University of Groningen Discovery of prognostic markers in laryngeal cancer treated with radiotherapy Bruine de Bruin, Leonie

Discovery of prognostic markers in laryngeal cancer treated with radiotherapy

Bruine de Bruin, Leonie

DOI:

10.33612/diss.143832673

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Bruine de Bruin, L. (2020). Discovery of prognostic markers in laryngeal cancer treated with radiotherapy.

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Chapter 1

1

GENERAL INTRODUCTION

Head and neck cancer

Head and neck cancer is the eight most common cancer in the world with an

estimated incidence of 890,000 cases per year in 2017

_{. It commonly refers to a}

collection of cancers, predominantly squamous cell carcinomas (HNSCC) arising

from the epithelial lining of the oral cavity, pharynx and larynx. This thesis focuses

on HNSCC and in particular laryngeal squamous cell carcinomas (LSCC).

Laryngeal squamous cell carcinoma

Of all HNSCC, 25% originate in de larynx. In recent years, around 700 cases of LSCC

were diagnosed annually in the Netherlands

_{. Smoking and alcohol consumption}

are the major risk factors for developing LSCC. Unlike in oropharynx carcinoma, in

LSCC high-risk HPV16 or HPV18 infections are rarely seen as a risk factor. Low-risk

HPV6 and HPV11 are strongly associated with recurrent respiratory papillomatosis

in the larynx but not detected in LSCC

_.

Treatment options of LSCC consist of surgery (endoscopic or external approach),

radiotherapy, and a combination of these treatments or chemoradiation

_{. Based on}

national and international guidelines, in our institute early stage LSCC patients are

treated with radiotherapy as single modality treatment with the exception of T

glottic LSCC in which transoral CO

laser microscopic surgery can be an alternative

with equivalent results compared to radiotherapy

_{. Although a meta-analysis}

revealed that there was insufficient evidence to establish a clear superiority for

laser surgery versus radiotherapy for T

-T

N

glottic cancer

, laser surgery has

the advantage of keeping all treatment modalities available for the treatment of

possible recurrences. In other cases of early stage (T

-T

) LSCC, hyperfractionated or

accelerated radiotherapy is the treatment of choice to preserve laryngeal function

(voice, swallowing and breathing). Locally advanced (T

-T

) disease is treated with

radiotherapy or combined chemoradiation, unless no functional larynx is present

or can be expected after treatment. In these cases, total laryngectomy needs to

be considered. In case chemoradiation is not feasible for patients with locally

advanced non-metastatic LSCC, the use of EGFR-targeted therapy (e.g. cetuximab)

is approved by the U.S. Food and Drug Administration (FDA) and European

Medicines Agency (EMA) in combination with radiotherapy based on studies

by Bonner et al.

_{. However, specifically in laryngeal cancer adding cetuximab}

to the radiation therapy does not seem to improve survival

_{. Patients with a}

positive lymph node status (N+) require definitive treatment of the neck, either by

comprehensive neck dissection or by definitive (chemo)radiation. For supraglottic

LSCC, elective bilateral treatment of the neck should be undertaken for T

or higher

stages. However, for patients with glottic disease sole treatment of the primary

site is sufficient and elective unilateral treatment of the neck is not needed until

tumors are stages T

or greater

.

Radiotherapy for the treatment of laryngeal cancer

Radiotherapy plays an important role in the treatment of LSCC patients. Efficacy of

radiotherapy relies on DNA damage, either via direct damage of DNA by ionizing

radiation or by free radicals that are generated and subsequently react with DNA,

which is the more common mechanism of DNA damage. This leads to single and

double strand DNA breaks, which ultimately result in apoptosis

_{. An increasing}

radiotherapy dose will lead to increasing cell death of neoplastic cells. However,

normal cells will also be damaged which leads to toxic side effects like dermatitis,

mucositis, pain, dysphagia and xerostomia. To prevent these side effects, patients

are treated with fractionated radiotherapy to enable normal tissues to recover and

repopulate. However, recovery and repopulation does not only occur in normal

tissue, but also in neoplastic cells. To reduce repopulation of neoplastic cells,

accelerated radiotherapy is given with a daily dose of 2 Gy for six fractions per

week for a total of 66-70 Gy in total.

The local control rate for T

-T

laryngeal carcinoma obtained with primary

radiotherapy is 70-95%

_{. In case of local recurrence, frequently a disabling}

total laryngectomy is required as a salvage surgery. Moreover, complications and

morbidity are considerably high in patients who undergo a salvage laryngectomy

after radiotherapy with/without chemotherapy

_.

Prognostic tumor-specific biomarkers for LSCC treated with radiotherapy

Recurrences in early-stage (T

-T

) LSCC treated with radiotherapy will result in a

disabling total laryngectomy. Given the high recurrence rate, prognostic factors

for the development of a local recurrence after primary radiotherapy are needed

to select the most optimal treatment for individual patients. Besides TNM

classification, anterior commissure involvement, sublocation in which supraglottic

cancers have worse prognosis than glottic cancers and a number of clinical factors,

like smoking and alcohol consumption habits, there is a lack of clinical prognostic

factors to predict worse local control after radiotherapy in LSCC

_.

1

In the past decades, research has concentrated on the identification of new

tumor specific biomarkers to predict clinical outcome. Especially markers involved

in tumorigenesis and tumor progression, such as genes associated with processes

like apoptosis, angiogenesis and cell growth, have been investigated extensively

in relation with radioresponse in LSCC

_.

Hypoxia as a prognostic factor

Among the several mechanisms proposed as potential causes of radioresistance,

hypoxia has been studied the most

_{. Hypoxia is characteristic for many solid}

tumors, including head and neck cancer

_{. It was already proposed in the 1950s}

that the radiosensitivity of tumors is limited by hypoxia

_{. The biological effect of}

radiotherapy depends on the degree of tissue oxygenation and hypoxic cells are

approximately threefold more resistant to radiation than well oxygenated cells

_.

Oxygen free radicals are highly reactive and are the primary source of

radiation-induced DNA damage. Therefore, oxygen is a potent radiosensitizer. Indirectly,

hypoxia-induced genomic changes may have impact on radiation resistance

by altering proliferation, cell cycle, apoptosis, angiogenesis and anaerobic

glycolysis

_{. In many tumor types, including head and neck cancer, hypoxia is}

associated with worse locoregional control

_.

Patients with a hypoxic tumor might benefit from hypoxic modification

during radiotherapy treatment. For example application of hypoxia sensitizers as

nitroimidazoles to radiotherapy, radiotherapy with carbogen and nicotinamide

(ARCON) or increased radiation dose to hypoxic areas or hyperbaric oxygen

treatment have been investigated in HNSCC

_.

However, assessing tumor hypoxia is a challenge. Direct measurement

of tumor oxygenation is possible with (Eppendorf) polarographic needle

electrodes

_{. Clinical studies demonstrated that this method could be used to}

predict tumor response and treatment outcome in patients with HNSCC treated

with radiotherapy

_{. However, a later study by the same researchers disagreed}

with the conclusions in their first study

_{. This can partly be explained by the}

spatial heterogeneity of hypoxia in the tumor. An important disadvantage of using

the needle electrodes is the restricted use to accessible tumors and intertumoral

heterogeneity of hypoxia known for many years

_.

Other, histological, methods to asses tumor hypoxia are the use of exogenous

(2-nitroimidazole compounds such as pimonidazole) and endogenous (HIF1α,

CA-IX, GLUT-1) hypoxia markers using immunohistochemistry

_{. The advantage is}

that presence of hypoxia could be related to histological morphology. Exogenous

markers are drugs, chemicals or even bacteria that, after administration to the

patient, specifically accumulate or are bio-reducible under hypoxic conditions.

Binding is ascertained in tissue biopsies using specific antibodies. One of the

clinically relevant markers is the 2-nitroimidazole pimonidazole

_{that is injected}

intravenously before biopsy taken or surgery. It is reductively activated and forms

protein products in mammalian cells at low pO

level

_{. The prognostic significance}

of pimonidazole was demonstrated by Kaanders et al. who showed worse

locoregional control in patients with high pimonidazole binding as compared to

low pimonidazole binding in advanced head and neck cancer

_{. The disadvantage}

of exogenous markers is that the chemical solution must be administered

intravenously to the patient before biopsy/surgery. Therefore, expression of

exogenous hypoxia markers can only be investigated in tissue samples from

patients who underwent this procedure preoperative and was performed only in

few retrospective studies.

Under hypoxic conditions, hypoxia inducible factor 1-alpha (HIF1α) is

upregulated, which leads to activation of transcription of many genes, including

carbonic anhydrases and glucose transporters. These proteins are involved in many

processes, such as angiogenesis, pH-regulation, cell proliferation, cell survival

and apoptosis, erythropoiesis, and energy and glucose metabolism

_{. Increased}

expression of HIF1α detected by immunohistochemistry has been associated with

poor locoregional control after radiotherapy in LSCC

_{. Carbonic anhydrase IX}

(CA-IX) catalyzes reversible hydration of carbon dioxide to carbonic acid thereby

maintaining a stable intracellular pH in hypoxic conditions. High expression of

CA-IX was found in many different tumor tissues. In vitro and in vivo studies

demonstrated that CA-IX was strongly induced by hypoxia in a broad range of

tumors. It was also shown that CA-IX positivity was associated with resistance to

radiation

_{. In contrast, low expression of CA-IX was predictive for response}

to accelerated radiotherapy with carbogen breathing and nicotinamide (ARCON),

a hypoxia target therapy, in laryngeal cancer

_{. Although CA-IX is a promising}

hypoxia marker, a weak correlation with pimonidazole

_{questioned its value as}

a marker for hypoxia. However, we cannot exclude that pimonidazole is poorly

associated with hypoxia and thus CA-IX is a good predictor because studies are

lacking to correlate expression with hypoxic tumor areas. GLUT-1 appeared to be

one of the prominent glucose transporters. Increased expression enables higher

cellular uptake of glucose and facilitates anaerobic glycolysis

_{. GLUT-1 expression}

1

in head and neck tumors is found at distance from vessels and adjacent to necrotic

areas, and indicates diffusion-limited hypoxia

_{. Furthermore, a correlation with}

oxygen electrode measurements and expression of pimonidazole and CA-IX was

found

_.

In summary, HIF1α, CA-IX and GLUT-1 are the most extensively studied

endogenous markers of tumor hypoxia. These biomarkers were associated with

worse survival and local control, almost regardless of the therapy provided.

Differences in outcome between various studies can be explained by spatial

heterogeneity in the distribution of hypoxia, many ways to score biomarker

expression levels and the use of different cutoff levels to define low/high expression

reported in literature. In addition, expression of many hypoxia-related genes may

be a complex function of hypoxia-dependent and -independent pathways

_{. For}

instance, HIF1α, CA-IX and GLUT-1 accumulation can be observed also under other

conditions than hypoxia, for example, hypoglycemia and acidosis

_suggesting

these markers are not robust hypoxia markers but might reflect a more aggressive

tumor phenotype

_.

Hypoxia imaging

Alternative and non-invasive methods to obtain information about the

oxygenation status with the possibility to use this for radiotherapy treatment

planning (for escalating the dose to the hypoxic regions) include the use of

radiologic and nuclear imaging techniques. During the past decades many PET

tracers for detecting the proportion of hypoxic cells in vivo have been developed.

Most widely used are the 2-nitroimidazoles like

_{F-labeled fluoromisonidazole}

(

_{F-FMISO) or}

_{F-fluoroazomycinarabinoside (}

_F-FAZA)-PET

_{. Several animal and}

human studies evaluated the use of radiolabeled nitroimidazoles for assessment

of the oxygenation status in solid tumors

_{. Clinical studies performed on}

patients with head and neck cancer demonstrated the potential prognostic value

of hypoxia imaging with

_{F-FMISO for radiotherapy outcome}

_{. In a small group}

of patients with HNSCC, pre-treatment tumor hypoxic fraction assessed using PET

imaging of

_{F-FAZA was predictive of survival following radiotherapy}

_{. However,}

the ideal hypoxia PET tracer should express only relevant oxygen concentrations

in viable cells and possess uniform and rapid cell entry (lipophilic molecule),

rapid clearance from normoxic cells (hydrophilic molecule) and yielding a high

target-to-background ratio. Overall, fluorinated nitroimidazoles have a low tumor

uptake relative to surrounding tissue and

_{F-FAZA has been shown to be superior}

to

_F-FMISO

_{. None of the radiotracers that have been used in clinical studies}

fulfill all properties and development of better protocols in existing radiotracers

and search in new tracers is still on-going. Furthermore, there is a lack of studies

verifying hypoxia in hypoxic subvolumes seen on hypoxia PET imaging.

Prognostic value of EGFR and PTEN

The epidermal growth factor receptor (EGFR) is a transmembrane protein that is

a receptor tyrosine kinase for extracellular ligands such as EGF. Ligand-binding

to EGFR induces activation of the intrinsic kinase domain and stimulation of

downstream signaling pathways such as the P13K/AKT pathway, regulating

numerous cellular responses such as increased cell proliferation, decreased

insensitivity to apoptosis, migration and differentiation

_{. Increased expression of}

EGFR is commonly observed in many human cancers including head and neck

_.

Furthermore, EGFR expression has been significantly correlated with poor local

control after radio- or chemotherapy in different cancer types, including head

and neck cancer

_{. Consequently, intensive research has focused on EGFR as}

potential target for cancer therapy. Although expression of EGFR is not a predictor

for response to cetuximab, a monoclonal antibody targeting EGFR

_{, patients with}

HNSCC independent of EGFR status also significantly benefit from treatment with

cetuximab

_{. Since 2006, the use of cetuximab is approved by the FDA and EMA in}

combination with radiotherapy for patients with locally advanced non-metastatic

HNSCC in case chemoradiation is not feasible

_.

Another mechanism for PI3K/AKT pathway activation is the loss of PTEN

(phosphatase and tensin homolog deleted on chromosome 10), a tumor suppressor

gene which opposes PI3K/AKT activation

_{. PTEN is the second most affected}

tumor suppressor gene after p53 and mutations/deletions in PTEN are found in a

variety of primary tumors including HNSCC

_{. In a cohort of patients with locally}

advanced HNSCC postoperatively treated with radiotherapy, overexpression of

PTEN was associated with increased radioresistance

_{, in line with an alternative}

role of PTEN in DNA damage repair

_.

DNA damage response markers as biomarkers for radiation response

During radiotherapy, DNA double strand breaks (DSBs) are introduced to cause

cell death. DSBs activate a complex DNA damage response (DDR) pathway that

controls cell cycle checkpoints, DNA repair and apoptosis

_{. Central in the DDR is the}

protein kinase ataxia telangiectasia mutated protein (ATM)

_{. DNA DSB induced}

1

by ionizing radiation leads to activation of ATM and subsequently phosphorylates

a variety of substrates including the checkpoint kinase 2 (Chk2) which is known

to prevent entry into mitosis. Both phosphorylated ATM and Chk2 are known to

activate the tumor suppressor gene p53 with cell cycle arrest and apoptosis

_.

In patients with cervical cancer treated with (chemo)radiation, high levels of

phosphorylated ATM were linked to poor locoregional disease-free survival and

inhibition of ATM sensitizes cell lines to radiation

_{. Although inhibition of ATM}

was also demonstrated to sensitize HNSCC cell lines to radiation

_{, only few}

studies using tumor tissues from patients with head and neck cancer showed no

relation between ATM expression and response to chemo(radiation)

_.

Epigenetic changes as prognosticator in LSCC treated with radiotherapy

DNA methylation is one of the major forms of epigenetic regulation of gene

expression. Among others, it plays an important role in tumorigenesis leading to

the epigenetic modulation of the expression of tumor suppressor genes involved

in cell cycle regulation, apoptosis, and DNA repair

_{. In cancer, DNA methylation}

becomes aberrant, causing global hypomethylation and local hypermethylation

in tumor suppressor genes as compared to normal cells

_{. DNA methylation was}

found to be associated with clinical outcome in HNSCC in our research group

and others

_.

DNA methylation is regulated by the DNA methyltransferase (DNMT) enzymes

such as DNMT1, DNMT3A and DNMT3B

_{. High expression of DNMT’s in a variety}

of tumors was associated with hypermethylation and oncogenic activation

_.

DNMT1 expression correlates well with aberrant DNA methylation in solid tumors,

including esophageal carcinomas resulting in poor prognosis in patients

_{. DNMT1}

expression positively correlated with radiation sensitization and longer survival of

esophageal squamous cell carcinoma patients

_{. Furthermore, positive staining}

for DNMT1 was significantly linked to lower rates of treatment response and

shorter survival of patients with pharyngeal squamous cell carcinoma treated with

surgery combined with adjuvant radiotherapy with/without chemotherapy, or

definitive concurrent chemoradiotherapy

_{. DNMT inhibitors were demonstrated}

to sensitize HNSCC cell lines to irradiation

_{. Epigenetic therapies, such as with}

DNMT1 inhibitors used in clinical practice (like azacitidine, decitabine, zebularine)

leading to hypomethylation of DNA, might offer new opportunities for modulating

the radiation resistance of tumors

_.

OUTLINE OF THIS THESIS

In early stage (T

-T

) LSCC radiotherapy is the preferred choice of treatment because

of laryngeal preservation. Despite the relatively high treatment response rate after

radiotherapy a considerable number of patients will develop a local recurrence,

which consequently requires salvage surgery (i.e. total laryngectomy) with high

morbidity and deterioration of quality of life. For individual patients, it would

be useful to predict local tumor control after radiotherapy on the pre-treatment

biopsy. In HNSCC many studies have been performed to identify prognostic tumor

biomarkers. However, diversity in staining protocols and in the composition of

study populations makes it difficult to compare the reported results and determine

the clinical value of these biomarkers as prognostic factors for local control.

In the first part of this thesis (Chapter 2 and 3), the feasibility of a hypoxia

PET tracer is investigated. In the second part of this thesis (Chapter 4, 5 and 6),

tumor biomarkers associated with local control are investigated in a well-defined

homogeneous cohort of early stage (T

-T

) glottic and supraglottic LSCC patients

all treated with curatively intended radiotherapy. For this purpose, we used

a bio-database covering 1286 patients diagnosed with LSCC at the department

of Otorhinolaryngology/ Head & Neck Surgery in the University Medical Center

Groningen (UMCG) between 1990 and 2008. Clinicopathological baseline

and follow-up data were available from the archives of the departments of

Otorhinolaryngology/ Head & Neck Surgery, Pathology and Radiation Oncology.

Formalin-fixed and paraffin-embedded pre-treatment tissue biopsies with

sufficient neoplastic cells were selected for immunohistochemical analysis.

Chapter 2 gives a comprehensive review on different animal and human

_{F-FAZA-PET studies and its potential use for individualized treatment in patients}

with hypoxic head and neck tumors. In Chapter 3, the accuracy of

_{F-FAZA-PET/CT}

scan in detecting hypoxic regions within the tumor using exogenous (pimonidazole)

and endogenous (HIF1α, CA-IX and GLUT-1) immunohistochemical markers is

investigated. For this purpose 11 patients were selected (outside previous mentioned

database) with an indication for total laryngectomy because of locally advanced or

recurrent laryngeal carcinoma. The prognostic value of EGFR and PTEN expression

on local control in 52 patients from our database with early-stage supraglottic

LSCC treated with radiotherapy, is evaluated in Chapter 4. The prognostic value

of the immunohistochemical expression of pATM, pChk2 and p53, all involved

in the ATM-associated DNA damage response pathway, is investigated in our

1

series of patients with early-stage LSCC treated with radiotherapy in Chapter 5.

Because DNA methylation is regulated by DNMT1, in Chapter 6 we investigate the

association between DNMT1 expression and local control in LSCC patients treated

with radiotherapy. Chapter 7 provides a summary, general discussion and some

future perspectives. Chapter 8 provides a Dutch summary of the results.

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