grant agreement no.: 228436 ulice · 6.3 penumbra 7 comparison of proton, ion, and neutron results...

Project co-funded by the European Commission within the FP7 (2007–2013) Grant agreement no.: 228436

ULICE Union of Light Ion Centres in Europe

Project type: Combination of CP & CSA

Integrating Activities / e-Infrastructures / Preparatory phase

Start date of project: 1st September 2009 Duration: 48 months

D.JRA 3.2 – Report of different methods available for measurement of radiobiological relevant parameters in patients

WP n° and title: WP 3 Biologically based expert system for individualised patient allocation

WP leader: Michael Baumann

Author(s): Reinhard Gahbauer, Alina Santiago

Contributor(s): Vincent Grégoire, Adrian Begg, Albert van der Kogel, Michael Baumann, Wolfgang Enghardt, Niels Bassler, Brita Singers

Pillar coordinator: Richard Pötter

Reporting period: September 2009 – February 2011, 18 months

Dissemination Level PU Public X PP Restricted to other programme participants (including the Commission Services) RE Restricted to a group specified by the consortium (including the Commission Services) CO Confidential, only for members of the consortium (including the Commission Services)

D.JRA 3.2 Dissemination level PU

ULICE -GA n°228436 of 33

CONTENTS AND SPECIFIC DOCUMENT STRUCTURE 1 Introduction

2 Patient selection

2.1 Clinical selection

2.2 Clinical exclusion criteria

2.3 Clinical inclusion or selection criteria

3 Resistance and improved dose distribution as indication for ions

4 Review of the high-LET clinical data

4.1 Fast neutron therapy

4.2 Light-ion therapy

5 Summary of radiobiological aspects of patient selection

6 Selection criteria based on dose distribution

6.1 Bragg Peak and fragmentation

6.2 RBE

6.3 Penumbra

7 Comparison of proton, ion, and neutron results by estimated biological equivalent dose

8 Tools for patient selection

8.1 Predictive methods as tools to quantify resistance in individual patients

8.2 Tabular summary of hypoxia imaging

8.3 MRI

8.3.1 DW-MRI

8.3.2 DCE-MRI

8.3.3 BOLD MRI

8.4 Functional imaging and dose painting

8.5 Molecular genetic profiling, genetic expression

8.5.1 Predictive assays

8.5.2 Intrinsic resistance

8.5.3 Hypoxia

8.5.4 Tumor initiating cells

8.5.5 Other comments

8.5.6 Current status and conclusions on predictive assays



LIST OF ABBREVIATIONS AND DEFINITIONS

FDG [18F]-Fluorodeoxyglucose

FMISO [18F]-Fluoromisonidazole

OER Oxygen enhancement ratio

PET Positron emission tomography

pO2 partial oxygen pressure

RBE Relative biological effectiveness

TCP Tumor control probability

NTCP Normal tissue complication probability

FAZA Fluoroazomycin arabinoside

FLT Fluoro-L-thymidine

Cu-ATSM Cu-Diacetyl-bis(N4-methylthiosemicarbazone)

EF5 2-(2-nitro-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide

BED BED = D·[(α/β + d)/(α/β + 2)]

Gy (RBE) RBE weighted dose in Gy



PUBLISHABLE SUMMARY

The deliverable reports D.JRA 3.2 and D.JRA 3.3, have been combined in the following document since, in view of the authors, the discussion of D.JRA 3.3 precedes D.JRA 3.2.

For two reasons a very careful and evidence based selection of patients is essential: 1) ion beam therapy is a very limited and expensive resource, 2) ion beam therapy may be advantageous or disadvantageous for some tumours.

The rationale for ion beam therapy is based on sound, fundamental first principles of physics and radiation biology, the latter confirmed by clinical evidence from earlier neutron therapy studies and more recently from ion beam trials in Berkeley, Japan and Europe. We reviewed the principles and clinical experience in the perspective of all available and evolving therapy modalities and emphasise that modern anatomic and functional imaging and therapy are in a phase of rapid and revolutionary change. Consequently today’s algorithms need to be continuously re-examined and opportunities for newer combined modality treatment need to be explored as the field develops.

Clinical evaluation will remain the most important initial selection tool and in the context of historical experience from neutrons or ion beam trials will optimally exclude or select the vast majority of suitable patients. As general and perhaps alternative therapeutic options increase it will become more important to develop tools to identify subsets of patients who are more likely to benefit from a specific form of therapy. This would of course be most important in cancers that are frequent, often successfully treated by conventional modalities but also have subsets of patients with tumours highly resistant to therapy.

The mechanisms of resistance were reviewed. Conceptually it is very attractive to search for biomarkers predictive of tumour or normal tissue response to help identify the optimum treatment modality in the beginning, allow to monitor the response and give early indications if changes are necessary. Such markers may then also be used to focus additional treatment (or dose) to regions in the tumour shown to be resistant (e.g. hypoxic) or to plan for protection of particularly sensitive normal structures (dose painting).

Since the conditions responsible for resistance often change as treatment progresses, non-invasive markers will be most useful. There are early indications that contrast and non contrast MRI techniques are becoming available to assess directly or indirectly changes in cellularity, vascularity or hypoxia.

PET, PET-CT has seen the most rapid evolution which promises to accelerate as more hospital based cyclotrons and isotopes/ tracers become available. PET techniques may still be easier to standardize to provide quantitative data for meaningful exchange of information between institutions.

Very few predictive tests are presently used in clinical practice, but some promising approaches are awaiting validation in the clinic.

Even more than in therapy, rapid and revolutionary developments are seen in the area of predictive markers, making this early review just a snapshot of the present situation very likely to change radically in the near future. This is of course even more true for molecular markers, which have conceptually a most promising and fascinating potential for the future. Many early approaches however await validation in the clinic.



1 Introduction

Due to high costs, ion treatment facilities will be a limited resource requiring optimum selection of those patients who might truly benefit from this technology. To this end, the rationale for ion treatment will be discussed by first principles to help define a patient population to be selected and evaluated in comparison to other treatment options. Modern tools to further and more quantitatively select among those patients will then be reviewed.

Recent history has shown that conventional treatment approaches have seen rapid and fundamental improvements. Examples are: in surgery (robotic and minimally invasive surgery), in chemotherapy (targeted drugs) and in radiation therapy (IMRT, protons, stereotactically focused radiation methods, brachytherapy and sensitizing agents as bioreductive drugs, hypoxic sensitizers, or chemotherapy). In the selection of patients for ion therapy, these developments may shift the indications and selection process with time and opportunities for combination therapies have to be contemplated as all of these technologies develop and mature [1]. A review at the present time must be seen as a snapshot of the current situation where all of the discussed treatment methods and tools for selecting patients are in rapid development and many of today’s assumptions may become irrelevant due to new developments in any of the discussed parameters. It may have been difficult to claim equivalence or equipoise between particle and conventional radiation therapy just ten years ago, however IMRT, stereotactic radiation techniques, image guidance and methods to adapt to changes with time and with motion (4D) have radically impacted and improved photon and and to some degree particle treatments, and have certainly reduced and in some indications eliminated the relative advantage of particles.

The issue of clinical studies to compare the outcome in terms of tumour control or normal tissue damage is not subject of this review. Goitein [2] and Suit [3] have recently summarized the issues of clinical trials very profoundly. Suit provided a review of the clinical results of protons and ions from which one may infer that some claims of superiority may or may not pass the test of time. One may surmise that any possible advantage of ions may well be hidden in clinical trials of disease sites, unless a subset of patients is selected who may by historical evidence or by predictive tests show characteristics suggesting possible benefits of ions versus protons. The focus of this review is to review the current state of the art to identify such patient groups.

Pommier and associates [4] provided a most interesting and important analysis and model of the impact of patient recruitment on cost-effectiveness in studies comparing the clinical benefit of ions to alternatives and they point to the importance of knowing not only the case mix of potential recruits, but more importantly the indications likely to benefit greatly, moderately or relatively little, which they ranked into seven priority levels.

Prostate cancer exemplifies some of these issues. Over the last 15 years the treatments have been revolutionized by advances in imaging, brachytherapy, IMRT or proton IMPT, image guidance, motion control, and medical treatments e.g. androgen suppression, etc. The neutron experience may suggest a benefit of high LET and thus ions. Pommier et al. [4] (table 1) have ranked prostate cancer at the lowest priority level. One has to consider that ions may not significantly change the overall outcome in survival of prostate cancer patients, given the effectiveness of alternative and conventional treatments and the shift to lower stage presentations as a result of PSA screening. Tools to possibly identify subsets who might benefit from high LET may be most useful and important in such high volume, high impact (cost) cancers.



If the threat to survival from tumour recurrence lessens with more effective treatment, the late consequences of treatment are given a higher weight in the cost benefit analysis. This is particularly true for pediatric cancers. The reduced risk of second malignancies in successfully treated children has been considered a special advantage of proton therapy over IMRT or ion therapy [5-9].

2 Patient selection

Regardless of tumour site, a clinical examination and evaluation will quantitatively be the most important factor in selecting patients for possible ion treatments.

2.1 Clinical selection A complete physical examination and history is essential, which includes a comprehensive oncological history with time of diagnosis, any previous treatment with information about response or time to progression.

2.2 Clinical exclusion criteria To truly benefit from ion therapy, the patient’s general health, performance status and comorbidities must be considered. Patients with poor life expectancy or patients with metastatic disease would rarely benefit. Exceptional cases may benefit, and if considered should be part of at least a registry or phase 1 or phase 2 clinical study.

2.3 Clinical inclusion or selection criteria Much can be learned about the natural history of a tumour from a detailed history of its behaviour in a given patient. In general a tumour could be considered based on its shown resistance to treatment, its location in proximity to a critical organ compromising other treatments or its observed slow growth pattern (see below). Rare tumours known to have a natural history dominated by local failure and late metastases may be considered for ion treatment on a registry, phase 1 or 2 studies.



3 Resistance and improved dose distribution as indication for ions

Modern treatment approaches have and will likely continue to increase the types of tumours and tumour sites that can be successfully treated with advanced conventional technology or combinations of multiple treatment modalities to achieve the ultimate goal of cure without complications.

Our first goal would therefore be the identification of those tumours where conventional approaches are limited in their efficacy or associated with serious morbidity. Only those tumours are then further evaluated for potential ion treatments.

We can identify four groups among these:

1) Tumours, shown to be successfully treated in previous high LET studies with neutrons or ions:

a. the neutron experience

b. (proton) / ion experience

2) Tumours, that might be expected to benefit from high LET radiation based on first principles. Some of these tumours may never have been selected to previous high LET trials because of poor dose distributions obtainable with neutrons or because of logistics with ion treatment facilities. The mechanisms of resistance are discussed below and include:

a. hypoxia

b. proliferation

c. repair characteristics

3) Tumours in or around sensitive normal structures. Such tumours may have a poor outcome from radiation treatment because of their location in or in proximity to sensitive normal structures. In these cases, the superior dose distributions obtainable with charged particles may be an important advantage. In some of these tumours, an additional preferential high LET advantage of tumour versus the surrounding tissue is considered as a criterion for ion therapy.

4) Tumours selected or rejected because of normal tissue consequences (protons versus ions or IMRT in pediatric cases).

5)

4 Review of the high-LET clinical data

4.1 Fast neutron therapy Most of the high-LET clinical data available today were obtained with fast neutrons. A short review concentrates on those sites where there is some consistent evidence of a therapeutic benefit. Some conclusions from the neon-ion pilot study at Berkeley are also briefly mentioned for comparison.

For inoperable, incompletely resected or recurrent salivary gland tumours, a significant advantage for neutrons was reported (table 1) [28, 29]. The local control reached 67 % for neutrons compared to only 24 % for comparable historical series treated with low LET techniques (Table 2). The complication rate, around 10 % was acceptable (Table 3). A randomized cooperative study, initiated by the RTOG and the MRC showed a significant advantage for neutrons compared to photons for loco-regional control at two years (76 % vs. 17 %, p < 0.005) and a trend towards improved survival (62 % vs. 25 %). Ten-year analyses continued to show a striking difference in loco-regional control (56 % for neutrons vs. 17 % for photons, p = 0.009), but both groups experienced a high rate of metastatic failure [28].

Neutron-beam therapy was recommended as the treatment of choice for patients with unresectable or recurrent malignant salivary gland tumours or in patients where radical resection would require facial nerve sacrifice [10, 28, 30, 31].The fact that salivary gland tumours are rather superficial explains why the neutron beams available at that time were suitable for treatment in spite of their inferior physical selectivity [29].



The typical slow growth rate of prostatic adenocarcinomas was the argument for exploring the value of fast neutrons for treatment [25, 32] . Later on, the low α/β ratio for some prostatic tumours provided the radiobiological basis for the expected benefit of high-LET radiation [33]. Among the numerous published data, only two randomized trials for treatment of locally advanced prostate tumours are considered here. In the United States, the RTOG (Radiation Therapy Oncology Group) compared "mixed beams" (a combination of photons and neutrons) to conventional photons. Loco-regional control, as well as survival, was significantly superior after mixed-beam irradiation [28, 32].

In order to confirm these conclusions the Neutron Therapy Collaborative Working Group (NTCWG) compared neutrons (alone) and conventional photons: stage T3-4, N0-1 tumours or high-grade (summed Gleason score > 6) T2 tumours. A significant difference (p < 0.01) was observed in "clinical" loco-regional failure, with actuarial 5-year failure rates of 11 % vs. 32 % after neutron and photon treatments, respectively [32]. Inclusion of routine post-treatment biopsies resulted in 5-year "histological" local-regional failure rates of 13 % and 32 %, respectively (p = 0.01) [32].

Due to the long natural history of recurrent prostate cancer, long follow-up is required to assess the ultimate impact of the improved local control on survival. However, PSA patterns confirmed the benefit of neutrons. Late sequelae were similar in the neutron and photon groups where the technical irradiation conditions were comparable (i.e., using multileaf collimators). The rates of colostomies as a function of collimator type are given in Table 4 [28]. Since these results were obtained, a number of novel techniques have been developed for the treatment of prostatic adenocarcinoma at different stages. These alternative techniques (such as IMRT and modern brachytherapy) may influence the proportion of patients potentially benefitting from high-LET techniques, but the conclusions of the above two clinical trials concerning the radiobiological advantage of high-LET radiation remain valid.

For some other tumour sites or types, such as slowly growing soft tissue sarcomas, fixed lymph nodes in the cervical area, locally advanced antrum tumours and some bronchus carcinomas, the available data showed a benefit whenever neutron therapy was applied under appropriate technical conditions. Randomized studies would be needed to confirm the benefit of high-LET [13, 27].

4.2 Light-ion therapy A pioneer ion-therapy program was initiated at Berkeley in the 1970s and 433 patients were treated between 1975 and 1992. The Berkeley program was limited by the availability of the machine and its complexity (which resulted in many unscheduled down-times) and as a consequence there was a patient recruitment problem. Nevertheless a great deal of valuable radiobiological and clinical information was obtained [11, 12, 30]. The clinical results are compared in Table 5 with some fast neutron therapy results [13]. Although the recruitments are not strictly comparable, it should be pointed out that tumour types or sites for which an advantage was found with neon ions are those for which an advantage was also found with fast neutrons. This suggests a specific "high LET" benefit.

More recently at GSI Darmstadt, Germany, a local tumour control rate for advanced salivary gland tumours of 77.5 % was reached at 5 years in patients treated by IMRT with carbon ion boost compared with 25 % after IMRT alone [12]. At the National Institute of Radiological Sciences (NIRS) at Chiba, Japan, a local control rate of 61 % was reported. For these salivary gland tumours local control rates of 24 % to 28 % were reported after photon irradiation [28, 29] .

Excellent results are also consistently reported for skull based chordomas with control rates above 80 -90 %. We also refer to the discussion on chordomas and chondrosarcomas by Suit [3] and tables 6 and 7, discussing the superior response of chordomas to high LET and the experience with protons and ion therapy for these two entities.



5 Summary of radiobiological aspects of patient selection

Radiobiological data indicate that high LET radiations are expected to be more effective than low LET for hypoxic, well differentiated and slowly growing tumours. Patient selection between low and high LET radiations is a radiobiological issue; it is not related to the technique or to the machine type. It is still complex and difficult because of the lack of accurate and robust predictive tests.

The large number of studies that has been very carefully undertaken to establish the clinical outcomes of fast neutron therapy still serves as the basis for the initial selection of patients for other more modern high-LET radiations. This approach was adopted by the two centers where the most patients have received carbon-ion therapy: Chiba, Japan and GSI Darmstadt, Germany. Both of these clinical teams had previous experience with fast neutron therapy.

Table 1. Review of the loco-regional control rates for malignant salivary gland tumours treated definitely with fast neutrons: inoperable, incompletely resected or recurrent. Patients treated post- operatively for microscopic residual disease are not included [Krüll et al., 1998]

Reference Number of

patients Loco-regional

control (%)

Saroja et al., 1987 113 71 (63 %)

Catterall and Errington, 1987 65 50 (77 %)

Battermann and Mijnheer, 1986 32 21 (66 %)

Griffin et al., 1988 32 26 (81 %)

Duncan et al., 1987 22 12 (55 %)

Tsunemoto et al., 1996 21 13 (62 %)

Maor et al., 1981 9 6

Ornitz et al., 1979 8 3

Eichhorn, 1981 5 3

Skolyszewski, 1982 3 2

Overall 310 207 (67%)



Table 2. Same type of patient series as in Table 1 (salivary gland), but treated with low-LET techniques (photon and/or electron beams, and/or radioactive implants) [Krüll et al., 1998].

Reference Number of

patients Loco-regional

control (%)

Fitzpatrick and Theriault, 1986 50 6 (12%)

Vikramet et al.,1984 49 2 (4%)

Borthne et al., 1986 35 8 (23%)

Rafla. 1977 25 9 (36%)

Fu et al., 1977 19 6 (32%)

Stewart et al., 1968 19 9 (47%)

Dobrowsky et al., 1986 17 7 (41%)

Shidnia et al., 1980 16 6 (38%)

Elkon et al., 1978 13 2 (15%)

Rossman, 1975 11 6 (54%)

Overall 254 61 (24%)



Table 3. European results in neutron therapy of salivary gland tumours. Severe radiation-related morbidity for patients reviewed in Table 1 [Krüll et al., 1998]

Reference n %

Catterall, 1987 8/65 11.8

Battermann and Mijnheer, 1986 4/32 12.5

Duncan et al., 1987 4/26 15.4

Kovács, 1987 2/15 13.3

Engenhart, 1994 2/49 4.1

Krüll et al., 1995 6/74 8.1

Lessel, 1995 6/38 15.8

Overall 32/299 10.7



Table 4. Neutron therapy of prostate cancer: bowel severe morbidity by treatment center and technical conditions [Lindsey et al., 1996]

Institution Colostomies

n %

University of Washington

50 MeV p-Be neutrons

Multileaf collimator

0/49 0 %

University of California

Los Angeles


Movable jaw collimator

2/25 8 %

M.D. Anderson Hospital


Fixed cone collimator

4/10 40 %



Table 5. Comparison of the clinical results obtained with different types of high-LET radiations: fast neutrons, and neon-ions at Berkeley [Wambersie et al., 2004b]

Tumor site (or type) Local control rates

Fast neutrons Neon ions

Salivary gland tumours 67 % (24 %) 80 % (28 %)

Paranasal sinuses 67 % 63 % (21 %)

Fixed lymph nodes 69 % (55 %)

Sarcomas 53 % (38 %) 45 % (28 %)

Prostatic adenocarcinomas 77 % (31 %) 100% (60 % -70 %)

( ) Local control rates currently obtained with conventional low-LET radiations for similar patient

series.



6 Selection criteria based on dose distribution Dose distribution advantages of ions have been extensively discussed in the literature and only some salient points are summarized here [3, 34-38].

6.1 Bragg peak in protons and ions and fragmentation: Protons and carbon ions share the advantage of Bragg peak radiation, i.e., a low and flat dose profile in the entrance plateau relative to the spread out Bragg peak (SOBP) and most importantly a finite range with an abrupt drop in dose at the end of the Bragg peak. In the case of ions, the differential between the biological dose in the entrance plateau versus the SOBP is enhanced due to the higher RBE in the latter. The magnitude of this RBE difference has been discussed and debated [2, 38]. A small dose due to fragmentation at the end of the SOBP is important for dosimetry, but of lesser importance for our discussion. The fragmentation tail is due to fragmentation of carbon by nuclear interactions in the path through tissue and consists of low or intermediate energy ions (high LET) [21, 39-42].

6.2 RBE Clinical selection based on high LET (RBE) biology and clinical experience was discussed above and it was emphasized that tumours will only benefit if they exhibit a higher RBE than normal tissues surrounding them.

Beyond such considerations of biological selectivity which were best demonstrated in neutron clinical trials, the RBE in ion therapy is a significant parameter resulting in the superior biological dose distributions claimed for ions. RBE determinations in ion therapy is highly complex, since RBE varies with LET along the beam path, being relatively low in the entrance plateau and highest in the SOBP. The magnitude and benefit of this always favourable ratio have been discussed and debated, particularly if dose rate effects are taken into consideration [2, 38].

6.3 Penumbra (80 – 20 % dose at field edge) The width of the penumbra depends on the type of beam spreading used (passive scattering, wobbling (uniform scanning), pencil beam scanning), on the collimation system and air gap between compensator and body surface, and it varies with depth. In principle, ions have a significantly smaller penumbra, since beam broadening by multiple Coulomb scattering is smaller by a factor of 4 for carbons versus protons. Plotting the penumbra vs. atomic number of the particle beam, the steepest decrease (by a factor of 2) was seen between proton and helium beams [3, 43]. This is a significant dose distribution advantage for ions, even though developments in proton delivery are expected to reduce that differential at least up to a depth of 15 cm [3].

7 Comparison of proton, ion, and neutron results by estimated biological equivalent dose

Much work has been done to compare clinical results from different types of radiation [3, 44-49]. Suit has provided a very thorough analysis of the historical experience with photons, neutrons, protons and ions [3] (tables 6-14). He points out significant issues making interpretation and comparative judgements difficult and perhaps tentative at this time. In general, as technologies evolved over the period, imaging, definitions of Gross Tumor Volumes (GTV) or Clinical Target Volumes (CTV) evolved and thus represent a significant uncontrolled variable. Better definition of target volumes would be most beneficial for treatments with superior dose distribution and has permitted different fractionation patterns to emerge even in photon therapy (stereotactic methods), further complicating comparisons.

Presenting the experience by estimated biologically weighted dose, a correlation of dose with tumor control probability (TCP) and normal tissue complication probability (NTCP) is shown, as is the beneficial effect of optimum dose distribution. Much less evident is the additional contribution of high LET even where it often has been assumed. The difficulty of normalizing trials to either a level TCP or level NTCP is described as a past and remaining challenge for clinical trials.

We present the tables from Suit, since they are more recent and include the analysis of estimated biologically weighted dose, but we also point to a series of similar excellent comparisons of photon



and hadron results in the literature, which do not contradict the results reproduced here and also could be briefly summarized as indicating that a true comparison is not possible with the available data, collected over a period of rapid technological change in all treatment modalities [46, 50-53].

NOTE: in the tables below: Gy (RBE) is the RBE weighted dose in Gy;

BED = nd x [(a/β + d)÷ (a/β + 2)];

Tables 6 and 7:

Comment on Tables 6: Skull based chordoma: favorable TCP vs NTCP for ions, numbers in high dose group small.

Comments on Table 7: Skull based chordoma, ions superior, skull based chondrosarcoma: Proton results seem superior



Tables 8 and 9:

Comment on Table 8: Uveal melanoma: Protons and ions yield equivalent TCP/NTCP. Stereotactic photons have similar control but higher enucleation rates.

Comment on Table 9: Head and neck cancer: advantage for squamous cell ca unclear, Ions favourable in adenoid cystic ca and malignant melanoma



Tables 10 and 11:

Comments Table 10: Non small cell lung cancer: comparable results for early stage cancers

Comments Table 11: Hepatocellular carcinoma: comparable results



Table 12:

Comments Table 12: Prostate cancer: advantage for ions, but satisfactory results with high dose XRT or brachytherapy

Comment on renal cell carcinoma: excellent results with ions.

Tables 13 and 14

Summary comments [3]: In principle, if there is a difference between ions and protons it would be due to a higher RBE/LET to tumour vs. normal tissue, dose distribution due to less penumbra with ions or due to different fractionation patterns, more easily adopted in ion therapy. With all caveats in this analysis, table 13 presents the highest TCPs for eight tumour types, highlighted in bold if there is more than a 9 % difference for one modality. It was concluded by the authors, that higher TCPs are seen at higher dose levels.



Tumors shown in table 14 show similar TCPs at similar biological doses. The results are not fully consistent with equivalent TCPs at similar dose levels, but they do indicate that dose is an important determinant of TCP. NTCP is at least of equal importance but much more difficult to analyse retrospectively. In the reported literature there seems to be less treatment morbidity related to ions.

The authors clearly state the constraints in the available outcome data, making it difficult to discern the contribution of high LET, dose distribution or fractionation and indicate the need for additional data accrued in a more standardized setting. For the purpose of our discussion, the data are consistent with an apparent high LET advantage in those tumours that benefitted from neutrons (melanoma, salivary gland, prostate, chordoma (slow growth rate).



8 Tools for patient selection

8.1 Predictive methods as tools to quantify resistance in individual patients Extensive research has explored the reason for resistance. We therefore reviewed the mechanisms of resistance and searched for any clinical or laboratory method that can help to predict treatment response in individual tumors.

From the discussion above we conclude that in selecting patients for ion therapy the largest bracket of potentially suitable patients would be first selected and/or excluded by clinical criteria, e.g. history and examination of the patient, experience with resistant tumors, historical evidence of response to high LET or observed tumor characteristics likely to benefit from high LET such as low grade or observed slow growth, particularly in tumors located in proximity of critical normal tissues benefitting from superior dose distribution and high LET. However, even not all patients with similar tumors will benefit from ion therapy.

CT, PET and MRI are important in the preselection of patients to define tumor extent and to exclude metastases. Here we shall only discuss the role of imaging in selecting those patients where a selective biological advantage may be expected from high LET radiation by providing non invasive methods to characterize tumors for:

1) Features of resistance, e.g. hypoxia and thus general selection for ions.

2) Spatial information about portions of a tumor with presumed higher resistance. With the ability to more precisely shape the dose distribution, such information could be valuable for possible dose painting. (see discussion below).

8.2 Hypoxia Because of the oxygen effect and the reduction of OER with increasing LET, tumors with a high proportion of hypoxic cells and poor reoxygenation patterns would in principle be an indication for high-LET therapy. Among various techniques capable of detecting hypoxic cells (see review in [54]), polarographic measurement (e.g. Eppendorf probe) is the only method for which a correlation with treatment outcome has been convincingly demonstrated in different human tumor types, e.g., cervix cancer, head and neck lymph nodes, and sarcomas. Nevertheless, until now, this technique has not been routinely used due to its relatively low sensitivity at low oxygen concentrations and some logistic constraints.

The use of the so-called hypoxic cell chemical markers (e.g., nitroimidazole, EF5, EF3) represents an attractive alternative to polarographic measurements (see review in [55]). However, so far no attempts have been made to use these markers for patient selection for high-LET radiation therapy. In principle, the use of these markers requires a representative tumour biopsy. Hence it is only feasible to assess the initial level of hypoxia, but is more difficult to assess reoxygenation where additional samples are needed during treatment. Non-invasive methods have been actively investigated. MRI signals have shown a correlation with Eppendorf probe measurements. PET imaging (e.g., with 18F-misonidazole) represents another promising approach [8, 29,13, 56]. In this review we focus on non-invasive methods:

The negative impact of chronic and acute hypoxia on TCP has long been known. It has also been shown that biological characteristics such as hypoxia, proliferation or perfusion of tissues show significant spatial and temporal variation in tissue and that the effects of hypoxia can be overcome by reoxygenation or redistribution of hypoxic regions. For these reasons, there is increasing interest in non-invasive methods, particularly imaging to characterize tumour tissue with respect to these characteristics at the beginning and during the early stages of treatment with the goal to either modify the type of treatment or the distribution of dose in the treated volume to high dose subvolumes for regions suspected to be resistant, a concept which is known as dose painting [57].

The most intensely studied methods for visualizing intra tumour radio-resistance are hypoxia tracers [25]. Several nitroimidazoles for PET imaging have been tested (FMISO, FAZA, EF-3, EF-5) to visualize inter patient and intra tumour hypoxia differences [57-61]. During the last decade FDG PET has become widely available and is well standardized for good reproducibility to visualize glucose metabolism. It has become one of the most important tools for diagnosis, staging, tumour delineation



and prognostic characterization. It has been shown that high FDG uptake is associated with a poorer treatment outcome with radiotherapy and surgery [57, 62-65]. It measures the uptake of glucose in cells by ATP independent glucose transporters. FDG is specific to this pathway, which is known to influence numerous other processes in the growth of cancer. In contrast to hypoxic tracers, FDG uptake in the tumour does not reflect a single biologic characteristic, but is correlated to radio-resistance, proliferation [66], cell density [67] and hypoxia [68, 69].

It has been shown that residual metabolic activity after treatment overlaps better than 70 % of the times with pre treatment uptake [57]. It remains to be validated that the intra tumour location with a high chance of relapse can be identified before treatment with a single FDG-PET CT and remains stable during radiation therapy. Only then would a ‘dose painting’ plan remain valid through the treatment course (see below).

8.2.1 Tabular summary of hypoxia imaging A recent comprehensive review by Sun [70] on tumour hypoxia imaging is summarized in tabular form :

Hypoxia 50 – 60 % of tumours exhibit macroscopic hypoxic regions, heterogeneously distributed

- Acute: perfusion limited

- Chronic: diffusion limited

Consequences of hypoxia [71, 72]: - cell cycle arrest, differentiation, apoptosis, necrosis

- promotion of tumour progression by enabling cells to overcome nutrient deprivation, escape or metastasize and by promoting unrestricted growth resulting from cellular changes to a more aggressive phenotype.

- radiation- and chemotherapy treatment failure

Measuring hypoxia Direct (invasive or non-invasive); for oxygen partial pressure (pO2), O2 concentration or

percentage: Polarographic needle electrode, phosphorescence imaging, near-infrared spectroscopy (NIRS), blood oxygen level dependent (BOLD) MRI and 19F MRI, electron paramagnetic resonance (EPR) imaging;

Indirect (invasive or noninvasive); using exogenous probes to measure molecular reporters of oxygen:

- with immunohistochemical staining (IHC) or

- antibodies against nitroimidazole derivatives (e.g., 2-nitroimidazole derivatives (misonidazole, pimonidazole, EF5, EF3);

Note: markers stain areas of chronic hypoxia and in severe hypoxia are more sensitive than polarographic needle electrode [73]. They are also useful for non-invasive PET imaging [74], see below.

Invasive methods either require insertion of a probe into tissue or sampling and thus have operator dependent variability and provide only partial information of the whole tumour region [70]. Limited in assessing the time course or variability of hypoxia in human tumours.



Measuring hypoxia by non-invasive imaging EPR spectroscopy (“tumour oximeter” to monitor changes, but lack of non-toxic paramagnetic marker); [75-77].

Photo acoustic tomography(PAT) (image blood oxygenation only); [78].

BOLD-MRI ( blood oxygenation only, not quantitative, sensitive to flow effects, hematocrit pH and temperature); [79] . 19F-MRI (spectroscopy) with perfluorocarbons (PFC) or fluorinated nitroimidazoles as contrast agents: (vascular oxygenation, able to detect changes in tumour oxygenation in response to radio-sensitizing and oxygen-augmenting treatments, but are sensitive to flow artefacts, and sensitive to temperature, dilution, pH, common proteins and blood. Risks are, e.g., embolism after PFC injections or toxicity profile of nitroimidazole) [80-86];

PET imaging of Hypoxia Commonly used isotopes are 18F, 124I, and 60-64Cu, which can be attached to markers, e.g. FMISO, EF5, FETA, IAZA, Cu-ATSM [87-90];

First generation marker 18FMISO has been shown to reflect hypoxia in glioma, head and neck cancer, renal cancer and non small cell lung cancer [63, 91, 92];

Second generation markers are more water-soluble and less easily degraded, like 18F-fluoroerythronitroimidazole (FETNIM) [89, 93], FETA and EF5 (complex labelling and low chemical yield) [56, 94]; 18 FAZA shows faster diffusion and faster clearance; 124 I-IAZG and 124 I glactoside are more suitable for xenograft models, because of low positron abundance and high energy, but show a poor image resolution, and higher exposure;

Dithiosemicarbazones (Cu-ATSM) offer simple chemistry, are rapidly cleared from aerobic and trapped in hypoxic cells. They are able to differentiate between dead, hypoxic, nonfunctional and viable tissue [95, 96]. 64Cu is Auger emitter and can cause DNA damage and induce apoptosis in hypoxic cells, a possible therapeutic benefit [97-99]. However, the efficacy of Cu-ATSM vary with tumour type, requiring further validation.

SPECT imaging of Hypoxia 123I-IAZA, the initial compound had inconsistent uptake. 123I-IAZXP and -IAZGP show promising results. 99m Tc with nitro-imidazoles and non-nitro imidazoles are increasingly used [100-103].

Imaging with multiple tracers Tissue oxygenation is very heterogeneous and impacts our ability to stratify patients or predict outcomes. Co-injection of tracers or co registration of images from different probes is a possible strategy. Examples of co-registration: 18 F-FAZA and 18 F-RGD allow hypoxia to be correlated with angiogenesis [104]: high F-FAZA and low F-RGD may indicate acute hypoxia and not yet stimulated angiogenesis; low F-FAZA together with high F-RGD may indicate averted hypoxia due to angiogenesis or hypoxia independent angiogenesis. 18 F-FMISO and 18 F-FDG correlate hypoxia with glycolysis, e.g. high glycolysis and low hypoxia might identify particularly aggressive tumours [105].

Optical imaging of endogenous markers associated with hypoxia (bioluminescence, phage display technology in biopsy specimens) Hypoxia-inducible factor 1, carbonic anhydrase IX; Proteins and genes whose expressions are associated with hypoxia are of interest for the possibility to combine various markers to create hypoxia-prognostic profiles [106, 107]. These imaging tools are useful to study the biology of hypoxia and mechanisms of response to experimental therapy.



8.3 Magnetic Resonance Imaging: MRI has become most important for the precise detection and delineation of malignancies for initial staging as well as for the planning of target volumes in therapy. ’Functional’ MRI provides additional information characterizing tissues, their biological state or metabolic processes.

8.3.1 Diffusion Weighted MRI: DW-MRI detects the molecular diffusion, i.e. the Brownian motion of water molecules in biological tissue and was first used in the early evaluation of stroke. The relative speed, independence on exogenous contrast and the ability to yield qualitative and quantitative information on tissues was recognized for a potential role in oncology. Lambrecht et al provided a review, initially from the perspective of head and neck cancer [109]. In biological tissues, the free movement of water molecules is limited or modified by the presence of cell membranes and macromolecules. i.e., the signal is defined by the motion of water molecules in extracellular, intracellular and intravascular space. Thus DW-MRI helps to characterize tissues based on their cellularity or integrity of cellular membranes, distinguishing hypercellular, hypocellular, necrotic, apoptotic or inflammatory tissues with sub-centimeters resolution for staging. Not free of diagnostic pitfalls it may best be seen as a problem solving technique complementing conventional imaging. In our context its potential in response prediction and assessment is of greatest interest for its ability to detect micro-structural changes with a high sensitivity for low levels of intra-tumoral cell loss, even in the absence of macroscopic tumour changes [110-113]. Particularly attractive may be the possibility to identify regions of poor tumour response within a given tumour for ‘dose painting’. If validated and standardized in the clinic, these possibilities and the promise of earlier diagnosis of tumour recurrence could make this technique very useful.

Identifying potential treatment failure early in the course of treatment is always important and in the context of ion beam therapy may help in selecting these patients for consideration of a high LET boost. It has also shown some usefulness in predicting metastases-free survival in melanoma patients after ion therapy [114].

8.3.2 Dynamic Contrast-Enhanced MRI Low vascular density and hypoxia are associated with poor treatment outcome. DCE MRI can provide anatomical and additional biological information. Based on their own work and a review of the literature, Rofstad [115] found inconsistent or weak correlations between DCE-MRI derived parameters and some biological characteristics of tumours, including microvascular density, expression of VEGF-A (vascular endothelial growth factor-A) and oxygen tension [116-120]. In some clinical studies, significant associations between DCE-MRI derived parameters and tumour regression, tumour control and disease free survival are reported [116, 121-123]. While DCE-MRI may provide useful prognostic information, the parameters derived from it appear to have too weak a correlation to permit individualized treatment plans. Rofstad concludes that the potential of this technique to show detailed information on the histomorphology of cervix carcinoma is limited by the significant morphological heterogeneity at the subvoxel level. In a study by Loncaster et al, combined MRI-derived enhancement and volume data were associated with large differences in outcome and pharmacokinetic modeling of DCE-MRI provided data that reflected tumour oxygenation and useful prognostic information [31]. Combined DCE-MRI and 18F-FDG PET parameters have been shown to be predictive of short term response to treatment [124].

8.3.3 BOLD MRI The potential of blood-oxygen-level-dependent MRI (BOLD) to provide non-invasive imaging of prostate cancer hypoxia and its possible role in ‘dose painting’ of hypoxic regions has attracted some investigations [125, 126].

8.4 Functional imaging and dose painting The size of tumours has long been a criterion to either increase the dose if safe and feasible or to consider additional treatments such as limited surgery etc. If imaging can provide information on tumour subvolumes at high risk of failure it is attractive to investigate the possibility of prescribing higher doses to such regions. Recent advances in imaging and radiation delivery are the preconditions for such attempts.



Dose painting is the prescription of nonuniform dose to the target volume based on functional or molecular images indicating high risk of relapse in tumour subvolumes based on hypoxia (e.g. FMISO-, Cu-ATSM-, FAZA-, EFS- PET), proliferation (e.g. FLT PET) or tumour burden (e.g. FDG, Choline). This may be executed by homogeneously increasing the dose to a biologic target volume or subvolume within a target by contours or segmentation or by dose-painting-by-numbers to shape the additional dose gradually, e.g., according to the PET voxel intensities. Preconditions are that the functional images are indeed related to treatment outcome and that there is spatial and temporal stability of the images before and during therapy. We refer to excellent recent reviews of the subject [58, 108].

8.5 Molecular, genetic profiling, genetic expression and hypersensitivities 8.5.1 Predictive assays [127-130] The decision on the best treatment for a patient is made primarily by a physician, based on clinical factors like tumor location, size, and stage, but there are also biological factors that determine the response of a tumor to radiotherapy and this causes a variability in outcome in patients with similar clinical features. In an ideal world one would be able to predict precisely a patient´s response to treatment, based on both clinical and biological factors and then select specific treatments, for instance a modified fractionation schedule, combination therapies, or high LET particle therapy. There is no assay in clinical use at the moment that can do this, or has been validated. It is known that resistance to conventional radiation therapy can be caused by 4 or 5 main factors:

1. Intrinsic radioresistance of the cancer cells

2. Tumor hypoxia

3. Rapid repopulation

4. Large number of tumor initiating (cancer stem) cells

5. Host factors (e.g. infiltrating cells such lymphocytes and macrophages which can affect vasculogenesis)

High LET therapy would theoretically provide an advantage for the first two (intrinsic radioresistance, hypoxia). For the other three, an advantage is less obvious or certain, although tumors with high numbers of cancer stem cells could be regarded as candidates for more effective high LET therapy.

In the past, predictive cell-based assays and functional assays were developed. Cell-based assays aim to measure cell kill, DNA damage, or cytogenetic damage. Examples are the colony forming assay to measure intrinsic radiosensitivity. Examples of functional assays are the polarographic electrode to measure oxygen tension, or uptake of thymidine analogs to estimate proliferation rates.

These assays have provided a great amount of useful information, like evidence on the influence of intrinsic radiosensitivity, hypoxia, and proliferation on radiotherapy outcome. However, some disadvantages exist, for example high technical complexity and long assay times. Surrogate assays have been developed to overcome these problems but preclinical results are too inconsistent to be translated into the clinical practice.

Table 1 (from [128]) summarizes direct predictive assays and indirect surrogate assays, some of them still used in the clinical praxis.



Genetic assays have been developed, based on genetic analysis at different levels: DNA, RNA, protein expression. Following, a brief summary of the present status, focusing on genetic assays. Table 2 summarizes new predictive assays based on genetic analysis at different levels: DNA, RNA, proteins.

8.5.2 Intrinsic radioresistance There are still no reliable genetic assays for intrinsic radioresistance. This includes expression profiling of mRNA or miRNA, or DNA copy number changes (amplifications or deletions using comparative genomic hybridization (CGH)), or promoter methylation status. Some studies on cell lines have produced gene signatures (sets of genes) correlating with radiosensitivity, but correlations are usually weak and none has been sufficiently validated.



8.5.3 Hypoxia There have been several studies looking at genes which are upregulated under hypoxia (studied at the mRNA level). Some of these signatures have been shown to correlate with outcome. This is not specific to radiotherapy (it would also apply to surgery and chemotherapy), although finding high tumor hypoxia would still be an indication for high LET therapy. The extent and time of hypoxia can also influence how dangerous the hypoxia is, and there are some indications that acute (short term) hypoxia is the more dangerous form, and some gene signatures have been described which monitor acute rather than chronic hypoxia. This is, however, still an area for further research and cannot be yet regarded as a reliable way to predict the extent and type of tumor hypoxia, although this is in a more advanced state than gene signatures for intrinsic radioresistance. There are also microRNAs (miRNA) which are upregulated under hypoxia, and studies have shown that expression of one of these miRNAs correlates with outcome. The advantage of miRNAs is that their expression can also be reliably assessed from paraffin embedded material, a considerable advantage. This is an area to watch out for.

8.5.4 Tumor initiating cells Researchers at NKI Amsterdam recently found a correlation between CD44 expression and local control after radiotherapy for head and neck cancer [131]. This could be related to the fact that CD44 has been described as a cancer stem cell marker in this disease. Another recent study also found that CD44 together with other markers was associated with bad prognosis in head and neck cancer [132]. It could therefore be argued that high CD44 expressing tumors would be candidates for alternative therapies such as high LET radiation.

8.5.5 Other comments Most studies to date on radiotherapy predictors have focused on head and neck and sometimes cervix cancer. There have also been studies on breast cancer given breast conserving therapy involving radiotherapy and on rectal cancer involving radiotherapy, although radiotherapy is not the primary modality at either of these sites. So far, none of these studies have produced reliable and validated predictors of outcome relevant to the radiotherapy.

There have been many studies showing that expression of particular markers, assessed using immunohistochemistry, have correlated with outcome after radiotherapy, including hypoxia and DNA repair related genes. Lack of independent validation, and sometimes conflicting results between different studies, mean that none can yet regarded as robust predictors of radiotherapy response.

8.5.6 Current status and conclusion on predictive assays In summary, this ULICE workgroup agrees with the recent conclusion by Begg [128]; there are presently no validated assays used to determine treatment choice. Cell based and functional assays have provided proof of principle that intrinsic radiosensitivity, hypoxia and proliferation are important determinants of outcome. The complexity and difficulty with standardization for reliable reproduction in different hospitals has hindered their use in practice and the search for surrogate assays has so far yielded inconsistent data in preclinical use for general adaptation. Attractive concepts and much work is in progress in the search for genetic assays, and their development for clinical practice is urgent. The paradigmatic example are the current tests used to determine estrogen receptor status in breast cancer. In ideal case, their usefulness would extend beyond the patient selection for ion beam therapy as tools to guide in the best selection of available modalities or combination of modalities, biological modifiers or appropriate aggressiveness of treatment strategies.



CONCLUSIONS

For two reasons a very careful and evidence based selection of patients is essential: 1) ion beam therapy is a very limited and expensive resource 2) ion beam therapy may be advantageous or disadvantageous for some tumours.

The rationale for ion beam therapy is based on physical properties and on sound, fundamental first principles of radiation biology, confirmed by clinical evidence from earlier neutron therapy studies and more recently from ion beam trials in Berkely, Japan and Europe. We reviewed the principles and clinical experience in the perspective of all available and evolving therapy modalities and emphasise that modern anatomic and functional imaging and therapy are in a phase of rapid and revolutionary change. Consequently today’s algorithms need to be continuously re-examined and opportunities for newer combined modality treatment need to be explored as the field develops.

Clinical evaluation will remain the most important initial selection tool and in the context of historical experience from neutrons or ion beam trials will optimally exclude or select the vast majority of suitable patients. As general and perhaps alternative therapeutic options increase it will become more important to develop tools to identify subsets of patients who are more likely to benefit from a specific form of therapy. This would of course be most important in cancers that are frequent, often successfully treated by conventional modalities but also have subsets of patients with tumours highly resistant to therapy.

The mechanisms of resistance were reviewed. Conceptually it is very attractive to search for biomarkers predictive of tumour or normal tissue response to help identify the optimum treatment modality in the beginning, allow to monitor the response and give early indications if changes are necessary. Such markers may then also be used to focus additional treatment (or dose) to regions in the tumour shown to be resistant (e.g. hypoxic) or to plan for protection of particularly sensitive normal structures (dose painting).

Since the conditions responsible for resistance often change as treatment progresses, non-invasive markers will be most useful. There are early indications that contrast and non contrast MRI techniques are becoming available to assess directly or indirectly changes in cellularity, vascularity or hypoxia.

PET, PET-CT has seen the most rapid evolution which promises to accelerate as more hospital based cyclotrons and isotopes / tracers become available. PET techniques may still be easier to standardize to provide quantitative data for meaningful exchange of information between institutions.

Very few predictive tests are presently used in clinical practice, but some promising approaches are awaiting validation in the clinic.

Even more than in therapy, rapid and revolutionary developments are seen in the area of predictive markers, making this early review just a snapshot of the present situation very likely to change radically in the near future. This is of course even more true for molecular markers, which have conceptually a most promising and fascinating potential for the future. Many early approaches however await validation in the clinic.



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