Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast.

Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases having matched primary and metastatic tumors) were profiled using gene sequencing, gene copy number analysis, whole genome expression, and protein expression. Whole genome expression analysis demonstrated consistent over-expression of genes involved in angiogenesis including VEGFA, Angiopoietin-2, VCAM1, PDGFRA, and PTTG1. EGFR protein overexpression was observed in 26/27 (96%) of cases with amplification of the EGFR gene in 8/24 (33%) cases. Two EGFR mutations were identified including EGFRvIII and a presumed pathogenic V774M mutation, respectively. The most common pathogenic mutations included TP53 (50%) and PIK3CA (15%). Cases with matched primary and metastatic tumors harbored identical mutations in both sites (PIK3CA/KRAS and RB1 gene mutations, respectively). Tumor expression of PD-L1 immunoregulatory protein was observed in 3/22 (14%) of cases. Overexpression of molecular biomarkers of increased angiogenesis, EGFR and immune checkpoints provides novel targeted therapy options in malignant phyllodes tumors of the breast.

Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast.

INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1alpha and its downstream targets in phyllodes tumors and fibroadenomas of the breast. METHODS: The expression of HIF-1alpha, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium. RESULTS: Only two fibroadenomas displayed low-level stromal HIF-1alpha reactivity in the absence of CAIX expression. Stromal HIF-1alpha expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1alpha expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1alpha overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032). CONCLUSION: These results indicate that HIF-1alpha expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1alpha most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1alpha and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1alpha and CAIX seem to be of minor relevance in breast fibroadenomas.

Stromal nitric oxide synthase (NOS) expression correlates with the grade of mammary phyllodes tumour.

BACKGROUND: Nitric oxide synthase (NOS), particularly endothelial and inducible forms (e/i-NOS), are expressed in various cancers, including breast cancer. In mammary fibroepithelial lesions, NOS expression in stromal cells has been reported to be lower in fibroadenomas than in phyllodes tumours. AIMS: To investigate NOS expression in phyllodes tumours of varying degrees of malignancy. METHODS: One hundred and sixty seven mammary phyllodes tumours (97 benign, 47 borderline malignant, and 23 frankly malignant) were evaluated for e-NOS and i-NOS expression by immunohistochemistry. Correlations with previously reported expression of stromal vascular growth factor (VEGF) and microvessel density were also performed. RESULTS: Stromal expression of e-NOS was absent, weak, moderate, and strong in 43%, 31%, 13%, and 13% of benign tumours; 17%, 26%, 13%, and 44% of borderline malignant tumours; and 17%, 35%, 13%, and 35% of frankly malignant tumours, respectively. Stromal expression of i-NOS was 77%, 18%, 4%, and 1% in benign tumours; 42%, 28%, 19%, and 11% in borderline malignant tumours; and 43%, 13%, 26%, and 18% in frankly malignant tumours, respectively. Stromal expression of both i-NOS and e-NOS was significantly different between the benign and malignant (borderline and frank) groups of phyllodes tumours (p < 0.0001). Furthermore, the expression of i-NOS correlated with stromal VEGF expression and microvessel density. The expression of NOS in the epithelial cells was strong, and showed no differences between the different groups of tumours. CONCLUSIONS: Higher stromal expression of NOS in phyllodes tumours is associated with malignancy, suggesting a possible role in malignant progression, particularly metastasising potential.

Stromal expression of vascular endothelial growth factor correlates with tumor grade and microvessel density in mammary phyllodes tumors: a multicenter study of 185 cases.

A retrospective review of 185 mammary phyllodes tumors (105 benign, 51 borderline, 29 malignant) from 4 centers was performed by immunohistochemistry to investigate the expression of vascular endothelial growth factor in the epithelial and stromal cells of mammary phyllodes tumors. The correlation of vascular endothelial growth factor with tumor grade, stromal cell nuclear pleomorphism, cellularity, mitotic rate, margin histomorphology, and the stromal microvessel density was evaluated. Vascular endothelial growth factor expression was found in the epithelium in 29% and in the stromal cells in 31% of cases. There was significant increase of vascular endothelial growth factor expression in the stromal cells with increasing degree of malignancy, but not the epithelium. Microvessel density in the stroma also showed significant correlation with tumor malignancy, and a correlation was shown with the stromal vascular endothelial growth factor expression. Statistical overlap of stromal vascular endothelial growth factor and microvessel density in predicting malignancy suggests that angiogenesis may be an effector mechanism for vascular endothelial growth factor. Assessment of stromal VEGF may be useful as an adjunctive diagnostic criterion in the histologic assessment of malignancy in phyllodes tumors.

Increased microvessel density in malignant and borderline mammary phyllodes tumours.

AIMS: Tumour vascularity is considered a prognostic indicator in breast carcinoma, but its utility in mammary phyllodes tumour has not been explored. The authors report the correlation between intratumoral microvessel density and the histological grade of phyllodes tumour. METHODS AND RESULTS: Forty cases of phyllodes tumour were reviewed for stromal cellularity, overgrowth, cytological pleomorphism, mitotic count and margin pattern. Using established criteria, these were diagnosed as benign (n=28), borderline (n=10) and malignant (n=2). Microvessel density was counted on CD31-stained slides as the number of vessels per high power field. For benign phyllodes tumour, the range was 7-26.2 (mean 13.1); for borderline phyllodes tumour the range was 17.2-32.5 (mean 22.4); for malignant phyllodes tumour the range was 25.9-33.3 (mean 29.6). The difference between the benign and borderline groups was significant (P < 0.0001) but that between the borderline and malignant groups was not, due to the small number of malignant cases. CONCLUSIONS: There is a significant difference in stromal microvessel density between benign and borderline phyllodes tumour. Although the small number of cases of malignant phyllodes tumour limits further interpretation, we believe that microvessel density can be used as an additional objective histological parameter in the evaluation of phyllodes tumour.

Phyllodes tumors of the breast.

Our objective was to study the color and pulse Doppler ultrasonography of phyllodes tumors of the breast. The data of 36 phyllodes tumors (19 benign, 5 borderline, and 12 malignant) were reviewed retrospectively. The median age of the patients was 41.5 years (range 13-62 years), and the tumors varied in size from 1.5 to 20 cm (median 4.6 cm). Most of the phyllodes tumors were lobulated masses with smooth margins, mildly hypoechoic internal echo texture, a heterogeneous internal echo pattern, and no microcalcification. Vessels were detectable on 35 of these tumors, with 51.4% having resistance index >0.700, 45.7% having pulsatility index >1.300, and 51.4% having V(max) >15 cm/s. The color Doppler characteristics were of no significant use in predicting the histological nature of the phyllodes tumors. If the sonographic features suggest a phyllodes tumor, a histological examination should be conducted to confirm the diagnosis.

Immunohistochemical profile of cystosarcoma phyllodes of the breast: a study of 23 cases.

Cystosarcoma phyllodes (CP) of the breast is a rare biphasic tumor composed of benign epithelium and a spindle cell stroma. Biologic behavior of CP cannot be predicted with certainty on the basis of morphologic criteria only. We studied immunohistochemical expression of basic fibroblastic growth factor (bFGF), urokinase, Ki67, p53 protein, and microvessel density in stromal and epithelial components of 14 low-grade CP (LCP) and 9 high-grade CP (HCP). bFGF was more often positive in LCP than in HCP. The stroma was positive for bFGF in 86% of LCP and 67% of HCP, and the epithelium was positive in 64% of LCP and 14% of HCP. Urokinase was positive in stromal cells of 86% of LCP and 93% of HCP. The epithelial positivity for urokinase in both groups resembled closely that of the stroma. p53 protein was more often positive in stromal cells of HCP (67%) than in LCP (50%). Ki67 was positive in the stroma of 43% of LCP and 89% of HCP and in the epithelium of 14% of LCP and 33% of HCP. There was no significant difference in microvessel density (MVD) in low- and high-grade lesions. Our study demonstrates that stromal Ki67 and p53 immunohistochemical positivity are more often associated with high-grade tumors. The positive immunostaining for bFGF, urokinase, Ki67, and p53 in stroma and epithelium of the majority of CP supports the existence of epithelial-stromal interactions and recognizes epithelium as an integral part of this tumor.

Proliferative activity and tumor angiogenesis is closely correlated to stromal cellularity of fibroadenoma: proposal fibroadenoma, cellular variant.

Fibroadenoma (FA) is the most common benign tumor of the breast in adult women. Some FA have a highly cellular stroma, making it difficult to differentiate from phyllodes tumors (PT). Forty-three FA were grouped into: (i) 27 conventional type (FACT) median stromal cellularity (SC) of highest cellular area (HCA), < or = 125 cells/1 high-power field (HPF); and (ii) 16 cellular variant (FACV) median SC of HCA, > 125 cells/1 HPF. These were studied for the proliferative activity of their stromal cells. Expression of c-fos, p53, basic fibroblast growth factor (bFGF), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor (VEGF) in the stromal cells were examined in the FA and 12 PT to determine whether it is possible to separate FACV from FACT. The proliferative activity of stromal cells was evaluated by the labeling index (LI) of proliferating cell nuclear antigen (PCNA). Conventional type fibroadenoma stromal cells had the lowest frequency of c-fos, p53, bFGF, FGFR and VEGF protein expression; PT stromal cells had the highest frequency of expression; and FACV stromal cells had an intermediate frequency of expression. Multivariate analysis demonstrated that bFGF and FGFR expression are significantly correlated with SC of FA. Separation of FACV from FACT by SC seems appropriate in revealing the phenotypic and biological differences of FA. The SC of FA seems to be regulated by bFGF and FGFR expression.

Tumour angiogenesis and p53 protein expression in mammary phyllodes tumors.

We examined 186 phyllodes tumors (106 benign, 51 borderline, 29 malignant) for angiogenesis by assessing stromal microvessel density by the hot spot method and assessing p53 protein expression; we correlated these factors with stromal cellularity, margin status, nuclear pleomorphism, mitosis, and stromal overgrowth. Increased degree of malignancy in phyllodes tumors is associated with increased patient age and tumor size. Microvessel density and p53 protein expression also showed a similar increase with malignancy. Using a logistic regression model, microvessel density was shown to be useful in predicting malignancy in phyllodes tumors, independent of key criteria of stromal overgrowth, nuclear pleomorphism, and mitosis. Microvessel density showed correlation with stromal cellularity and margin status, suggesting an interrelationship between these parameters. P53 protein expression showed a positive correlation with microvessel density, suggesting possible overlap in the underlying mechanism of these two factors in the pathogenesis of phyllodes tumors. The numbers of recurrences and metastases are small in our series, and no significant difference was demonstrated in microvessel density and p53 protein expression compared with the primary. We conclude that microvessel density and p53 are useful as independent criteria in evaluating malignancy in phyllodes tumors.

Aggressive giant fibroepithelial lesion with unusual vascular stroma--a case report.

The stroma of fibroadenoma and phyllodes tumor usually consists of fibroblastic proliferation. Rarely the stroma contains bundles of smooth muscle. Pseudoangiomatous hyperplasia of the mammary stroma has been described in fibroadenomas. However, true benign vascular stroma has not been reported. We report a case of a 34-year-old Chinese woman who presented with a large mass occupying the entire left breast. Left mastectomy was performed and showed a large, well-circumscribed, lobulated, rubbery-firm tumor measuring 13 x 10 x 6 cm. Microscopic examination revealed a fibroepithelial tumor formed by an organoid pattern of ductal structures with a very striking stromal appearance composed of extensive vascular proliferation and that demonstrated strong immunoreactivity for CD31, CD34, and Factor VIII. Ultrastructural examination revealed intercellular junctions, basal lamina, pinocytotic vesicles, and Weibel-Palade bodies in the cells lining the vascular spaces, confirming their endothelial nature. These findings rule out the diagnosis of pseudoangiomatous hyperplasia. The patient developed local recurrence a year later, and the resection showed malignant phyllodes tumor with ductal carcinoma in situ. The extensive vascular stroma noted in the primary tumor may have played a role in the malignant transformation of the epithelial and stromal components in this tumor.

Massive infarction of a recurrent phyllodes tumor of the breast: MRI-findings.

We report the imaging findings in an infarcted phyllodes tumor of the breast. A 40-year-old woman presented ten months after surgery for a benign phyllodes tumour with a palpable lump under the scar. We performed clinical examination, mammography, sonography, and MRI. The radiological diagnosis was a postoperative hematoma with granulation tissue. Surgery was performed. Histological diagnosis revealed an infarcted phyllodes tumour. This case illustrates the radiological presentation of a completely infarcted phyllodes tumour. As far as we know, imaging findings of an infarcted phyllodes tumour have not been reported yet.

Immunohistochemical studies on expression of human vascular smooth muscle myosin heavy chain isoforms in normal mammary glands, benign mammary disorders and mammary carcinomas.

The expression of myosin in normal and diseased mammary glands of 199 Japanese women was evaluated immunohistochemically by the avidin-biotin peroxidase complex method using antibodies to three human smooth muscle myosin heavy chain isoforms derived from the vascular smooth muscle: myosin SM1 is expressed consistently from fetal stage to adulthood, myosin SM2 appears only in well-differentiated smooth muscle after birth, and myosin SMemb is more abundant in embryonic aortas. SM1 was expressed in myoepithelial cells of normal mammary glands and fibrocystic diseases and in myoepithelial-like tumor cells in the basal layer of fibroadenomas and phyllodes tumors. SM2 was expressed only in the myoepithelial cells of mammary glands in breastfeeding women. SMemb was expressed more intensely in the cytoplasm of luminal epithelial cells in larger fibroadenomas (P< 0.01), or in the cytoplasm of carcinoma cells in invasive ductal carcinomas with metastasized lymph nodes (P< 0.001) and in those of higher histological grade (P<0.0001). Multivariate logistic analysis showed a significant correlation only between the expression of SMemb and histological grade (P< 0.0001), which is a prognostic factor of mammary carcinomas. These findings suggested the possible prognostic value of SMemb.