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Abstract
Aims Phyllodes tumours (PTs) are relatively common fibroepithelial tumours comprising epithelial and stromal component. Usually, PTs show a spindle cell morphology with a fibroblast phenotype, while some tumour cells exhibit epithelioid morphological features and sarcomatoid transformation. However, the molecular characteristics of this morphology subset remain unclear. This study aimed to summarise the clinicopathological, morphological and molecular characteristics of seven cases of PT with epithelioid features.
Methods Morphological and clinicopathological characteristics were observed and retrieved. Immunohistochemistry, immunofluorescence and electron microscope were performed on seven cases of epithelioid PT to explore immunophenotypic and ultrastructural characteristics. Transcriptomic and proteomic analyses were conducted to compare differentially expressed genes and proteins between epithelioid PT and classical PT.
Results Patients with epithelioid PT exhibit a high recurrence rate (42.8%). Morphologically, in addition to having epithelioid cytological features, neoplastic stromal cells exhibit moderate to marked atypia and often exhibit sarcomatoid transformation, similar to the characteristics of borderline PT. Transcriptomic and proteomic analyses demonstrated that epithelioid PTs are distinct from classical PTs in gene expression and protein abundance levels. Immunohistochemical analysis showed that among all differentially expressed proteins, epithelioid PT showed abnormal p16/retinoblastoma expression patterns, similar to those of malignant PT.
Conclusions Epithelioid PT has unique morphological characteristics, biological behaviour and protein expression profile, which meets the diagnostic criteria of borderline PT and is prone to sarcomatoid transformation. It may be a special morphological subgroup of borderline PT and has partial characteristics of malignant PT, which should be taken seriously in pathological diagnosis and clinical management.
- MORPHOLOGY
- PROTEINS
- DIAGNOSIS
- Breast Neoplasms
Data availability statement
Data are available on reasonable request.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
The stromal cells of classic phyllodes tumour (PT) exhibit a spindle cell morphology, which serve as the basis for morphological diagnosis and tumour grading, while a subset of tumour cells exhibit epithelioid morphological features. The molecular characteristics of this epithelioid subset, as well as their distinctions from classical PTs, remain unclear. Further investigation at the genetic and protein levels is warranted.
WHAT THIS STUDY ADDS
This study provides a comprehensive summary of the clinicopathological, morphological and molecular characteristics observed in seven cases of PT exhibiting epithelioid features. Epithelioid PT demonstrates distinct morphological characteristics, biological behaviour and gene and protein expression profiles that differentiate it from classic PT. It exhibits some features of malignant PT and shows a propensity for sarcomatous transformation.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This study proposes for the first time that epithelioid PT may be an independent morphological subtype, supplementing the morphological spectrum of PT. This finding is anticipated to aid pathologists in diagnostic practices and to prompt clinicians to consider proactive treatment strategies for epithelioid PT.
Introduction
According to the 2019 WHO Classification of Breast Tumors,1 phyllodes tumour (PT) is a fibroepithelial breast tumour type primarily characterised by significant stromal cell proliferation and extrusion of breast ducts into leaf-like structures.1 The interstitial components of PT are mainly composed of spindle cells that differentiate towards myofibroblasts. However, morphological variations can also be observed in PT, warranting further exploration to prevent misdiagnosis and mismanagement. Wabik et al2 described a special PT type, periductal stromal sarcoma, whose morphological manifestations included interstitial cells surrounding the duct, no lobulated structure, unclear boundaries and infiltration of the surrounding fat. Furthermore, Lee et al3 described a malignant PT case with endometrioid morphology, with typical lobular structures in some areas and endometrioid morphology in others. In previous studies, we observed three cases of PT with epithelioid morphology of tumour stromal cells.4 Although the pathological diagnostic criteria and tumour grade of PTs are based on spindle stromal cell evaluation, breast PTs with evident epithelioid cell morphology can also be observed. However, whether PTs exhibiting epithelioid morphology are a coincidental phenomenon or a distinct subgroup of mammary PTs remains unclear. This study aimed to summarise the morphological and clinicopathological characteristics of cases of PT with epithelioid features via immunohistochemical, immunofluorescence, electron microscope and transcriptomic and proteomic analyses.
Material and methods
Tissue sample selection
We collected seven cases of PT with epithelioid features, of which cases 1–3 were briefly described in morphology in previous studies.4 In this study, we further conducted electron microscopy, transcriptomics and proteomics analysis on these cases. The diagnosis of classic breast PTs is based on the criteria of the 2019 WHO Classification of Breast Tumors. The 2019 WHO classification does not describe epithelioid PT, so we diagnose epithelioid PTs if the proportion of epithelioid stromal cells exceeds 90%, but still classify epithelioid PT according to the WHO histological grading criteria. Myofibroblastoma, desmoid fibromatosis, inflammatory myofibroblastic tumour, rhabdomyosarcoma, metaplastic carcinoma and leiomyosarcoma were ruled out. Borderline PTs was diagnosed in this group because all histological features required for the diagnosis of malignant PTs were not met, especially insufficient mitoses. Paraffin samples from six epithelioid PT tissues (cases 1–6), two adjacent normal tissues and eight classic PT tissues (one benign, six borderline and one malignant PT) were obtained for proteomic and transcriptomic analyses. Two senior pathologists or associate professors confirmed their diagnoses.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed in all cases using the automated immunohistochemical staining platform (Leica Bond-III, Australia) with the following markers (all from Labvision, USA): cytokeratin (CK (PAN); Clone MX005), vimentin (VIM; Clone MX034), B-cell lymphoma 2 (Bcl-2; Clone MX022), CD34 (Clone QBEnd/10), epidermal growth factor receptor (EGFR; Clone EP22), Ki-67 (Clone EP22), P63 (Clone MX013), CK5/6 (Clone MX040), CD45 (Clone PD7/26+2B11), cytokeratin 8 (CK8; Clone MX049), p16 (Clone MX007), retinoblastoma (Rb; Clone 1F8) and oestrogen receptor (Clone SP1), desmin (Clone MX046), myogenin (Clone MX078), myoD1 (Clone MX007). We combined the intensity and extension of immunohistochemical staining to quantify the expression values of all antibodies using a 1–3 scoring.
Immunofluorescent double staining
Immunofluorescent double staining for CK8 and myosin (diluted 1:50) was performed. The sections were blocked and incubated overnight at 4°C with primary antibodies. After washing with phosphate-buffered saline, the sections were incubated with fluorescence-conjugated secondary antibodies (1:200; Alexa Fluor 568, Thermo Fisher, USA) for 1 hour at 25–30°C. The sections were washed and counterstained with the nuclear dye 4,6-diamino-2-phenylindole, and images were visualised using a confocal microscope (LSM710, Carl Zeiss, Germany).
Transmission electron microscopy
A tissue mass of 1 mm3 was placed in precooled 2.5% glutaraldehyde and fixed for 12 hours at 4°C, fixed with 1% osmium at 20–25°C for 1 hour and washed with 0.1 mol of phosphate buffer. The tissue was soaked in epoxy propane mixed with resin (1:1) at 20–25°C for 2 hours. The tissue was then saturated with pure resin for 12 hours at 20–25°C, embedded with fresh pure resin and polymerised at 45°C for 12 hours. After polymerising at 60°C for 48 hours, the tissue was cut into semithin slices, stained with toluidine blue and selected for preparing ultrathin sections. Ultrathin sections were prepared using an ultrathin slicer, transferred to a 200-mesh copper net and dried. The copper mesh with ultrathin sections was fixed to a staining silicon plate and coated with 3% lead citrate and 5% uranium acetate. After drying the copper mesh at 20–25°C, the ultrastructure of tumour cells was observed using transmission electron microscopy (JEM-1400, JEOL, Tokyo, Japan).
Transcriptomics and proteomics
Materials and methods related to transcriptomics and proteomics can be found in online supplemental file 1.
Supplemental material
Results
Patient clinical information
The clinical data of the seven patients are presented in table 1. The patients were young females aged 17–39 years old. The breast mass presents primarily as a unilateral lesion, with a maximum diameter of 2.0–18.2 cm. Three patients (cases 1–3) underwent simple tumour resection and experienced local recurrence after 14, 10 and 7 months, respectively. The other three patients (cases 4–5 and 7) underwent a mastectomy and exhibited no recurrence afterwards. However, patient six was lost during follow-up. In addition, due to the replacement of the medical information system, we are unable to confirm the specific surgical procedure of patient six.
Clinicopathologic data
Histopathological and immunohistochemical findings
The major morphological characteristics of seven epithelioid PTs are summarised in table 2.
Summary of morphological characteristics of seven epithelioid phyllodes tumours
Case 1
At low power, the tumour exhibited a multinodular growth pattern. Microscopically, the tumour comprised two components (figure 1A). One was a glandular structure with irregular shapes and sizes arranged in tubular or branched shapes. The lumen was lined with a layer of epithelial cells with abundant eosinophilic cytoplasm and no evident atypia. Another component was epithelioid stromal cells, which were abundant and diffused around the glands. The tumour stromal cells overgrew and intermingled with the glands, compressing and deforming the glands. However, typical leaf-like structures were not observed in the primary tumours. The neoplastic stromal cells exhibited epithelioid characteristics with prominent nucleoli, abundant cytoplasm and mitotic images (figure 1B). Immunohistochemically, CK labels the gland structure. The epithelioid stromal cells were positive for EGFR (figure 1C) and Bcl-2 (figure 1D).
Case 1. Primary case. (A) Hyperchromatic mononuclear cell interspersed between irregular glands. (B) Epithelioid cells with prominent nucleoli, abundant cytoplasm and mitotic cells are around the glands. (C) Epithelioid neoplastic stromal cells are epidermal growth factor receptor positive. (D) Epithelioid neoplastic stromal cells are BCL-2 positive. (E) Double-labelling immunofluorescence, cytokeratin 8 red fluorescence and myosin green fluorescence showed the presence of peripheral myoepithelial glands, even though some glandular structures were interspersed and the myoepithelium was still relatively intact. The tumor stromal cells were not stained. (F) Electron microscopic examination: tumour cells are mostly round, with irregular nuclei, evident nucleoli and chromatin edge clustering; myoepithelial cells are present (white star), and individual myoepithelial cells are isolated between stromal tumour cells (red asterisk). (G) No connection was observed between the tumour cells and varying amounts of collagen fibres. (H) The tumour cells had microfilaments, dense bodies and an abundant rough endoplasmic reticulum, demonstrating the characteristics of myofibroblast differentiation. (I) Recurrence was experienced after 14 months. The stromal cells grow around the gland as multilayered rosettes with myoepithelial cells. (J) Typical leaf-like structures are observed locally after recurrence.
Moreover, immunofluorescence and electron microscopic examinations were performed in case 1. Double staining of the myoepithelial and gland markers, myosin and CK8, respectively, revealed the presence of an intact myoepithelium, even in scattered small nests. The tumor stromal cells were not stained (figure 1E). Electron microscopy revealed that the tumour cells were mostly round, with irregular nuclei, evident nucleoli and chromatin edge clustering (figure 1F). Ultrastructural analysis revealed the presence of collagen fibres, microfilaments, dense bodies and abundant rough endoplasmic reticulum in the cytoplasm, revealing the characteristics of myofibroblast differentiation (figure 1G–H). Compared with classic PTs, epithelioid types reduce collagen production.
After 14 months, the tumour recurred. In the recurrent tumours, atypical stromal cells grew in the form of multilayered rosettes around the glands in the presence of myoepithelial cells (figure 1I). In addition, typical leaf-like structures were observed in the recurrent cases (figure 1J). Immunohistochemically, CK8 and p63 staining revealed the presence of glandular and myoepithelial cells, respectively.
Case 2
The primary tumour exhibited a multinodular growth pattern at low magnification (figure 2A). At high power, glands and stromal cells were interwoven and distributed. Mild stromal cell proliferation was observed around the lumina with a sleeve-shaped growth pattern (figure 2B). The pericanalicular stromal cell morphology was round or oval instead of spindle-shaped, with typical epithelioid cell morphology and moderate atypia, including a high nuclear/cytoplasmic ratio, prominent large and vacuolated nucleoli, and an irregular nuclear membrane (figure 2C). Mitotic activity was observed frequently. Immunohistochemical staining of stromal epithelioid cells with CD34 positive supported PT diagnosis (figure 2D).
Case 2. Primary case. (A) Borderline epithelioid phyllodes tumour with nodular morphology in the first specimen. (B) Enlargement of the epithelial–mesenchymal junction area. (C) The nuclei of epithelioid stromal cells in the first specimen are large and vacuolated, nucleoli are evident, and the nuclei of glandular epithelial cells are similar to those of stromal cells. (D) Stromal cells are CD34 positive. (E) The recurrence case still shows nodular structure. (F) Cell hyperplasia in the epithelial–mesenchymal junction area after recurrence, resembling periductal stromal sarcoma. (G) Interstitial cells still exhibit an epithelioid morphology, accompanied by increased nuclear atypia and mitosis. (H) Immunohistochemistry showed positive Bcl-2 expression in stromal cells.
After 10 months, the patient experienced local recurrence. Recurrent cases exhibited a nodular growth pattern with the formation of local leaf-like structures (figure 2E). The increased cell density in the epithelial–mesenchymal junction after recurrence presented a local periductal stromal sarcoma pattern (figure 2F). Interstitial cells remained in the form of epithelioid cells, accompanied by increased nuclear atypia and mitotic activity (figure 2G). Immunohistochemistry showed positive Bcl-2 expression in interstitial cells (figure 2H).
Case 3
The boundary of the tumour was unclear, and two different morphological regions were observed. In some areas, epithelial and stromal components were significantly proliferative and mixed in arrangement (figure 3A). No evident dysplasia was observed in the epithelial components. Interstitial cells exhibited significant atypia with enlarged vacuolar nuclei, prominent nucleoli and increased mitotic figures (figure 3B). In another region, sarcomatoid transformation was observed, with atypical spindle cells, bizarre nuclei and distinct mitotic figures (figure 3C,D). Immunohistochemically, CK8 and P63 are distributed in the glandular epithelium and myoepithelium. Positive vimentin expression in stromal epithelioid cells confirms its mesenchymal origin. Epithelioid cells were slightly positive for Bcl-2, suggesting PT diagnosis. However, we did not obtain tissue sections from this patient after recurrence and could not elucidate the morphological characteristics after recurrence.
Cases 3 and 4. (A) The glandular epithelial components are arranged and mixed with epithelioid stromal components. (B) Epithelioid stromal cells have enlarged vacuolated nuclei and prominent nucleoli. (C–D) Fibrosarcoma-like components are observed in another region, accompanied by significant cellular atypia and mitosis.(E) The neoplastic stromal cells proliferate diffusely, and the irregular glands mimic invasive growth. (F) Formation of the early leaf-like structures. (G–H) Immunohistochemistry revealed that the stromal cells are positive for Bcl-2 and epidermal growth factor receptor.
Case 4
In this case, the tumour boundary was unclear with local adipose tissue infiltration. Neoplastic stromal cells proliferated significantly and diffusely, whereas the glandular components were reduced (figure 3E). The epithelial–mesenchymal junction area expanded and formed early leaf-like structures locally (figure 3F). Neoplastic stromal cells proliferated significantly, compressing the gland and presenting a pseudo-infiltrating morphology. Tumour stromal cells still exhibited an epithelioid morphology, with chromatin vacuoles and visible small nucleoli distinct from mitosis. Immunohistochemically, positive Bcl-2 (figure 3G) and EGFR expressions (figure 3H) in stromal cells supported the diagnosis of PT.
Case 5
In this case, the epithelioid stromal cells proliferated significantly, mixed with the benign atrophic ductal components and locally compressed the ducts to form a leaf-like structure (figure 4A,B). Epithelial stromal cells exhibited significant atypia, vacuolar nuclei and distinct mitotic figures (figure 4C). A specific pleomorphic liposarcoma transformation was observed in a partial region. Neoplastic lipoblasts were evident and atypical. Immunohistochemical staining for CK8 and P63 revealed the structures of the glandular epithelium and myoepithelium. The epithelioid stromal cells were positive for CD34 and negative for Bcl-2.
Cases 5, 6 and 7. (A) Local formation of leaf-like structures. (B) Diffuse proliferation of tumour stromal cells. (C) Tumour stromal cells are epithelioid with significant atypia. (D) Multinodular pattern around the glands. (E) Periductal epithelioid stromal cell proliferation and duct compression. (F) Sarcomatoid transformation component infiltrating adipose tissue. (G) Epithelioid stromal cells proliferate diffusely and are distributed around the ducts with atypia. (H) Irregular glands mixed with epithelioid tumour cells mimicking invasive growth. (I) Sarcomatoid transformation area.
Case 6
The epithelioid stromal cells grew in a multinodular pattern around the glands (figure 4D). In this case, the mass boundary was irregular, with tumour cells infiltrating the surrounding adipose tissue. Periductal epithelioid stromal cells proliferated, resulting in duct compression into the early leaf structures (figure 4E). The glands in the focal area were irregular and branched, mimicking the infiltration growth pattern, whereas the myoepithelium was still present around the glands. Stromal cells still present an epithelioid cell morphology with abundant eosinophilic cytoplasm, vacuolar nuclei and distinct nucleoli. The sarcomatoid transformation was also observed (figure 4F). The sarcoma components had infiltrated the adipose tissue, suggesting that the lesion was highly malignant. Immunohistochemically, CK outlines the morphological pattern of gland pseudo-infiltration. P63 confirmed the presence of myoepithelial cells. The neoplastic stromal cells were positive for EGFR and Bcl-2. The sarcomatoid transformation area was positive for vimentin expression.
Case 7
The neoplastic stromal cells proliferated significantly, were diffusely distributed around the ducts (figure 4G) and were segmented into nodules by fibrous grounding tissue. Early-stage leaf-like structures and a high density of epithelioid stromal cells were observed in the epithelial–stromal junction. The epithelioid stromal cells exhibited evident atypia, vacuolised nuclei and distinct nucleoli (figure 4H). The compressed glands showed an irregular morphology resembling pseudo-infiltration (figure 4H). In addition, sarcomatoid transformation occurred locally in this case, with epithelioid cell morphology, focal necrosis and pathological mitosis (figure 4I).
Epithelioid and classical breast PTs demonstrated significant differences in gene expression and protein abundance
We explored whether epithelioid breast PTs are a distinct type of breast PTs at the molecular level. We collected tumour tissues from six epithelioid PT cases. For comparison, tumour tissues were collected from eight classical PT cases (one benign, six borderline and one malignant cases). Using RNA-Seq and Data-independent acquisition (DIA)-based microproteomic technologies, we obtained the transcriptomes and proteomes of epithelioid and classical breast PTs. The clustering and differential analysis revealed that epithelioid and classical breast PTs differed significantly in terms of gene expression and protein abundance(figure 5). Particularly, the proteome delineated these two types. Differential analysis showed that epithelioid and classical PTs had more highly expressed genes and proteins than normal tissues. Genes highly expressed in the epithelioid type were mainly enriched in protein phosphorylation modifications. The top 100 genes significantly upregulated and downregulated in epithelioid PT compared with the classical type are summarised in online supplemental tables 1A,B respectively. These results demonstrated that epithelioid PTs differ from classical PTs in gene expression and protein abundance levels, and represent a unique type of breast PTs.
Molecular differences between epithelioid and classical phyllodes tumours (PTs). (A) Clustering of samples. We applied principal component analysis method on transcriptomic (left) and proteomic (right) data of all samples separately in order to cluster the samples. Each dot in the figure represents a sample, and the different colours filled represent the sample types. The clustering results show that epithelioid and classical samples are clearly separated (marked by purple and pink circles). Distribution of differentially expressed genes (B) and differentially expressed proteins (C) between the groups. Based on the fold change as well as the Q value, we obtained the significantly differentially expressed genes and proteins. Each point in the graph represents a gene or a protein, with red marking significantly up-regulated genes or proteins and blue significantly down-regulated ones. The red lines show the thresholds of log2 fold change as well as Q value. The numbers after ‘up’ and ‘down’ show the number of significantly upregulated and downregulated genes or proteins, respectively. (D) Gene ontology functional enrichment for differentially expressed proteins between epithelioid and classical PTs. (E) Overlap among the three groups with upregulated proteins. We extracted proteins with significant differences in both gene expression and protein abundance between groups. In particular, we were interested in proteins that were highly expressed in epithelioid compared with normal tissue (E_up vs N), highly expressed in classic compared with normal tissue (C_up vs N), highly expressed in epithelioid compared with classic (E_up vs C) and highly expressed in classic compared with epithelioid (C_up vs E). Two Venn diagrams show the overlap of differential proteins between these groups. (F) Heatmap of protein abundance levels across different samples and the ratios between different groups.
Next, we attempted to identify the protein markers of epithelioid PTs. By applying log2 fold change with an absolute value >2 and a Q value<0.01, we performed a more stringent filtering of differential proteins. We identified 21 proteins that were more highly expressed in classical and epithelioid PTs than those in normal tissues. In addition, we identified four proteins that were highly expressed only in epithelioid PTs: HAUS1, MAPKAPK2, CDKN2A and TIMP1.
Epithelioid PT represents a unique morphological subgroup with an immunophenotype similar to that of malignant PT
We further verified the expression of these four genes and the downstream gene Rb of CDKN2A in classic PT (seven benign, eight borderline and five malignant samples) and epithelioid PT (five samples) using immunohistochemistry. These results demonstrate that p16 and Rb exhibit unique expression patterns. Among benign and borderline PTs, p16 and Rb were negative or partially weakly positive. Malignant PT showed three immune phenotypes, including one case of diffuse p16+/Rb−, one case of p16−/diffuse Rb+ and three cases of diffuse p16+/diffuse Rb+. The expression patterns of p16/Rb in epithelioid PT are similar to those in malignant PT, including three cases of diffuse p16+/Rb−, one case of p16−/diffuse Rb+ and one case of p16−/Rb− (online supplemental table 2 and figure 1). Epithelioid and malignant PTs exhibited abnormal expression of p16 and/or Rb proteins. Therefore, we conducted a statistical analysis of the different types of classical and epithelioid PTs. The results showed significant differences among the four groups (table 3; p=0.001). No significant difference was observed between epithelioid and malignant PTs (online supplemental table 3). Therefore, our results suggest that epithelioid PT has an immunophenotype similar to that of malignant PT.
Comparison of p16 and Rb expression patterns among four groups
Discussion
In this study, we summarised the morphological characteristics of seven epithelioid PT case with a high recurrence rate (42.8%, 3/7). Similar epithelioid cell morphology can be observed in other stromal tumours, such as prostate stromal sarcoma.5 6 Osaki et al5 present a rare case of prostate stromal sarcoma with mixed epithelioid and spindle cell morphology, exhibiting obvious atypia and pleomorphism. The research findings indicate that stromal tumours with epithelioid morphology seem to suggest more aggressive biological behaviour, consistent with our research findings.
The main histopathological features of breast PT are the excessive proliferation of spindle stromal cells and the formation of leaf-like structures. The mesenchymal cells in this study were epithelioid and distributed diffusely in the stroma or around glands. The morphology of the early stage was similar to that of periductal stromal sarcoma.7 Although typical leaf-like structures were not observed in the early stages of epithelioid PT, the immunophenotype of tumour cells, such as EGFR expression, was similar to that of general malignant PT.8 Furthermore, multilayered rosette patterns and leaf-like structures were observed in the recurrent cases. Therefore, the morphological features of PTs are not limited to spindle cells, overgrowth of mesenchymal cells and leaf-like structures. Epithelioid morphology, lack of leaf-like structures and abundance of glands should also be considered. Additionally, early epithelioid PTs exhibit abundant glands associated with tumours, and the glandular structure is interwoven with neoplastic stromal cells, similar to papillary or glandular infiltration changes. This presents as a special morphology of pseudo-epithelioid infiltration, which requires high vigilance and should be differentiated from breast-infiltrating cancers. The presence of an intact myoepithelium around these irregular glands, as confirmed by immunohistochemistry, suggests that the glands are benign.
Additionally, differences in gene expression and protein abundance levels were observed between the classical and epithelioid PT in transcriptome and proteome data. Given that our transcriptomic data were obtained from formalin-fixed paraffin-embedded tissues and some genes may have been degraded, we considered the credibility of the proteomic data. Notably, most samples included in this study were borderline PTs, and the similarities and differences between gene expressions or protein abundance in epithelioid, benign and malignant PTs remain unknown. Overall, our data suggest that epithelioid PTs behave as distinct subgroups of borderline PTs. The clinical phenotypes, including recurrence and metastatic potential, warrant long-term follow-up studies.
Further immunohistochemical validation showed that p16 and Rb were negative or partially weakly positive in benign and borderline PTs. Epithelioid PT is similar in immunophenotype to classical malignant PT, with abnormal expression of p16 and/or RB. Consistent with our findings, Cimino-Mathews et al9 also described a group of malignant PTs with p16/RB alterations and found an inverse relationship between p16 and RB expressions. Kuijper et al10 and Karim et al11 found an increase in p16 and Rb expressions using immunohistochemistry with increasing PT grades. These results suggest that epithelioid PT has partial characteristics of malignant PT. Esposito et al12 found no significant association between p16 expression using immunohistochemistry and PT grade but did not investigate Rb expression.
Notably, p16 and RB are key regulators of cell cycle and proliferation.13–15 When growth factors bind to their corresponding receptors, they promote cell cycle-dependent protein kinase phosphorylation of RB through signal transduction. Phosphorylated RB dissociates from E2F, and E2F promotes cell cycle-related protein expression, leading to the cell cycle transition from the G1 to S phase. The protein p16 is an inhibitor of cyclin-dependent kinase D1, and it inhibits the functions of Rb and entry into the cell cycle. Abnormal expression of p16 and/or Rb in epithelioid PT may affect the proliferation of tumour cells by regulating the cell cycle. Furthermore, due to the small sample size, our results may have limitations. Further research is needed to expand the sample size to confirm our results and confirm the role of p16/Rb in epithelioid PT.
Conclusion
This study is the first to report the clinicopathological and molecular features of seven PT cases with epithelioid morphology. Epithelioid PT differs from classical PT using transcriptomics and proteomics, but has a high recurrence rate, moderate atypia, often accompanied by sarcomatoid transformation and an immunophenotype similar to that of malignant PT. It may be a special morphological subgroup of borderline PT and predisposes to malignant transformation, which should be paid attention to in pathological diagnosis and clinical management.
Data availability statement
Data are available on reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants. This study was approved and supervised by the research ethics committee of Shenzhen Hospital of Chinese Traditional Medicine (approval number: K2022-020-02). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We would like to express our sincere gratitude to the late Professor Gong Xiyu, my breast pathology supervisor, for providing inspiration for the concept of this study. In addition, we thank Dr Jintao Hu, Shenzhen People’s Hospital, and Dr Hongping Tang, Shenzhen Maternity and Child Healthcare Hospital, for providing two interesting cases.
Supplementary materials
Supplementary Data
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Footnotes
Handling editor L C Collins.
Contributors MS, GM and XG performed study concept and design. MS, LZ and JB performed development of methodology and writing, review and revision of the paper. MS, LZ, XL and HX provided acquisition, analysis and interpretation of data and statistical analysis. XY, XJ and YL performed immunohistochemistry, transmission electron microscopy and immunofluorescence experiments. All authors read and approved the final paper. MS is the guarantor.
Funding This work was supported by the Shenzhen Science and Technology Innovation Project under grant JCYJ20220531092402004.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.