We showed here that local irradiation of patients during the course of head and neck cancer radiotherapy resulted in gross changes in the low-molecular-weight component of the serum proteome after the end of RT. The relatively smaller changes in the serum proteome that were detected during the first phase of RT could be explained by a lag period and/or certain dose threshold-requirements of radiation-induced processes. This observation was coherent with the results of a study performed on a smaller group of patients treated due to larynx cancer. In the present study we focused on dose effects and aimed to assess the importance of clinically low and intermediate doses of radiation delivered to large fractions of normal tissues. Two previous studies concerning the impact of local cancer irradiation on the serum proteome, which revealed the importance of radiation doses, focused on maximum doses delivered to tumor volume and based their conclusions on rather heterogenous clinical material[11, 12]. Here, we analyzed homogenous material obtained from a relatively large group of patients where no cancer surgery or chemotherapy was applied. Hence, the heterogeneous factors that could interfere with the obtained results were excluded. We observed that large overall extent of massive changes induced upon the irradiation apparently impaired ability to detect more refined quantitative correlations with radiation doses. Nevertheless, association of specific changes in abundance of serum components with doses and volumes of irradiated tissue was found. Although correlations identified between specific proteome features and different parameters reflecting radiation doses possess moderate statistical power when analyzed separately, reliable conclusions could be drawn based on the general patterns of observed association. We noted that effects of low-to-medium doses delivered to large volumes of normal tissue were apparently more frequent than effects of high doses delivered to target volume of tumor, and concluded that effects of such low-to-medium doses could be observed at the level of whole body response to radiotherapy.
We found that massive changes of serum proteome profiles observed during and soon after the end of radiotherapy, i.e. “early” effects of irradiation, were somehow reversed/compensated by “late” changes, observed at longer times of the follow-up. This apparently reflected process of return to the initial steady-state level (e.g. accumulation and subsequent healing of radiation damage). However, “late” radiation-induced changes observed during the follow-up were relatively weaker and not always fully reversed the stronger changes induced during the treatment. As a consequence, radiation-related changes could be observed months after the end of radiotherapy and abundances of several serum components were significantly different when samples collected more than one year after the treatment, were compared to samples collected before the radiotherapy. Similar observation was made in a group of breast cancer patients where changes in serum proteome profiles related to the adjuvant radio/chemotherapy were detected one year after the treatment. Such results clearly indicated that toxic effects of anti-cancer therapy are long-lasting and could be detected as specific features of serum proteome many months after the treatment. Importantly, this finding indicates the possible applicability of MS-based analyses of serum proteome profiles in retrospective bio-dosimetry of radiation exposure. On the other hand, one should note that serum proteome components involved in a dose/volume-related “early” radiation response of the organism did not show significant association with long-term efficacy of the treatment (i.e. tumor eradication vs. re-growth) when a group of patients with similar prognosis treated with unified radiotherapy protocol was analyzed.
Model presented in current study is extremely complex and could be affected by many different processes ongoing in the patient’s organism. However, we assumed that accumulation and subsequent healing of radiation-induced damage (acute reactions, mucositis, hematological reactions, etc.) would have the major influence on general therapy-related changes observed at the level of serum proteome. In fact, we found an association of serum proteome features with the intensity of radiation-induced acute mucosal reaction, which was in accordance with the apparent correlation between the observed radiation toxicity and the doses of radiation delivered to normal tissues. We found the strongest correlation between the mucosal reaction and volume irradiated at 30-40 Gy (i.e., at 0.7-1.0 Gy dose fractions). Of note, changes of serum proteome profiles observed two weeks after the start of RT, which putatively accompanied escalation of mucosal reaction, also showed the most frequent correlations with volume of tissue irradiated at similar “intermediate” dose fractions (0.7-1.0 Gy). Several peptide ions registered in the low-molecular-weight fraction of serum proteome that changed their abundance in samples from irradiated patients were hypothetically annotated in the proteomic knowledge base EPO-KB as fragments of proteins related to the inflammation, immunity and defense. Among such proteins, there were acute phase proteins (fibrinogen, haptoglobin, hepcidin, SAA), complement factors (CO3, CO4A), protease inhibitors (AACT, ANT3, CYTC, ITIH4), cytokines (CCL13, CXCL7, OSTP, PLF4, S10AC) and antibacterial defensins (DEF1, DEF3, DEFB1) (see Additional file1: Table S1 and Table S7 for details). These processes are potentially involved in response to radiation, and several proteins related to acute phase and inflammation were previously reported in serum samples collected after radiotherapy of cancer patients. Hence, we postulate that radiation-induced features of the serum proteome, detected in post-treatment samples, correspond to the direct toxic effects of radiation and/or healing of the acute reaction.
Due to dosimetrical superiority of IMRT plans this treatment modality constantly replaces the more “traditional” radiotherapy techniques. Here, we observed that irradiation of large volumes of normal tissue with doses of radiation considered as “therapeutically irrelevant”, which is typical for IMRT, may have some biological effects. Their potential importance for output of the treatment is not as obvious, because exposure of increased volume of normal tissues could hypothetically be either detrimental (e.g. inflammation reactions related to radiation toxicity) or beneficial (e.g. radiation-induced stimulation of immune reactions). Hence, their clinical implications need to be carefully considered when complete data of long-term effects concerning the IMRT implemented in large cohorts of patients are available for analyses.