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

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10.33612/diss.143832673

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Chapter 2

Head and neck tumor hypoxia

imaging by

18

F-fluoroazomycin-arabinoside (

18

_F-FAZA)-PET:

a review

BFAM van der Laana,#_{, J Pruim}c_{, RJHM Steenbakkers}d

a_{Department of Otorhinolaryngology/Head and Neck Surgery,University of Groningen,}

University Medical Center Groningen, Groningen, The Netherlands

b_{Department of Pathology and Medical Biology,University of Groningen, University Medical}

Center Groningen, Groningen, The Netherlands

c_{Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University}

Medical Center Groningen, The Netherlands and Department of Nuclear Medicine, Tygerberg Hospital, Stellenbosch University, Stellenbosch, South-Africa

d_{Department of Radiation Oncology,University of Groningen, University Medical Center}

Groningen, Groningen, The Netherlands †Current affiliation: Department of Otorhinolaryngology/Head and Neck Surgery, Hospital St

Jansdal, Harderwijk/Lelystad, The Netherlands

#_{Current affiliation: Department of Otorhinolaryngology/Head and Neck Surgery, Haaglanden}

Medical Center, The Hague, The Netherlands

ABSTRACT

Tumor hypoxia is known to be associated with poor clinical outcome; therefore, patients with hypoxic tumors might benefit from more intensive treatment approaches. This is particularly true for patients with head and neck cancer. Pre-treatment assessment of hypoxia in tumors would be desirable, not only to predict prognosis but also to select patients for more aggressive treatment.

As an alternative to the invasive polarographic needle electrode method, there is the possibility of using PET with radiopharmaceuticals visualizing hypoxia. Most hypoxia imaging studies on head and cancer have been performed using 18_F-labeled

fluoromisonidazole (18_{F-FMISO). A chemically related molecule,} 18

F-fluoroazomycin-arabinoside (18_{F-FAZA), seems to have superior kinetic properties and may therefore}

be the radiopharmaceutical of choice.

This minireview summarizes the published literature on animal and human

18_{F-FAZA-PET studies. Furthermore, future perspectives on how individualized}

treatment could be applied in patients with hypoxic head and neck tumors are discussed, for instance the use of hypoxia sensitizers or special intensity modulated radiation therapy techniques achieving tumor subvolume dose escalation.

2

INTRODUCTION

Tumor hypoxia

Tumor hypoxia is a well-recognized adverse prognostic factor in patients with solid tumors treated with radiotherapy1 _{and is associated with a number of unfavorable}

biological characteristics, including increased genetic instability, increased invasiveness, enhanced metastatic potential and decreased radiosensitivity2,3_.

Poorer clinical outcome of hypoxic tumors has been observed in patients with head and neck cancer treated by radiotherapy4,5 _{and surgery}6_{. From this point of}

view, patients with hypoxic tumors might benefit from more intensive treatment approaches to counterbalance the radioresistance. It has been estimated that tumor hypoxia is present in about half of the solid tumors regardless of the tumor volume or histological type7_{. Unfortunately, patients with head and neck cancer}

tend to present themselves with tumors in advanced stages, which increase the likelihood of developing hypoxia8_.

Detection of hypoxia

Although tumor hypoxia is usually defined as a tumor region with a partial oxygen pressure (pO₂) less than 10 mm Hg9_{, there is no consensus over the interpretation}

and analysis of hypoxia-positive areas of different imaging modalities. For instance, some of the PET studies use the tumor-to-background ratio (T/B), others the tumor-to-muscle ratio (T/M) and there are also self-developed scoring systems. Unfortunately, these differences make it difficult to compare studies.

Polarographic needle electrode sampling is considered the gold standard to assess hypoxia in vivo10,11_{. However, assessing tumor hypoxia using the Eppendorf}

electrodes is invasive, requires sophisticated skills and technical demands and may be subject to sampling error because of the known heterogeneity in tumor hypoxia12,13_{. In addition, the results of these measurements cannot be used for}

radiotherapy treatment planning, for example, for escalating the dose to hypoxic regions within a tumor.

Therefore, assessing tumor hypoxia with metabolic imaging techniques such as PET, using specific radiopharmaceuticals visualizing hypoxia, is an attractive alternative, at least theoretically. Several studies have been published and several different radiopharmaceuticals have been applied. Due to the different analysis techniques applied, however, it is difficult to compare studies even using the same PET radiopharmaceuticals (Table 1).

Table 1. Summary of studies on human 18_F-FAZA-PET

Publication No. patients Tumor site (n) Definition of

hypoxic volume Percentage of patients with increased 18_{F-FAZA uptake}

(ie, hypoxia) Grosu et al.16 _and

Souvatzoglou et al.43 18 Head and neck ₍₁₈₎ T/M ≥ 1.5* 83

Postema et al.44 _{50 Head and neck}

(9) Lymphoma (21) High-grade glioma (7) Lung (13) Visual inspection and T/B ratio ≥ 1.2 6614 100 54 Schuetz et al.45 _{15 Cervix (15) T/M ≥ 1.2† 33}

Shi et al.46 _{5 Head and neck}

(5) Different kinetic models 80 Garcia-Parra et al.47 _{14 Prostate (14) T/B ratio‡ 0}

Mortensen et al.48 _{40 Head and neck}

(40) T/M ≥ 1.4§ 63 Bollineni et al.49 _{11 Lung (11) T/B ratio ≥ 1.2 and}

T/B ratio ≥ 1.4 100 *Tumor SUV/muscle SUVmean ratio ≥ 1.5.

†Tumor SUVmax/muscle SUVmax ratio ≥ 1.2.

‡Tumor SUVmax/benign prostate SUV.

§Tumor SUVmax/muscle SUV mean ratio ≥ 1.4.

18_{F-FMISO for hypoxia imaging}

A number of PET radiopharmaceuticals for hypoxia imaging have been identified, of which the group of nitroimidazoles is the largest. These compounds undergo reduction under hypoxic conditions and form highly reactive oxygen radicals. After binding to intracellular macromolecules, they are trapped inside the hypoxic cells. Among the radiolabeled nitroimidazoles, 18_{F-FMISO is the most frequently}

used. Troost et al.14 _{found a significant correlation between} 18_{F-FMISO-PET imaging}

of tumor hypoxia in head and neck cancer and the extrinsic hypoxic cell marker pimonidazole. Pimonidazole is a nitroimidazole-like robust exogenous hypoxia marker, which needs to be injected 2 hours before biopsy. The distribution of pimonidazole is visualized by immunohistochemistry. In another validation study, only a week correlation was found between 18_{F-FMISO uptake and pO}

2 histography15.

These apparently conflicting results are most likely due to heterogeneity in intratumoral hypoxia16_{. In a study of 73 patients with head and neck cancer,}

hypoxia, defined by increased 18_{F-FMISO uptake, was found in almost 80% of}

the patients. Moreover, increased 18_{F-FMISO uptake found to be associated with}

significant worse overall survival17_{. Rischin and co-workers}18 _{also found} 18_F-FMISO

2

tirapazamine (a hypoxic cytotoxin), was found to be effective in patients with hypoxic tumors as assessed by 18_{F-FMISO-PET. Pre-treatment dynamic} 18

F-FMISO-PET scanning has also found to be successful in predicting radiotherapy outcome in non-small cell lung cancer and head and neck cancer19,20_{. Moreover, a later study}

of the same group21 _{showed that the same imaging technique is also suitable}

for following the radio-induced reoxygenation of head and neck cancer during radiotherapy. A recent study by Zips et al.22 _{investigated the change of hypoxia}

and the predictive value of it for survival during radiotherapy in patients with head and neck cancer using 18_{F-FMISO-PET scan. Each patient has been scanned before}

and three times during radiotherapy. The scan parameters performed at week 1 (8-10 Gy) and 2 (18-20 Gy) strongly correlated with the local progression-free survival endpoints, suggesting good prognostic value of 18_{F-FMISO-PET at these}

time points and not at baseline.

Most of the studies investigating hypoxia by PET scans using static scans, as technically static scans are easier to perform. However, dynamic scans are more informative as they allow kinetic modeling and estimation of some rate constants, which can discriminate between tumor and background.

Alternatives to 18_F-FMISO

Although hypoxia imaging using 18_{F-FMISO-PET is feasible and has prognostic value,}

there are also some disadvantages using this PET radiopharmaceutical. One of the problems is the relatively short half-life time (110 minutes) of 18_{F-FMISO, which}

hampers late imaging that could enhance good contrast between hypoxia and normal tissues23_{. There is ongoing intensive research in order to find alternative}

hypoxia PET radiopharmaceuticals with better kinetics. 18_{F-HX4, another new}

potential marker for hypoxia PET scanning has recently been described24_{. Preclinical}

studies showed advantageous biodistribution and dosimetry properties, which make 18_{F-HX4 a promising hypoxia radiopharmaceutical candidate. A pilot PET}

study on hypoxia imaging using 18_{F-HX4 as a radiopharmaceutical in head and neck}

cancer patient has recently been published25_{. Although this study included only 12}

patients, the data are promising; higher sensitivity, specificity, faster clearance, and shorter injection-imaging time were found compared to 18_F-FMISO. 18_F-EF5

is another nitroimidazole that has been evaluated in imaging hypoxia in patients with head and neck squamous cell carcinoma. Data showed that the later uptake and binding of 18_{F-EF5 are hypoxia specific. The optimal scanning time and the}

also been assessed by 18_F-FETNIM27_{. In a later study, high uptake of} 18_F-FETNIM

seemed to correlate with poorer radiotherapy outcome, although firm conclusions are difficult to draw due to small study population28_{. Beside these nitroimidazoles,}

other molecules have been also tested, like the dithiosemicarbazones. One of them is 64_{Cu-ATSM, which is also a PET radiopharmaceutical developed to accumulate in}

hypoxic tumors. It found to be feasible for radiotherapy planning in a pilot study, and the biokinetic properties seems to be superior to 18_{F-FMISO, as a hypoxic}

tumor subvolume could be identified as early as 10 minutes after injection29_.

Purpose of the present review

In this review, we will focus on the possible applicability of PET scanning with a relatively new hypoxia radiopharmaceutical, 18_{F-FAZA, in radiotherapy treatment}

planning based on tumor hypoxia determination in head and neck cancer. More specifically, the purposes of this review are to assess whether hypoxia as determined with 18_{F-FAZA-PET correlates with histological or biological parameters,}

to investigate whether the presence of hypoxia as demonstrated with 18_F-FAZA

predicts outcome and to investigate how 18_{F-FAZA could be used in clinical practice.}

PROPERTIES OF

_F-FAZA

18_{F-FAZA is, like} 18_{F-FMISO, a 2-nitroimidazole compound, but sugar-coupled}

(Figure 1). As already mentioned, 2-nitroimidazole compounds undergo reduction under hypoxic conditions, forming highly reactive oxygen radicals. Subsequently, they bind to macromolecules in the intracellular compartment and trapped inside hypoxic cells30_{. When} 18_{F-FAZA is labeled with the radioisotope} 18_{F, it can be}

detected by a PET scanner.

Kumar et al31 _{were the first that reported on the synthesis of} 18_{F-FAZA by}

fluorination of 1-α-D-(2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole with DAST followed by deprotection. Their main objective was to develop a PET imaging compound that was similar to the SPECT compound 123_{I-IAZA, but that}

was less lipophilic than 18_{F-FMISO. Theoretically, a less lipophilic compound may}

produce higher perfusion and faster clearance from blood resulting in a better hypoxia-background-ratio. In a rat model, they showed that 3_{H-FAZA had similar}

biodistribution, tumor uptake, and pharmacokinetics as 123_{I-IAZA, but was indeed}

2

IN VITRO

_{F-FAZA EXPERIMENTS AND}

_F-FAZA-PET

XENOGRAFT MODELS

As all new radiopharmaceuticals, 18_{F-FAZA is also intensively investigated in}

both in vitro and in vivo experiments. The detection of hypoxia-dependent radiopharmaceutical accumulation is fundamental, just like exact characterization of pharmacokinetic features.

In the first in vitro study by Busk et al.32_{, oxygenation-dependent} 18_F-FAZA

retention was compared to that of 18_{F-FDG in different carcinoma lines.} 18_F-FAZA

accumulation was measured after radiopharmaceutical incubation in different oxygenation conditions. Significant 18_{F-FAZA retention was observed after 3 hours}

of anoxia and no binding in nonhypoxic cells. Furthermore, it showed superior in vivo hypoxia specificity compared with 18_{F-FDG. This study concluded that} 18_F-FAZA

has excellent in vitro characteristics for hypoxia imaging. Another study33 _compared

the hypoxia-selective uptake of 18_{F-FAZA to that of} 18_{F-FMISO, as the mostly used}

hypoxia radiopharmaceutical, and found no differences in the vitro experiments with tumor cell lines, but in the in vivo animal PET study the elimination of

18_{F-FAZA was found to be faster. This feature seems to be an advantage in imaging,}

but the concentration of 18_{F-FAZA was lower, which suggests a lower sensitivity}

of 18_{F-FAZA. In a later animal PET study, obvious superior biokinetics for} 18_F-FAZA

were found as compared to 18F-FMISO, for example, significantly higher T/M and tumor-to-blood ratios34_.

Beck et al35 _{evaluated the predictive value of} 18_{F-FAZA in a hypoxic xenograft}

tumor model and found that delayed tumor growth by radiotherapy can be further enhanced by adding the hypoxic sensitizer tirapazamine to radiotherapy. This effect has only been observed in hypoxic tumors, but not in normoxic ones. High

18_{F-FAZA uptake was found to be an independent negative prognostic factor for}

tumor progression. Furthermore, 18_{F-FAZA-PET was able to predict the success of}

the hypoxia sensitizing treatment of tirapazamine and radiotherapy.

The distribution of 18_{F-FAZA in xenograft tumors was examined by Busk et al.}36_.

The 18_{F-FAZA-PET images were compared to the spatial distribution of} 18_{F-FAZA using}

autoradiography, to polarographic hypoxia mapping using Eppendorf electrode measurements, and to pimonidazole fluorescence imaging. The consistency of

18_{F-FAZA distribution with hypoxia was verified using these various methods. The}

spatial link between 18_{F-FAZA and pimonidazole was further justified by the same}

research group37_{. Interestingly, the same paper describes substantial spatial overlap}

but also areas of mismatch of 18_{F-FAZA and the endogenous hypoxia marker}

GLUT-1. The authors explained these conflicting data by regional blood flow changes in solid tumors and the latency of endogenous hypoxia markers. Namely, acute decrease in tumor perfusion can result in 18_{F-FAZA retention without accumulation}

of GLUT-1, or on the contrary, reoxygenated regions with the presence of GLUT-1 have no 18_{F-FAZA accumulation. There is of course one more explanation, that is,}

the possible unreliable features of endogenous hypoxia markers, which is already described earlier38_.

The same research group published their data on dynamic 18_{F-FAZA-PET imaging}

in three squamous cell carcinoma xenograft models39_{. In this study, 3 hours after} 18_{F-FAZA administration, high tumor-to-reference tissue ratios were found. At the}

same time, using pimonidazole and 18_{F-FAZA autoradiography, a strong correlation}

was observed between hypoxic cell density and 18_{F-FAZA concentration. These}

results confirmed that late 18_{F-FAZA imaging provides a reliable measure of}

hypoxia, but the time-activity curves were more dependent on tumor type than on the extent of hypoxia. These conflicting data are explained to be the result of the generic 2-compartment model applied in the study, which assumes homogeneous tumors; however, intratumoral hypoxia is known to be heterogeneous.

An interesting study by Meier et al.40 _{tried to answer the question whether}

different breathing protocols affect oxygenation status of xenograft tumors models. The eventual hypoxic areas have been visualized using 18_{F-FAZA-PET in}

2

visualization of tumor hypoxia, this study also focused on the reversibility of

18_{F-FAZA binding in normoxic muscle applying three different breathing protocols.}

The uptake and clearance of 18_{F-FAZA has found to be oxygen supply dependent}

in both examined carcinomas, but it remained constant in normal muscle tissue.

18_{F-FAZA accumulation showed correlation with the hypoxia immunostaining.}

These results confirm that 18_{F-FAZA is a reliable hypoxia biomarker.}

Recently, a Danish group compared the prognostic value of 18_F-FAZA-PET

scanning and Eppendorf needle sampling in an animal model41_{. Oxygenation status}

of subcutaneously grown tumors in mice has been defined with both methods before single dose radiation. Both 18_{F-FAZA-PET and Eppendorf pO}

2 histography

have found to be prognostic of therapy response.

The change of hypoxia during radiotherapy remains an interesting issue. Busk et al.42 _{monitored tumor hypoxia change in xenograft models before and after}

fractionated radiotherapy using 18_{F-FAZA-PET. Although during treatment, overall}

radiopharmaceutical accumulation changed in the experimental animals there was no signs of re-oxygenation. These results prove the high reproducibility of hypoxia PET scanning with 18_{F-FAZA but contradict the results of other human studies,}

where significant changes were observed during therapy22_.

Based on these in vitro and xenograft model experiments 18_{F-FAZA-PET seems}

to be a reliable and reproducible imaging technique to visualize hypoxia.

HUMAN

_{F-FAZA-PET STUDIES}

To the best of our knowledge, only eight studies have been published evaluating

18_{F-FAZA-PET in humans}16,43-49_{. The main results of these studies are summarized}

in Table 1. All 11 head and neck cancer patients included in the publication by Souvatzoglou et al.43 _{were also analyzed in the publication by Grosu et al.}16_{, but}

the latter study included an additional seven patients. Therefore, both publications are considered as one study.

In the study of Grosu et al.16_, 18_{F-FAZA-PET was performed before radiotherapy}

in patients with head and neck cancer. Hypoxic areas were identified in the primary tumor in 15 of 18 patients. In four of these patients, the hypoxic area was diffusely dispersed throughout the primary tumor. Therefore, no single area of hypoxia could be identified in these patients. Postema et al.44 _{found large differences in the}

were seen in all patients with high-grade gliomas. For the other tumor sites there were significantly less hypoxic areas, especially in patients with lymphomas; only 14% of these subjects had increased 18_{F-FAZA uptake. Unfortunately, the reasons}

for these large differences in hypoxia between tumor sites are not discussed. In the study by Schuetz et al.45_, 18_{F-FAZA-PET scans were performed before, during}

and after external beam radiotherapy in patients with cervical cancer. Five of the 15 patients showed increased 18_{F-FAZA uptake with inhomogeneous patterns.}

Due to the small number of patients, 18_{F-FAZA-PET could not be used to predict}

treatment outcome. In the study by Shi et al.46_{, five patients with head and neck}

cancer were scanned by dynamic PET using 18_{F-FAZA and} 15_O-H

2O. The results of

this study show that different kinetic models provide different estimates of tumor hypoxia, and different models may even rank patients differently. Accordingly, although pharmacokinetic analysis may be superior to static scans, additional studies are required in order to assess the true value of kinetic analysis in this respect. In the recently published study using 18_{F-FAZA-PET by Garcia-Parra et al.}47_,

14 patients with prostate cancer were scanned before a prostatectomy. They found no existing clinically relevant hypoxic area’s in localized prostate tumors that could be possibly detected by 18_{F-FAZA-PET, meaning the limited value of} 18_{F-FAZA in}

this specific tumor type. A recent human 18_{F-FAZA-PET study}48 _{investigated the}

hypoxia using 18_{F-FAZA-PET in forty patients with head and neck cancer treated by}

(chemo)radiotherapy. Tumors were located at different primary sites and tumor stage varied between patients. Scans were made before and during therapy. There were 25/40 hypoxic tumors before and 6/13 during treatment. Significantly poorer prognosis was observed of patients with hypoxic tumors (disease-free survival, 60%), compared to non-hypoxic counterparts (disease-free survival, 93%). Despite the relatively low patient population and the diversity of the tumors sites that were investigated, this study further strength the idea that 18_{F-FAZA-PET scan is a reliable}

method for hypoxia imaging with prognostic potential. The most recent human

18_{F-FAZA-PET study compared} 18_{F-FAZA-PET with} 18_{F-FDG-PET scans in 11 patients}

with stage III-IV non-small cell lung cancer just before chemoradiotherapy49_{. They}

concluded that 18_{F-FAZA-PET imaging is able to detect heterogeneous distributions}

of hypoxic subvolumes within homogeneous 18_{F-FDG background. Furthermore,}

no correlation between 18_{F-FAZA and} 18_{F-FDG uptake was found. The authors of this}

study claim that 18_{F-FAZA provides additional information on tumor hypoxia to}18

F-FDG and might be used to develop a tool for guiding individualization of treatment of advanced non-small cell lung cancer.

2

FUTURE PERSPECTIVES

Prognosis/patient selection

As shown earlier, 18_{F-FAZA-PET can be used to identify tumor regions with hypoxia.}

Theoretically, such knowledge can be used to make a better estimation of prognosis, but also to select patients for some kind of hypoxia-targeted treatment. For example, accelerated radiotherapy combined with the hyperoxic gas carbogen and nicotinamide (ARCON) is recently investigated in phase 3 trials50-53_{. For head}

and neck cancer, ARCON showed no benefit compared with accelerated radiation, except for regional control in T2-T4 laryngeal cancer51, but a significant gain in

regional control rate was observed among the patients receiving ARCON with equal levels of toxicity53_{. In this study, no hypoxic imaging has been performed}

before the treatment, because at the time of patient selection no hypoxic image tool was available. For the future, 18_{F-FAZA-PET may be able to select patients with}

hypoxic tumors, which might indeed benefit from ARCON and consequently the true value of ARCON defined.

Tirapazamine is a drug with selective cytotoxicity for hypoxic cells. Recently, in a phase 3 trial54_{, 861 patients with locally advanced head and neck cancer}

were randomly assigned to receive chemoradiation or chemoradiation with tirapazamine. No benefit was found in terms of overall survival, failure-free survival, time to locoregional failure, or quality of life. Although in this study no advantage was found, in a substudy18 _{tirapazamine was found to be effective in}

patients with hypoxic tumors as assessed by 18_{F-FMISO-PET. Again, this finding}

suggests that introducing hypoxia imaging would better help selecting patients for additional treatment.

Radiotherapy dose escalation

In the past, several attempts have been made to overcome tumor hypoxia, such as the use of hyperbaric oxygen, radiosensitizers, vasodilators or hypoxic cytotoxins such as tirapazamine combined with (chemo)radiation. Unfortunately, generally speaking, these combined approaches have come to the expense of increased acute and late radiation-induced side-effects55_.

Another approach, which may overcome hypoxic tumor resistance, is increasing the radiation dose. However, increasing the radiation dose may also increase radiation-induced side effects, especially in the head and neck area where critical structures, such as the spinal cord, carotid arteries and parotid glands,

are in close proximity to the tumor. Ideally, only hypoxic tumor cells or hypoxic cell islets should receive such a higher dose. Currently, with modern radiotherapy techniques like intensity-modulated radiotherapy, it has become possible to intensify radiation dose in specific subvolumes within the gross tumor volume56_.

It has been hypothesized that 18_{F-FAZA-PET/CT can be used to guide radiotherapy}

in order to substantially increase the dose to hypoxic tumor subvolumes16_{. This}

might improve local control and subsequently survival of patients with locally advanced head and neck squamous cell carcinoma. To the best of our knowledge, few clinical data are available so far to prove this hypothesis. In addition, little is known about the way hypoxic areas behave during the radiotherapy course: hypoxic areas may disappear and subsequently appear at different locations. To achieve the most optimal hypoxic tumor subvolume dose escalation, several strategies are developed (e.g., gradual dose escalation using intensity-modulated radiation therapy with a simultaneous integrated boost, intensity modulated arc therapy, stereotactic boost, or protons). Additional information is required with regard to these possible changes.

CONCLUSIONS

Based on this review, we conclude that 18_{F-FAZA-PET is feasible to detect tumor}

hypoxia and to have superior biokinetics compared to 18_F-FMISO. 18_{F-FAZA is a}

promising PET radiopharmaceutical for visualization of tumor hypoxia, although clinical studies must still confirm the exact role of 18_{F-FAZA-PET scanning in}

head and neck oncology. Also, the superior hypoxia PET radiopharmaceutical is not known yet. Therefore, a clinical study directly comparing different hypoxia radiopharmaceuticals (e.g., 18_F-FMISO, 18_F-FAZA, 18_{F-HX4) in the same patients}

2

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