Borderline phyllodes tumour of the breast with eosinophilic inclusion bodies: Case report and molecular sequencing

Introduction and importance The presence of eosinophilic inclusion bodies in the breast is very rare and fewer than 20 cases were described in the literature. Herein we report the first case of borderline phyllodes tumour associated with this kind of cells. To the best of our knowledge, this is also the first time that a molecular sequencing is made targeting the stroma cells with inclusion bodies. Case presentation A 33-yr-old woman presented a large mass in the right breast. Imaging techniques by mammogram and ultrasonographic examination were performed. After multidisciplinary approach, a breast conserving surgery has been decided. Microscopic analysis, immunohistochemical stains and molecular tests were performed on the lesion. The proposed diagnosis is borderline phyllodes tumour with eosinophilic inclusion bodies. Clinical discussion Inclusion bodies are typically found in the infantile digital fibromatosis. Finding them in extradigital fibromatosis is rare. Their signification is still unclear. Some studies suggest a disturbance in the metabolism of proliferating myofibroblasts. Conclusion The presence of inclusion bodies in breast tumour do not seem to have a prognosis impact. It might be interesting to perform others molecular tests on lesions with eosinophilic inclusion bodies to discover potential mutations.


Introduction
Eosinophilic intracytoplasmic inclusion is a pathognomonic feature of infantile digital fibromatosis (IDF) [1].It is a rare, superficial fibromatosis characterized by little, firm, painless, pink-red nodules on the fingers and toes of children.The pathogenesis of Idiopathic Digital Fibromatosis (IDF) is still unclear and many studies have not found an association with a genetic syndrome or systemic disease.Eosinophilic inclusion bodies in extradigital fibromatosis are uncommon.Some cases have reported intracytoplasmic inclusion bodies in cervical polyps [2], in leiomyosarcoma [3] or in phyllodes tumour of the breast [4].To the best of our knowledge, we report for the first time a phyllodes tumour with borderline features associated with eosinophilic intracytoplasmic inclusions.This is also the first that a molecular sequencing is made in this context.

Case presentation
Here we report a case of a 33-yr-old woman who presented a large mass in the right breast.Her surgical history includes a breast conserving surgery (BCS) in Cambodia in 2019 for a benign lesion without more precise details.She is not using contraception.In her family history, her mother's cousin had breast cancer at the age of 50.The breast examination revealed a large mass with a bluish spot on the skin in the upper external quadrant of the right breast (Fig. 1).
There was an enlargement of the areola and a retraction of the nipple.The examination of the left breast was unremarkable.Bilateral mammogram was performed and showed dense breasts.In the right breast, we found a mass measuring 8 cm and occupying the entire upper quadrant.The lesion was relatively well delimited.Rare microcalcifications were found but were not suspicious.In the left breast, there were scattered microcalcifications but no suspicious focus.Ultrasound of the right breast showed a vascularised mass measuring 15 × 4.7 cm (Fig. 1).Microbiopsy was performed.Histologically, biopsy cores showed a biphasic tumour with a fibroepithelial proliferation and small glandular structures.The fibrous compartment was fibrous, focally myxoid and contained a few irregularly arranged fibroblasts.The proliferation index evaluated by Ki67 (MIB-1, Dako) was estimated at 10 %.The lesion was classified as B3 in the Nottingham system [5].
After multidisciplinary approach, a BCS has been decided.The patient had no post-operative complications.
Macroscopically, the resected lesion measured 11 × 10 × 7 cm, was relatively well delimited and appeared as a multinodular, gray-white, homogeneous mass on the cut surface (Fig. 2).
The lesion was excised with a microscopic free margins.The distance between the tumour and the closest margin was evaluated at 2 mm.
The diagnosis of borderline phyllodes tumour with eosinophilic inclusion bodies was made.
Next-Generation Sequencing (NGS) targeting stromal cells with inclusions was done.The library construction was performed using two custom Ampliseq panel targeting in 58 genes (Table 1).Sequencing was performed using the Ion S5 GeneStudio instrument (ThermoFisher Scientific, Waltham, MA).Molecular tests revealed no mutations.

Discussion and conclusion
Intracytoplasmic eosinophilic inclusion is a pathognomonic feature in infantile digital fibromatosis (IDF) that was first described by Reye in 1965 [6].IDF is a dermal fibroblastic and myofibroblastic lesion typically found as firm nodules on digits of infants and children.Intracytoplasmic inclusion bodies in stromal cells are a classic histopathologic finding and can be highlighted with special stains like Masson's trichrome [1].However their presence in extradigital fibromatosis and in adults is rare.Fewer than 20 cases were described.Some authors have reported intracytoplasmic inclusion bodies in cervical polyps [2], in leiomyosarcoma [3] or in phyllodes tumour of the breast [4].In the literature, 5 cases were described in phyllodes tumour of the breast.The clinical and histological features of these cases were described in Table 2.
All the 5 cases were diagnosed as benign phyllodes tumour [4,7-10].Our case is the first one to describe a borderline phyllodes tumour with those inclusion bodies.Ortega and al. reported a case of benign       phyllodes tumour of the breast with inclusion bodies by fine-needle aspiration (FNA) [9].Dey and al. described a case where the needle core biopsy (NCB) showed epithelial lobular units with spindled stromal cells [4].They found multiple intracytoplasmic, homogenous eosinophilic inclusions in the spindled stromal cells.Their diagnosis was phyllodes tumour with intracytoplasmic inclusions.
The inclusion bodies in our case were positive for SMA and vimentin.In different published cases, the spindled stromal cells are positive for SMA, desmin or vimentin [4,7,8,11,12].In contrast there is a difference between cases in positivity for SMA, with either a positivity within the inclusion or at its periphery [11,12].The principal differential diagnosis is a rhadoid differentiation in phyllodes tumour [13].In rhadoid differentiation there is a cohesive large epithelioid cells with irregular nuclei, prominent nucleoli and a large paranuclear intracytoplasmic eosinophilic inclusions.Most of the time there is a positive staining of myogenin on those cells.In contrast our lesion was composed of spindled stromal cells containing round eosinophilic intracytoplasmic inclusion bodies.Also the rhabdoid differentiation was invalidated by the negative staining of myogenin in our case.
Some studies performed electronic microscopy on the inclusion bodies [7,8,10].The core of those was composed of granular material; while in the periphery, it showed a microfibrillary structure.Actin positivity and fibrillary appearance on ultrastructural examination suggest a mechanism of disturb regulation of proliferating myofibroblastic cells.We did not perform electronic microscopy but our laboratory realised molecular test of the lesion.To the best of our knowledge, molecular sequencing by NGS have not yet been conducted for lesions with inclusion bodies.We tried to analyse the area in which the inclusions were numerous.NGS (panels of 58 genes) revealed no mutations.In the literature, genetic alterations of TERT promoter and MED12 are usually observed in phyllodes tumour [14].In borderline or malignant phyllodes tumour TP53 alteration and/or EGFR amplification can be found.In our case there were no alteration of TP53, TERT promoter and no EGFR amplification and we could not test the presence of a MED12 mutation with our panel.The presence of inclusion bodies in breast tumour do not seem to have a prognosis impact.In the literature only one case reported a patient's follow-up.She underwent a surgery for a fibroepithelial tumour of the breast with inclusion bodies and there was no recurrence 5 years after surgery [3].

Fig. 1 .
Fig. 1.Clinical and imaging techniques. A. Mass with a bluish spot on the skin in the upper quadrant of the right breast.B. Mammogram C. Ultrasound of the lesion in the right breast.

Fig. 5 .Fig. 6 .
Fig. 5. Inclusion bodies stained red by Masson's trichrome (arrows) (HE ×40).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
N.M.Lam et al.