The role of c-MYC expression in the diagnostic and clinical confirmation of radiation-induced angiosarcoma: A case report and a review of literature

Introduction. Angiosarcomas (AS) arising from vascular tissue, accounting for
 3.3% of all sarcomas, have a poor prognosis. Radiation-induced AS is a rare
 late complication of radiotherapy treatment and is characterized by a gene
 expression profile such as amplification of the MYC oncogene, by which we
 can distinguish primary from the secondary induced tumor. Case report, At
 77-year-old patient, with early-stage endometrial adenocarcinoma, the
 radical hysterectomy with bilateral salpingo-oophorectomy was initially
 done. According to pathological risk factors, the postoperative external
 beam conformal radiotherapy (CRT) of the pelvis was administered with
 concomitant brachytherapy. Six years after the treatment, on the anterior
 abdominal wall, in the region of the postoperative irradiation field and
 surgical scar, an infiltrative angiosarcoma of the skin and subcutaneous
 adipose tissue, was histologically confirmed. The patient received six
 cycles of mono-Adriamycin chemotherapy with verified partial regression.
 Additional immunohistochemical analysis (IHH) of c-MYC, Ki67 and CD34
 expression showed a high proliferative index (Ki67 around 60%) and c-MYC
 positivity indicating the molecular pattern of radiation-induced AS.
 Furthermore, the high proliferative index could explain a good response to
 chemotherapy. Conclusion. The novel postoperative radiotherapy techniques
 provide better survival and local control in risk- endometrial cancer groups
 with a decrease of irradiation complications. These patients with longer
 survival, are in a higher risk of developing radiation-induced tumours as
 late side-effects of radiotherapy. When assessing the probability of
 radiation-induced AS, IHH analysis of c-MYC expression could distinguish
 secondary from others AS if Cahan?s criteria are fulfilled.


Introduction
Angiosarcoma (AS) is rarely occurring malignancy that arises from vascular tissue and has a poor prognosis. The incidence of all soft tissue sarcomas in Europe ranges from 3.3 per 100,000 in Eastern Europe to 4.7 per 100,000 in Northern Europe and it is reported that 3.3% of all sarcomas are angiosarcomas 1 . By site AS are divided as follows: soft tissue AS, bone AS and cutaneous AS. Furthermore, cutaneous AS are divided as scalp and face AS, AS in the contest of lymphedema (Stewart-Treves syndrome), epithelioid AS and radiationinduced AS 2 . 5 AS is characterized by diverse but recurrent chromosomal abnormalities and mutations of genes involved in angiogenesis and endothelial cell receptors 3 . The patterns of mutation are so distinct that it could distinguish secondary, mostly radiation-induced AS from primary tumours 4 .
The first set of criteria used for the diagnosis of radiation-induced malignancy (RIM) were established by Cahan et al. in 1948 5 . Today a modified Cahan's criteria are used that encompasses the following: i) Radiation-induced malignancy must arise in the boundaries of the irradiation field; ii) Duration of the latent period between proposed induced malignancy and previous irradiation must be greater than 4 years; iii) Primary malignancy and induced malignancy must be biopsied and must be of different histology, iv) The tissue from the induced malignancy arose must be metabolically and genetically normal before irradiation.
From a molecular point of view around 100 genes are deregulated during secondary AS development including upregulation of MYC, KIT and RET genes, as well as concomitant upregulation of MYC and FLT4 and downregulation of CDKN2C gene. Similar genetic patterns are present in other radiation-induced tumors suggesting the distinct tumorigenic mechanism of radiation 6 .
In our study, we analyzed the clinical problem of distinguishing the primary from secondary-radiation induced angiosarcoma by immunohistochemical analysis of c-MYC and other markers expression in the case of a patient with endometrial cancer treated with adjuvant radiotherapy.

Clinical features
The patient was 77 years old with no prior or family history of malignancy. Initial staging

Pathological and IHH analysis
Specimen of skin measured 105x40x30mm was sent. On the cross-section, we revealed fields of haemorrhage in subcutis.
Sections of 4µ thickness were sampled. Standard histological analysis was performed using The same blocks of tissue, previously prepared for classical pathohistology, were used for immunohistochemical analysis.

Discussion
Secondary AS occurs as radiation-induced, but also in patients with chronic lymphoedema due to prior lymphadenectomy (after breast surgery) or in patients that have a chronically altered lymph drainage for other reasons (i.e. Stewart-Treves syndrome) 7,8 .
Radiation-induced secondary malignancies are rare but important late side effects of radiotherapy (RT) and have an impact on optimal treatment decision-making especially with expected survival longer than 5 years.
The adverse effects of radiation vary depending on the technique and dose applied and they are generally divided into early and late adverse effects. Early toxic effects of radiation on healthy tissue are due to acute inflammation (radiation colitis, cystitis, radio-dermatitis etc).
On the other hand, the late adverse effects are caused by micro-vascular damage, chronic inflammation and radiation-induced genetic instability. Whereas early adverse effects are reversible and have a good prognosis when treated medically, late effects are permanent and generally are less responsive to medications or lifestyle changes.
The development of contemporary RT techniques such as CRT (4 field box-technique) and intensity-modulated radiotherapy (IMRT) have improved target dose coverage and reduced early and late treatment toxicities 9 . Some studies, however, found no reduction of gastrointestinal and genitourinary toxicities of radiation when CRT is used and even suggested a positive correlation between the development of early and late toxicities in organs which receive a relatively high dose of radiation during the CRT treatment 10, 11, 12 . Nevertheless, contemporary radiation techniques (CRT and IMRT) made possible escalation of treatment dose which increased the number of long-term cancer survivors, patients at increased risk of developing late adverse effects of radiotherapy including RIM 13 .
Endometrial carcinoma is one of the most common gynecologic malignancies worldwide, with standard treatment protocol in early operable stages which includes radical hysterectomy followed by adjuvant radiotherapy if postoperative histology assesses risk factors for local recurrence (high-grade carcinomas or deep myometrial and cervical stroma invasion) 14,15 . Adjuvant RT provides a significant improvement of local control and diseasefree survival after 5 years of 90% for intermediate-risk patients and around 80% for highrisk patients (high-grade tumors or myometrial invasion) 16 .In our patient, the adjuvant RT was performed due to microscopic invasion of more than half of myometrium to achieve better local control (according to hospital protocol at the period).
Modern CRT allows more precise irradiation of a targeted volume, with an sparing effect on normal tissue, however with these techniques a larger volume of normal tissue is irradiated with a lower dose. RIM arises mainly in the irradiated tissue or the nearby tissues due to collateral radiation exposure 17,18 . A large cohort study by Chaturvedi et al. has shown that after radiation treatment of gynecological malignancies (external beam RT and brachytherapy) there is a secondary malignancy incidence increase of 12 % compared with the cohort that did not receive radiation treatment. RIM was detected with a median follow up of 12.2 years. The most common RIM observed were anal, colo-rectal and 9 gynecological malignancies 19 . Concerning endometrial carcinoma treatment, (PORTEC)-1 trial showed that 22% of the patients that received RT developed secondary neoplasm after 15 years, while 16% of non-irradiated patients developed secondary neoplasms 20 .
In several clinical series, comparing c-MYC gene amplification and expression between secondary AS of the skin and primary AS showed that c-MYC expression is statistically significantly more prevalent in secondary AS 21,22 . Also, a c-MYC expression is present in secondary angiosarcoma associated with chronic lymphedema (Stewart-Treves sy) 23 .
Nevertheless, in a minority of primary skin angiosarcoma c-MYC expression could be found too as well as in primary AS of other sites 24 .
The study by Styring et al. underlined the role of MYC, KIT and RET genes upregulation in the pathogenesis of radiation-induced AS and it's diagnostic application as а basis for therapeutic use of kinase inhibitors in these sarcomas. This study also found that over 100 genes are significantly deregulated between primary and secondary angiosarcomas, for example: upregulation of FLT4 a tyrosine kinase receptor for vascular endothelial growth factor and other vascular-specific receptor tyrosine kinases, like TIE1, KDR and FLT1.
This somatic mutation pattern could be of diagnostic importance since it is common in radiation-induced sarcomas and other radiation-induced malignancies 6 .
In the end, it should be noted that the overall prognosis of radiation-induced AS is poor 25 .
Although surgical treatment is the therapy of choice in other sarcomas, it seems that reoccurrence of the AS, including radiation-induced ones is high, even when radical excision is made 25 . This could be explained by the multifocality of the tumour so truly negative resection margin is hard to be achieved 26 . Haematogenic spread in lung, pleura and bone as well as spread to regional lymph nodes are possible 27,28 . At the time, Doxorubicin-based chemotherapy remained the standard treatment for metastatic or unresectable AS, but other chemotherapeutic agents such as Taxanes showed activity against AS 29 . Overall response rates to chemotherapy are variable from 20% to 60% 30 . In our patient high proliferative index (Ki67 around 60%) and c-MYC positivity could explain a good response to chemotherapy.
Target therapy could be a potential new approach in the treatment of radiation-induced AS such as tyrosine kinase inhibitors sorafenib, brivanib and sirolimus, as well as KIT inhibitor imatinib, anti-VEGF antibody bevacizumab and thalidomide but the experience is still limited and further studies are necessary 26 .

Conclusion
The development of novel radiotherapy techniques provided longer survival and better local control of high-risk endometrial cancer. Longer survival of these patients put them in a higher risk of developing RIM and other late side-effects of RT. When assessing the probability of radiation-induced AS, IHH finding of c-MYC expression could help to distinguish secondary from others AS if Cahan's criteria are fulfilled. Additional HH analysis to other molecular markers such as KIT or RET kinases and VEGF expression could be of diagnostic and therapeutic importance, in the era of target therapy in oncology. Clinical presentation initially, before chemotherapy with mono-ADM (left) and after chemotherapy (right).