Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity.

Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with liposarcomatous components. EGFR amplification was heterogeneous and present only in the non-heterologous component of one tumor with liposarcomatous differentiation. The results identify novel pathways involved in the pathogenesis of malignant phyllodes tumors, which significantly increase our understanding of tumor biology and have potential clinical impact.

The expression of succinate dehydrogenase in breast phyllodes tumor.

The purpose of this study is to investigate the expression of succinate dehydrogenase (SDH)A, SDHB, and HIF-1α in phyllodes tumors and the association with clinic-pathologic factors. Using tissue microarray (TMA) for 206 phyllodes tumor cases, we performed immunohistochemical stains for SDHA, SDHB, and HIF-1α and analyzed their expression in regard to clinicopathologic parameters of each case. The cases were comprised of 156 benign, 34 borderline, and 16 malignant phyllodes tumors. The expression of stromal SDHA and epithelial- and stromal- SDHB increased as the tumor progressed from benign to malignant (P⟨0.001). There were five stromal SDHA-negative cases and 31 stromal SDHB-negative cases. SDHB negativity was associated with a lower histologic grade (P=0.054) and lower stromal atypia (P=0.048). Univariate analysis revealed that a shorter disease free survival (DFS) was associated with stromal SDHB high-positivity (P=0.013) and a shorter overall survival (OS) was associated with high-positivity of stromal SDHA and SDHB (P⟨0.001 and P⟨0.001, respectively). The multivariate Cox analysis with the variables stromal cellularity, stromal atypia, stromal mitosis, stromal overgrowth, tumor margin, stromal SDHA expression, and stromal SDHB expression revealed that stromal overgrowth was associated with a shorter DFS (hazard ratio: 24.78, 95% CI: 3.126-196.5, P=0.002) and a shorter OS (hazard ratio: 176.7, 95% CI: 8.466-3691, P=0.001). In conclusion, Tumor grade is positively correlated with SDHA and SDHB expression in the tumor stroma in phyllodes tumors of the breast. This result may be attributed to the increased metabolic demand in high grade tumors.

The expression of redox proteins in phyllodes tumor.

This study aimed to investigate the associations between the expression of redox-related proteins which regulate reactive oxygen species (ROS) production and the histologic factors in phyllodes tumor (PT). We used tissue microarrays to analyze 193 PTs and performed immunohistochemical staining against five redox-related proteins including catalase, thioredoxin reductase (TxNR), glutathione S-transferase π (GST π), thioredoxin interacting protein (TxNIP), and manganese superoxide dismutase (MnSOD). We then compared the immunohistochemical results and histologic parameters. The 193 PTs were classified as benign (n = 145, 75.1 %), borderline (n = 33, 17.1 %), and malignant (n = 15, 7.8 %). With worsening histologic grade, the expression of catalase, TxNR, TxNIP, and MnSOD in the stromal component increased (P < 0.001), and GST π and MnSOD expression in the epithelial component increased (P = 0.014, and 0.038). Significant associations were found between the expression of catalse-TxNR, catalase-TxNIP, catalase-MnSOD, TxNR-TxNIP, TxNR-MnSOD, and TxNIP-MnSOD in both the epithelial and stromal components (P < 0.05). This study confirmed that the stromal expression of catalase, TxNR, TxNIP, and MnSOD increased with worsening histologic grade in PT, reflecting the change in ROS production during the malignant transformation of PT.

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.

Telomerase activity in phyllodes tumours.

AIMS: To investigate telomerase, a ribonucleoprotein that synthesizes telomeres. Recent evidence suggests that telomerase reactivation is associated with the acquisition of immortalization and malignancy. METHODS: Using a sensitive PCR-based assay (the TRAP assay), we examined telomerase activity in two recurrent phyllodes tumours in two patients. RESULTS: Both tumours expressed telomerase activity. Histological examination of the lesions, according to the criteria proposed by Azzopardi and Salvadori, revealed a malignant phyllodes tumour in one patient and a benign phyllodes tumour in the other patient. CONCLUSIONS: Our findings suggest that telomerase activity may have a potential role as a prognostic marker in predicting the clinical behaviour of these rare tumours.

Telomerase activity in human breast tumors.

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is not detected in normal somatic cells; thus, with each cell division, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode. The current model gaining support is that telomerase activity in germline and immortal cells maintains telomere length and thus compensates for the "end-replication problem." PURPOSE: Our objective was to determine when telomerase activity is reactivated in the progression to malignant breast cancer and if knowledge of telomerase activity may be an indicator for the diagnosis and potential treatment of breast cancer. METHODS: Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 140 breast cancer specimens (from 140 patients), four phyllodes tumors (from four patients), 38 noncancerous lesions (20 fibroadenomas, 17 fibrocystic diseases, one gynecomastia; from 38 patients), and 55 adjacent noncancerous mammary tissues (from 55 of the 140 breast cancer patients). In addition, 33 fine-needle-aspirated breast samples (from 33 patients) were analyzed. RESULTS: Among surgically resected samples, telomerase activity was detected in 130 (93%) of 140 breast cancers. Telomerase activity was detected in 68% of stage I primary breast cancers, in 73% of cancers smaller than 20 mm, and in 81% of axillary lymph node-negative cancers. Moreover, the activity was detected in more than 95% of advanced stage tumors but in only two (4%) of 55 adjacent noncancerous tissues. While telomerase activity was not detected in any of 17 specimens of fibrocystic disease, surprisingly low levels of telomerase activity were detected in nine (45%) of 20 fibroadenomas. Among samples obtained by fine-needle aspiration, 14 (100%) of 14 patients whose fine-needle-aspirated specimen contained telomerase activity and who subsequently underwent surgery were confirmed to have breast cancer. Multivariate analysis of 125 specimens from patients for whom data were available on age at surgery, stage of disease, tumor size, lymph node status tumor histology, and menopausal status indicated that stage classification exhibited the strongest association with telomerase activity (for stage I versus stages II-IV: odds ratio = 1.0 versus 73.4; 95% confidence interval = 2.0-959.0; P = .02). CONCLUSION: Telomerase activity was detected in more than 95% of advanced stage breast cancers. It was absent in 19%-32% of less advanced cancers. Since a determination of any association between telomerase activity and patient survival is not possible at the present time, it remains to be determined whether lack of telomerase activity predicts for favorable outcome.

Dipeptidyl peptidase IV expression identifies a functional sub-population of breast fibroblasts.

The immunocytochemical distribution of the cell-surface enzyme dipeptidyl peptidase IV (DPP IV) has been studied in the human breast at the light and ultrastructural level. The presence of the enzyme was demonstrated on the cell membranes of interlobular fibroblasts, whilst intralobular fibroblasts were DPP-IV-negative. A fluorograph, after immunoprecipitation of 35S-methionine-labelled proteins of fibroblasts from primary breast cultures with an anti-serum to DPP IV, demonstrated a band at 135 kDa consistent with the presence of the enzyme. The clear delineation of 2 functionally distinct subpopulations of breast fibroblasts was maintained in benign fibro-adenomas and cystosarcoma phyllodes, both tumour types having growth characteristics of intralobular stroma. This observation has important implications for both normal breast biology and for breast carcinogenesis.

[Sarcoma phyllodes of the breast. Apropos of a case withan ultrastructural and histoenzymological study]. / Sarcome phyllode mammaire. A propos d'un cas avec étude ultrastructurale et histo enzymologique.

One case of cystosarcoma phyllodes is reported. Histologically, the neoplasm was composed of two elements: benign epithelial cells and malignant stromal cells. The stromal cells, spindle shaped, grow in solid sheets or in sparcely cellular, myxomatous and alcianophilic areas. Scanning electron microscopy demonstrates a mucous secretion, showing porelike openings and mucous droplets on the surface of tumoral cells.By transmission electron microscopy, sarcomatous cells look like very polymorphous. Secretory cells (showing dilated granular endoplasmic reticulum and abundant lysosomes) are mixed with primitive mesenchymal cells and with intermediate, myoepithelial cells. The myoid origin of these cells is demonstrated by the bundless of myofilaments terminated in marginal plaques on the plasma membrane, the numerous pinocytic vesicles and basal lamina investing irregularly the cells. Histoenzymology corroborates these findings, disclosing a high activity of alkaline phosphatase. Discovery of ambiguous myoepithelial cells associated with undifferentiated cells, likewise reported in epitheliomas, is of a great histogenetic interest. It suggests development of mammary neoplasm from stem cells coming under local of general influences, unknown today, but perhaps responsible of epithelial or conjunctive differentiation. The presence of a contralateral carcinoma in our case is consistent with this hypothesis.

Histochemistry of estrogen sulfatases in human breast diseases.

Two estrogen sulfatases, arylsulfatase C-estrone sulfatase (ASC-ES) and d-equilenin sulfatase (EqS) were demonstrated histochemically in the normal human female breast, in benign breast diseases and in infiltrating mammary ductal carcinomas to study their significance in the pathogenesis of epithelial proliferations. By hydrolyzing estrone sulfate, the amount of which in female blood is about ten times greater than that of estradiol or estrone, estrogen sulfatases can produce a high local concentration of estrogens. A simultaneous azo-coupling method for histochemical demonstration of ASC-ES is described in the present study; EqS was demonstrated by a previously described method. Estrogen sulfatases were not found in the normal female breast. Both estrogen sulfatases were found in epithelial cells in some examples of mastopathic disease and in fibroadenomas, while ASC-ES was found in periductal fibroblasts. In some cases of infiltrating ductal carcinomas, estrogen sulfatases were present in carcinoma cells. In most of these tumors ASC-ES activity was observed in fibroblasts around infiltrative cell cords. There was no correlation between the presence of estrogen sulfatases and of hormone receptors in carcinomas. It is concluded that estrogen sulfatases play no role in the early stages of benign or malignant epithelial proliferations. However, the induction of estrogen sulfatases may promote epithelial proliferation in some cases if estrogen receptors are present in epithelial cells.

Histochemically demonstrable enzyme activities and their independence of the hormone receptor content in female breast carcinoma.

Acid phosphatase, leucine aminopeptidase, monoamine oxidase and non specific esterase activities were histochemically demonstrated in specimens derived from 15 infiltrating ductal carcinomas of female breast. The relative areas occupied by the enzyme-positive carcinoma cells were visually estimated and, in the cases of leucine aminopeptidase, assessed morphometrically. All enzyme activities were found to be subject to major variations within a single carcinoma and between individual carcinomas, and the activity of any single enzyme was independent of that of three others. None of the enzyme activities correlated with the estrogen and progesterone receptor values, nor the histological grade of malignancy of the tumour. Thus, histochemically demonstrable enzyme activities seem to be of no use in predicting the hormone receptor content in infiltrating ductal carcinomas of the female breast.