgements
The work presented in this thesis was performed at the Department of Cancer Genetics, Institute for Cancer Research at the Norwegian Radium Hospital.
I would like to express my gratitude towards the University of Oslo for admitting me to the ph.d program. The financial support from Kreftforeningen is very much
appreciated. I will also remain thankful to all the patients participating in this project and thus contributing to the increased knowledge of non-small cell lung cancer.
Åslaug Helland, my main supervisor and academic inspiration. Thank you for including me in your research group and for all your support. Thank you for being positive, for always being available for a meeting and for pushing me forward when things were going slow. Thank you for believing in me. Your knowledge and interest have been essential in the work in this thesis and is very much appreciated.
Odd Terje Brustugun, my co-supervisor. I highly appreciate your encouragement, input and guidance. Thank you for sending me to the ETOP meeting in Lugano, a truly unforgettable educational (and travel) experience.
Cecilie Kiserud, thank you for getting me started, for introducing me to statistics and the use of SPSS, and for teaching me the basics of writing an academic paper. I also appreciate your continued interest in my work.
I would like to acknowledge the valuable contributions of all my co-authors. I am grateful for the fruitful collaborations with the Department of Medical Physics, Ingerid Skjei Knudtsen and Eirik Malinen, and with the Department of Cell Therapy, Jon Amund Kyte and Anne Fåne.
Further, I have to thank all my colleagues at the Department of Cancer Genetics, and especially Anne-Lise Børresen-Dale, Therese Sørlie and Gry Geitvik for being responsible for a creative and engaging work environment. Thank you, Vilde and Janne Beathe for good discussions and always good company. Thank you, Daniel for valuable IT-support and for improving my attempts at making decent figures. Thank you, Vandana for help with the statistics. Thank you, Helga for your great sense of humour and for always making me laugh.
Rita, you are a wonderful co-worker. Thank you for teaching me laboratory work, for explaining me qPCR and array-data, for always being friendly and supportive, and for useful scientific discussions.
6
Ingjerd, thank you for all your help in retrieving tissue samples and patient
information. Thank you for always taking an active interest in my work and in my life in general. Thank you for coming to my office in the clinic and asking me on how I am doing. Thank you for crying with me when my mother passed away, I will never forget.
Maria, you have been the best part of my years at the institute. Thank you for being a fantastic colleague and friend! Thank you, Mette for many enjoyable coffee (and chocolate) breaks as well as good friendship.
To my colleagues at the Department of Oncology, I am grateful for your continued support and interest. Thank you for the warm welcome back into the clinic.
I would like to express my deepest gratitude to my mother for teaching me the importance of hard work and always doing your best. To my dear aunt Liv, I am forever grateful for your encouragement during my whole life and for being a fantastic role-model for female accomplishment. You are both sorely missed. Thank you also to my father for always good company, for taking an interest in my work and for many, many discussions.
Lastly, I would like to thank my husband Martin for all your love and support, and for always being proud of my accomplishments. To my two wonderful sons, Petter and Arne, I love you – you make me happy every single day.
Oslo, April 2019 Hanne A. Eide
7 Abbreviations
ACTB Actin-beta
AIS Adenocarcinoma in situ
ALK Anaplastic lymphoma kinase
BRAF B-Raf proto-oncogene
CAF Cancer-associated fibroblast
CCL C-C motif chemokine ligand
CDKN2A Cyclin-dependent kinase inhibitor 2A
CEA Carcinoembryonic antigen
COPD Chronic obstructive pulmonary disease
CRP C-reactive protein
CT Computed tomography
CTLA-4 Cytotoxic T-lymphocyte antigen-4 CXCL C-X-C motif chemokine ligand
DNA Deoxyribonucleic acid
ECOG Eastern Cooperative Oncology Group EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay FFPE Formalin-fixed paraffin-embedded GCP-2 Granulocyte chemotactic protein 2 G-CSF Granulocyte-colony stimulating factor
GM-CSF Granulocyte-macrophage colony-stimulating factor Gro-a Growth-regulated protein alpha
Gro-b Growth-regulated protein beta
Gy Gray
HMGA2 High mobility group AT-hook 2
HR Hazard ratio
IFN-g Interferon gamma
IHC Immunohistochemistry
IL Interleukin
IP-10 Interferon gamma-induced protein 10
I-TAC Interferon Inducible T-cell alpha chemoattractant LDCT Low dose computed tomography
MCP Monocyte chemotactic protein
MDC Macrophage derived chemokine
MIF Macrophage migration inhibitory factor MIG Monokine induced by gamma interferon
MIP Macrophage inflammatory protein
MMP Matrix metalloproteinase
MPIF Myeloid progenitor inhibitory factor mRNA Messenger ribonucleic acid
MTV Metabolic tumour volume
MYCN V-myc avian myeolocymatosis viral oncogene neuroblastoma derived homolog NF-κB Nuclear factor kappa B
NLR Neutrophil-lymphocyte ratio
NOS Not otherwise specified
NSCLC Non-small cell lung cancer
NSE Neuron-specific enolase
OS Overall survival
OUS Oslo University Hospital
8
PDGF-BB Platelet-derived growth factor subunit B
PD1 Programmed cell death 1
PD-L1 Programmed cell death ligand 1
PFS Progression free survival
Pro-GRP Pro-gastrin-releasing peptide
RECIST Response Evaluation Criteria in Solid Tumours
RNA Ribonucleic acid
ROS Reactive oxygen species
ROS1 ROS proto-oncogene 1
RT Radiotherapy
RT-qPCR Reverse transcription quantitative polymerase chain reaction SDF-1a + b Stromal-derived factor 1alpha + beta
SCLC Small cell lung cancer
SUV Standardized uptake values
TAM Tumour-associated macrophage
TARC Thymus and activation regulated chemokine
TCGA The Cancer Genome Atlas
TECK Thymus Expressed Chemokine
Th-cell Helper T-cell
ThoRaT Thoracal Radiotherapy and Tarceva®
TIL Tumour infiltrating lymphocyte
Tis Tumour in situ
TKI Tyrosine kinase inhibitor
TLG Total lesion glycolysis
TMA Tissue microarray
TNF-a Tumour necrosis factor alpha
TNM Tumour node metastasis, classification of malignant tumours TRAIL TNF-related apoptosis-induced ligand
TTF-1 Thyroid transcription factor-1
UICC Union for International Cancer Control VEGF Vascular endothelial growth factor
WHO World Health Organization
9 Aims
The overall aim of the studies presented in this thesis was to identify biomarkers in patients with non-small cell lung cancer (NSCLC). Divided into 4 subprojects both tumour tissue and serum samples obtained from lung cancer patients were
investigated.
The work conducted is considered explorative and hypothesis-generating.
The specific aims were:
To characterise the MYCN, HMGA2 and CDKN2A pathway in NSCLC tumours (paper I).
To assess the prognostic potential of mRNA expression of MYCN, HMGA2, CDKN2A and DICER1 in NSCLC tumours (paper I).
To study blood-based levels of cytokines and matrix metalloproteinases obtained from NSCLC patients with advanced disease and compare the concentrations with a cohort of patients suffering from chronic obstructive pulmonary disease (paper II).
To investigate the dynamics of cytokines and matrix metalloproteinases in serum collected from NSCLC patients during a course of radiotherapy (paper III).
To investigate the dynamics of 18F-FDG PET/CT parameters SUVmax, MTV and TLG in NSCLC patients during a course of radiotherapy and further to study possible associations to the cytokines/matrix metalloproteinases in serum (paper III).
To study the prognostic value of protein levels of TP53, MDM2, BCL2, EGFR, HMGA2 and PD-L1 in NSCLC tumours in patients with locally advanced and advanced disease treated with radiotherapy (paper IV).
10 List of papers
I. Hanne A. Eide, Ann Rita Halvorsen, Maria Moksnes Bjaanæs, Hossein Piri, Ruth Holm, Steinar Solberg, Lars Jørgensen, Odd Terje Brustugun, Cecilie Essholt Kiserud and Åslaug Helland.
The MYCN-HMGA2-CDKN2A pathway in non-small cell lung carcinoma - differences in histological subtypes.
BMC Cancer. 2016 Feb 8;16:71. doi: 10.1186/s12885-016-2104-9
II. Hanne A. Eide, Ann Rita Halvorsen, Vandana Sandhu, Anne Fåne, Janna Berg, Vilde Drageset Haakensen, Elin H. Kure, Odd Terje Brustugun, Cecilie Essholt Kiserud, Jon Amund Kyte and Åslaug Helland.
Non-small cell lung cancer is characterised by a distinct inflammatory signature in serum compared with chronic obstructive pulmonary disease.
Clinical & Translational Immunology. 2016 Nov; 5(11):e109. doi:
10.1038/cti.2016.65
III. Hanne A. Eide, Ingerid Skjei Knudtsen, Vandana Sandhu, Ayca M. Løndalen, Ann Rita Halvorsen, Azadeh Abravan,Elin H. Kure, Trond V. Bogsrud, Odd Terje Brustugun, Jon Amund Kyte, Eirik Malinen, and Åslaug Helland.
Serum cytokine profiles and metabolic tumor burden in patients with non-small cell lung cancer undergoing palliative thoracic radiotherapy.
Advances in Radiation Oncology. 2018 Feb 13;3(2):130-138. doi:
10.1016/j.adro.2017.12.007.
IV. Hanne A. Eide, Marius Lund-Iversen, Martina Landschoof Skrede, Elin Kure and Åslaug Helland
PD-L1 protein expression in patients with locally advanced and advanced non- small cell lung cancer receiving radiotherapy.
Manuscript
11 Introduction
Globally, lung cancer constitutes a major disease burden. In Norway, about 60
patients are diagnosed with lung cancer every week. In the treatment of non-small cell lung cancer (NSCLC) radical surgery remains the gold standard, although recently developed treatment modalities such as stereotactic radiotherapy can also cure a NSCLC patient with a limited disease burden. A majority of patients with NSCLC are diagnosed in more advanced stages and many patients will die within a few years following the lung cancer diagnosis. Indeed, the World Health Organization (WHO) presents lung cancer as the fifth deadliest disease of all diseases worldwide.
Unfortunately, no symptom is lung cancer specific.
It is evident that smoking cessation could prevent a substantial number of lung cancer patients in the forthcoming decades. Screening with low dose CT scans for early detection of lung cancer is further an important initiative, eagerly discussed worldwide and now even implemented in the USA. The lung cancer diagnostic pathway
(pakkeforløp) ensures fast track diagnostics and start of treatment within a defined set of days for all lung cancer patients in Norway. Moreover, the national guidelines for diagnostics and treatment of lung cancer are frequently updated and easily available through the Norwegian lung cancer group website providing the foundation for personal based treatment decisions.
For many years cancer research focused primarily on the malignant cell, translating cell experiments into the clinical setting. Genetic instability is regarded as an
underlying trait in cancer development. In a minority of NSCLCs, driver mutations are discovered and tailored treatment is now an option for selected patient groups. It is however, increasingly recognized that the cancer cells interact with, and even shapes, the constituents of the surrounding tumour microenvironment, making an impact both in tumour development and in the outcome of treatment. As a consequence, new promising treatment opportunities, like immunotherapy, have entered the clinic with encouraging results.
12
There used to be a saying among physicians that lung cancer could not be diagnosed too late due to lack of efficient treatment options. Contrary, developments in recent years have changed the way we look upon NSCLC. An increase in options for
personalized treatment and prolonged survival, even in patients with advanced stages of disease, is seen as a result of countless scientific achievements all united in an effort to unveil the mystery of lung cancer and its remedy.
Background
Lung cancer epidemiology
In Norway, 3214 patients were diagnosed with lung cancer in 2017 [1]. Lung cancer is a common cancer, only surpassed by prostate cancer in men, and breast and colorectal cancer in women. The distribution of lung cancer among men and women is
approaching 1:1, with an incidence of 1705 and 1509 in men and women,
respectively. In comparison, the male to female ratio was 3.8 in the mid-1980s [1].
The Cancer Registry of Norway has in previous years expressed a deep concern that the incidence rate of lung cancer in women has continued to increase, while a decline is seen in men [2]. Historically, the incidence of lung cancer among men in Norway reached a plateau in the 1990s. The rate of lung cancer in men has declined by 6.5%
comparing the 5-year time period 2013-2017 to the previous one (Figure 1). Among women, declining rates under the age of 60, and a flattening of the rates in the age group 60-69 is now reported. The only group with still increasing rates is the women above 70 years old where a 9.2% increase is seen in the last 5-year period compared to the preceding time period [1]. The number of patients diagnosed with advanced
disease has remained stable for many decades constituting 40 - 50% of the total lung cancer incidence [1].
13
Figure 1. Age adjusted incidence rates for lung cancer in Norway in 5-year intervals from 1957 to 2016 [2].
Lung cancer is a deadly disease and the mortality rates reflect the incidence rates.
Lung cancer caused 2234 (20%) of 10944 cancer related deaths in 2016, representing the most frequent cause of cancer related death in men and women in Norway [1]. A calculation of human years lost due to cancer, found that lung cancer alone was responsible for almost as many years of life lost, as breast, prostate and colon cancer combined [3]. According to the Cancer Registry of Norway, 7507 individuals
remained alive after being diagnosed with lung cancer at the end of 2016, 34% of which were diagnosed more than five years ago. Indeed, a recent paper on nation-wide trends in lung cancer in Norway describes an increase of 136% in lung cancer
prevalence from 2000 to 2016 [4].
Globally, lung cancer is a common cancer, with an estimated 1.8 million new lung cancer cases among a total cancer incidence of 14.1 million [5]. Geographical
differences in incidence and mortality in various types of cancer is related to the status of economic development in different countries [4]. In less developed countries lung cancer is the most frequent cancer in men, while it is surpassed by prostate cancer in more developed countries (Figure 2). In women, lung cancer is the third most
0 10 20 30 40 50 60 70 80
Women Men
14
common cancer, passed by breast and colorectal cancer in developed countries and by breast and cervical cancer in less developed countries (Figure 2). In Norway, a similar pattern in cancer incidence as our fellow developed countries is seen [1]. Lung cancer causes approximately 1.6 million deaths worldwide [6]. For men in developed and less developed countries, and for women in developed countries, lung cancer resides as the number one cancer killer. In less developed countries breast cancer has the highest mortality rate in women, while lung cancer comes second (Figure 2) [5].
Figure 2. (a) Estimated numbers (thousands) of new cancer cases (incidence) and deaths (mortality) in men in more developed and less developed regions of the world in 2012. (b) Estimated numbers (thousands) of new cancer cases (incidence) and deaths(mortality) in women in more developed and less developed regions of the world in 2012 [5].
Reprinted with permission.
15 Aetiology
The pattern of smoking in the general population is closely linked to the historical trends in the lung cancer incidence and is further a predictor of the lung cancer burden in the future.
Lung cancer used to be an uncommon disease, and prior to the 20th century considered as a rarity among physicians. Concurrent with a rise in the incidence of lung cancer during the first half of the twentieth century a suspicion of a malicious effect of cigarette smoking gradually developed. Isaac Adler proposed in 1912 smoking to be blamed for a growing incidence of lung tumours in the publication Primary Malignant Growths of the Lungs and Bronchi. However, it was not until the 1950s and the
epidemiological studies by Wynder and Graham in the USA and the British Doctor study by Doll and Hill, that convincing statistical evidence of a link between smoking and lung cancer was established [7–10]. These epidemiological studies were further supported by findings from animal experiments demonstrating tumour growth following skin exposure of tobacco [11], as well as the discovery of cancer causing chemicals in the cigarette smoke [10]. The health risk of cigarette consumption became during the last half of the 20th century unequivocal. It is now estimated that smoking causes approximately 80 – 90% of lung cancers. Furthermore, second hand smoking is also shown to represent a substantial risk factor for lung cancer death [12, 13].
The trends in lung cancer incidence in Norway also reflect the different phases of the smoking epidemic. According to Statistics Norway, 12% of the population were daily smokers in 2016, representing a marked drop from > 30% in the 1990s (Figure 3) [14]. The estimated lag time between smoking prevalence and mortality rates of lung cancer is 20-30 years. A decline in cigarette consumption among Norwegian men has been observed since the early 1970s, while the decrease in women did not start until 1990s, thus accounting for the decline observed in lung cancer incidence in men the recent years, while a continuous increase is still seen in Norwegian women.
16
Figure 3. Percentages of daily smokers in Norway, age 16-74 years old [14].
Lung cancer also occurs in non-smokers. Second hand smoking, radon gas emanation, occupational exposure to asbestos dust and nickel, as well as outdoor pollution is listed as possible causes of lung cancer in non-smoking patients [15–17]. If 90% of the total cancer incidence is caused by smoking, 308 Norwegian patients have, according to the statistics, developed lung cancer due to other causality in 2016 – the incidence of non-smoking associated lung cancer thus surpasses the total incidence of cervical cancer, testicular cancer and thyroid cancer in Norway in 2016. Human papilloma virus is previously investigated in lung cancer, but is less likely of significance in lung cancer development [18].
Diagnosis
No symptoms are lung cancer specific. The suspicion of lung cancer often arise in a person of somewhat advanced age (>50 years), frequently with a smoking history, presenting with a cough, chest/shoulder pain, dyspnea, weight loss, hoarseness or hemoptysis [19]. These lung cancer symptoms however, may very well resemble other smoking-relating diseases such as chronic obstructive pulmonary disease (COPD) and
0 5 10 15 20 25 30 35 40 45 50
1976 1980 1984 1988 1992 1996 2000 2004 2008 2012 2016
Women, daily smokers Men, daily smokers
17
pulmonary fibrosis, making the diagnosis challenging. It is further important to remember that lung cancer can develop in non-smokers and/or in younger patients.
Lung cancer may also cause few specific symptoms, especially in early stages.
Approximately 40% of lung cancer patients are diagnosed with advanced stage disease [1], most likely reflecting the lack of specific symptomatology in lung cancer disease.
In order to diagnose lung cancer, a thorough review of the patient’s history, followed by a clinical evaluation, serum tumour markers, a bronchoscopy, CT-scans and biopsies from the primary tumour or possible metastases are often utilized in the clinic. Supplementary procedures such as ultrasound, MRIs, PET-scans or drainage of pleural effusions are performed depending on the results of the initial investigations [19].
The cancer pathway for lung cancer (pakkeforløp) was established in Norway in January 2015 with the intent of providing a national standard for procedures and tests in order to diagnose lung cancer if lung cancer is suspected [20]. The cancer pathway defines the number of calendar days each assessment ideally should take. The aim is to complete the evaluation within 28 calendar days. Further, once the diagnosis of lung cancer has been made, surgical treatment or radiotherapy should commence within 14 calendar days, or medical treatment within 7 calendar days depending on which treatment is recommended. Finally, it is expressed that the treatment is to be decided in accordance with the patient’s own wishes.
Screening for lung cancer has been much debated around the world as well as in the Nordic countries. Early diagnosis by low dose computed tomography (LDCT) showed a 20% reduction in lung cancer mortality in a large randomized trial investigating American heavy smokers in the National Lung Screening Trial [21, 22]. This study provided the basis for implementing annual LDCT screening in the USA.
Comparably, in Europe, a plethora of studies regarding lung cancer screening are either currently ongoing or recently completed. The NELSON trial being the largest
18
European trial to date, has yet to publish mortality data, although the results so far in regards to detection rates and lung cancer stage distribution are concurrent with the results of the American National Lung Screening trial [23–26]. A pooling of seven European trials is planned after the publication of the NELSON mortality results and will then include over 36 000 participants [27]. Nevertheless, implementation of CT screening is already recommended by the European Respiratory Society, the European Society of Radiology, and the European Society of Medical Oncology and by Swiss University Hospitals [28]. Moreover, representatives of the Nordic Thoracic Oncology group have recently joined together in forming an expert study group to prepare a Nordic protocol as a common platform for implementation of future screening programs in the Nordic countries [28].
LDCT have a high sensitivity but a low specificity for the detection of lung cancer, resulting in a somewhat high false positive rate possibly causing psychological distress in the subjects and a definitive increased diagnostic work load. Consensus criteria for lung nodule identification still need to be established. Other potential harms of screening are the diagnosis of cancer that would otherwise not be
symptomatic or cause death in a patient. Definition of the risk-population is further a challenge, a cut-off in age and tobacco exposure being the most used in studies so far.
Additionally, the optimal screening interval is yet to be determined. Ultimately, it is the evaluation of the benefit of mortality reduction together with the expected cost- effectiveness of screening that will be decisive.
Classification and staging of non-small cell lung cancer
Lung cancer is divided into two main subtypes; non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) where NSCLC constitutes approximately 85% of the lung cancer cases. NSCLC are further divided into main groups adenocarcinomas, squamous cell carcinomas and large cell carcinomas. Traditionally, the subdivision of NSCLC had little clinical or therapeutic significance due to lack of distinguishable treatment options. Treatment was determined by disease stage only. However, in the last decades, clinical trials have demonstrated that responses to chemotherapy can
19
indeed depend on the histological subtype [29]. Also, with the introduction of targeted therapies, histological differentiation has become even more important with an impact on tailoring the NSCLC treatment.
A majority of the adenocarcinomas arise in the small bronchi, bronchioles or in the alveolar epithelial lining and is therefore more likely to reside in the periphery of the lungs [30]. Contrary, the squamous cell carcinomas tend to develop in the central areas of the lung, frequently arising in the major bronchi [30, 31]. Squamous cell carcinomas are heavily linked to smoking, whereas adenocarcinomas are also common in lung cancer in non-smoking patients. Adenocarcinomas will typically consist of mucus producing cells organized in glandular structures. Squamous cell carcinomas consist of keratinizing tumour cells, while large cells with no glandular structures or keratinization constitute the large cell carcinomas. Within the
adenocarcinomas and the squamous cell carcinomas numerous sub-classifications exists [32]. A mix of subtypes is even not unusual, fully underscoring the
heterogeneity of the NSCLC disease. The pathologist makes a classification based on the dominant subtype. Of note, the diagnosis of large cell carcinoma can only be made on resected tumour tissue.
Immunohistochemically staining of biopsies or cytology material is used to guide the subtyping of NSCLC tumours in addition to the morphological tissue assessment. In adenocarcinomas, thyroid transcription factor (TTF1) and cytokeratin 7 is more likely to be positive in a majority of the samples, while squamous cell carcinomas are best characterised by cytokeratin 5/6 and p63 staining, however this is not absolute [33]. If it is not possible to classify the lung cancer tumours based on morphology or
immunohistochemistry, the term not otherwise specified (NOS) are used. The NSCLC not otherwise specified declined from 24% to 13% in the time period from 2000 to 2016 [4]. It is currently recommended in Norway to test all NSCLC tumours of non- squamous origin for epidermal growth factor receptor (EGFR) mutation as well as anaplastic lymphoma kinase (ALK)- and ROS1-fusionprotein. Further, all NSCLC tumours are to be investigated in regards to programmed death ligand 1 (PD-L1) by
20 immunohistochemistry [19].
A shift in the histopathological subgroups in NSCLC is observed since the 1980s in Norway [34]. Previously, the squamous cell carcinomas were the dominating subtype in men, but have now been surpassed by the adenocarcinomas. In women
adenocarcinomas has remained as the main NSCLC entity over decades, but a definitive increase is seen also in women. In 2016 adenocarcinomas accounted for 50% of all lung cancers [4]. The increasing incidence of the adenocarcinomas are concurrent with trends in the western part of the world and may be related to changes in smoking patterns and/or in the composition of cigarettes [35, 36].
The extensiveness of the lung cancer disease in a patient is vital for the decision- making process regarding adequate treatment, as well as an important indicator of disease prognosis. The Tumour-Node-Metastasis (TNM) system is a standardized framework evaluating the primary tumour size and extension, lymph node affection and distant metastases. The eight edition of the TNM classification of lung cancer [37]
were implemented in January 2017 in Norway. The lung cancer patients investigated in this thesis however, are classified according to the seventh edition. The main changes from the seventh to the eight edition comprise a further differentiation in subgroups based on tumour size, and categories are now created for single and multiple extrathoracic metastases (Table 1) [37]. The nodal descriptors remain unchanged, but it is clear that quantification of nodal disease has prognostic impact [38].
21
Table 1. Changes in descriptors introduced in the eight edition of the TNM classification of lung cancer compared with the seventh edition (table adapted from [38])
Descriptor Seventh Edition Eight Edition
Tumour 0 cm
(pure lepidic adenocarcinoma
≤ 3 cm in total size)
T1a if ≤ 2 cm; T1b if > 2-3 cm Tis (AIS)
≤ 0.5 cm invasive size
(lepidic predominant adenocarcinoma
≤ 3 cm in total size)
T1a if ≤ 2 cm; T1b if > 2-3 cm T1mi
≤ 1 cm T1a T1a
> 1-2 cm T1a T1b
> 2-3 cm T1b T1c
> 3-4 cm T2a T2a
> 4-5 cm T2a T2b
> 5-7 cm T2b T3
> 7 cm T3 T4
Bronchus > 2 cm from carina T3 T2
Total atelectasis/pneumonitis T3 T2
Invasion of diaphragm T3 T4
Invasion of the mediastinal pleura T3 No longer a T
descriptor Node
NX, N0, N1, N2, N3 No change Metastasis
Metastases within the thoracic cavity M1a M1a
Single extrathoracic metastasis M1b M1b
Multiple extrathoracic metastases M1b M1c
Abbreviations: AIS, adenocarcinoma in situ; mi, minimally invasive adenocarcinoma; Tis, tumour in situ
The TNM classification can be clinical (cTNM) based on radiology and clinical assessment, or pathological (pTNM) following surgical resection. Finally, based on the TNM classification a staging system of NSCLC from stage I to stage IV is devised. In the eight edition multiple modifications in staging groups was made compared to the seventh edition, making the staging of lung cancer more complex than it was before (Table 2).
22
Table 2. Changes in staging introduced in the eight edition of the TNM classification of lung cancer compared with the seventh edition (table adapted from [19, 38].
Seventh Edition Eight Edition
T N M T N M
Occult carcinoma
TX N0 M0 Occult
carcinoma
No changes
Stage 0 Tis N0 M0 Stage 0 No changes
Stage IA T1a,b N0 M0 Stage IA1 T1mi N0 M0
T1a N0 M0
Stage IA2 T1b N0 M0
Stage IA3 T1c N0 M0
Stage IB T2a N0 M0 Stage IB T2a N0 M0
Stage IIA T1a,b N1 M0 Stage IIA T2b N0 M0
T2a N1 M0
T2b N0 M0
Stage IIB T2b N1 M0 Stage IIB T1a,b,c N1 M0
T3 N0 M0 T2a,b N1 M0
T3 N0 M0
Stage IIIA T1, T2 N2 M0 Stage IIIA T1a,b,c N2 M0
T3 N1, N2 M0 T2a,b N2 M0
T4 N0, N1 M0 T3 N1 M0
T4 N0 M0
T4 N1 M0
Stage IIIB T4 N2 M0 Stage IIIB T1a,b,c N3 M0
Any T N3 M0 T2a,b N3 M0
T3 N2 M0
T4 N2 M0
Stage IIIC T3 N3 M0
T4 N3 M0
Stage IV Any T Any N M1a,b Stage IVA Any T Any N M1a
Any T Any N M1b
Stage IVB Any T Any N M1c
Abbreviations: mi, minimally invasive adenocarcinoma; Tis, tumour in situ
Treatment and survival in patients with non-small cell lung cancer
The five-year relative survival of lung cancer (all stages) increased from 13.8% to 17.8% in men and from 19.5% to 24.4% in women in the period 2013-2017 compared to the previous five-year period in Norway (Figure 4) [1]. For patients with localized disease, eligible for treatment with surgery or stereotactic radiotherapy with curative intent, the five-year relative survival was 56.6% for men and 67% for women. For advanced stages, the long-term survival remains poor with 2% of male and 3.6% of female lung cancer patients alive after 5 years. An increase in one- and two-year survival is however, observed for all stages [19].
23
Figure 4. Five year relative survival by period of diagnosis 1977-2016 [2].
The main modalities in the management of cancer were developed during the twentieth century; surgery, radiotherapy and chemotherapy. Since the turn of the millennium however, new treatment options such as targeted therapy and
immunotherapy, have emerged into clinical practice.
Treatment of patients with NSCLC depends on the histological subtype and staging of the disease. The general condition of the patients, the patients’ age, additional disease, as well presentation of symptoms, is taken into consideration in the decision-making process. Finally, the patients’ preferences are taken into the account. In Norway, consensus based diagnostics and treatment are incorporated into national guidelines where it is recommended that the patients eligible for treatment with curative intent should be debated in multidisciplinary teams [19].
Surgery
Surgical resection remains the gold standard of treatment in patients with technically operable NSCLC disease, mainly stage I and II, and a few selected patients in stage III
0 5 10 15 20 25
Women Men
24
(T3-4 N0-1 M0). The aim of surgery is the complete removal of the malignant lung tumour and lymph node involvement. The patients eligible for surgery will have to be medically fit in order to survive the surgery, and to tolerate the potential long-term side-effects as a consequence of loss of lung tissue. Approximately 20% of the lung cancer patients undergo surgery every year in Norway [39].
Patients with advanced NSCLC disease (Any T N2-3 M+), will in general not benefit from surgery. The exception is oligo-metastatic disease i.e. a solitary brain or an adrenal gland metastasis, eligible for resection or stereotactic radiotherapy, where curative lung surgery should still be evaluated as a possibility [40].
Radiotherapy
Radiotherapy has remained an effective cancer treatment during many decades and it is estimated that about half of all cancer patients are eligible for treatment with
radiotherapy at some point during the management of their disease [41]. Radiotherapy is currently of use both as a curative and a palliative treatment option in NSCLC.
Depending on the indication several radiotherapy techniques and fractionation schedules are available in the clinic.
Conventional radiotherapy, in addition to chemotherapy, is recommended following surgery if a N2 affected lymph node is identified during surgery (pN2) and should also be considered if the resection margins are incomplete in the case of pN0/pN1,
especially if further surgery is not advised. Significant increased survival and less local tumour regrowth is seen in previous studies with postoperative radiotherapy [42–
44]. Radiotherapy prior to surgery is in general not advised, though exceptions can be made in tumours located at the top of the lungs with risk of local nerve/vasculature involvement (known as Pancoast tumours), or in locally advanced lung tumours with chest wall invasion (T3 N0).
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Stereotactic body radiotherapy has emerged as a treatment option in inoperable NSCLC patients with limited disease; in stage I, or in T2b/T3 tumours without nodal involvement. Using extreme hypo-fractionated irradiation, studies have demonstrated local tumour control above 90% with low grade toxicity [45, 46]. Retrospective analyses show comparable results with surgery in regards to overall survival [47].
Randomized trials comparing surgical treatment and stereotactic radiotherapy have been difficult to conduct due to poor accrual. A pooled analysis of two randomized trials, including 58 stage I NSCLC patients, randomized to either surgery or
stereotactic radiotherapy, showed a 3-year disease specific survival of 95% and 79%
in the radiotherapy and the surgery group, respectively [48].
Stage III NSCLC patients in good general condition, with locally advanced disease not eligible for surgery, are recommended conventional radiotherapy with curative intent. If possible, the radiotherapy will be combined with chemotherapy. Norwegian patients receiving conventional curative radiotherapy > 50 Gray (Gy) in the time period 1993-2001 had a five-year relative survival of 10% [49]. Stage III patients with negative prognostic factors and patients with distant metastatic disease (stage IV) will be offered palliative treatment. Negative prognostic factors entail high performance status and weight loss, but patients age, tumour size and burden of metastatic disease are also taken into consideration [19, 50].
Finally, irradiation can effectively alleviate troublesome symptoms caused by metastases located in bone, brain or within the thoracic cavity, and many NSCLC patients receive valuable palliation by radiation therapy. The treatment will in general consist of hypo-fractionated radiotherapy with a lower total radiation dose compared to curative treatment.
Chemotherapy
Chemotherapy as a single modality will not cure the NSCLC patient. A benefit of adjuvant chemotherapy however, is shown following surgery in stage II and IIIA patients. Four courses of cisplatin and vinorelbine is currently offered to Norwegian
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NSCLC patients with a performance status of ECOG 0-1 [51, 52]. Further, concurrent use of platinum-based chemotherapy and conventional radiotherapy with curative intent are recommended to fit stage III patients. A meta-analysis from 2010 showed a five-year survival of approximately 15 % with concurrent radio-chemotherapy
investigating mainly stage III patients [53]. Adding chemotherapy to preoperative radiotherapy has also been shown to increase local control in Pancoast tumours of the lung [54].
Chemotherapy is established as an essential part of palliative treatment and is recommended to NSCLC patients with a PD-L1 expression in less than 50% of the tumour cells and no EGFR or ALK alterations present in the tumour tissue. Different treatment doublets containing cisplatin/gemcitabine, cisplatin/paclitaxel,
cisplatin/docetaxel or carboplatin/paclitaxel have previously been compared in a large randomized trial in the USA where no significant differences were found [55]. The Norwegian standard of choice is a combination of carboplatin and vinorelbine, 3-4(6) courses. The aim of palliative treatment is mainly prevention and/or alleviation of cancer related symptoms. In addition, chemotherapy may prolong life in patients with advanced NSCLC [56]. Previous studies by the Norwegian Lung Cancer Study Group investigating the efficacy of palliative intended chemotherapy in patients with
advanced NSCLC demonstrated a median overall survival of approximately 7 months [57–59]. In general, a response rate of roughly 30 % is seen in palliative treatment with chemotherapy in NSCLC patients.
Second-line chemotherapy can be offered to motivated patients in good general health experiencing disease progression following first-line treatment. Recommended
regimens include docetaxel or pemetrexed monotherapy. Pemetrexed has been shown superior and is recommended in a second line setting in tumours of non-squamous origin [60]. Maintenance therapy with pemetrexed is also an option following the initial platinum doublet. A study by the Norwegian Lung Cancer Group is currently investigating whether immediate maintenance treatment with pemetrexed prolongs
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survival compared to observation and then commencement of therapy at disease progression [61].
Targeted therapy
A deeper understanding of the pathogenesis of NSCLC has led to the development of treatment directed towards defined molecular targets within the disease. Indeed, NSCLC is known to have a substantial amount of genomic alterations compared to many other types of cancer [62]. NSCLC patients with advanced disease of non- squamous origin are routinely tested for EGFR mutations and ALK rearrangements in the clinic. If a molecular alteration is discovered, the patients will be recommended targeted treatment.
Activating mutations in the EGFR gene is found in 7.5 % of the NSCLC tumours, mainly in adenocarcinomas, and is more likely to be present among women and in non-smokers [63]. The EGFR Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved as first line treatment in advanced NSCLC in Norway, regardless of which type of EGFR mutation is present in the tumour tissue. In general, the EGFR TKIs demonstrate a progression free survival of 9 to 13 months in the first line setting of advanced NSCLC [64]. Many patients however, treated with EGFR inhibitors in the first line setting, will develop an EGFR T790M mutation during the course of treatment, resulting in an ineffective treatment [65]. The EGFR inhibitor osimertinib has been shown efficacious in both untreated, as well as in previously treated patients with a verified T790 mutation [66, 67], but is currently not approved for use in the Norwegian public health system.
Rearrangements including the ALK gene are, in addition to EGFR mutations, most frequent in adenocarcinomas and non-smokers, and is present in 2-5 % of the NSCLC tumours. Several studies have shown superior effect of the TKIs crizotinib and
ceritinib compared to chemotherapy in advanced NSCLC [68–70]. Currently,
crizotinib used in the first and ceritinib in the second line setting is reimbursed in the public health system [19]. Recently, a third agent, alectinib, was approved and is now
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available in the first line setting of treatment in ALK-positive NSCLC. A study comparing alectinib and crizotinib have shown a median progression free survival of 25.7 months in the alectinib group compared to 10.4 months in the crizotinib group [71]. Interestingly, a significant difference was also seen in the incidence of brain metastases where 12 % of the patients treated with alectinib developed brain metastasis compared to 45 % in the crizotinib group.
BRAF mutations or ROS1 dysregulation can separately be detected in 1-2 % of the NSCLC tumours, usually in adenocarcinomas [72, 73]. Treatment with BRAF-/MEK- inhibitors in patients with a BRAF (V600) mutation, or treatment with the TKI
crizotinib in ROS1 positive patients have been shown efficacious in advanced NSCLC [74, 75]. Crizotinib is available for reimbursement in the public health system in Norway, while the decision on BRAF-/MEK inhibitors in the treatment of NSCLC is pending [19, 76]. ROS1 aberrations are now recommended to be tested in all NSCLC tumours of non-squamous origin, but BRAF mutations are as of yet not routinely tested in the clinic.
Immunotherapy
Recent advances in the field of immunotherapy have made a definitive impact in the treatment of NSCLC. All patients with NSCLC are currently tested for PD-L1 expression in the tumour tissue. If PD-L1 is expressed in at least 50% of the tumour cells, and neither an EGFR mutation nor an ALK/ROS1 translocation is detected, first line palliative treatment with immunotherapy (Pembrolizumab) is recommended [19].
It is expected that approximately 1/3 of the NSCLC patients have a PD-L1 expression exceeding 50 % in the tumour tissue, and should thus be offered immunotherapy following the diagnosis of advanced disease. In the Keynote 024 study, the PD-1 inhibitor pembrolizumab, demonstrated an overall survival of 10.3 months compared to 6 months in NSCLC patients treated with platinum-based chemotherapy [77].
Moreover, the response rate in Keynote 024 was shown superior with a 44.8%
response in the pembrolizumab group versus 27.8 % response in the chemotherapy group [77].
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Immunotherapy is also a treatment option in the second line setting in NSCLC patients in good general condition (ECOG 0-1), with PD-L1 present in the tumour tissue, but in less than 50% of the tumour cells. The PD-1 inhibitor nivolumab was the first immunotherapy agent to demonstrate a superior effect compared to docetaxel in patients with advanced NSCLC experiencing disease progression following first line treatment [78, 79]. Pembrolizumab, as well as the PD-L1 inhibitor atezolizumab have shown similar results [80, 81]. Due to cost effectiveness atezolizumab are currently recommended by the Norwegian lung cancer group in the second line setting [19].
Recently, pembrolizumab in addition to a platinum doublet in previously untreated metastatic NSCLC was demonstrated superior to a chemotherapy – placebo
combination [82, 83]. In February 2019, pembrolizumab was declined for use in this setting in the public health system in Norway [76].
In stage III locally advanced, unresectable NSCLC, immunotherapy has also proven efficacious. In the PACIFIC-study patients were treated with the PD-L1 inhibitor durvalumab in the adjuvant setting following chemoradiotherapy. A median progression-free survival of 16.8 months was shown in the durvalumab group compared to 5.6 months in patients treated with placebo [84]. A later analysis also conferred a benefit in overall survival where the median time to death or distant metastasis was 28.3 months in the durvalumab cohort and 16.2 months in the placebo cohort [85]. Adjuvant treatment with durvalumab is currently under evaluation for reimbursement within the public health system in Norway [76].
Of note, immunotherapy in general has a favourable toxicity profile compared to chemotherapy [77–81].
30 The development of cancer - tumour biology
What is cancer? Cancer is uncontrolled cell division and growth that can lead to the formation of malignant tumours capable of local invasion and possibly distant spread to other parts of the body. Untreated, cancer can kill you.
A genetic and epigenetic disease
Most likely, cancer is caused by a combination of hereditary as well as environmental factors, and molecular alterations at the genetic, epigenetic and protein level are important parts of the carcinogenesis. Moreover, cancer develops in a step-wise manner where molecular aberrations accumulate over time leading to abnormal signalling patterns and further to dysfunctional cell division and growth, surpassing the normal control mechanism of the cells.
Biological features essential in the carcinogenesis were reviewed in Hanahan and Weinberg’s widely known papers Hallmarks of Cancer, and Hallmarks of Cancer: The Next Generation [86, 87]. The main hypothesis discussed in these seminal papers was;
even though the sequence of events may vary, the acquisitions of some common traits are necessary for tumour formation, growth and invasion in all types of cancer
development. The capability of cancer cells to induce and sustain its own proliferative signalling, to bypass anti-growth signals, to develop resistance against cell death as well as the acquired mastery of limitless replication were considered important steps in tumorigenesis, addressed in the initial publication from 2000 [86]. Further, the ability to generate vasculature through angiogenesis, the potential of local invasion and ultimately distant formation of metastases were recognized as distinct hallmarks in the cancer development [86]. In the second paper published in 2011, the hypothesis was further expanded, and deregulation of cellular energetics as well as the evasion of the immune system were included as hallmarks. Genomic instability/mutations and tumour-promoting inflammation were introduced as underlying, or rather facilitating traits, in the cancer development (Figure 5) [87].
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Figure 5. The Hallmarks of Cancer proposed by Hanahan and Weinberg in 2011. Modified from [87].
The DNA molecules encode the genetic information essential in the development and functioning of the living organisms. According to the “the central dogma of molecular biology” the DNA molecules, consisting of chains of nucleotides, direct its own replication to produce RNA, which in turn directs the translation to proteins [81].
The DNA of tumour cells can contain many aberrations, from point mutations to large chromosomal alterations. Genome instability constitutes the backbone of cancer development [87]. For many years, the subdivision of NSCLC tumours was based solely on pathological characteristics, recent advances in genomic profiling platforms however, have indeed expanded our view and a wide variety of genetic alterations have now been discovered. A direct comparison of 21 different tumour types published in 2014 showed a high frequency of somatic mutations in lung
adenocarcinomas and squamous cell carcinomas compared to other tumour entities [62]. The identification of genetic alterations has fuelled the search for personalized treatment options and led to a paradigm shift in NSCLC treatment.
Epigenetic mechanisms do not alter the nucleotide sequences of the DNA, but have the potential to modify gene expression, hence affecting the phenotype but not the
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genotype of the cells. Examples of epigenetic regulation mechanisms include DNA methylation and histone modification [88]. DNA methylation is the most studied epigenetic event and refers to the attachment of methyl groups to the cytosine
nucleotide within the DNA. Increased or decreased methylation can lead to a silencing of genes or an increased gene expression, respectively. Changes in DNA methylation occurs frequently in cancer and can influence the carcinogenesis through regulation of oncogenes or tumour suppressor genes, amongst other [89]. Contrary to genetic
alterations epigenetic changes constitutes a dynamic modification.
MicroRNAs are small functional non-coding RNAs made up by 19 – 25 nucleotides participating in gene regulation by inhibiting translation or inducing mRNA
degradation [90, 91]. Thus, microRNAs function as negative regulators of gene expression. They are implicated in a plethora of cellular processes and are considered important players in cancer development and progression [92]. Deregulation of the microRNA machinery has been reported in various cancers [93] and considerable research focus on identifying microRNAs as cancer biomarkers. The lethal-7 (let-7) microRNAs refer to a family of several microRNAs described as tumour suppressors, often found down-regulated in cancer in general, and in lung cancer in particular [94].
Altered expression of let-7 microRNAs are shown related to resistance to EGFR inhibitors [95]. Moreover, reduced levels of let-7s is associated to poor survival in patients with lung cancer [96].
Many hypotheses are proposed regarding how the malignant tumours progress and how the heterogeneity shown in an abundance of tumours actually develops. The hypothesis of clonal evolution suggest that tumour cells acquire favourable genetic and epigenetic traits leading to a positive selection [97]. Contrary, the stem cell hypothesis is based on stem cells obtaining a malignant potential and developing into cancer cells, or even that the cancer cells itself acquire stem-cell like attributes, such as self-renewing cell division [98].
33 The tumour microenvironment
Traditionally, cancer research focused solely on the malignant cell. As a consequence, a majority of our knowledge on tumour biology was derived from different cell
experimental settings, translated into the clinic. As early as the mid-1800s however, a connection between cancer and the surrounding tissue was suggested when Virchow discovered a tendency of tumour growth at sites of chronic inflammation [99]. In the last decades, the relationship between the malignant tumour and the tumour
microenvironment has been revisited and certainly gained a lot of attention. Today, a causality is widely recognized [100]. Indeed, inflammation is considered a facilitator of the hallmark functions in the carcinogenesis [87]. To target the non-malignant cells in the tumour environment or the means of communication between the cells residing in a tumour is a novel approach to the treatment of cancer.
The tumour microenvironment can contain a variety of cells; immune cells,
fibroblasts, adipocytes and cells comprising the blood and lymph vessels. In addition, the extracellular matrix, the interstitial fluids and an assortment of molecules such as growth factors, proteinases and cytokines are a part of the tumour microenvironment (Figure 6) [100, 101]. The exact components of the tumour microenvironment vary from site to site, and from cancer to cancer. The interaction between the genetically aberrant tumour cells and the surrounding tissue can lead to a reprogramming of the stroma and facilitate carcinogenesis by contributing to inflammation, immune suppression, generation of pre-metastatic niches and the establishment of distant metastasis [102]. Further, the tumour microenvironment may play a part in resistance to cancer treatment. As a consequence, cancer cannot any longer be considered just as a collection of autonomous malignant cells.
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Figure 6. The lung cancer microenvironment [103]. Carcinoma cells are closely associated with the extracellular matrix, mesenchymal cells such as fibroblasts, vasculature and immune cells. ECM, extracellular matrix; PDGR, platelet derived growth factor; VEGF, vascular endothelial growth factor, CXCL, CXC motif chemokine ligand; CXCR2, CXC motif chemokine receptor type 2; PDL1, programmed cell death1 ligand; PD1, programmed cell death 1. Reprinted with permission.
During tumorigenesis the cells comprising the tumour microenvironment undergo different changes. Cancer-associated fibroblasts (CAFs) are non-malignant cells in the tumour stroma; their main precursors are normal locally residing fibroblasts. CAFs accumulate in tumours and contribute to an inflammatory pro-carcinogenic
microenvironment [104]. Studies have suggested that the presence of CAFs in early stage NSCLC tumours are associated with a higher risk of disease recurrence [105, 106]. During tumour development the expansion of new blood and lymphatic vessels will also lead to the recruitment of macrophages and other immune cells. The
macrophages can in turn be transformed into tumour-associated macrophages (TAMs). TAMs can promote tumour progression in a number of ways, including angiogenesis, immune suppression and metastasis, as well as to contribute to treatment resistance [107]. The number of TAMs in the stroma of NSCLC tumour tissue is proposed as a predictor of survival, with a high density of TAMs indicative of a poor prognosis [108, 109].
35 The immune system
Tumour cells and immune cells interact in a dynamic process. The immune system protects against cancer development, but research shows that the immune system also actively contributes in shaping the phenotype of the malignant tumour, thus
representing possible targets for treatment. The composition of the immunological tumour microenvironment is heterogeneous and differences are shown between various cancer types as well as between tumours in patients with the same type of cancer. Some tumours have a poor infiltration of immune cells while others are seen highly infiltrated with lymphocytes. The degree of lymphocyte infiltration provides prognostic information in NSCLC, where a strong positive correlation has been shown associated with a positive clinical outcome [110]. This also holds true for other solid cancers [111]. Hence, current research is focused on creating an “immunoscore” as a supplement to the TNM-classification [112, 113].
The immune system is subdivided in the innate and adaptive immune system. The first line of defence against any microorganism is the physical barriers of the human body, the skin and mucous membranes. The innate immune system constitutes the second line of defence, comprising phagocytes (macrophages, neutrophils and dendritic cells), natural killer cells, mast cells, basophils, eosinophils and the complement system proteins. The third level of defence is the adaptive immune system, representing a more specific, tailored immune response as well as the immunological memory. The main players in the adaptive immune system are the lymphocytes; B and T cells [114].
T cells are divided into two major groups according to the T cell receptor (TCR) they express; T cells and T cells. T cells are further sub-classified according to effector function into CD8 cytotoxic T cells (CTLs) and CD4 helper T (Th) cells which is then divided into Th1, Th2, Th17, T regulatory (Treg) cells and natural killer T (NKT) cells.
In 1909 Paul Ehrlich hypothesized that the host defence may prevent neoplastic cells developing into tumours. Later, in the 1950s the concept of immunosurveillance was introduced by Burnet and Thomas, based on immune-mediated rejection of
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transplanted tumours in mice. Just a few years later, the theory lost momentum
however, due to lack of convincing experimental evidence [115, 116]. In 2004 the 3E theory was proposed, now introducing three separate stages in cancer
immunosurveillance and editing; elimination, equilibrium and escape by activation of the innate and adaptive immune responses (Figure 7) [117].
According to the 3E theory most cells undergoing malignant transformation will express tumour specific neoantigens. Subsequently these cells will be detected as
“non-self” and be destroyed by the innate and adaptive immune cells in the host. At this point the balance is tipped toward anti-tumour immunity. Some cells however, have a high proliferative rate and are able to compensate for the ongoing elimination by the immune system, thus rendering an equilibrium in immune-escape and
elimination, and a stable tumour size (tumour dormancy) remains. Finally, some malignant cells may overcome the elimination and are able to escape the regulation of the immune system. As a consequence, a tumour can now grow in an uncontrolled manner. In conclusion, the immune system not only protects against cancer
development (immunosurveillance), it will also shape the character of the emerging tumour by selecting transformed cancer cells that can evade the surveillance
(immunoediting) [117, 118]. The work by Schreiber and colleagues on the 3E theory launched a paradigm shift in the field of cancer immunology, and evading immune destruction was indeed included as a hallmark in cancer development in the revised Hallmarks of Cancer published in 2011 [87].
Various mechanisms utilized in the escape phase has since been proposed including downregulation of mechanisms involved in immune recognition, increased immune resistance, and the development of a tumour microenvironment in favour of tumour growth and development [119].
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Figure 7. The three Es of cancer immunoediting; elimination, equilibrium and escape [112]. CD8+, cytotoxic T cells; CD4+, T helper cells; NK, natural killer cells; NKT, natural killer T cells; DC, mature dendritic cells; Treg, regulatory T cells; MDSC, myeloid-derived suppressor cells; γδ, gamma delta T cells. Reprinted with permission.
It has become increasingly evident that the immune system plays a pivotal role in cancer development, and modulations of the immune system thus represent interesting treatment opportunities. One mechanism that has gained a lot of attention in recent years is the immune checkpoints. The PD-1/PD-L1, and the cytotoxic T-lymphocyte antigen-4 (CTLA-4) checkpoint inhibitors have been proven efficacious in several solid cancers, and immunotherapy is now readily available in the clinic. In normal circumstances the immune checkpoints play a vital part in the maintenance of self- tolerance by preventing auto-immunity and protects normal tissue from destruction when the immune system otherwise is activated [120]. Immune checkpoints are however, also exploited in tumours, facilitating immune escape.
Multiple checkpoints are discovered and represent promising targets for development of therapy. As of yet the cytotoxic T-lymphocyte associated protein 4 (CTLA4) and the PD-1/PD-L1 pathways have the received the most attention. PD-1 is a surface
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receptor expressed on many cell types, including activated T cells, B cells and natural killer cells [121]. Interaction with its ligands, PD-L1 or PD-L2, will elicit responses leading to a downregulation of T cell activation [122, 123]. The PD-L1 ligand can be expressed on a variety of cell types, such as T cells, epithelial cells and endothelial cells, but also be upregulated on tumour cells, including non-small lung cancer cells, thus aiding tumour escape from T cell destruction [124]. Two classes of antibodies targeting the PD-1/PD-L1 axis are developed; PD-1 and PD-L1 inhibitors. Blockade of these targets have demonstrated antitumour activity, by releasing the brakes on activated T-cell immunosuppression.
The PD-1 and PD-L1 inhibitors have entered the clinic as the new standard of care in advanced NSCLC, although a problem with efficacy remains. Many NSCLC patients will not respond to the treatment, and the response rates are reported to be
approximately 20 – 40 % in NSCLC patients [77–81]. The search for predictive biomarkers beyond PD-L1 expression in the tumour cells is currently ongoing.
Further, combinations with other immune check point inhibitors, as well as
chemotherapy, targeted therapy or radiation therapy with an aim of amplifying the immune response are currently being explored [82–85, 125, 126]. In addition, different aspects of immune-modulations altogether, including cancer vaccines and engineered T cells (CAR-T) cells represent interesting approaches for the future treatment of cancer [127].
Cytokines and metalloproteinases
Cytokines and matrix metalloproteinases (MMPs) constitute an important part of cell to cell crosstalk, and are mediated through multiple, complex regulatory networks.
Cytokines are a group of small proteins including interferons, interleukins,
chemokines, mesenchymal growth factors, tumour necrosis factors and adipokines, generally transiently expressed in response to stimuli. Most cytokines are soluble proteins, although some function as integral membrane proteins. Every cell in the human body, with the exception of the red blood cells, can produce and respond to
