Radiotherapy and chemoradiation remain the major treatment modalities of head and neck cancer either as a primary treatment or in postoperative adjuvant setting.
Despite the clear therapeutic benefits, acute and late side effects of radiation and chemoradiation still limit the quality of life of patients. Furthermore, there is significant heterogeneity in response to radiation, both in tumours and healthy tissues. It would therefore be of great value for the clinicians to predict the response not only in the primary tumour volume, but also in the surrounding normal tissues.
Radiation damage in any tissue occurs primarily through single or double DNA strand break (DSB), which lead to cell death due to chromosomal aberrations and apoptosis if not repaired. Phosphorylated histone H2AX (called γ-H2AX) is an indirect protein marker of DSB. It has been shown that DSB in cell nucleus triggers H2AX phosphorylation in position Ser139 (
In this paper, we suggest that assessment of γ-H2AX levels after irradiation could serve as a marker of sensitivity to radiation damage in normal tissue, enabling the estimation of the individual damage repair potential. Peripheral blood lymphocytes (PBL) could serve as effective and easily accessible surrogates of normal tissue. Previous studies have focused on lymphocytes irradiated
The main objective of the study was to examine the clinical usefulness of assessing radiation effects based on the γ-H2AX evaluation in the PBL. The secondary goal was to assess the changes of γ-H2AX expression in PBL according to the severity of radiation-induced mucositis (M) during radiotherapy or chemoradiation. Mucositis is evaluated in clinic using many scales, most widely used are Common Terminology Criteria for Adverse Events v.4 (CTCAE) and European Organisation for Research and Treatment of Cancer/Radiation Therapy Oncology Group (EORTC/RTOG) scales.6,7
Table 1 presents grading of toxicity, using oral mucositis as an example, with two most commonly used scales.
Grading of radiation-induced oral mucositis according to CTCAE v4 and EORTC/RTOG scales.6,7
Mucositis (oral) | Grade 1 | Grade 2 | Grade 3 | Grade 4 | Grade 5 |
---|---|---|---|---|---|
Asymptomatic/mild symptoms, intervention not indicated | Moderate pain not interfering with oral intake, modified diet indicated | Severe pain interfering with oral intake | Life-threating consequences, urgent intervention indicated | Death | |
Irritation, may experience slight pain, not requiring analgesic | Patchy mucositis that may produce inflammatory serosanquinitis discharge, may experience moderate pain requiring analgesia | Confluent, fibrinous mucositis, may include severe pain requiring narcotic | Ulceration, haemorrhage or necrosis | N/A |
In this prospective study, we included patients with head and neck cancers treated with postoperative or definitive radiotherapy or chemoradiation. Patients treated with palliative intent were excluded from the study. The study was approved by the Ethics Committee of University of Medical Sciences in Poznan (No723/14) and written informed consent was provided by all the participants. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Patients were treated with adjuvant radiotherapy or chemoradiation to 60-66 Gy, if the known features of risk of relapse were present in pathology report.
Patients with inoperable tumors were treated mostly with concomitant chemoradiation to dose 70 Gy (fractions of 2 Gy once daily, 5 times a week) concurrently with cisplatin 40 mg/m2 once a week or 100 mg/m2 every three weeks of treatment, on days 1, 21, and 42 of radiotherapy (fraction 1, 16, 31). In every patient mucositis was evaluated once a week, independently, by radiation oncologist and head and neck surgeon, using the Common Terminology Criteria for Adverse Events version 4 (CTCAE v.4) and the Radiation Therapy Oncology Group (RTOG) scales.6,7 For the purpose of this study, grading results based on the CTCAE scale were employed because it is more commonly used in clinic. The maximum score observed during treatment was used for correlation with γ-H2AX level.
The level of γ-H2AX was analyzed in PBL. The first blood sample was collected before treatment, to measure basic level of γ-H2AX in every patient. The second sample was taken 45 minutes after the first fraction and then once a week, 45 minutes after radiotherapy fraction. The sampling time was selected according to observation from a pilot study, showing that the level of γ-H2AX in PBL was highest 45 min after irradiation. In patients treated with concurrent chemotherapy, the blood sample was taken the day after chemotherapy. The collected blood samples were stabilized by Transfix (Cytomark, UK) administration according to manufacturer protocol. H2AX phosphorylation levels in the leucocytes were examined by flow cytometry according to manufacturer protocol (Apoptosis, DNA Damage, and Cell Proliferation Kit, BD, USA). Briefly, isolated leucocytes (centrifuging 1200g at 20°C for 20 min) were fixed with BD Cytofix/ Cytoperm Fixation/Permeabilization Solution for 30 min at room temperature. Subsequently, cells were incubated with BD Cytofix/Cytoperm Plus Permeabilization Buffer for 10 min at 4⍛C, and re-fixed, with BD Cytofix/Cytoperm Fixation/Permeabilization Solution (5 min, at room temperature). Next, the cells were incubated with Alexa Fluor 647 Mouse Anti-H2AX antibody (pSer139). Staining with isotype control (APC Mouse IgG2b κ Isotype Control; Becton Dickinson, USA) was performed to assess the threshold of positive staining. Data acquisition was performed using a BD Accuri C6 Plus flow cytometer (BD Biosciences, USA), and analyzed using FlowJo software (FlowJo, LLC, USA).
Statistical analysis was performed using STATISTICA (StatSoft, Inc. USA version 12, 2014). The differences between groups were assessed with the use of Chi-square test (Table 2). Two-sided Welch’s t-test, and two-way ANOVA were applied to assess the statistical significance of differences between γ-H2AX levels. The result were evaluated at α = 0.05 significance level.
Demographics, staging and treatment information of studied patients
Characteristics | Total | M 0-2 | M 3 | ||
---|---|---|---|---|---|
N = 50 (%) | N = 35 (%) | N = 15 (%) | |||
Male | 44 (88) | 30 (85.7) | 14 (93.3) | 0.44 | |
Female | 6 (12) | 5 (14.2) | 1 (6.6) | ||
Parotid | 5 (10) | 5 (14.3) | 0 (0) | ||
Oral cavity | 11 (22) | 7 (20) | 4 (26.7) | ||
Larynx | 14 (28) | 10 (28.6) | 4 (26.7) | ||
Hypopharynx | 4 (8) | 4 (11.4) | 0 (0) | 0.27 | |
Oropharynx | 12 (24) | 6 (17.1) | 6 (40) | ||
CUP | 2 (4) | 1 (2.8) | 1 (6.7) | ||
Nasalcavity/paranasal sinuses | 2 (4) | 2 (5.7) | 0 (0) | ||
T1 | 4 (8) | 2 (5.7) | 2 (13.3) | ||
T2 | 17 (34) | 11 (31.4) | 6 (40) | ||
T3 | 8 (16) | 7 (20) | 1 (6.7) | 0.49 | |
T4 | 19 (38) | 14 (40) | 5 (33.3) | ||
Tx | 2 (4) | 1 (2.8) | 1 (6.7) | ||
N0 | 26 (52) | 19 (54.3) | 7 (46.7) | ||
N1 | 4 (8) | 4 (11.4) | 0 (0) | 0.25 | |
N2 | 20 (40) | 12 (34.3) | 8 (53.3) | ||
Yes | 28 (56) | 22 (62.85) | 6 (40) | 0.13 | |
No | 22 (44) | 13 (37.1) | 9 (60) | ||
Yes | 33 (66) | 26 (74.3) | 7 (46.7) | 0.06 | |
No | 17 (34) | 9 (25.7) | 8 (53.3) | ||
Yes | 34 (68) | 21 (60) | 13 (86.7) | 0.06 | |
No | 16 (32) | 14 (40) | 2 (13.3) | ||
Diabetes type II | 6 (12) | 5 (14.3) | 1 (6.7) | ||
Hypertension | 17 (34) | 12 (34.3) | 5 (33.3) | 0.09 | |
Ischemic heart disease | 11 (22) | 4 (11.1) | 7 (46.7) |
CUP = cancer unknown primary; M = mucositis; grade 0-2 or grade 3 (of mucositis).
No grade 4 mucositis was observed. The statistical differences between groups were assessed with the use of Chi-square test.
Fifty patients with head and neck cancers treated with adjuvant or primary radiotherapy or chemoradiation were included in the study. Mild (grade 1) to moderate (grade 2) mucositis was observed in 35 patients and severe (grade 3) in 15. No cases grade 4 mucositis were observed. No patient was lost from follow up. Median follow-up was 51 months (range, 2–60 months), median overall survival (OS) for whole group reached 51 months. For patients with mild and severe mucositis median OS was 50.5 and 55 months respectively. We did not observe any unexpectedly severe acute reaction
The difference in basic level of γ-H2AX between the groups with mild and severe mucositis was statistically insignificant (p = 0.25) (Figure 1A). However, we observed a significantly lower level of H2AX phosphorylation in lymphocytes in week 7 (p = 0.011) in the group with severe mucositis in comparison to the mild one (Figure 1B).
Interestingly, we noticed an opposite statistical trend in week 2 (p = 0.065) (Figure 1C). Analyzing the kinetics of γ-H2AX during treatment, we observed a statistically significant difference (p = 0.0088) between groups with mild and severe mucositis (Figure 2A, Table 3, Table S1 in Supplementary material). Between treatment weeks 4 and 7, levels of γ-H2AX decreased significantly in the group with severe mucositis and increased in patients with mild side effects. The observed difference is not caused by the expected decreasing of peripheral lymphocytes level during radiotherapy which was similar in both groups (Figure 2B).
Level of γ-H2AX in peripheral blood lymphocytes before and during treatment
Week of radiotherapy | Mild mucositis | Severe mucositis | No. of samples | |||
---|---|---|---|---|---|---|
Mean | SD | Mean | SD | |||
Before | 10.64 | 16.38 | 11.40 | 11.71 | 48 | 0.868 |
Week 1 | 8.48 | 8.99 | 11.24 | 10.78 | 48 | 0.347 |
Week 2 | 7.22 | 9.86 | 13.27 | 11.94 | 48 | 0.065 |
Week 3 | 9.45 | 11.46 | 12.44 | 12.16 | 48 | 0.403 |
Week 4 | 10.81 | 14.10 | 11.87 | 15.46 | 48 | 0.812 |
Week 5 | 14.95 | 19.88 | 11.97 | 19.60 | 47 | 0.624 |
Week 6 | 18.25 | 20.96 | 8.90 | 15.62 | 47 | 0.121 |
Week 7 | 20.50 | 23.13 | 3.81 | 4.78 | 39 | 0.011* |
Week 8 | 13.23 | 10.82 | 5.46 | 8.83 | 8 | 0.268 |
SD = standard deviation
Further analysis showed no statistical differences in γ-H2AX levels neither between the groups treated with concomitant treatment and radiotherapy alone nor between groups with and without common comorbidities like hypertension or diabetes, and habits like smoking. Furthermore, we did not notice correlation between γ-H2AX levels and survival, staging or tumor primary site.
The aim of this study was to test the usefulness of γ-H2AX as a clinically feasible predictor of severity of acute side effects caused by radiotherapy
γ-H2AX is a marker of DNA damage and kinetics of repair. Thus, residual γ-H2AX can be valuable for evaluation of radiosensitivity and accumulation of unrepaired DNA damage. Due to difficulties in obtaining mucosa or skin cells during radiotherapy for
To assess γ-H2AX levels, we employed flow cytometry. Although sensitivity of flow cytometry has been questioned by Beaton
The only significant difference in γ-H2AX levels between patients with mild and severe mucositis was found in the seventh week of radiotherapy. Our results are similar to findings of Vasireddy
Other published reports analysing H2AX phosphorylation during acute phase of radiotherapy side effects are conflicting. Fleckenstein
Many publications assess γ-H2AX formation and its 24-hours kinetics after one dose of irradiation
We also did not find significant differences in baseline γ-H2AX levels between the individual patients before start of radiotherapy or chemoradiation. The mean baseline level of γ-H2AX was higher for patients with severe mucositis but observed difference was not statistically significant.
An interesting finding from our study is a rapid decrease in γ-H2AX levels after fourth week of therapy in group with severe mucositis in contrast to mild mucositis group, where an increase was observed. This phenomenon can be explained by ineffective DNA damage repair system (DDR) responsible for recognition and repair of DSB in most sensitive patients and changes in the conformation of chromatin after irradiation. Such changes can affect DSB signaling after the next fraction, resulting in lower phosphorylation of H2AX or lower detection of phosphorylated histones.15 However, this phenomenon has been observed when the next fraction was applied 5–6 hours following the first. So far little is known about repair processes and γ-H2AX formation when the time between fractions is longer than 12 hours. Bouquet
Finally, we did not find γ-H2AX levels to be predictive for severe risk of mucositis before or early during treatment. Kinetics of γ-H2AX during treatment could potentially allow for such discrimination by evaluation of changes in subsequent γ-H2AX levels after 4 weeks of therapy. To our knowledge, this is the only study evaluating the kinetics of γ-H2AX during 7-week treatment.
In our study, we also considered selected, patient-related factors with potential impact on severity of mucositis like comorbidities, age and staging. Smoking was not included in the analysis as it was very challenging to obtain reliable information about smoking during treatment. We did not find any correlation between γ-H2AX levels and studied factors, as well as cisplatin use or radiotherapy setting. Similarly to the Werbrouck
The limitations of current
In this study, we tested the PBL of relatively large group of patients during radiotherapy and chemoradiation. In line with other studies, we were not able to prove that assessment γ-H2AX with flow cytometry is a method ready to be used clinically for identification of patients with high risk for mucositis before or early during treatment.9,11 The probable reason for this is the multifactorial clinical and radiobiological background of acute mucositis, which depends among others on site of disease, total dose, dose per fraction, irradiated volume, comorbidities, concomitant chemoradiation, hydration, nutrition, and repair potential of normal tissue.5 Furthermore, circulating PBLs receive variable doses of radiation and highly damaged cells might be eliminated from circulation, potentially biasing the results.
Based on the results presented, it is not yet possible to predict the severity of mucositis induced by radiation using γ-H2AX. However, preliminary results indicating significant differences in kinetics of γ-H2AX levels between groups after fourth week of treatment encourage further studies.