Predicting Normal Tissue Radiotoxicity in Radiotherapy Patients Based on the Individual Radiosensitivity Using Three-Color Fluorescence In Situ Hybridization:
A Literature Review
Dwi Ramadhani
1,2,3*, Isnaini Farida
3, Arum Wulansari
3, Wiwin Mailana
3, Hartini Ahadiyatur Ru’yi
3, Syarifatul Ulya
3, Sofiati Purnami
3, Nastiti Rahajeng
3, Devita Tetriana
3, Iin Kurnia
2,3, Mukh Syaifudin
2,31 Doctoral Program for Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
2 Research and Technology Center for Radioisotope, Radiopharmaceutical and Biodosimetry, Research Organization for Nuclear Energy, Jakarta, Indonesia
3 Research and Technology Center for Safety, Metrology and Nuclear Quality, Research Organization for Nuclear Energy, Jakarta, Indonesia
*Corresponding author:
Dwi Ramadhani
Research and Technology Center for Radioisotope, Radiopharmaceutical and Biodosimetry, Research Organization for Nuclear Energy, Jakarta, Indonesia
INTRODUCTION
The individual response to ionizing radiation exposure is differently affected and could be influenced by many factors such as age, smoking habits, diseases, and genetic background. The clear evidence of variability in individual response to radiation exposure could be seen in radiotherapy (RT) patients [1]. Nowadays, the improvement of RT techniques and the use of radio- sensitizers in RT treatment has a significant impact on the outcome of RT and the quality of life of RT patients [2]. However, approximately 5–10% of RT patients developed radiation toxicity (radiotoxicity) in normal
tissue surrounding the tumor area, such as burns and radio-dermatitis in the irradiated area [1–4]. The variability of normal tissue reaction in RT patients is due to differences in individual radiosensitivity [5].
The terminology of radiosensitivity used worldwide recently is being subjected to debate among researchers and experts. Foray et al. [6] proposed three different terminologies related to individual responses to radiation exposure. The first is radiosensitivity, defined as proneness to harming tissue after exposure to a high dose of radiation, particularly in RT. The second is radio- susceptibility, defined as the proneness to having cancer after exposure to ionizing radiation, even at low radiation A R T I C L E I N F O
Received : 01 January 2021 Reviewed : 27 January 2021 Accepted : 21 May 2021 Keywords:
cancer, chromosome, FISH, radiosensitivity, radiotherapy
A B S T R A C T
Background: The variability of clinical response in radiotherapy (RT) patients revealed that individual radiosensitivity exists in humans. Although several techniques for radiosensitivity assessment are available, fluorescence in situ hybridization (FISH) has proven to be the most reliable cytogenetic technique. This study will review the use of three-color FISH to evaluate the individual radiosensitivity in cancer patients, particularly breast and prostate cancer patients.
Also, it will explain factors that should be considered when using this assay in RT patients.
Methods: We used the “radiosensitivity”, “fluorescence in situ hybridization”, “FISH”, and
“cancer” as medical subject headings. Non-English articles were excluded. Only articles written in English with a full-text PDF format could be found and using three-color FISH for individual radiosensitivity prediction in radiotherapy patients as subjects were included in this review.
Results: In total, 1,905 articles were retrieved from PubMed and PubMed Central databases from 1990 to 2020. The articles were screened, and those that met the inclusion criteria and did not meet the exclusion criteria were reviewed. Finally, we evaluated eight articles in this review.
Conclusions: Appropriate assays such as three-color FISH for individual radiosensitivity assessment could optimize the effectiveness of RT treatment and predict the severity of normal tissue toxicity reactions in RT patients.
then defined based on in vitro chromosomal sensitivity to radiation exposure, expressed as the number of B/M normalized to B/M in an irradiated blood sample (Figure 3). The radiation dose used in this protocol is 2 Gy since this value represents the single fraction dose used in RT treatment. Several studies using three-color FISH have been performed to evaluate the radiosensitivity of cancer patients undergoing RT and its association with the severity of normal tissue reactions. This article reviews how three-color FISH is used to identify the individual radiosensitivity in cancer patients who received the RT treatment, particularly breast and prostate cancer patients, and some aspects that need to be considered when using this assay in RT patients.
doses of exposure. The third is radio-degeneration, defined as non-cancer effects induced by radiation exposure such as cataracts or circulatory disease. Britel et al. [7], in their publication, reinforced the separate use of radiosensitivity, radio-susceptibility, and radio- degeneration terminologies in radiation biology.
Interestingly, Wojcik et al. [8] disagree with the separation use of radiosensitivity, radio-susceptibility, and radio- degeneration terminologies proposed by Foray et al. [6]
and Britel et al. [7]. They stated that it might be premature to make clear distinctions among phenomena described on these three terminologies.
Despite the contradictory opinions on the radiosensitivity terminology, a clinical experience reveals that the severity of normal tissue reaction could limit the RT dose to the tumor [9]. Since the degree of severity depends on the individual radiosensitivity, testing to find out the radiosensitivity of RT patients could help much in personalizing RT treatment. In this case, the RT patients are identified as being highly radiosensitive. Reduction of total RT doses or even replacing the RT treatment with surgery and/or chemotherapy might be an option to avoid adverse effects in RT patients. In contrast, for RT patients with low sensitivity to radiation, the escalation of RT doses could be applied to enhance the effectiveness of RT treatment [5]. The term individual radiosensitivity is highly related to RT patients [10].
Nowadays, several assays for radiosensitivity quantification are available. The clonogenic and cytogenetics assays are the oldest methods that are accessible for individual radiosensitivity quantification.
One cytogenetic assay used to evaluate the individual radiosensitivity is Fluorescence In Situ Hybridization (FISH). FISH is a molecular cytogenetic that was developed in the 1980s. The FISH protocol consists of several steps. The first is the denaturation of target DNA and the probe into single strands. The probe is short sequences of single-stranded DNA that match with the sequence of target DNA. The next step is the hybridization of the probe with a target of DNA that commonly incubates overnight. The last step is post- hybridization washes using saline-sodium citrate (SSC) solutions enriched with a non-ionic detergent that could reduce the unspecific signals of the unbound probe.
The analysis of the FISH signals is then performed using an epifluorescence microscope equipped with a set of filters (Figure 1) [11,12].
In 2009, Distel et al. developed a protocol of individual radiosensitivity evaluation using three-color FISH [13]. In brief, the individual radiosensitivity is defined based on the breakpoints per metaphase (B/M) value. The B/M value is calculated using specific rules.
First, translocations, dicentrics, and ring chromosomes are scored as two breaks. Second, the open breaks and acentric fragments with one color only are scored as one break (Figure 2). The individual radiosensitivity is
Figure 1. The basic sequential steps of FISH [11].
Figure 2. Representative images of translocation (tr) (a), dicentric chromosomes (dc) (b and c), ring (rc) (d), open break (br) (e), and acentric fragment (af) (f).
Figure 4. The flowchart for inclusion and exclusion
IdentificationScreeningEligibilityIncluded
Record retrieved from Pubmed using keywords:
1. “radiosensitivity”, “fluorescence in situ hybridization”, “cancer” (n = 16) 2. “radiosensitivity”, “FISH”, “cancer”
(n = 18)
Record retrieved from PMC using keywords:
“radiosensitivity”,
“fluorescence in situ hybridization”,
“cancer”
(n = 1871)
Record screened (n = 20)
Record excluded (n = 12) Full-text article assessed
for eligibility (n= 8)
Article included in review (n= 8)
Figure 3. The outline of individual radiosensitivity evaluation using three-color fluorescence in situ hybridization (FISH) was proposed by Distel and colleagues [13].
Database MeSH Results
Pubmed ((radiosensitivity[Title/Abstract]) AND fluorescence in situ hybridization[Title/
Abstract])) AND (cancer[Title/Abstract]) 16 articles
Pubmed ((radiosensitivity[Title/Abstract]) AND (FISH[Title/Abstract])) AND (cancer[Title/
Abstract]) 18 articles
PMC (((radiosensitivity) AND fluorescence in situ hybridization) AND cancer) 1871 articles Table 1.
Search strategy
Therefore, we reviewed all studies that used three-color FISH to assess the individual radiosensitivity in RT patients and how this assay could be used to predict the normal tissue radiotoxicity in RT patients.
Three-color FISH for evaluating individual radiosensitivity in breast cancer patients
The first study that attempts to use three-color FISH to evaluate the individual radiosensitivity in cancer patients among all articles included for review is the study performed by Dunst and colleagues in 1995 [20].
Unfortunately, the article was written in German. Thus, we only retrieved the information from the abstract of the article. In this study, Dunst et al. assessed the individual radiosensitivity of 16 cancer patients, including 14 breast cancer patients that had undergone definitive or postoperative curative radiotherapy for cancer (examined at 1 to 108 months upon receiving a completed radiotherapy treatment). The three-color FISH on chromosomes 1, 2, and 4 were used in this study.
As a result, four patients had an increased individual radiosensitivity since they showed poor tolerance to radiotherapy treatment. One of the patients with increased clinical radiation sensitivity was a breast cancer patient with an acute skin reaction above average with subsequent fibrosis after irradiation. The 0.7 and 2 Gy doses were used as in-vitro irradiation doses. The frequency of radiation-induced breaks/1,000 mitoses in the four patients with increased individual radiosensitivity showed a statistically higher compared to the 12 patients with normal radiation tolerance.
Later in 1997, the same investigators assessed 48 blood samples from cancer patients treated with radiotherapy, and 28 patients out of 48 were breast cancer patients [15]. Twelve cancer patients were categorized as the “high or extreme responders”. Among the “high responder” patients, one was the breast cancer patient with a severe skin reaction. The three-color staining on chromosomes 1, 2, and 4 and two different dose points (0.7 and 2 Gy) was used again in this study.
However, in this study, the investigators aimed to find out in more detail about the impact of complex chromosomal rearrangements (CCR) on the individual radiosensitivity prediction, particularly in cancer patients.
Interestingly, the investigators found that the total number of B/M in “high responder” patients was statistically higher than that in patients with average clinical reaction. Moreover, Dunst et al. [22] evaluated 52 cancer patients, including 41 breast cancer patients.
Interestingly, two breast cancer patients were identified to have severe radiation reactions and categorized as radiosensitive groups. Like their previous studies, Dunst et al. used three-color FISH on chromosomes 1, 2, and 4 in this study that approximately accounted for 22%
of the human genome. Two different dose points (0.7
METHODS
The authors used several medical subject headings (MeSH) as follows “radiosensitivity”, “fluorescence in situ hybridization”, “FISH”, and “cancer” in PubMed and PubMed Central (PMC) databases (Table 1). All articles from 1990 to 2020 were included. Manual curation was then performed to evaluate all the articles retrieved using several MeSH combinations. The articles were discarded if they had uncorrelated titles, and all sections of the abstract were quickly skimmed to find out the possibility to be included in this review. Non-English articles were then excluded except for complete information that could be extracted from the abstract.
Full-text PDF format could not be found, and articles not using three-color FISH for individual radiosensitivity prediction or not using cancer patients as subjects in the study were also excluded.
RESULTS
In total, 16 and 18 articles were extracted from the Pubmed database using two different keyword combinations. From the PMC database, a total of 1,871 articles were retrieved. The manual curation resulted in 20 articles that could be included in this review. The repeated articles were then excluded, and finally, only eight articles were included for review (Figure 4) and carefully evaluated.
DISCUSSION
It is well known that normal tissue radiotoxicity in RT patients can occur in weeks, months, or even years after radiation exposure. This phenomenon is considered as the dose-limiting factor in RT treatment, especially when toxicity compromises the patient’s quality of life [5,22,23]. The RT treatment is aimed to diminish the tumor with a minimal impact on the quality of life of cancer patients. Some biomarker tests are available nowadays to evaluate the individual radiosensitivity of RT patients and predict which individuals are at risk of normal tissue radiotoxicity events. The individual radiosensitivity prediction is an important step in the personalization of RT treatment and also for the protection of those that are occupationally exposed to radiation [24–26]. However, no consensus existed for their usefulness, and an independent validation might be essential to perform [27]. Until now, an extensive study for predicting normal tissue radiotoxicity based on individual radiosensitivity of RT patients has never been performed in our country. Thus, the utilization of the cytogenetic approach in evaluating the individual radiosensitivity in RT patients, particularly using three- color FISH, might be interesting to perform in the future.
and 2 Gy) were used in this study. The mean of B/M in all doses of the radiosensitive group that experienced Grade 3 and 4 radiation reactions was statistically higher than that of patients without tissue radiation reaction or patients with mild to moderate radiation reaction (Grade 1–2).
In 2006, Distel et al. [23] assessed 222 individuals (31 healthy subjects, 159 cancer patients) with Ataxia Telangiectasia Mutated (ATM) and Nijmegen breakage syndrome (NBS1) heterozygotes. Also, there were subjects with ATM and NBS1 homozygotes subjects.
Cancer patients in this study comprised of non-exposed patients meant that the blood samples from this group were collected before radiotherapy and from pre- exposed patients that donated their blood samples during the regular post-treatment follow-up visits. Of the total of 159 cancer patients, 119 suffered from breast cancer. In this study, to identify the sensitive individual, the authors used a B/M cut-off point of more than three standard deviations (3 SDs) from the mean of B/M in the control group. Using this method, they found that 32% of non-exposed cancer patients and 44% of pre-exposed cancer patients were categorized as sensitive patients. Since most non-exposed and pre- exposed cancer patients were breast cancer patients, it is most likely that the sensitive patients were breast cancer patients.
Huber et al. [24] in 2011 evaluated the chromosomal radiosensitivity of 47 breast cancer patients who received radiotherapy treatment exclusively after surgical lumpectomy without any additional chemotherapeutic treatment using FISH staining on chromosomes 1, 4, and 12. Interestingly, the authors found that the translocation levels could be used as a cytogenetic marker for individual radiosensitivity evaluation in breast cancer patients. The Protocol for Aberration Identification and Nomenclature Terminology (PAINT) nomenclature system was used to score the staining patterns. In the PAINT system, each color is designated by a letter starting alphabetically with the counterstain. The centric chromosomes were labeled with capital letters, and acentric chromosomes were labeled with non-capital letters [25].
In their study, the chromosome pairs 1, 4, and 12 appeared in green color (FITC) while the other chromosomes appeared in red since they used the Propidium Iodide (PI) as counterstain. The centromeres were stained in blue (AMCA). Chromosomal translocations defined as stained chromosomes with one centromere and two different colors visible simultaneously in the chromosomal arm were categorized as t(Ab) and t(Ba) using the PAINT classification system (Figure 5). Huber et al. [24] found that 4 out of 47 breast cancer patients exhibited a statistically higher translocation level in the
in vitro irradiated lymphocytes with 3 Gy exposure.
Among these four patients, three patients showed a severe side reaction of radiotherapy and a premature side reaction after exposure to 10 Gy of irradiation.
Based on this finding, the authors stated that a correlation between cellular radiosensitivity measured as chromosome aberration yield in peripheral lymphocytes and acute clinical side reactions existed.
They also assumed that translocation level is suitable to identify cancer patients having a short response time to radiation-induced skin reactions.
Three-color FISH for evaluating individual radiosensitivity in prostate cancer patients
Studies from Neubauer et al. [21], Dunst et al. [22], and Distel et al. [23] also assessed the individual radiosensitivity in prostate cancer patients. As already explained previously, these authors used three-color FISH to evaluate the individual radiosensitivity in prostate cancer patients. One prostate cancer patient in Neubauer et al. [21] and Dunst et al. [22] studies were categorized as a radio-sensitive group and had a higher B/M value compared to those with mild or moderate normal tissue reactions. There are two studies from Schmitz et al.
[26] and Beaton et al. [27] that specifically evaluated prostate cancer patients. In Schmitz et al. [26] study, ten prostate cancer patients with severe clinical side effects after radiotherapy (PS) and ten patients without side effects (P0) along together with 11 male age- matched healthy donors (HS) were evaluated. Three- color FISH was applied to chromosomes 2, 11, and 17 using commercial directly labeled whole chromosome probes. The chromosome aberration scoring method used in this study was the Savage and Simpson (S&S) system. Chromosomal aberrations were classified as
Figure 5. The schematic image of translocation scoring in Huber et al. [24]
dicentrics, translocations, centric rings, and acentric fragments. Interestingly, the total aberration yield showed no statistically significant difference among any donor groups (PS, P0, and HS). The authors stated that the in vitro radiation dose more than 2 Gy (3‒4 Gy) used in their study might be more suitable to reveal differences in chromosomal radiosensitivity among prostate cancer patients [19].
In contrast to Schmitz and colleagues’ findings, Beaton et al. [27] in 2013 examined 10 prostate cancer patients exhibiting grade 3 late proctitis (defined as a radiosensitive group) and 10 grade 0 patients having a significantly higher rate of chromosome damage using three-color FISH in the radiosensitive group of prostate cancer patients [20]. In detail, Beaton et al. used the three-color FISH for chromosomes 1, 2, and 4. The PAINT system was used for chromosome aberration scoring.
The authors used 0 and 4 Gy in vitro irradiation doses in this study. They found significant differences in the frequencies of color junctions per cell, deletions per cell, dicentrics per cell, acentric per cell, damage in chromosome 1 per cell, and chromosome 2 per cell between two cohorts of prostate cancer patients.
A longitudinal study by Fahrig in 2018 examined patients who experienced severe side effects after additive RT for prostate cancer and died from the complications 25 months after RT (21). In this study, the authors tested the three-color FISH in lymphocytes at 13 months after the patient was treated with RT.
Also, the three-color FISH was applied to fibroblast derived from skin biopsy at 15 months after the patient was treated with RT. Like Beaton et al. [27] study, FISH staining was applied to chromosomes 1, 2, and 4. The 0 and 2 Gy doses were used as in-vitro irradiation doses.
The finding of three-color FISH revealed that the B/M value in the patient increased even compared to the B/M value of RT-treated cancer patients (0.98 vs. 0.50) found in Distel et al. [13] study. A similar finding is also shown in three-color FISH applied to fibroblast.
The B/M value resulting from 2 Gy irradiation was 0.91.
In the end, based on their case reports, the authors concluded that the identification of cancer patients with increased radiosensitivity is important in the view of radiation protection for the patients.
Three-color FISH for evaluating individual radiosensitivity in other cancer patients
Four out of 8 articles by Dunst et al. [20], Neubauer et al. [21], Dunst et al. [22], and Distel et al. [23]
assessed breast and prostate, bladder, head and neck, lung, rectal, bladder, M. Hodgkin, oropharyngeal, and bronchial cancer patients. Overall, all the authors of these studies showed that three-color FISH is correlated with high clinical reactions and might predict individual radiosensitivity in other cancer types.
Technical consideration of evaluating the individual radiosensitivity using three-color FISH (Number of metaphases to be analyzed and suitable in vitro irradiation dose)
Regarding the required minimum number of metaphases that must be analyzed to obtain a reliable measurement of B/M value, Keller et al. [29] in 2004 published an article with a detailed explanation on this matter. Since cancer patients often have lymphocytopenia that reduces the stimulating effect of phytohemagglutinin (PHA) on lymphocytes, it is challenging to obtain enough evaluable metaphases. Based on their investigation, Keller et al. [29] suggested that to provide reliable results using the three-color FISH, scoring 500 metaphases is justifiable for calculating the spontaneous aberrations (B/M). The evaluation of a minimum of 150 metaphases is also recommended when counting aberrations (B/M) after a dose of 2 Gy. In the viewpoint of radiation dose to be used in predicting radiosensitivity using three-color FISH, a dose equivalent to fractionated radiotherapy such as 2 Gy is the most used in several studies. In 2004, Keller et al. [29] found that analyzing breaks per mitosis (B/M), translocations, and complex aberrations levels could give the best distinction between healthy individuals and hypersensitive patients [30].
CONCLUSIONS
The normal tissue tolerance surrounding the tumor area of RT patients proves that variability of normal tissue reaction in RT patients is existing. The variability of normal tissue reaction in RT patients is considerably due to differences in individual radiosensitivity of cancer patients. Among many techniques available for individual radiosensitivity assessment, the three-color FISH offers a promising result. Several previous studies successfully identified a high responder group in the RT patient cohort by this assay. Overall, three-color FISH could optimize the effectiveness of RT treatment and predict the severity of normal tissue toxicity reactions in RT patients.
DECLARATIONS Competing interest
The authors declare no competing interest in this study
Acknowledgment
The authors gratefully acknowledge the National Nuclear Energy Agency of Indonesia (Badan Tenaga Nuklir Nasional) for providing administrative support in this work. The first author also acknowledges the SAINTEK scholarship program of the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia.
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