AND SECONDARY MALIGNANCIES FOLLOWING RADIOTHERAPY

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General regards
Radiotherapy (RT) is an integral part of multidisciplinary cancer management and is indicated by evidence-based guidelines in up to more than 50% of all cancer patients 1 . State of the art RT techniques such as intensity-modulated radiotherapy (IMRT), image-guided radiotherapy and proton therapy, have decreased the risk of cancer recurrences, improved target dose coverage with dose escalation, reduced treatment toxicities and improved survival.
Potentially adverse effects of these treatments can reduce the quality of life and lead to morbidity and even mortality in cancer survivors. Regarding radiotherapy treatment, toxic effects are generally divided into early and late effects.
Early toxic effects of radiation on healthy tissue are reversible and develop due to acute inflammation, whereas late adverse effects mostly remain permanent and are caused by chronic inflammation, microvascular damage, fibrosis, and radiationinduced genetic instability 2 .
Long-term cancer survivors treated with RT treatment are at greater risk of developing late effects, including the development of radiation-induced malignancy (RIM) 2 . In 1948, Cahan et al. defined a radiation-induced sarcoma, while nowadays, in practice, modified Cahan's criteria for the definition of RIM are used 3,4 . These criteria include that RIM must arise within the treatment field, with a significant latent period and have different histology than primary malignancy (moreover, the origin of tissue of RIM must be metabolically and genetically normal before irradiation).
Cancer patients are generally at higher risk for developing a second malignancy when compared to the general population. However, subsequent neoplasms may not be associated with prior cancer treatment, and RIMs make only a small proportion of the second malignancies 5 .

Etiology and contributing factors for developing RIM
The risk for developing RIM after RT treatment varies upon multiple factors, such as i) the patient's age at the time of radiation; ii) genetic susceptibility; iii) patients' 4 family history of cancer; iv) lifestyle and environmental factors; v) the organ and tissue site receiving radiation; vi) RT treatment modality; vii) dosimetric characteristics of the RT plan 2,6 .
Children are considered to be 10 times more sensitive to the carcinogenic effect of radiation than adults. Several studies found that pediatric cancer patients who underwent RT have a greater risk of developing RIM than adults 7,8 . These malignancies may lead to a decrease in the overall survival after the treatment of primary cancer 2,9 . When assessing the risk of developing RIM regarding gender, studies have shown that females are at greater risk compared to males 10 . Unfortunately, the irradiation of the breast tissue during RT treatment in Hodgkin's lymphoma is well known as a risk factor in inducing breast cancer. Previously published studies have reported that menopausal-and ovarian function status in correlation with age in female patients, affect the risk for developing RIM in cancer survivors treated with chest RT for Hodgkin's lymphoma. Namely, early menopause, as well as ovarian disfunction, at a younger age may reduce the risk for breast cancer as a radiationinduced malignancy 11 .
The dose-response relationship for radiation carcinogenesis as well as longterm effects of radiation on the development of RIM in human are explored in Japanese atomic bomb survivors, in whom leukemia was initially diagnosed with a latent period of 5-10 years, and afterward solid tumors with a latent period of 10-60 years (figure 1). The latent period for developing RIM in irradiated patients is reported to be similar to that in Japanese atomic bomb survivors and the risk continues to increase with decades gained after the exposure 7,12 .
Radiotherapy patients receive a high dose of radiation at a low volume and significantly lower doses at larger volumes, however RIMs can arise from the highdose irradiated tissues, as well as from the low-dose irradiated tissues, e.g., organs that are distant to the radiation field 13 .
Cancer patients are often treated with combined treatment modalities, so it may be difficult to define the specific effect of a particular agent. Exposure to chemotherapeutic agents may be associated with an increased risk of secondary malignant neoplasms, such as anthracyclines and alkylating agents with sarcoma 14 , alkylating agents with carcinomas 15 , cisplatin-based therapy with solid tumors after testicular nonseminomas 16 . Treatment-related myeloid neoplasms including therapy-related acute myeloid leukemia and therapy-related myelodisplastic 5 syndrome may be linked with exposure to alkylating agents, as well as topoisomerase (TOP) II inhibitors 17 .

Site of radiation
For the adult population, clinical data on RIM development are best reported for breast and prostate cancer due to the high rate of long-term survival. To assess the incidence of developing RIMs, an appropriate control group should be available, that is often difficult to provide; notable exceptions are prostate and cervical cancer, where patients treated with surgery provide control groups.

Breast cancer
Radiotherapy is an essential adjuvant part of breast cancer treatment, which reduces disease recurrence and improves overall survival. However, RT can also be associated with an increased risk for second cancer in exposed sites.
Radiation-induced sarcoma is a rare complication of breast irradiation with an increased risk of appearance over time after radiotherapy 18,19 . According to Salminen et al. 18 , the most common site of radiation-induced sarcoma was breast soft tissue (figure 2), while the prevalent histological subtype was angiosarcoma.
According to the large meta-analyses conducted by Grantzau and Overgaard 20 , RT treatment of breast cancer has significantly increased the risk of second non-breast cancers with a relative risk (RR) of 1.22. The risk remained elevated even 5 years after diagnosis, with a RR of 1.12. The most common second cancer sites were lung and esophageal cancers and soft tissue sarcoma. The estimated RRs for these sites were 1.23, 1.17, and 2.41, respectively. After a latency time of at least five years from breast cancer diagnosis, the incidence of second cancer gradually increased. A significant association between RT of breast cancer and second thyroid cancer was not found.
Gonsalez et al. 21 estimated the long-term cancer risk of all solid cancers on a large cohort of patients after breast cancer radiotherapy. In the study, second cancer sites were divided into three dose groups (high: 1+Gy; medium: 0.5-0.99Gy; and low: 0.5Gy; dose sites) according to the mean organ dose from the RT treatment.
Estimated RRs were increased for the group of sites that received the highest radiation exposure (1+ Gy: lung, esophagus, pleura, bone, and soft tissue; ~1Gy: contralateral breast cancer), while for lower dose sites RRs were not elevated. They even found that most of the second solid cancers were also related to other risk factors such as lifestyle and genetic factors.
Regarding secondary sarcomas, both studies also showed that RRs were especially highly elevated for angiosarcomas.
In the study by Mladenovic et al. 22 tumor response and long-term outcome were analyzed in 134 patients with non-inflammatory locally advanced breast cancer treated with preoperative radiotherapy. Their results of pathological complete tumor response to preoperative RT were in agreement with similar previously conducted trials. The occurrence of second malignancy was detected as breast cancer in the contralateral breast in two patients, and papillary thyroid cancer in one patient.

Prostate cancer
Radiation-induced malignancies are reported in long-term survivors of prostate cancer. Fontenot et al. 23 estimated that proton therapy reduced the risk of RIM by 26% compared to 39% with contemporary IMRT in prostate cancer patients. When comparing the risk for developing second malignancy treated with radiotherapy versus surgery, Brenner et al. 24 published that RT significantly increased the risk of second malignancies by about 6% (p = 0.02). For patients who survived for ≥ 5 and ≥ 10 years, the increased RR was 15%, and 34%, respectively. The vast majority of second solid cancer sites were bladder, rectum, and lung cancers, as well as sarcomas within the treatment field, while no significant increase in rates of leukemia was noted.

Gynecological malignancies
Chaturvedi et al. 25 reported that irradiated cervical cancer patients were at increased risk of secondary malignancies even after 40 years of follow-up when compared to the general population. The risk of second cancers was increased at sites that are close to the cervix, including anal, colorectal, and genitourinary sites.
Rodriguez et al. 26 analyzed the risk of developing colorectal cancer among long-term survivors of cervical cancer who received RT. Results of the study implied that RIM of the colon and rectum may occur 8 years after RT for cervical cancer.
Furthermore, these patients treated with radiation at a young age should start screening for colorectal cancer earlier than the age recommended for low-risk individuals (approximately 8 years after the treatment).
Post-Operative Radiation Therapy in Endometrial Carcinoma (PORTEC)-1 trial, in which patients were randomly assigned into irradiated (postoperative external beam RT) and observational group, has shown that after 15 years of followup 22% of patients were diagnosed with second primary cancer in RT group, versus 7 16% in observational group. The most common cancer type in irradiated patients was gastrointestinal cancer 27 .

Lymphoma
Many studies have published an increased incidence of secondary malignancies in patients treated with RT for Hodgkin's and non-Hodgkin lymphoma. Factors contributing to the higher incidence of radiation-induced malignancies in Hodgkin's lymphoma patients are relatively young age of patients, high curability, large irradiation field, technique used in past decades, and combined therapeutic modalities, including cytotoxic drugs. The majority of second malignancies were thyroid, breast, lung, and stomach cancer as well as sarcoma 28 . Moreover, RT increases the risk of developing both, solid tumors and leukemia for non-Hodgkin lymphoma survivors 29 .

Pediatric malignancies
With an increasing number of long-term cancer survivors of childhood malignancy, the occurrence of second malignant neoplasms (SMNs) has risen.
Primary cancer treatments, including radiotherapy and chemotherapy, are associated with risk for SMN after primary childhood cancer.
The Childhood Cancer Survivor Study (CCSS) analyzed the incidence and risk factors of subsequent neoplasms after treatment of childhood cancer. It was reported that the cumulative incidence of all subsequent neoplasms was 20.5% thirty years after diagnosis, whereas radiation exposure was associated with an increased risk of second malignancies. Regarding subsequent neoplasm subtype, the 30-year cumulative incidences were 7.9%, 9.1%, and 3.1% for SMNs (excluding nonmelanoma skin cancer), nonmelanoma skin cancer, and for meningiomas,

Inheritance patterns of radiation-induced tumors and toxicities
Radiation sensitivity is not a monogenetic trait but rather a polygenetic trait where the majority of the population is located in the middle of the normal distribution of expression. People with mutations that could be associated with higher sensitivity to radiation, such as mutations concerning DNA repair mechanisms, might have a higher risk of developing early or late toxicities, and a possibility for deceleration of the treatment dose can be consider 32 .
Nowadays the carcinogenic effect of radiation is well documented. Genetic susceptibility plays an important role in the pathogenesis of radiation-induced malignancies. Radiation can cause DNA damage to normal cells, which might lead to genomic instability and finally, but rarely to RIM. A recent study from University of Utah School of Medicine in 2017th showed that 13% of patients who received radiotherapy for breast cancer developed a secondary malignancy with a median follow-up of 8.9 years, and it was estimated that only 3.4% of secondary malignancies were attributable to radiation therapy 33 .
Although radiation sensitivity is largely explained as a polygenic trait The most commonly studied heterozygous mutations are ATM and BRCA heterozygotes 34,35,36 . It is well known that homology-directed repair route mediated by products of BRCA genes is activated in only 15% of DNA double-strand brakes caused by therapeutic radiation, so it should be no surprise that the clinical data consistently demonstrate no increased risk in BRCA heterozygous patients treated with standard adjuvant radiation regimens 36 . On the other hand, ATM serine/threonine kinase is directly involved in double-strand brakes repairs, but the clinical data on its importance in developing radiation sensitivity is contradicted. It seems that there is no increased risk for developing radiation toxicities in 9 heterozygote carriers of pathogenic ATM mutations, but in SEER clinical trial in breast cancer patients, an increased risk of developing contralateral breast carcinoma was observed in irradiated group (probably due to scattering radiation) 35,37 .
Pathological ATM mutations are still not a contraindication for radiotherapy treatment. Notably, individuals with genetic syndromes with an increased risk of developing several types of cancer should be monitored for SMNs after RT treatment.
To develop personalized medical approach in RT, with better tumor response and lower radiation toxicity, without dose escalation, many trials explore molecular signature concept of radiosensitivity. It would be beneficial to identify predictive biomarkers of initial response to radiotherapy, that could be helpful to predict clinical outcome in patient treated with RT. Tanic et al. 38 published that MAP3K4 gene could be a potential biomarker of response to RT and a potential target for radiosensitising combination therapy.
In a genome-wide association study, Best et al. 39 identified two variants (rs4946728 and rs1040411 noncoding single nucleotide polymorphisms located between PRDM1 and ATG1 genes) on chromosome 6q21 associated with the risk for SMNs after RT in pediatric Hodgkin's lymphoma survivors. This data indicates the significance of genetic susceptibility to second cancer etiology.

Somatic mutations and their effect on tumor radiobiology
Testing for somatic mutations (mutations present in the tumor itself) is the most frequent scenario when cancer patients come in contact with genetic testing.
Mutations in tumors occur at a higher rate than in normal tissues due to genetic instability, and their genetic information is different from genetic information of the patient. These mutations have implications mainly for tumor response and tumor treatment decisions 32 .
The somatic mutation pattern (genetic signature) could also be of diagnostic importance. Several studies have shown that radiotherapy could have its' molecular signature on the treated area, which can be detected in secondary malignancies that develop later. For example, upregulation of MYC, RET, and FLT4 with downregulation of CDKN2C and PRDM1 genes are frequent in radiation-induced sarcomas and other radiation-induced malignancies 40,41 .
On the other hand, somatic mutations of key genes involved in DNA repair mentioned above can change the radiobiological behavior of the tumor. Usually, pathogenic mutations involved in DNA repairs such as ATM or BRCA 1 and 2 could be expressed at a higher rate and/or be of higher penetrance in the tumor more than in the normal tissue due to loss of heterozygosity. In such a scenario, a full expression of recessive mutations and/or full penetrance of dominant mutations such as ATM and BRCA, respectively, can make the tumor more radiosensitive 42 . On the other hand, somatic mutations in k-RAS can make a tumor more radioresistant 43 .

Type of radiation and its' role in the development of RIM
Nowadays different types of ionizing radiation are used for cancer treatment that can be divided roughly into two groups: photon and particle radiation. High energy LET radiation is more likely to produce cell death and mutation than low LET radiation 47,48 .
Studies that compare the incidence of secondary malignancy in long-term cancer survivors following proton and photon beam therapy are limited, however available data suggest a lower incidence of RIM in patients treated with proton beam therapy when compared to photon therapy 49 . Chung et al. 50 published a comparative analysis of incidence rates of secondary malignancies after radiation for cohorts of proton and photon treated patients. After a median follow-up of 6.7 and 6.0 years in proton and photon treated groups, the rate of second malignancies, was lower among patients treated with proton radiation compared to patients treated with photon RT (5.2% vs. 7.5%, respectively).

Role of radiation technologies in the development of RIM
With the advances in RT, from conventional and three-dimensional conformal radiotherapy (3D-CRT) to IMRT and volumetric-modulated arc therapy, radiation is delivered to the targeted areas more precise with dose escalation and the organs at risk are better spared. As a complex radiation technique, IMRT compared to 3D-CRT is associated with better organ risk management and decreased frequency of acute and chronic treatment toxicities, followed by improved quality of life after treatment 51,52. The study by Hall and Wuu showed that the move from 3D-CRT to IMRT can lead to increase in RIMs 53 . The rationale for this theory is that IMRT requires many fields, irradiating a larger volume of healthy tissue (so-called "lowdose bath"). Also, IMRT requires twofold to a threefold larger number of monitor units to deliver a preset dose compared with 3D-CRT. This larger number of monitor units leads to X-ray leakage and distant tissue irradiation. Considering that IMRT and volumetric-modulated arc therapy are fairly modern techniques and that development of radiation-induced malignancies takes years and even decades, there are few studies that compare risk in RIM development between novel techniques and 3D-CRT. However, we can assess that risk using different models. For example, the concept of organ equivalent dose can be used to calculate the risk for RIM development in different tissues when three-dimensional dose distribution radiotherapy techniques are used 54 .
Image-guided brachytherapy based on MRI, with radioactive source Ir-192, has become a standard treatment in gynecological malignancies, and it provides precise information about radiation dose distribution, target volume coverage, doses delivered to organs at risk while decreasing the toxicity 55 .

Conclusion
As the number of long-term cancer survivors after radiotherapy increases, the RIMs are becoming a relevant clinical problem in a long-term follow-up. Radiationinduced malignancies are important late adverse effects of RT that can directly impact patient management and treatment decision-making. These facts could modify initial work-up, treatment, and follow-up protocols. Inclusion of genetic testing, further investigation of novel RT techniques, additional screening and surveillance strategies should be added to the overall cancer care.