Precise diagnosis of breast phyllodes tumors using Raman spectroscopy: Biochemical fingerprint, tumor metabolism and possible mechanism.

BACKGROUND: Breast fibroadenomas and phyllodes tumors are both fibroepithelial tumors with comparable histological characteristics. However, rapid and precise differential diagnosis is a tough point in clinical pathology. Given the tendency of phyllodes tumors to recur, the difficulty in differential diagnosis with fibroadenomas leads to the difficulty in optimal management for these patients. METHOD: In this study, we used Raman spectroscopy to differentiate phyllodes tumors from breast fibroadenomas based on the biochemical and metabolic composition and develop a classification model. The model was validated by 5-fold cross-validation in the training set and tested in an independent test set. The potential metabolic differences between the two types of tumors observed in Raman spectroscopy were confirmed by targeted metabolomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 204 patients with formalin-fixed paraffin-embedded (FFPE) tissue samples, including 100 fibroadenomas and 104 phyllodes tumors were recruited from April 2014 to August 2021. All patients were randomly divided into the training cohort (n = 153) and the test cohort (n = 51). The Raman classification model could differentiate phyllodes tumor versus fibroadenoma with cross-validation accuracy, sensitivity, precision, and area under curve (AUC) of 85.58 % ± 1.77 %, 83.82 % ± 1.01 %, 87.65 % ± 4.22 %, and 93.18 % ± 1.98 %, respectively. When tested in the independent test set, it performed well with the test accuracy, sensitivity, specificity, and AUC of 83.50 %, 86.54 %, 80.39 %, and 90.71 %. Furthermore, the AUC was significantly higher for the Raman model than that for ultrasound (P = 0.0017) and frozen section diagnosis (P < 0.0001). When it came to much more difficult diagnosis between fibroadenoma and benign or small-size phyllodes tumor for pathological examination, the Raman model was capable of differentiating with AUC up to 97.45 % and 95.61 %, respectively. On the other hand, targeted metabolomic analysis, based on fresh-frozen tissue samples, confirmed the differential metabolites (including thymine, dihydrothymine, trans-4-hydroxy-l-proline, etc.) identified from Raman spectra between phyllodes tumor and fibroadenoma. SIGNIFICANCE AND NOVELTY: In this study, we obtained the molecular information map of breast phyllodes tumors provided by Raman spectroscopy for the first time. We identified a novel Raman fingerprint signature with the potential to precisely characterize and distinguish phyllodes tumors from fibroadenoma as a quick and accurate diagnostic tool. Raman spectroscopy is expected to further guide the precise diagnosis and optimal treatment of breast fibroepithelial tumors in the future.

Single cell profiling of female breast fibroadenoma reveals distinct epithelial cell compositions and therapeutic targets.

Fibroadenomas (FAs) are the most common breast tumors in women. No pharmacological agents are currently approved for FA intervention owing to its unclear mechanisms and a shortage of reproducible human models. Here, using single-cell RNA sequencing of human FAs and normal breast tissues, we observe distinct cellular composition and epithelial structural changes in FAs. Interestingly, epithelial cells exhibit hormone-responsive functional signatures and synchronous activation of estrogen-sensitive and hormone-resistant mechanisms (ERBB2, BCL2 and CCND1 pathways). We develop a human expandable FA organoid system and observe that most organoids seem to be resistant to tamoxifen. Individualized combinations of tamoxifen with ERBB2, BCL2 or CCND1 inhibitors could significantly suppress the viability of tamoxifen-resistant organoids. Thus, our study presents an overview of human FA at single-cell resolution that outlines the structural and functional differences between FA and normal breast epithelium and, in particular, provides a potential therapeutic strategy for breast FAs.

Oestrogen And Progesterone Receptors' Expression Pattern In Fibro-Adenoma Of The Female Breast.

BACKGROUND: Fibro-adenoma is the most common benign condition of the female breast comprising about 68% of all breast lumps. Fibroadenoma is an independent risk factor for the development of breast cancer. Complex fibroadenoma has a 2-3-fold increased risk ratio and simple fibroadenoma has 1.49 times increased risk ratio of developing cancer than the normal population over a period of 20 years. This study aimed to qualitatively check the frequency of oestrogen receptor-positive and progesterone receptor-positive cases of fibroadenoma in our region. METHODS: This cross-sectional study was conducted in the pathology department of Ayub Medical College, Abbottabad from June 2020 to December 2021. Biopsy confirmed cases of fibroadenoma were examined using immune-histochemical stains to score qualitatively the expression pattern of ER and PR. Data was analyzed and assessed using SPSS version 25. A p-value of ≤0.05 was considered statistically significant. RESULTS: The mean age of patients who presented with fibro-adenoma was 24.5±9.29 years with a median age of 21.5 years. In most cases, oestrogen receptor expression was mild 23 (54.76%) whereas progesterone receptor expression was severe 19 (45.23%). On chi-square test, the pattern of progesterone receptor expression for the category of hormone intake showed significant differences. Whereas, the pattern of oestrogen receptor expression for the categories of marital status, history of hormone intake, history of menstrual cycle and type of fibroadenoma showed no statistically significant difference. CONCLUSIONS: Further study into the pathogenesis of fibroadenoma is required to understand the role of ER and PR and explore the therapeutic potential of such drugs that affects these receptors. Cabling.

Comparative Proteomic Profiling of Secreted Extracellular Vesicles from Breast Fibroadenoma and Malignant Lesions: A Pilot Study.

Extracellular vesicles (EVs) shuttle proteins, RNA, DNA, and lipids crucial for cell-to-cell communication. Recent findings have highlighted that EVs, by virtue of their cargo, may also contribute to breast cancer (BC) growth and metastatic dissemination. Indeed, EVs are gaining great interest as non-invasive cancer biomarkers. However, little is known about the biological and physical properties of EVs from malignant BC lesions, and even less is understood about EVs from non-malignant lesions, such as breast fibroadenoma (FAD), which are clinically managed using conservative approaches. Thus, for this pilot study, we attempted to purify and explore the proteomic profiles of EVs from benign breast lesions, HER2+ BCs, triple-negative BCs (TNBCs), and continuous BC cell lines (i.e., BT-549, MCF-10A, and MDA-MB-231), combining experimental and semi-quantitative approaches. Of note, proteome-wide analyses showed 49 common proteins across EVs harvested from FAD, HER2+ BCs, TNBCs, and model BC lines. This is the first feasibility study evaluating the physicochemical composition and proteome of EVs from benign breast cells and primary and immortalized BC cells. Our preliminary results hold promise for possible implications in precision medicine for BC.

Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers.

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no ß-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

Organochlorine Pesticide Tissue Levels in Benign and Malignant Breast Disease: A Comparative Exploratory Study.

Exposure to organochlorine pesticides (OCPs) may be a risk factor for breast cancer (BC). Their role may be more relevant in developing countries such as India, where an abundance of these products is used for agricultural purposes. The present study compares OCP tissue levels in patients who underwent BC surgery (group A) or patients who had surgery for excision of breast fibroadenoma (group B). We perform OCP level quantification using a PerkinElmer, Inc. (Waltham, MA) gas chromatograph (GC) that is equipped with a 63Ni selective electron-capture detector. Significantly higher breast tissue OCP levels are present in the study population, indicating significant exposure. We detect 18 different types of OPCs in study subjects, with six OPCs (γ-hexachlorocyclohexane [HCH], δ-HCH, endrin, endosulfan-II, p,p'-dichlorodiphenyldichloroenthane [DDD], and p,p'-dichlorodiphenyltrichloroenthane [DDT]) present in all subjects. Endosulfan-II, p,p'-DDT, and p,p'-DDD tissue levels are significantly higher in BC patients than in those with fibroadenoma. Higher tissue levels of OCPs (α-HCH) are significantly associated with the presence of extracapsular spread (1.42 vs. 0.91; p = 0.04) and higher disease stage (early BC vs. locally advanced BC; 18.90 vs. 11.90; p = 0.04). The present pilot study indicates higher OCP tissue levels in northern India BC patients compared to patients with fibroadenoma.

P53 Expression in benign Breast Disease Development: A Systematic Review.

BACKGROUND: Benign breast disease (BBD) is one of main breast cancer risk factors. Dysfunctions on p53 protein, which has a genome protective role, have been related to breast cancer developments. However, its role on BBD development is still unclear. METHODS: A systematic review of literature was proceeded according to PRISMA-P guidelines. PubMed, BVS, MEDLINE and Scholar Google were used as databases, complemented by a manual search in articles references.  Articles searches were conducted from May to July 2019 and publications in English, Spanish and Portuguese were selected. P53 expression was set as outcome among women with BBD and were included only articles with good quality according STROBE tools. Data concerning p53 expression frequencies were independently extracted by two review authors, and eligible articles were synthesized. RESULTS: From 12 studies selected for this review, the majority analyzed p53 expression in non-proliferative lesions and general p53 expressions ranged from 0 to 100%. P53 expression was more frequently observed in cases series studies (91.7%) and in studies conducted in Occidental Europe (41.7%). P53 expression was more frequent among tissues with fibrocystic disease (22.5%) and fibroadenoma (22.5%). CONCLUSION: When compared with all breast tissues types, benign breast disease corresponds to 34.39% of p53 expression. Second outcomes were not evaluated because the heterogeneity observed in selected studies. In addition, more studies considering ethnicity and benign breast disease classification should also be considered for further analysis.

A case-control study of Metallothionein-1 expression in breast cancer and breast fibroadenoma.

The overexpression of Metallothionein-1 (MT-1) may play an important role in breast cancer; however, few studies have compared MT-1 expression between breast cancer and fibroadenoma. A cross-sectional controlled study was performed in 66 premenopausal women, aged 20-49 years, who had been histologically diagnosed with breast fibroadenoma or breast cancer. The patients were divided into two groups: group A, control (fibroadenoma, n = 36) and group B, study (breast cancer, n = 30). Immunohistochemistry was performed on tissue samples of fibroadenoma and breast cancer patients to evaluate the expression of metallothionein using an anti-MT-1 polyclonal antibody (rabbit polyclonal anti-metallothionein-Catalog Number biorbyt-orb11042) at a dilution of 1:100. The data were analyzed using NOVA (p < 0.05). Microscopic analysis showed a higher concentration of anti-MT-1-stained nuclei in breast cancer tissues than in fibroadenoma tissues. The mean proportion of cells with anti-MT-1-stained nuclei was 26.93% and 9.10%, respectively, in the study and control groups (p < 0.001). Histological grade 3 tumors showed a significantly higher MT-1 expression than hitological grade 1 (p < 0.05), while breast tumors negative for estrogen-, progesterone- and HER2- receptors had a significantly higher MT-1 expression than positive breast tumors positive for these parameters (p < 0.05). MT-1 protein in women of reproductive age was significantly higher in breast cancer than in fibroadenoma in this study. Furthermore, there was higher MT-1 immunoreactivity in more aggressive tumors.

Patients With Invasive Ductal Carcinoma Have Reduced Levels of Decorin Expression in Their Breast Tissue Compared to Patients With Fibroadenoma While Plasma Decorin Remains Unchanged.

BACKGROUND: Reduction in the level of tissue decorin is a hallmark of many types of cancer including breast carcinoma. However, reduced decorin expression has also been reported in several types of benign tumors to the extent that it has been proposed as a tissue marker to differentiate malignant from benign tumors. The aim of this study was to investigate the potential role of plasma decorin to distinguish breast cancer from fibroadenoma, the second most common type of benign tumor, after fibrocystic disease. METHODS: From 35 patients recruited in this study, 24 were affected with invasive ductal carcinoma, either grade II (n = 14) or grade III (n = 10). The other 11 patients had fibroadenoma lesions in their breasts. Tissue decorin mRNA and protein levels were assessed with real-time qPCR and Immunohistochemical analysis. ELISA was employed to measure plasma levels of decorin. RESULTS: The mean plasma decorin in cancer patients was measured to be 5.42 ± 1.83 ng/mL while fibroadenoma patients had an average of 4.22 ± 1.17 ng/mL decorin in their plasma. The difference was not significant. However, the mean expression level of decorin mRNA calculated by the 2-ΔΔCt method was 5.6-fold lower in the biopsied tissue specimens of IDC patients versus fibroadenoma, as expected. Consistent reduction in protein abundance was observed in the studied tissue sections. CONCLUSION: We have shown that tissue decorin is a reliable marker, unaffected by patient disease stage, to differentiate IDC from fibroadenoma. However, plasma decorin does not seem to have diagnostic value in this regard.

Genetics and genomics of breast fibroadenomas.

Fibroadenomas of the breast are benign fibroepithelial tumours most frequently encountered in women of reproductive age, although they may be diagnosed at any age. The fibroadenoma comprises a proliferation of both stromal and epithelial components. The mechanisms underlying fibroadenoma pathogenesis remain incompletely understood. In the clinical setting, distinguishing cellular fibroadenomas from benign phyllodes tumours is a common diagnostic challenge due to subjective histopathological criteria and interobserver differences. Recent sequencing studies have demonstrated the presence of highly recurrent mutations in fibroadenomas, and also delineated the genomic landscapes of fibroadenomas and the closely related phyllodes tumours, revealing differences at the gene level, which may be of potential adjunctive diagnostic use. The present article provides an overview of key studies uncovering genetic and genomic abnormalities in fibroadenomas, from initial karyotype reports revealing myriad cytogenetic aberrations to next-generation sequencing-based approaches that led to the discovery of highly recurrent MED12 mutations. A thorough understanding of these abnormalities is important to further elucidate the mechanisms by which fibroadenomas arise and to refine diagnostic assessment of this very common tumour.

Interaction with the GDF8/11 pathway reveals treatment options for adenocarcinoma of the breast.

Breast adenocarcinoma continues to be the most frequently diagnosed tumor entity. Despite established therapy options, mortality for breast cancer remains to be as high as 40,000 patients in the US annually. Thus, a need to develop a patient-oriented, targeted therapy exists. In this study, we investigated the interaction of breast adenocarcinoma with the ubiquitously present protein Follistatin and subsequently the GDF8/11 pathway. We analyzed primary histological samples from adenocarcinoma patients for expression of Follistatin and GDF8/11. Furthermore, expression levels of Follistatin and GDF8/11 in MCF7 were compared with MCF10a cells. From the resulting data, GDF8 and Follistatin were used as chemotherapeutic agents in MCF7 cells and their migratory, proliferative behavior and viability were measured. From the experiments, we were able to detect a significantly increased expression of Follistatin and GDF8/11 in the low malignant breast adenocarcinoma (G1) as compared to benign breast fibroadenoma. Interestingly, a decrease was demonstrated in higher grade malignancies. These findings were accompanied by the clinical observation that increased expression of Follistatin and GDF8 is associated with a higher overall survival rate of breasts cancer patients. Substitution of GDF8 and Follistatin reduces the viability of the MCF7 cells and disrupts the migrative and proliferative potential. In summary, MCF7 cells show high chemosensitivity to Follistatin and especially GDF8 and both proteins might serve as targets to improve systemic treatment in breast cancer. In contrast to most established chemotherapy regimens Follistatin and GDF8 show no cytotoxicity to other organs.

Molecular alterations in lesions of anogenital mammary-like glands and their mammary counterparts including hidradenoma papilliferum, intraductal papilloma, fibroadenoma and phyllodes tumor.

Lesions affecting anogenital mammary-like glands (AGMLG) are histopathologically very similar to those seen in the breast but whether this morphological similarity is also reflected at the genetic level is unknown. To compare the underlying molecular mechanisms in lesions of AGMLG and their mammary counterparts, we analyzed the mutational profile of 16 anogenital neoplasms including 5 hidradenomas papilliferum (HP), 1 lesion with features of HP and fibroadenoma (FA), 7 FA, 3 phyllodes tumors (PhT)) and 18 analogous breast lesions (6 intraductal papillomas (IDP), 9 FA, and 3 PhT) by high-coverage next generation sequencing (NGS) using a panel comprising 50 cancer-related genes. Additionally, all cases were analyzed for the presence of a mutation in the MED12 gene. All detected mutations with allele frequencies over 20% were independently validated by Sanger sequencing (concordance: 100%). Mutations in PIK3CA, AKT1, MET, ABL1 and TP53 genes were found in lesions of AGMLG and also their mammary counterparts. The PI3K-AKT cascade plays a role in tumors arising at both sites. It appears that some histopathologically similar anogenital and breast lesions develop along similar molecular pathways.

Cytokine profiles of tumor supernatants in invasive ductal cancer and fibroadenoma of the breast and its relationship with VEGF-A expression in the tumors.

Interrelations between cytokines, produced by invasive ductal carcinoma (IDC) and fibroadenoma (FA) of the breast, and angiogenic growth factor VEGF-A, expressed in IDC and FA, were investigated. The analysis of the cytokine profiles of IDC and FA was performed by cultivation of tumor biopsy specimens in vitro. Testing of the cytokine-producing reserve of the tumors for production of VEGF-A was conducted by culturing samples of IDC and FA in a medium containing polyclonal activator (a complex of phytohemagglutinin, concanavalin A, and lipopolysaccharide). Levels of cytokines and growth factors (IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1ß, IL-1Ra, TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF-A) and MCP-1 (monocyte chemoattractant protein-1) in tumor supernatants were determined by an ELISA. Expression of VEGF-A was analyzed in tumor biopsy specimens by immunohistochemical analysis. In the IDC supernatants, the concentrations of IL-17, IL-18, and IFN-γ were higher and the concentrations of IL-10 and MCP-1 were lower in comparison with the FA supernatants. We observed negative correlations between the macrophage infiltration and VEGF-A concentration in the IDC supernatants (r = -0.508; P = 0.011) and between VEGF-A expression and the IDC vascularization degree (r = -0.423, P = 0.039). Spontaneous expression of VEGF-A in samples of IDC significantly exceeded the VEGF-A expression in FA. There was no difference between IDC and FA in VEGF-A expression after treatment with the polyclonal activators. Our results indicate that greater malignancy may have a paradoxical effect that is controlled by cytokines and characterized by weakening of tumor angiogenesis during overproduction of VEGF-A. These findings point to complex mechanisms of positive and negative regulation of tumor angiogenesis by cytokines that are produced by the tumor and by cells in its microenvironment, whose cytokine profiles may change at different stages of tumor progression.

Fibroadenoma with "immature-like" type of usual ductal hyperplasia.

We herein report a case of the breast fibroadenoma with foci of so-called immature variant of the conventional ductal hyperplasia. This type of usual ductal hyperplasia is histologically characterised by encircling intraductal proliferation of large cells with pale to amphophilic cytoplasm and large nuclei which vary in shape and in staining quality of the chromatin. We showed here, using the cytokeratin immunohistochemistry, that the proliferating cells were not of immature but rather mature immunohistochemical phenotype. Because of the presented discordance between immature histology and mature immunohistological profile we suggest that this rare type of usual ductal hyperplasia should be called "immature-like".

Phyllodes Tumor of the Breast: Histopathologic Features, Differential Diagnosis, and Molecular/Genetic Updates.

CONTEXT: -Phyllodes tumor (PT) of the breast is a rare fibroepithelial neoplasm with risks of local recurrence and uncommon metastases. The classification proposed by the World Health Organization for PTs into benign, borderline, and malignant is based on a combination of several histologic features. The differential diagnosis between PT and fibroadenoma and the histologic grading of PT remain challenging. In addition, the molecular pathogenesis of PT is largely unknown. OBJECTIVE: -To provide an updated overview of pathologic features, diagnostic terminology, and molecular alterations of PT. DATA SOURCES: -Current English literature related to PT of the breast. CONCLUSIONS: -Phyllodes tumor shows a wide spectrum of morphology. There are no clearly distinct boundaries between PT and fibroadenoma. Strict histologic assessment of a combination of histologic features with classification can help to achieve the correct diagnosis and provide useful clinical information. The genomic landscapes of PT generated from genomic sequencing provide insights into the molecular pathogenesis of PT and help to improve diagnostic accuracy and identify potential drug targets in malignant PT.

Loss of imprinting of IGF2 in fibroadenomas and phyllodes tumors of the breast.

Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) is thought to be implicated in the pathogenesis of some tumors by upregulating IGF2 mRNA but its role in the pathogenesis of fibroadenomas (FAs) and phyllodes tumors (PTs) of the breast is yet to be studied. LOI of IGF2 was investigated in 25 FAs and 17 PTs which were heterozygous for Apa I polymorphism, and was found to be present in 13 FAs and 12 PTs. IGF2 mRNA expression was more upregulated in FAs and PTs than in paired surrounding normal tissues and laser microdissection showed that IGF2 mRNA expression was significantly higher in the stromal than the epithelial cells. LOI was not associated with upregulation of IGF2 mRNA, nor were MED12 mutations and methylation status of the differentially methylated region 0 (DMR0) of IGF2. These results demonstrate that IGF2 mRNA expression is more upregulated in FAs and PTs than in normal tissues, especially in their stromal cells, but such an upregulation is not related to LOI of IGF2, and that hypomethylation of DMR0 is unlikely to be involved in induction of LOI.

IMP3 Is Strongly Expressed in Malignant Phyllodes Tumors of the Breast: An Immunohistochemical Study.

BACKGROUND: Phyllodes tumors (PTs) of the breast are rare biphasic neoplasms and are classified as benign, borderline, or malignant. Many biological markers have been studied to discriminate between different grades of PTs. IMP3 is a member of the insulin-like growth factor II mRNA binding protein (IMP) family and is expressed in developing tissues during embryogenesis, whereas in adult tissues it is found only at low or undetectable levels. IMP3 is considered a marker of biological aggressiveness in many cancers, including breast and lung. The aim of this study was to evaluate the immunohistochemical expression of IMP3 in a series of PTs and to determine its association with histological grade and clinical outcome. MATERIALS AND METHODS: We reviewed retrospectively 62 cases of PTs including their recurrences and 20 cases of fibroadenoma. PTs have been classified as benign in 40 cases, borderline in 13 cases, and malignant in 9 cases. RESULTS: There were significant differences in IMP3 expression: in malignant PTs IMP3 expression was higher (56% of cases) than in borderline (15%) and benign cases (5%), (P = .001). Fibroadenoma showed no expression for IMP3. IMP3 expression was different in cases with recurrence than cases without recurrence. Furthermore, 3 of the recurrences had a higher histological grade with a positive IMP3 expression compared with the primary tumor. CONCLUSIONS: This is the first study evaluating the IMP3 immunohistochemical expression in PTs. Its expression correlates with histological grade and could be used in the differential diagnosis of fibroepithelial tumors and in predicting a more aggressive behavior.

Genomic landscapes of breast fibroepithelial tumors.

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

Second harmonic generation microscopy is a novel technique for differential diagnosis of breast fibroepithelial lesions.

Breast fibroepithelial lesions, including fibroadenomas and phyllodes tumours, are commonly encountered in clinical practice. As histological differences between these two related entities may be subtle, resulting in a challenging differential diagnosis, pathological techniques to assist the differential diagnosis of these two entities are of high interest. An accurate diagnosis at biopsy is important given corresponding implications for clinical decision-making including surgical extent and monitoring. Second harmonic generation (SHG) microscopy is a recently developed optical imaging technique capable of robust, powerful and unbiased label-free direct detection of collagen fibril structure in tissue without the use of antibodies. We constructed tissue microarrays emulating limited materials on biopsy to investigate quantitative collagen signal in fibroepithelial lesions using SHG microscopy. Archived formalin-fixed paraffin-embedded materials of 47 fibroepithelial lesions (14 fibroadenomas and 33 phyllodes tumours) were evaluated. Higher collagen signal on SHG microscopy was observed in fibroadenomas than phyllodes tumours on SHG imaging (p<0.001, area under the curve 0.859). At an automated threshold (2.5 million positive pixels), the sensitivity and specificity of the SHG microscopy for fibroadenoma classification was 71.4% and 84.4%, respectively. To corroborate these findings, we performed immunohistochemistry on tissue array sections using collagen I and III primary antibodies. Both collagen I and III immunohistochemical expressions were also significantly higher in fibroadenomas than in phyllodes tumours (p<0.001). In conclusion, label-free collagen quantitation on SHG microscopy is a novel imaging approach that can aid the differential diagnosis of fibroepithelial lesions.

MicroRNA-10a is reduced in breast cancer and regulated in part through retinoic acid.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules that play a critical role in mRNA cleavage and translational repression, and are known to be altered in many diseases including breast cancer. MicroRNA-10a (miR-10a) has been shown to be deregulated in various cancer types. The aim of this study was to investigate miR-10a expression in breast cancer and to further delineate the role of retinoids and thyroxine in regulation of miR-10a. METHODS: Following informed patient consent and ethical approval, tissue samples were obtained during surgery. miR-10a was quantified in malignant (n = 103), normal (n = 30) and fibroadenoma (n = 35) tissues by RQ-PCR. Gene expression of Retinoic Acid Receptor beta (RARß) and Thyroid Hormone receptor alpha (THRα) was also quantified in the same patient samples (n = 168). The in vitro effects of all-trans Retinoic acid (ATRA) and L-Thyroxine (T4) both individually and in combination, on miR-10a expression was investigated in breast cancer cell lines, T47D and SK-BR-3. RESULTS: The level of miR-10a expression was significantly decreased in tissues harvested from breast cancer patients (Mean (SEM) 2.1(0.07)) Log10 Relative Quantity (RQ)) compared to both normal (3.0(0.16) Log10 RQ, p < 0.001) and benign tissues (2.6(0.17) Log10 RQ, p < 0.05). The levels of both RARß and THRα gene expression were also found to be decreased in breast cancer patients compared to controls (p < 0.001). A significant positive correlation was determined between miR-10a and RARß (r = 0.31, p < 0.001) and also with THRα (r = 0.32, p < 0.001). In vitro stimulation assays revealed miR-10a expression was increased in both T47D and SK-BR-3 cells following addition of ATRA (2 fold (0.7)). While T4 alone did not stimulate miR-10a expression, the combination of T4 and ATRA was found to have a positive synergistic effect. CONCLUSION: The data presented supports a potential tumour suppressor role for miR-10a in breast cancer, and highlights retinoic acid as a positive regulator of the microRNA.