Human Pathology (2015) 46, 327–333
www.elsevier.com/locate/humpath
Case study
Benign phyllodes tumor of the breast recurring as a malignant phyllodes tumor and spindle cell metaplastic carcinoma☆,☆☆ Kristen E. Muller DO a,c , Laura J. Tafe MD a,c , Francine B. de Abreu PhD a,c , Jason D. Peterson MS a,c , Wendy A. Wells MD a,c , Richard J. Barth MD b,c , Jonathan D. Marotti MD a,c,⁎ a
Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 Department of Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 c Geisel School of Medicine at Dartmouth, Hanover, NH 03755 b
Received 31 July 2014; revised 13 October 2014; accepted 22 October 2014
Keywords: Phyllodes tumor; Metaplastic breast cancer; Molecular testing; FBXW7; Immunohistochemistry
Summary We report a unique case of a 59-year-old woman diagnosed with a benign phyllodes tumor (PT), which recurred twice in the same location over a 7-year period: first as a malignant PT and then as a malignant PT with coexisting spindle cell metaplastic breast carcinoma (MBC). The MBC was differentiated from the malignant PT by expression of cytokeratins (CKs) AE1/AE3, CK MNF-116, CK 5/6, and p63. Somatic mutation analysis using a next-generation sequencing platform revealed a shared mutation in F-box and WD repeat domain containing 7, a tumor suppressor gene that encodes a ubiquitin ligase–associated protein, in the original benign PT and the first recurrent malignant PT. Chromosomal microarray analysis showed shared genetic gains and losses between the malignant PT and MBC. This case highlights the utility of immunohistochemistry to differentiate malignant PT from spindle cell MBC, describes a novel mutation in PT, and demonstrates a biologic relationship between these 2 entities. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
☆ Competing interests: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. ☆☆ Funding/Support: This work was supported by the Department of Pathology Translational Research Program and the Pathology Shared Resource of the Dartmouth-Hitchcock Medical Center and Norris Cotton Cancer Center. ⁎ Corresponding author. Department of Pathology, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. E-mail address: [email protected] (J. D. Marotti).
http://dx.doi.org/10.1016/j.humpath.2014.10.014 0046-8177/© 2015 Elsevier Inc. All rights reserved.
Phyllodes tumors (PT) are uncommon biphasic fibroepithelial lesions of the breast, characterized by proliferation of both the epithelial and stromal components and comprise less than or equal to 1% of all primary breast tumors [1]. The current World Health Organization classification system proposes a 3-tiered grading system: benign, borderline, and malignant, with most PTs (75%) characterized as benign [1]. Classification is based on a constellation of histologic features including tumor border, stromal cellularity, stromal atypia, mitotic activity, stromal overgrowth, and malignant heterologous elements [1]. The treatment for all PT includes at least surgical excision with
328 negative margins, as positive margins are strongly associated with local recurrence [2]. Patients with borderline and malignant PT may also benefit from adjuvant radiotherapy [3]. Although PTs were once considered to be purely “stromal driven,” there is increasing evidence that the epithelium is not a bystander and that the epithelial-stromal interaction is likely critical for the development and progression of PT [4]. Furthermore, PT epithelium can harbor chromosomal abnormalities [5] and can give rise to an array of benign and malignant changes, including both in situ and invasive carcinoma. Interestingly, the invasive carcinoma can consist of metaplastic breast carcinoma (MBC), which has only been recognized in rare isolated case reports [6-11]. In most of these cases, the metaplastic component was either monophasic squamous cell carcinoma or spindle cell carcinoma [6-11]. Differentiating the malignant stromal component of PT from a spindle cell MBC can be diagnostically challenging, as there is potential for considerable histologic overlap [6]. Specifically, both can show malignant spindle cells, frequent mitosis, squamous differentiation, and heterologous chondroid or osteoid elements. We herein report an unusual case of a 59-year-old woman originally diagnosed with a benign PT, which recurred twice over a 7-year period with eventual transformation into a malignant PT with coexisting spindle cell MBC. We performed somatic mutation analysis using a next-generation sequencing platform and chromosomal microarray analysis (CMA) on the tumors to evaluate for underlying genetic alterations that may be contributing to the progression of the PT and development of MBC. In addition, the case highlights the utility of immunohistochemical studies to distinguish malignant PT from spindle cell MBC.
2. Case study 2.1. Clinical findings A 59-year-old postmenopausal woman with no family history of breast or ovarian cancer presented for a selfpalpated right breast mass. A 3.5 cm well-circumscribed mass was identified in the upper outer quadrant of the right breast during a diagnostic mammogram. An excisional biopsy revealed a benign PT, focally present at the margins. A reexcision showed two microscopic foci of residual benign PT, which were completely excised with negative margins. Nineteen months after the initial presentation, the patient palpated a mass in the right breast in the area of the prior lesion. A mammogram revealed a new 3.8 cm mass at the site of the previous benign PT. This lesion was excised, and histologic findings were consistent with a malignant PT. Reexcision margins were negative for tumor. The patient received adjuvant radiotherapy and was followed up with annual mammograms and examinations. Four years later, a screening mammogram revealed a new density in the right lateral breast in the prior operative site, and an ultrasound
K. E. Muller et al. confirmed a 1.8 cm mass. The patient's workup at that time, which included a bone scan and computed tomography scans of the chest, abdomen, and pelvis, was negative for metastatic disease. After a core needle biopsy that showed a malignant spindle cell lesion concerning for MBC, the patient underwent a right simple mastectomy with sentinel node excision. The final diagnosis was spindle cell MBC arising in a recurrent malignant PT.
3. Materials and methods 3.1. Immunohistochemical analysis Immunohistochemical staining was performed on 4-μmthick formalin-fixed, paraffin-embedded tissue sections using standard automated methodology (Leica Bond; Leica Microsystems, Bannockburn, IL). Antibodies tested included cytokeratins (CKs) AE1/AE3 (clone AE1/AE3, 1:140; Biogenex, Freemont, CA), CK 5/6 (clone D5 & 16B4, 1:200; Leica Concentrate, Rocklin, CA), CK MNF-116 (clone MNF116, 1:200; Dako, Carpenteria, CA), p63 (clone 4A4, 1:200; Biocare, Concord, CA), and CD31 (clone JC70A, 1:150; Dako). Standard immunohistochemical protocols were followed with control slides as appropriate.
3.2. Microdissection and DNA extraction Unstained slides from formalin-fixed, paraffin-embedded tissue blocks were obtained, and areas of interest were macrodissected from each. DNA was extracted with either the Gentra Puregene Kit (for next-generation sequencing) or the QIAamp DNA FFPE Tissue Kit (for chromosomal microarray) (Qiagen, Valencia, CA), according to the manufacturer's instructions. DNA was quantified using the PicoGreen method Quant-iT PicoGreen(r) dsDNA Kit, Invitrogen, Paisley, UK.
3.3. Next-generation sequencing Sequencing was performed on the patient's benign PT, the first recurrent malignant PT, and the second recurrent PT and coexisting MBC using Life Technology's Ion Torrent AmpliSeq Cancer Hotspot Panel version 2 (Grand Island, NY), which has been previously validated and described [12]. The panel consists of 207 amplicons covering more than 20 000 bases of 50 genes with known cancer associations.
3.4. CMA We performed high-resolution genome-wide DNA copy number and single nucleotide polymorphism analysis on the first malignant PT and the MBC using the Affymetrix OncoScan FFPE Assay (Santa Clara, CA) according to the manufacturer's protocol. There was insufficient tissue to test
Phyllodes tumor and metaplastic carcinoma the second malignant PT. The Nexus Express and Chromosome Analysis Suite software packages were used for analysis (Affymetrix).
4. Results 4.1. Histopathologic findings The original right breast mass consisted of a 3.0 cm wellcircumscribed, gray, multilobulated mass. Histologic sections revealed a circumscribed lesion with a pericanalicular growth pattern, low-to-moderate stromal cellularity, minimal stromal cell atypia, absent stromal overgrowth and mitoses, and focal stromal condensation around ducts and cleft-like spaces, consistent with a benign PT (Fig. 1). The first recurrence 19 months later grossly measured 3.0 cm and contained an irregular cystic cavity with solid foci of tanpink soft tissue and pale yellow gelatinous material. Microscopic examination revealed a malignant PT with marked stromal cellularity and stromal cell atypia, greater than 10 mitoses/10 high-power fields, and pushing and infiltrating margins (Fig. 2).
329 Heterologous elements and stromal overgrowth were not present. Prior surgical site changes were noted; however, residual benign PT was not identified. Core biopsy of the second recurrence 4 years later showed malignant spindle cells with increased mitotic activity and necrosis. Immunohistochemical stains performed on the core biopsy demonstrated focal immunoreactivty for CK AE1/AE3, CK MNF-116, CK 5/6, and CK MNF-116 as well as focal p63 immunoreactivity. A diagnosis of “malignant spindle cell lesion” was rendered, and the possibility of an MBC was raised. The patient's subsequent mastectomy specimen contained a 3.4 cm firm, white mass with irregular borders. The mass was comprised predominantly of highly atypical spindle cells arranged in short fascicles with increased mitotic figures (5-9 mitoses/10 high-power fields), pushing and infiltrating borders, and necrosis. Similar to the core biopsy, the spindle cells in the mastectomy specimen were focally immunoreactive for CK AE1/AE3, CK 5/6, and CK MNF-116 and displayed focal nuclear staining for p63 (Fig. 3). The spindle cells were negative for CD31. A single microscopic section contained a 0.2 cm adjacent nodule of PT that was morphologically similar to the malignant PT in the patient's previous excision. This
Fig. 1 Original benign PT. A, Solitary solid lesion with smooth borders on ultrasound. B, Well-circumscribed fibroepithelial neoplasm with cystic components. C, Prominent leaf-like architecture and areas of hypocellular stroma. D, Bland stromal spindle cells without mitoses, nuclear atypia, or stromal overgrowth.
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Fig. 2 First recurrent malignant PT. A, Lobulated fibroepithelial neoplasm with leaf-like architecture, pushing borders, and a variably cellular stroma. B, Hypercellular stroma composed of pleomorphic spindle cells. C, Nuclear atypia and numerous mitoses (arrowheads) are present.
focal residual PT lacked immunoreactivity for the above CKs and p63. One sentinel lymph node was negative for malignancy. The final diagnosis was spindle cell MBC arising in a recurrent malignant PT (Fig. 3).
growth factor receptor (EGFR), v-myc avian myelocytomatosis viral oncogene homolog (MYC) or Cyclin-dependent kinases regulatory subunit 1 (CKS1B).
5. Discussion 4.2. Molecular findings Next-generation sequencing revealed a shared F-box and WD repeat domain containing 7 (FBXW7) mutation in the benign PT and the first recurrent malignant PT. Both tumors contained a single nucleotide change, c.1394GNA, resulting in an amino acid change from arginine to histidine in a known mutational hotspot positioned at Arg465 (p.R465H). The second malignant PT recurrence and coexisting MBC lacked any identifiable mutations with this panel. Between the first malignant PT and MBC, the OncoScan microarray showed a number of shared alterations including gains in 5p and complete gains of chromosomes 7 and 8; losses in 4p, 9p, 18p and 22q; and deletion of chromosome 10. In addition, the MBC had deletions in 1p and 11p, gain of 1q, complete gain of chromosome 5, and loss of chromosome 18 as well as additional smaller deletions (Fig. 4). Specific gene amplification was not detected in Epidermal
We report a case of spindle cell MBC arising in a recurrent malignant PT and detail the immunohistochemistry, somatic mutation, and CMA findings. The distinction between malignant PT and spindle cell MBC is critical because both treatment options and prognosis differ; the two entities share common histologic patterns and can be challenging to differentiate without ancillary studies. This case highlights the morphologic overlap that can be seen and the utility of immunohistochemical studies in this setting. When a predominant spindle cell component is present, the differential diagnosis should include (but is not limited to) a PT with stromal overgrowth versus spindle cell MBC. It is important to assess the epithelial component, as the presence of in situ carcinoma favors the diagnosis of MBC over PT [13]; however, occasionally, PT may also contain in situ carcinoma. The spindle cells in both lesions can have either low- or highgrade cytologic atypia, and both can have spindle cells that are
Phyllodes tumor and metaplastic carcinoma
331
Fig. 3 Metaplastic carcinoma arising in recurrent PT. A, Two distinct lesions, a spindle cell lesion with central necrosis (left) and a separate nodule of residual PT (right, circled). B, Residual malignant PT. C, Hypercellular areas of malignant spindle cells arranged in short fascicles. The malignant spindle cells are immunoreactive for a panel of CKs and p63: CKAE1/AE3 (D), CK5/6 (E), and p63 (F).
sarcomatoid in appearance. Furthermore, heterologous elements, most commonly of osteoid or chondroid differentiation, can be present in both malignant PT and MBC. These mesenchymal elements can range from benign metaplasias to osteosarcomatous and chondrosarcomatous in morphology. Immunohistochemistry can play a critical role differentiating between PT and MBC. The immunoprofile of MBC includes variable expression of broad spectrum and high molecular weight CK markers such as AE1/AE3, MNF-116, and 5/6 [14]. Perhaps, more importantly, the nuclear antigen p63 as a marker for MBC has been described with a sensitivity and specificity of 86.7% and 99.4%, respectively [15]. In contrast, the stromal cells of PT typically express CD34, vimentin, desmin, B cell lymphoma 2 (bcl-2), and variable S100 and historically have been thought to be negative for epithelial markers [14]. Although CK expression is the most
useful in distinguishing these lesions, it too has its pitfalls. MBC may show only selective and focal CK immunoreactivity, which is why a broad panel of keratin markers is required to demonstrate unequivocal epithelial differentiation. On the other hand, Chia et al [16] showed that stromal cells in malignant PT have, on occasion, demonstrated focal CK staining. In this same study, no PT was positive for p63. In our case, the malignant spindle cells were immunoreactive for a panel of CK immunostains as well as p63 supporting the diagnosis of spindle cell MBC arising within recurrent malignant PT. Here, not unlike prior studies, we saw a relative paucity of somatic mutations in PT. Tan et al [17] did not identify any mutations in 20 PT using OncoScan, and Korcheva et al [18] describe the absence of activating mutations in 26 PT using the Sequenom, San Diego, CA MassARRAY system. Interestingly, in our case, the benign PT and the first
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Chromosome
Malignant PT MBC
Chromosome
Malignant PT MBC
Fig. 4 Chromosomal microarray findings on the OncoScan array for the malignant PT and the MBC. Chromosomes 1 to 22 and X and Y are shown; the blue bars indicate gains, the red bars losses, and the golden bars loss of heterozygosity. The two tumors share several gains and losses with some additional copy number changes in the MBC.
recurrent malignant PT harbored the same FBXW7 p.R465H mutation. The FBXW7 gene, located on chromosome 4, encodes for the F box/WD repeat-containing protein 7. This protein binds directly to cyclin E, targeting cyclin E for ubiquitin-mediated degradation and has a cumulative mutation frequency in human cancers of 6% [19]. The second malignant PT and coexisting MBC, which arose after radiation therapy and a span of 4 years, lacked any mutations tested on the 50 gene “hotspot” panel. Although a speculation, the absence of the FBXW7 mutation in the second malignant PT and coexisting MBC could have been due to proliferation of a malignant PT clone that lacked the mutation (tumoral heterogeneity) after radiation therapy, which then gave rise to the MBC. Alternatively, the mutation could have reverted back to the wild-type sequence. Such “revertant mutations” can also be radiation associated. In this study, the CMA findings revealed multiple copy number changes in the first malignant PT and the MBC. Both tumors showed a number of shared gains and losses: gain in 5p and complete gains of chromosomes 7 and 8; losses in 4p, 9p, 18p and 22q as well as deletion of chromosome 10. The MBC additionally had deletions in 1p and 11p and gain of 1q, complete gain of chromosome 5, and loss of chromosome 18 as well as a number of smaller deletions. These findings support the theory that the first malignant PT and the MBC are biologically related and that accumulation of additional chromosomal abnormalities might be driving the MBC. Jones et al [20] report recurrent large copy number changes in malignant phyllodes, some of which were also seen in our cases, including gains in 1p, 5p, and chromosomes 7 and 8 and deletion of 9p, 10p, and chromosomes 6 and 13. In summary, this unusual case documents the histologic progression of a benign to malignant PT and development of coexisting spindle cell MBC. Both the benign and malignant PT contained a shared mutation in the FBXW7 gene, which is a previously undescribed molecular alteration in PT.
Although the relevance of the FBXW7 mutation remains unclear, the role of ubiquitin-like and ubiquitin-associated domains in PT warrants further investigation in a larger series of cases. Furthermore, MBC arising in the setting of a malignant PT is an extremely rare occurrence; however, an appropriate immunohistochemical panel is composed of multiple CKs and p63 can aid in distinguishing between these two lesions.
References [1] Tan PH, Tse G, Lee A. Fibroepithelial tumours. In: Lakhani SR, Ellis IO, Schnitt SJ, et al, editors. WHO Classification of Tumours of the Breast. 4th ed. Lyon, France: International Agency for Research on Cancer (IARC); 2012. p. 143-7. [2] Tan PH, Thike AA, Tan WJ, et al. Predicting clinical behavior of breast phyllodes tumours: a nomogram based on histological criteria and surgical margins. J Clin Pathol 2012;65:69-76. [3] Barth Jr RJ, Wells WA, Mitchell SE, et al. A prospective, multiinstitutional study of adjuvant radiotherapy after resection of malignant phyllodes tumors. Ann Surg Oncol 2009;16:2288-94. [4] Jara-Lazaro AR, Tan PH. Molecular pathogenesis of progression and recurrence in breast phyllodes tumors. Am J Transl Res 2009;1:23-34. [5] Sawyer EJ, Hanby AM, Ellis P, et al. Molecular analysis of phyllodes tumors reveals distinct changes in the epithelial and stromal components. Am J Pathol 2000;156:1093-8. [6] Ramdass MJ, Dindyal S. Phyllodes breast tumour showing invasive squamous-cell carcinoma with invasive ductal, clear-cell, secretory, and squamous components. Lancet Oncol 2006;7:880. [7] Sharma R, Usmani S, Siegel R. Primary squamous cell carcinoma of breast in background of phyllodes tumor—a case report. Conn Med 2009;73:341-3. [8] Kinkor Z, Sticova E, Sach J, et al. Sarcomatoid (metaplastic) spindle cell carcinoma arising in a phylloid tumor with massive squamous metaplasia—a case report and review of the literature. Cesk Patol 2012;48:156-60. [9] Hunger E, Turk R, Wurster K. Malignant cystosarcoma phylloides and squamous cell carcinoma of the breast—a rare tumour combination. Geburtshilfe Frauenheilkd 1984;44:640-2.
Phyllodes tumor and metaplastic carcinoma [10] de Rosa G, Ferrara G, Goglia P, et al. In situ and microinvasive carcinoma with squamoid differentiation arising in a phyllodes tumor: report of a case. Tumori 1989;75:514-7. [11] Leible S, Gogg-Kammerer M, Sommersacher A, et al. Metaplastic breast carcinomas: are they of myoepithelial differentiation? Immunohistochemical profile of the sarcomatoid subtype using novel myoepithelial markers. Am J Surg Pathol 2005;29:347-53. [12] Tsongalis GJ, Peterson JD, de Abreu FB, et al. Routine use of the Ion Torrent AmpliSeq™ Cancer Hotspot Panel for identification of clinically actionable somatic mutations. Clin Chem Lab Med 2014; 52:707-14. [13] Davis WG, Hennessy B, Babiera G, et al. Metaplastic sarcomatoid carcinoma of the breast with absent or minimal overt invasive carcinomatous component: a misnomer. Am J Surg Pathol 2005;29: 1456-63. [14] Dunne B, Lee AH, Pinder SE, et al. An immunohistochemical study of metaplastic spindle cell carcinoma, phyllodes tumor and fibromatosis of the breast. HUM PATHOL 2003;34:1009-15.
333 [15] Koker MM, Kleer CG. p63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma. Am J Surg Pathol 2004;28:1506-12. [16] Chia Y, Thike AA, Cheok PY, et al. Stromal keratin expression in phyllodes tumours of the breast: a comparison with other spindle cell breast lesions. J Clin Pathol 2012;65:339-47. [17] Tan WJ, Lai JC, Thike AA, et al. Novel genetic aberrations in breast phyllodes tumours: comparison between prognostically distinct groups. Breast Cancer Res Treat 2014;145:635-45. [18] Korcheva VB, Levine B, Beadling C, et al. Immunohistochemical and molecular markers in breast phyllodes tumors. Appl Immunohistochem Mol Morphol 2011;19:119-25. [19] Akhoondi S, Sun D, von der Lehr N, et al. FBXW7/hCDC4 is a general tumor suppressor in human cancer. Cancer Res 2007;67:9006-12. [20] Jones AM, Mitter R, Springall R, et al. Phyllodes Tumour Consortium. A comprehensive genetic profile of phyllodes tumours of the breast detects important mutations, intra-tumoral genetic heterogeneity and new genetic changes on recurrence. J Pathol 2008;214:533-44.
www.elsevier.com/locate/humpath
Case study
Benign phyllodes tumor of the breast recurring as a malignant phyllodes tumor and spindle cell metaplastic carcinoma☆,☆☆ Kristen E. Muller DO a,c , Laura J. Tafe MD a,c , Francine B. de Abreu PhD a,c , Jason D. Peterson MS a,c , Wendy A. Wells MD a,c , Richard J. Barth MD b,c , Jonathan D. Marotti MD a,c,⁎ a
Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 Department of Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 c Geisel School of Medicine at Dartmouth, Hanover, NH 03755 b
Received 31 July 2014; revised 13 October 2014; accepted 22 October 2014
Keywords: Phyllodes tumor; Metaplastic breast cancer; Molecular testing; FBXW7; Immunohistochemistry
Summary We report a unique case of a 59-year-old woman diagnosed with a benign phyllodes tumor (PT), which recurred twice in the same location over a 7-year period: first as a malignant PT and then as a malignant PT with coexisting spindle cell metaplastic breast carcinoma (MBC). The MBC was differentiated from the malignant PT by expression of cytokeratins (CKs) AE1/AE3, CK MNF-116, CK 5/6, and p63. Somatic mutation analysis using a next-generation sequencing platform revealed a shared mutation in F-box and WD repeat domain containing 7, a tumor suppressor gene that encodes a ubiquitin ligase–associated protein, in the original benign PT and the first recurrent malignant PT. Chromosomal microarray analysis showed shared genetic gains and losses between the malignant PT and MBC. This case highlights the utility of immunohistochemistry to differentiate malignant PT from spindle cell MBC, describes a novel mutation in PT, and demonstrates a biologic relationship between these 2 entities. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
☆ Competing interests: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. ☆☆ Funding/Support: This work was supported by the Department of Pathology Translational Research Program and the Pathology Shared Resource of the Dartmouth-Hitchcock Medical Center and Norris Cotton Cancer Center. ⁎ Corresponding author. Department of Pathology, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. E-mail address: [email protected] (J. D. Marotti).
http://dx.doi.org/10.1016/j.humpath.2014.10.014 0046-8177/© 2015 Elsevier Inc. All rights reserved.
Phyllodes tumors (PT) are uncommon biphasic fibroepithelial lesions of the breast, characterized by proliferation of both the epithelial and stromal components and comprise less than or equal to 1% of all primary breast tumors [1]. The current World Health Organization classification system proposes a 3-tiered grading system: benign, borderline, and malignant, with most PTs (75%) characterized as benign [1]. Classification is based on a constellation of histologic features including tumor border, stromal cellularity, stromal atypia, mitotic activity, stromal overgrowth, and malignant heterologous elements [1]. The treatment for all PT includes at least surgical excision with
328 negative margins, as positive margins are strongly associated with local recurrence [2]. Patients with borderline and malignant PT may also benefit from adjuvant radiotherapy [3]. Although PTs were once considered to be purely “stromal driven,” there is increasing evidence that the epithelium is not a bystander and that the epithelial-stromal interaction is likely critical for the development and progression of PT [4]. Furthermore, PT epithelium can harbor chromosomal abnormalities [5] and can give rise to an array of benign and malignant changes, including both in situ and invasive carcinoma. Interestingly, the invasive carcinoma can consist of metaplastic breast carcinoma (MBC), which has only been recognized in rare isolated case reports [6-11]. In most of these cases, the metaplastic component was either monophasic squamous cell carcinoma or spindle cell carcinoma [6-11]. Differentiating the malignant stromal component of PT from a spindle cell MBC can be diagnostically challenging, as there is potential for considerable histologic overlap [6]. Specifically, both can show malignant spindle cells, frequent mitosis, squamous differentiation, and heterologous chondroid or osteoid elements. We herein report an unusual case of a 59-year-old woman originally diagnosed with a benign PT, which recurred twice over a 7-year period with eventual transformation into a malignant PT with coexisting spindle cell MBC. We performed somatic mutation analysis using a next-generation sequencing platform and chromosomal microarray analysis (CMA) on the tumors to evaluate for underlying genetic alterations that may be contributing to the progression of the PT and development of MBC. In addition, the case highlights the utility of immunohistochemical studies to distinguish malignant PT from spindle cell MBC.
2. Case study 2.1. Clinical findings A 59-year-old postmenopausal woman with no family history of breast or ovarian cancer presented for a selfpalpated right breast mass. A 3.5 cm well-circumscribed mass was identified in the upper outer quadrant of the right breast during a diagnostic mammogram. An excisional biopsy revealed a benign PT, focally present at the margins. A reexcision showed two microscopic foci of residual benign PT, which were completely excised with negative margins. Nineteen months after the initial presentation, the patient palpated a mass in the right breast in the area of the prior lesion. A mammogram revealed a new 3.8 cm mass at the site of the previous benign PT. This lesion was excised, and histologic findings were consistent with a malignant PT. Reexcision margins were negative for tumor. The patient received adjuvant radiotherapy and was followed up with annual mammograms and examinations. Four years later, a screening mammogram revealed a new density in the right lateral breast in the prior operative site, and an ultrasound
K. E. Muller et al. confirmed a 1.8 cm mass. The patient's workup at that time, which included a bone scan and computed tomography scans of the chest, abdomen, and pelvis, was negative for metastatic disease. After a core needle biopsy that showed a malignant spindle cell lesion concerning for MBC, the patient underwent a right simple mastectomy with sentinel node excision. The final diagnosis was spindle cell MBC arising in a recurrent malignant PT.
3. Materials and methods 3.1. Immunohistochemical analysis Immunohistochemical staining was performed on 4-μmthick formalin-fixed, paraffin-embedded tissue sections using standard automated methodology (Leica Bond; Leica Microsystems, Bannockburn, IL). Antibodies tested included cytokeratins (CKs) AE1/AE3 (clone AE1/AE3, 1:140; Biogenex, Freemont, CA), CK 5/6 (clone D5 & 16B4, 1:200; Leica Concentrate, Rocklin, CA), CK MNF-116 (clone MNF116, 1:200; Dako, Carpenteria, CA), p63 (clone 4A4, 1:200; Biocare, Concord, CA), and CD31 (clone JC70A, 1:150; Dako). Standard immunohistochemical protocols were followed with control slides as appropriate.
3.2. Microdissection and DNA extraction Unstained slides from formalin-fixed, paraffin-embedded tissue blocks were obtained, and areas of interest were macrodissected from each. DNA was extracted with either the Gentra Puregene Kit (for next-generation sequencing) or the QIAamp DNA FFPE Tissue Kit (for chromosomal microarray) (Qiagen, Valencia, CA), according to the manufacturer's instructions. DNA was quantified using the PicoGreen method Quant-iT PicoGreen(r) dsDNA Kit, Invitrogen, Paisley, UK.
3.3. Next-generation sequencing Sequencing was performed on the patient's benign PT, the first recurrent malignant PT, and the second recurrent PT and coexisting MBC using Life Technology's Ion Torrent AmpliSeq Cancer Hotspot Panel version 2 (Grand Island, NY), which has been previously validated and described [12]. The panel consists of 207 amplicons covering more than 20 000 bases of 50 genes with known cancer associations.
3.4. CMA We performed high-resolution genome-wide DNA copy number and single nucleotide polymorphism analysis on the first malignant PT and the MBC using the Affymetrix OncoScan FFPE Assay (Santa Clara, CA) according to the manufacturer's protocol. There was insufficient tissue to test
Phyllodes tumor and metaplastic carcinoma the second malignant PT. The Nexus Express and Chromosome Analysis Suite software packages were used for analysis (Affymetrix).
4. Results 4.1. Histopathologic findings The original right breast mass consisted of a 3.0 cm wellcircumscribed, gray, multilobulated mass. Histologic sections revealed a circumscribed lesion with a pericanalicular growth pattern, low-to-moderate stromal cellularity, minimal stromal cell atypia, absent stromal overgrowth and mitoses, and focal stromal condensation around ducts and cleft-like spaces, consistent with a benign PT (Fig. 1). The first recurrence 19 months later grossly measured 3.0 cm and contained an irregular cystic cavity with solid foci of tanpink soft tissue and pale yellow gelatinous material. Microscopic examination revealed a malignant PT with marked stromal cellularity and stromal cell atypia, greater than 10 mitoses/10 high-power fields, and pushing and infiltrating margins (Fig. 2).
329 Heterologous elements and stromal overgrowth were not present. Prior surgical site changes were noted; however, residual benign PT was not identified. Core biopsy of the second recurrence 4 years later showed malignant spindle cells with increased mitotic activity and necrosis. Immunohistochemical stains performed on the core biopsy demonstrated focal immunoreactivty for CK AE1/AE3, CK MNF-116, CK 5/6, and CK MNF-116 as well as focal p63 immunoreactivity. A diagnosis of “malignant spindle cell lesion” was rendered, and the possibility of an MBC was raised. The patient's subsequent mastectomy specimen contained a 3.4 cm firm, white mass with irregular borders. The mass was comprised predominantly of highly atypical spindle cells arranged in short fascicles with increased mitotic figures (5-9 mitoses/10 high-power fields), pushing and infiltrating borders, and necrosis. Similar to the core biopsy, the spindle cells in the mastectomy specimen were focally immunoreactive for CK AE1/AE3, CK 5/6, and CK MNF-116 and displayed focal nuclear staining for p63 (Fig. 3). The spindle cells were negative for CD31. A single microscopic section contained a 0.2 cm adjacent nodule of PT that was morphologically similar to the malignant PT in the patient's previous excision. This
Fig. 1 Original benign PT. A, Solitary solid lesion with smooth borders on ultrasound. B, Well-circumscribed fibroepithelial neoplasm with cystic components. C, Prominent leaf-like architecture and areas of hypocellular stroma. D, Bland stromal spindle cells without mitoses, nuclear atypia, or stromal overgrowth.
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Fig. 2 First recurrent malignant PT. A, Lobulated fibroepithelial neoplasm with leaf-like architecture, pushing borders, and a variably cellular stroma. B, Hypercellular stroma composed of pleomorphic spindle cells. C, Nuclear atypia and numerous mitoses (arrowheads) are present.
focal residual PT lacked immunoreactivity for the above CKs and p63. One sentinel lymph node was negative for malignancy. The final diagnosis was spindle cell MBC arising in a recurrent malignant PT (Fig. 3).
growth factor receptor (EGFR), v-myc avian myelocytomatosis viral oncogene homolog (MYC) or Cyclin-dependent kinases regulatory subunit 1 (CKS1B).
5. Discussion 4.2. Molecular findings Next-generation sequencing revealed a shared F-box and WD repeat domain containing 7 (FBXW7) mutation in the benign PT and the first recurrent malignant PT. Both tumors contained a single nucleotide change, c.1394GNA, resulting in an amino acid change from arginine to histidine in a known mutational hotspot positioned at Arg465 (p.R465H). The second malignant PT recurrence and coexisting MBC lacked any identifiable mutations with this panel. Between the first malignant PT and MBC, the OncoScan microarray showed a number of shared alterations including gains in 5p and complete gains of chromosomes 7 and 8; losses in 4p, 9p, 18p and 22q; and deletion of chromosome 10. In addition, the MBC had deletions in 1p and 11p, gain of 1q, complete gain of chromosome 5, and loss of chromosome 18 as well as additional smaller deletions (Fig. 4). Specific gene amplification was not detected in Epidermal
We report a case of spindle cell MBC arising in a recurrent malignant PT and detail the immunohistochemistry, somatic mutation, and CMA findings. The distinction between malignant PT and spindle cell MBC is critical because both treatment options and prognosis differ; the two entities share common histologic patterns and can be challenging to differentiate without ancillary studies. This case highlights the morphologic overlap that can be seen and the utility of immunohistochemical studies in this setting. When a predominant spindle cell component is present, the differential diagnosis should include (but is not limited to) a PT with stromal overgrowth versus spindle cell MBC. It is important to assess the epithelial component, as the presence of in situ carcinoma favors the diagnosis of MBC over PT [13]; however, occasionally, PT may also contain in situ carcinoma. The spindle cells in both lesions can have either low- or highgrade cytologic atypia, and both can have spindle cells that are
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Fig. 3 Metaplastic carcinoma arising in recurrent PT. A, Two distinct lesions, a spindle cell lesion with central necrosis (left) and a separate nodule of residual PT (right, circled). B, Residual malignant PT. C, Hypercellular areas of malignant spindle cells arranged in short fascicles. The malignant spindle cells are immunoreactive for a panel of CKs and p63: CKAE1/AE3 (D), CK5/6 (E), and p63 (F).
sarcomatoid in appearance. Furthermore, heterologous elements, most commonly of osteoid or chondroid differentiation, can be present in both malignant PT and MBC. These mesenchymal elements can range from benign metaplasias to osteosarcomatous and chondrosarcomatous in morphology. Immunohistochemistry can play a critical role differentiating between PT and MBC. The immunoprofile of MBC includes variable expression of broad spectrum and high molecular weight CK markers such as AE1/AE3, MNF-116, and 5/6 [14]. Perhaps, more importantly, the nuclear antigen p63 as a marker for MBC has been described with a sensitivity and specificity of 86.7% and 99.4%, respectively [15]. In contrast, the stromal cells of PT typically express CD34, vimentin, desmin, B cell lymphoma 2 (bcl-2), and variable S100 and historically have been thought to be negative for epithelial markers [14]. Although CK expression is the most
useful in distinguishing these lesions, it too has its pitfalls. MBC may show only selective and focal CK immunoreactivity, which is why a broad panel of keratin markers is required to demonstrate unequivocal epithelial differentiation. On the other hand, Chia et al [16] showed that stromal cells in malignant PT have, on occasion, demonstrated focal CK staining. In this same study, no PT was positive for p63. In our case, the malignant spindle cells were immunoreactive for a panel of CK immunostains as well as p63 supporting the diagnosis of spindle cell MBC arising within recurrent malignant PT. Here, not unlike prior studies, we saw a relative paucity of somatic mutations in PT. Tan et al [17] did not identify any mutations in 20 PT using OncoScan, and Korcheva et al [18] describe the absence of activating mutations in 26 PT using the Sequenom, San Diego, CA MassARRAY system. Interestingly, in our case, the benign PT and the first
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Malignant PT MBC
Chromosome
Malignant PT MBC
Fig. 4 Chromosomal microarray findings on the OncoScan array for the malignant PT and the MBC. Chromosomes 1 to 22 and X and Y are shown; the blue bars indicate gains, the red bars losses, and the golden bars loss of heterozygosity. The two tumors share several gains and losses with some additional copy number changes in the MBC.
recurrent malignant PT harbored the same FBXW7 p.R465H mutation. The FBXW7 gene, located on chromosome 4, encodes for the F box/WD repeat-containing protein 7. This protein binds directly to cyclin E, targeting cyclin E for ubiquitin-mediated degradation and has a cumulative mutation frequency in human cancers of 6% [19]. The second malignant PT and coexisting MBC, which arose after radiation therapy and a span of 4 years, lacked any mutations tested on the 50 gene “hotspot” panel. Although a speculation, the absence of the FBXW7 mutation in the second malignant PT and coexisting MBC could have been due to proliferation of a malignant PT clone that lacked the mutation (tumoral heterogeneity) after radiation therapy, which then gave rise to the MBC. Alternatively, the mutation could have reverted back to the wild-type sequence. Such “revertant mutations” can also be radiation associated. In this study, the CMA findings revealed multiple copy number changes in the first malignant PT and the MBC. Both tumors showed a number of shared gains and losses: gain in 5p and complete gains of chromosomes 7 and 8; losses in 4p, 9p, 18p and 22q as well as deletion of chromosome 10. The MBC additionally had deletions in 1p and 11p and gain of 1q, complete gain of chromosome 5, and loss of chromosome 18 as well as a number of smaller deletions. These findings support the theory that the first malignant PT and the MBC are biologically related and that accumulation of additional chromosomal abnormalities might be driving the MBC. Jones et al [20] report recurrent large copy number changes in malignant phyllodes, some of which were also seen in our cases, including gains in 1p, 5p, and chromosomes 7 and 8 and deletion of 9p, 10p, and chromosomes 6 and 13. In summary, this unusual case documents the histologic progression of a benign to malignant PT and development of coexisting spindle cell MBC. Both the benign and malignant PT contained a shared mutation in the FBXW7 gene, which is a previously undescribed molecular alteration in PT.
Although the relevance of the FBXW7 mutation remains unclear, the role of ubiquitin-like and ubiquitin-associated domains in PT warrants further investigation in a larger series of cases. Furthermore, MBC arising in the setting of a malignant PT is an extremely rare occurrence; however, an appropriate immunohistochemical panel is composed of multiple CKs and p63 can aid in distinguishing between these two lesions.
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