Histological comparison between irradiated and non-irradiated breasts in breast reconstruction

1 Hospital Daher Lago Sul, Brasília, DF, Brazil. Conflicts of interest: none. Introduction: The treatment of breast cancer includes not only curative therapies but also breast reconstruction. Radiotherapy, an adjuvant strategy, provides favorable outcomes by reducing the rate of recurrence of the disease. This study aimed to compare histological differences between irradiated and non-irradiated breasts in the same patient. Methods: This is a prospective cohort study of patients undergoing breast reconstruction with prosthesis or expander under pectoralis major muscle flap that compared histological skin patterns, subcutaneous cell tissue, pectoralis major muscle, and implant capsule of irradiated and non-irradiated breasts in paired samples of the same patient. All patients included in this study were irradiated in only one breast. The results of the anatomopathological analysis were compared to clinical findings and intraoperative macroscopic aspects. Results: The study included a total of 7 patients with a mean age of 52.15 years. The main histological findings in the skin and subcutaneous cellular tissue of the irradiated breast were as follows: epidermal hyperplasia, flattening of the papillary layer, atrophy of the skin appendages, vascular congestion in fatty tissue, high density of skin collagen fibers, hyalinization, and reduction of elastic fibers in the deep dermis and unidirectional alignment of collagen fibers. The main histological findings for the capsule and pectoralis major muscle in the irradiated breast were as follows: lower density of elastic fibrosis, perivascular fibrosis, synovial metaplasia, skeletal muscle sequestration at the interface with the capsule, capsular hyalinization, and capsular fribrosclerosis. Conclusion: We found common histological changes in irradiated breasts in most patients. These findings are compatible with the clinical and macroscopic changes observed. This study presents itself as a pilot for the development of further studies investigating the physiopathological mechanisms of the described histological changes. ■ ABSTRACT


INTRODUCTION
Since the 1990s, the treatment of breast cancer has included not only curative therapies but also breast reconstruction, which has increased patients' interest in getting protected from cancer and obtaining better aesthetic results. Many studies have reported that breast reconstruction has no negative impact on cancer safety, reassuring patients and motivating them to perform the procedure 1,2 .
Some of the skin changes after irradiation have been investigated, and early effects (up to 90 days after the onset of radiation) include dehydration, pigmentation changes, loss of skin appendages, erythema, and desquamation. Delayed histological changes (after 90 days) include atrophy or hyperplasia of the epidermis, hypocellular fibrosis of the dermis, sclerotic vascular changes, and absence of pilosebaceous units (appendages). However, it is still unclear whether these changes are associated with the difficulties and complications of breast reconstruction with expander/ implant 11,12 . The susceptibility of the skin to changes after irradiation can be determined genetically. This concept is reinforced by individual differences in changes caused by radiation and developed complications 13,14,15 .
This study aimed to describe and compare histological differences between skin, subcutaneous cell tissue, pectoralis major muscle, and capsule of irradiated and non-irradiated breast implant in the same patient and to guide further studies to analyze possible methods of prophylaxis and treatment of complications. Currently, there are no studies that address such comparisons on all tissue layers.

Patients and tissue collection
This was a prospective cohort study of patients who underwent breast reconstruction with prosthesis or expander under pectoral major muscle flap, between January and August 2019, at the Daher Lago Sul Hospital, Brasília (DF). The histological patterns of skin, subcutaneous cellular tissue, pectoralis major muscle, and capsule of irradiated and non-irradiated breast implant were compared in paired samples of the same patient. All patients signed an informed consent form authorizing and agreeing to undergo surgical procedures and anatomopathological examinations and to record them for scientific purposes. The study was submitted to Plataforma Brasil, and the manuscript was validated by the Research Ethics Committee of the Health Sciences Teaching and Research Foundation/FEPECS/SES/DF, whose CAAE number is 15942719.6.0000.5553.
Surgeries were performed at the Daher Lago Sul Hospital-DF and the Brasília Hospital-DF. All selected patients had a history of previous radiotherapy in another institution, following the current reference scheme (50 Gy), which consists of 25 radiotherapy sessions for five weeks plus an additional dose on the tumor bed. Patients older than 18 years undergoing breast reconstruction for treatment of capsular contracture or other complications of adjuvant radiotherapy or contralateral symmetrization were included. Patients that were included in this study received irradiation in only one breast.
Biopsy tissues were collected during breast reconstruction. Samples of skin and subcutaneous cell tissue with length, width, and depth ranging between 0.5 and 1.0 cm were taken bilaterally from the submammary sulcus.
Samples of capsular tissue and pectoralis major muscle with dimensions 1.0 × 1.0 cm were taken from the site of greatest cicatricial retraction in the irradiated breast and in the inferomedial portion near the submammary sulcus of the non-radiated breast.
Anatomopathological analysis was performed, and the results were compared to the clinical findings of physical examination and the transoperative macroscopic aspects such as color, elasticity, vascularization, healing, and tissue sensitivity, to establish correlations between the clinical/histological variables and radiation.

Histological approach
The histological processing of tissue specimens was performed according to the method described by Huanget et al., in 2016 16 . Briefly, tissue specimens were fixed in buffered formaldehyde, embedded in paraffin, and stained by hematoxylin-eosin, Masson's trichrome, and Voerhoff elastic fibers for light microscopy. The histological evaluation was performed by a single pathologist in the Diagnose laboratory, Brasília (DF).
Quantitative and qualitative aspects of the epidermis were evaluated. Panniculus and implant capsule in the dermis were quantitatively and qualitatively evaluated for collagen, cellularity, inflammation, and vascularization.

RESULTS
The data from 7 patients, all female, with a mean age of 52.15 years (ranging from 34 to 68 years) were analyzed. They received 25 sessions of conventional radiotherapy in one breast, with a total dose of 50 Gy. The mean time between the last radiotherapy session and breast reconstruction was 54.14 months (ranging from 7 to 204 months). One of the postoperative complications of the first surgery was capsular contracture, which affected all patients and led to the second surgical period. One patient had suture dehiscence on both breasts, which was resolved with local bandages. None of them had infections.
The main histological findings in the skin and subcutaneous cellular tissue in the irradiated breast were as follows: epidermal hyperplasia (71.42%), flattening of the papillary layer (85.71%), atrophy of the skin appendages (100%), vascular congestion in fatty tissue (71.42%), high density of skin collagen fibers (100%), hyalinization of vascular walls (85.71%), reduction of elastic fibers in the deep dermis (85.71%), and unidirectional alignment of collagen fibers (100%), as shown in Table 2. These findings were observed in the samples of irradiated skin with considerable differences.
All patients had similar anatomical findings, which were consistent with clinical findings. It should be mentioned that cases 5 and 7 had more discreet anatomopathological findings, occasionally having a mixed pattern of presentation with areas containing normal tissue in the irradiated breast. Figures 1, 2, 3, and 4 show the main histological differences between irradiated and non-irradiated breasts. Hypovascularized muscle; SD: Skin dyschromia; "-" (not found); "+/-" (found moderately); "+" (found).

Case 1
A 36-year-old patient, without comorbidities, diagnosed with invasive ductal carcinoma in the right breast, undergoing neoadjuvant chemotherapy + bilateral mastectomy without preservation of nippleareola-complex, with right axillary dissection + immediate reconstruction with retromuscular remote valve expanders, in February 2016, without complications. to clinical treatments, she underwent microsurgical reconstruction of the right breast with transverse flap of the rectus abdominis muscle in June 2019, as shown in Figure 5.

Case 2
A 41-year-old patient with no comorbidities, diagnosed with invasive carcinoma in the left breast, undergoing left mastectomy with sentinel lymph node biopsy (negative) and contralateral prophylactic mastectomy + immediate reconstruction with retromuscular silicone implants with lower pole amputation (Torek) in December 2017, without complications. She underwent adjuvant radiotherapy in the left breast (last session in August 2018), and the second surgical period in June 2019 for left capsulotomy + implant repositioning, fat grafting for symmetrization and correction of scars in axillary extensions (reconstruction of nipple-areola complex programmed in the third period) without complications as shown in Figure 6.

DISCUSSION
As a result of early detection and improvement in treatments such as surgery, hormonal therapies, chemotherapy, and radiotherapy, the mortality rate for breast cancer has been decreasing since the 1950s. Therefore, more patients with breast cancer are surviving and remaining with sequelae that must be treated. In this study, we sought to describe the histological differences of skin, subcutaneous cell tissue, implant capsule and pectoralis major muscle between irradiated and non-irradiated breasts of the same patient. Previous studies have addressed only the skin and subcutaneous region.
The findings of epidermis hyperplasia, flattening of the papillary layer, atrophy of the skin appendages, high density of the skin collagen fibers, and presence of unidirectional collagen fibers had already been reported 11,12 . Atrophy of skin appendages is of particular clinical importance, as loss of sebaceous tissue and sweat glands lead to dehydrated skin, resulting in the need for long-term skincare using moisturizers. Chronic radiation dermatitis is reported to be associated with fibroblast atypia, which is not seen in other types of fibrosis, such as third-degree burn scars 12.13 . The present study corroborates these findings. Other findings included the following: reduction of elastic fibers in the deep dermis, vascular congestion in fatty tissue, and hyalinization of vascular walls. Although histological changes in dermatitis due to radiation have already been described 12,13 , these have been considered to be less important clinically with acceptable side effects. However, this does not apply when planning breast reconstruction with silicone expander/implant. Archambeau et al., in 1995 11 , found skin changes due to irradiation progressing for up to 10 years. Given this fact, the delay in indicating reconstruction after radiotherapy does not increase safety. This finding could explain the mild findings found in cases 5 and 7, in which the last radiotherapy session was more recent (10 and 8 months, respectively). One of the main complications of radiotherapy is fibroproliferation of the capsular tissue around the implant with a resulting capsular contracture. This leads to an inadequate expansion with distortion and undesirable aesthetic results, sometimes causing additional surgery. Currently, the pathogenetic mechanism of fibroproliferation and capsular contracture induced by radiation is unknown. The correct anatomical description of the observed changes can help in the development of new studies, unraveling the biochemical mechanisms involved in this pathogenesis.
Understanding the pathogenesis of the fibroproliferative process, which starts with the expansion of the tissue previously subjected to radiotherapy, may probably lead to the discovery of prevention strategies or clinical treatment. For example, COX-2 selective inhibitors are commercially available and were effective in partially decreasing cell proliferation in fibrosis models mediated by increased catenin levels 17 . There is great potential to explore treatment protocols in an animal model and eventually in clinical trials.
Encapsulation occurs as a result of an inflammatory response to the presence of the foreign body, and fibrosis progresses to nearby tissues. When fibrosis progresses excessively, due to the persistence of the inflammatory response and exposure to external risk factors, contracture occurs around the thickened capsule 18 .
Therefore, breast reconstruction with implants is performed under the assumption that if radiotherapy is administered, capsular contracture will be recognized as a fundamental limitation, and many studies will be conducted to find solutions to this question. Kim et al., in 2018 19 , confirmed that the infiltration of myofibroblasts was promoted in irradiated mice, suggesting that this phenomenon acts as a catalyst to accelerate the progression of contracture. We did not find this type of cellular infiltration in any of the samples of irradiated breast.
Some studies reported using coverage with acellular skin matrix and some medications such as montelukast, antileukotrienes, and steroids to reduce the occurrence of capsular contracture around textured implants 20,5 . Although it is consensus that radiation can induce fibroproliferation in skin and subcutaneous tissues 11,13 , the relative occurrences of specific molecular mechanisms are still unclear.
We believe that dry skin may be related to atrophy of the skin appendages. The loss of skin elasticity was related to the reduction of elastic fibers in the deep dermis, epidermal hyperplasia, flattening of the papillary layer, high density of skin collagen fibers, and unidirectional alignment of collagen fibers. Hypovascularized fat was related to subcutaneous vascular congestion and hyalinization of vascular wall. The thickened and contracted capsule was related to lower density of elastic fibers, capsular hyalinization, capsular fibrosclerosis, and synovial metaplasia. Hypotrophic and hypovascularized muscle was related to perivascular fibrosis and skeletal muscle sequestration by capsule.
With the data obtained so far, it is not possible to establish a cause and effect relationship, but we will continue to include new patients into the study and try to optimize the quantitative analysis of the information to get to this point. Since each histological evaluation was performed between the breasts of the same patient and not between two different groups, even a small number of patients provided significant results.

CONCLUSION
We found common histological changes in irradiated breasts in most patients. These findings are compatible with the clinical and macroscopic changes observed. This is a descriptive study that presents itself as a pilot for the development of new studies investigating the physiopathological mechanisms related to the described histological changes, thus proposing methods of prophylaxis and treatment for the complications of radiotherapy.

COLLABORATIONS AB
Analysis and/or data interpretation, Final manuscript approval, Realization of operations and/or trials