Cancer stem cell and epithelial-mesenchymal transition markers predict worse outcome in metaplastic carcinoma of the breast.

Metaplastic breast carcinomas are known to overexpress markers of epithelial-mesenchymal transition and cancer stem cells. We evaluated their immunohistochemical expression, correlating with clinicopathological parameters and survival outcomes. The study cohort comprised 63 cases diagnosed at the Department of Pathology, Singapore General Hospital. Tumor size, grade, lymph node stage, and metaplastic components were reviewed. Immunohistochemistry was performed on sections cut from tissue microarray blocks. Antibodies to ER, PR, HER2, CK14, EGFR, 34ßE12, cancer stem cell markers (CD44, CD24, ALDH1A1), epithelial-mesenchymal transition markers (Twist and E-cadherin), were applied. Survival outcomes were correlated with immunohistochemical findings. T2 tumors accounted for 74.7 % of cases, with grade 3 tumors predominating (71.4 %). Triple negativity occurred in 87.3 %, and basal-like subtype in 69.8 % of tumors. CD44+, CD44+CD24-, ALDH1A1+, loss of membranous E-cadherin (Ecadloss) and positive Twist expression was found in 82.5, 73.0, 77.8, 54.0, and 57.1 % of tumors, respectively. Combinational phenotypes of CD44+EcadlossTwist+, CD44+CD24-EcadlossTwist+, and ALDH1A1+EcadlossTwist+ were observed in 28.6, 25.4, and 2.6 % of tumors. Histologic grade was significantly correlated with E-cadherin loss (p = 0.042), Twist positivity (P = 0.001), CD44+EcadlossTwist+ (P = 0.010), CD44+CD24-EcadlossTwist+ (P = 0.018), and ALDH1A1+EcadlossTwist+(P = 0.010). Lymph node stage was significantly associated with CD44+EcadlossTwist+(P = 0.044) and CD44+CD24-EcadlossTwist+ (P = 0.044). Basal-like phenotype was significantly correlated with CD44 expressing (P = 0.004) and CD44+CD24- tumors (P = 0.049). Tumors harboring CD44+EcadlossTwist+ and CD44+CD24-EcadlossTwist+ phenotypes disclosed early recurrence (P = 0.027, P = 0.006) and poorer overall survival (P = 0.037, P = 0.006), respectively. Expression of cancer stem cell and epithelial-mesenchymal transition markers in metaplastic breast cancers correlates with adverse pathological parameters and outcome.

Expression of FGFR1 is an independent prognostic factor in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) are clinically aggressive tumors with limited treatment options. We examined the clinicopathological associations and prognostic implications of FGFR1 and FGFR2 expression in TNBCs. Tissue microarrays constructed from TNBCs were immunostained with FGFR1 and FGFR2, and scored by intensity and percentage of tumor cells stained per intensity for each subcellular compartment, which were correlated with clinicopathological parameters and survival. Cell migration following siRNA-mediated silencing of the FGFR1 gene in TNBC cell lines was also performed. 714 cases were informative for FGFR1 and FGFR2 immunostaining. Thresholds were defined as at least 1 % of cells stained and H-score of 100 or more. Proportions positive by each threshold were, respectively, 89.9 %, 7.1 % for FGFR1 (cytoplasm); 36.8 %, 7.8 % for FGFR2 (cytoplasm); and 33.5 %, 5.2 % for FGFR2 (membrane). Significant associations included FGFR1 and FGFR2 immunostaining for lobular subtype, FGFR2 immunostaining with lower grade, and more basal-like cancers with H-scores of 100 or more FGFR1 immunostaining. Multivariate Cox regression analysis showed FGFR1 expression in TNBCs to be independently prognostic for overall survival (OS) at both thresholds. Cases completely negative (less than 1 % staining) for FGFR1 immunostaining showed improved OS, while those with H-score of 100 or more immunostaining had the worst OS. Cell line studies revealed up-regulation of the FGFR1 gene in the MDA-MB-231 and Hs578T TNBC cells, and specific knockdown of FGFR1 expression significantly reduced cell migration in MDA-MB-231 cell line. In conclusion, FGFR1 expression in TNBCs is independently prognostic of OS, and H-score of 100 or more FGFR1 immunostaining may define tumors that have treatment potential via FGFR signaling inhibition.

CD117 expression in breast phyllodes tumors correlates with adverse pathologic parameters and reduced survival.

CD117 (c-kit) is a type III receptor tyrosine kinase encoded by the KIT gene. Deregulation of expression and mutations in the gene are implicated in various tumors. Reports of CD117 expression in phyllodes tumors have been controversial. We aim to investigate the protein expression of CD117 and mutations in the KIT gene in phyllodes tumors, and correlate the findings with pathological parameters and clinical outcome. A total of 272 cases were included in this study. CD117 expression was investigated by immunohistochemistry on tissue microarray sections. Toluidine blue staining was performed to indicate mast cells. Overall, 28 (10%) cases were CD117 positive. CD117 expression was significantly associated with tumor grade (P<0.001), increased stromal hypercellularity (P=0.003), stromal atypia (P=0.01), and stromal mitotic activity (P<0.001), permeative microscopic margins (P=0.002), and presence of hemorrhage (P=0.001). Expression was also associated with poorer overall survival (P=0.003). Nineteen cases were further selected for mutation screening through the Affymetrix OncoScan platform. No mutation of the KIT gene was found. Despite a lack of mutations in the KIT gene, CD117 protein expression is associated with unfavorable pathological parameters and poorer prognosis, suggesting an underlying role in the biology of phyllodes tumors.

Novel genetic aberrations in breast phyllodes tumours: comparison between prognostically distinct groups.

Phyllodes tumours of the breast are uncommon fibroepithelial neoplasms which pose management challenges due to difficulties in accurate prediction of clinical behaviour, as histological assessment has its limitations. Molecular studies have improved the understanding of these rare tumours but such findings are scant. We aimed to investigate genetic aberrations in phyllodes tumours stratified according to clinical behaviour, to identify potential genes contributing to disease progression. Twenty phyllodes tumours were separated into prognostically distinct categories depending on whether they had recurred/metastasized within the follow-up period. DNA extracted from FFPE materials was subjected to Affymetrix OncoScan™ FFPE Express molecular inversion probe microarray platform for analysis of copy number changes and mutational status. Results were cross validated with Sanger sequencing, FISH and immunohistochemistry. A higher number of chromosomal aberrations were observed in cases which recurred/metastasized, with median events of 19 compared to 3.5 in cases which did not recur/metastasize. High-level amplification and homozygous deletions were detected exclusively in the former group. Regions of high-level amplification included MDM4 (1q32.1), RAF1 (3p25), EGFR (7p12) and PDZD2 (5p13.3). EGFR amplification was confirmed on FISH and accompanied by intense EGFR immunostaining. Regions of homozygous deletion included CDKN2A (9p21) and MACROD2 (20p12.1). Homozygous deletion of 9p21 which involved CDKN2A was accompanied by loss of protein expression. No mutations were identified in all samples. These findings provide insights into identifying target genes and pathways exploited by phyllodes tumours, which would aid future development of individualised therapy.