Mouse-INtraDuctal (MIND): an in vivo model for studying the underlying mechanisms of DCIS malignancy.

Due to widespread adoption of screening mammography, there has been a significant increase in new diagnoses of ductal carcinoma in situ (DCIS). However, DCIS prognosis remains unclear. To address this gap, we developed an in vivo model, Mouse-INtraDuctal (MIND), in which patient-derived DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Similar to human DCIS, the cancer cells formed in situ lesions inside the mouse mammary ducts and mimicked all histologic subtypes including micropapillary, papillary, cribriform, solid, and comedo. Among 37 patient samples injected into 202 xenografts, at median duration of 9 months, 20 samples (54%) injected into 95 xenografts showed in vivo invasive progression, while 17 (46%) samples injected into 107 xenografts remained non-invasive. Among the 20 samples that showed invasive progression, nine samples injected into 54 xenografts exhibited a mixed pattern in which some xenografts showed invasive progression while others remained non-invasive. Among the clinically relevant biomarkers, only elevated progesterone receptor expression in patient DCIS and the extent of in vivo growth in xenografts predicted an invasive outcome. The Tempus XT assay was used on 16 patient DCIS formalin-fixed, paraffin-embedded sections including eight DCISs that showed invasive progression, five DCISs that remained non-invasive, and three DCISs that showed a mixed pattern in the xenografts. Analysis of the frequency of cancer-related pathogenic mutations among the groups showed no significant differences (KW: p > 0.05). There were also no differences in the frequency of high, moderate, or low severity mutations (KW; p > 0.05). These results suggest that genetic changes in the DCIS are not the primary driver for the development of invasive disease. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Elevated NRAS expression during DCIS is a potential driver for progression to basal-like properties and local invasiveness.

BACKGROUND: Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives DCIS to invasive breast cancer is unclear. Basal-like invasive breast cancers are aggressive. We have previously shown that NRAS is highly expressed selectively in basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers. METHODS: Wilcoxon rank-sum test was performed to assess expression of NRAS in DCIS compared to invasive breast tumors in patients. NRAS mRNA levels were also determined by fluorescence in situ hybridization in patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and association of NRAS mRNA levels with DCIS and invasive breast cancer was assessed by paired Wilcoxon signed-rank test. Pearson's correlation was calculated between NRAS mRNA levels and basal biomarkers in the TMAs, as well as in patient datasets. RNA-seq data were generated in cell lines, and unsupervised hierarchical clustering was performed after combining with RNA-seq data from a previously published patient cohort. RESULTS: Invasive breast cancers showed higher NRAS mRNA levels compared to DCIS samples. These NRAShigh lesions were also enriched with basal-like features, such as basal gene expression signatures, lower ER, and higher p53 protein and Ki67 levels. We have shown previously that NRAS drives aggressive features in DCIS-like and basal-like SUM102PT cells. Here, we found that NRAS-silencing induced a shift to a luminal gene expression pattern. Conversely, NRAS overexpression in the luminal DCIS SUM225 cells induced a basal-like gene expression pattern, as well as an epithelial-to-mesenchymal transition signature. Furthermore, these cells formed disorganized mammospheres containing cell masses with an apparent reduction in adhesion. CONCLUSIONS: These data suggest that elevated NRAS levels in DCIS are not only a marker but can also control the emergence of basal-like features leading to more aggressive tumor activity, thus supporting the therapeutic hypothesis that targeting NRAS and/or downstream pathways may block disease progression for a subset of DCIS patients with high NRAS.

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion.

INTRODUCTION: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated ß-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized. METHODS: Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers. RESULTS: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification. CONCLUSION: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance.

In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in ß-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3ß overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3ß isoform caused mammary epithelial cell death and a significant reduction in ß-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3ß.

Emerging functions of microRNA-146a/b in development and breast cancer: microRNA-146a/b in development and breast cancer.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression through translational repression or mRNA degradation. These molecules play critical roles in regulating normal developmental processes, but when deregulated, are causally linked to the pathogenesis of numerous diseases, including cancer. MicroRNA-146a and -146b are encoded by two different genes, but differ by only two bases and appear to function redundantly in many systems. Initial studies branded miR-146a/b as important mediators of inflammatory signaling, documenting the ability of these miRNAs to influence differentiation, proliferation, apoptosis and effector immune mechanisms within the hematopoietic system. Numerous contemporary studies now implicate miR-146a/b as pleiotropic regulators of tumorigenesis, as a polymorphism in miR-146a and altered expression of both miR-146a/b have been linked with cancer risk, tumor histogenesis and invasive and metastatic capacity in diverse cancers. Despite the numerous reports concerning miR-146a/b in human cancers, the mechanistic contributions of these miRNAs in both normal and neoplastic mammary gland development and biology remains poorly characterized.

Calcifying fibrous tumor.

Calcifying fibrous tumor is a rare benign mesenchymal neoplasm. The etiology and pathogenesis of this tumor are uncertain. It has wide anatomical distribution. The tumor is most commonly found in the soft tissues of the extremities in younger individuals. However, in middle-aged patients, it tends to affect the visceral locations more commonly. In visceral location, it can mimic aggressive lesions clinically. The purpose of this report is to describe a case of calcifying fibrous tumor in a 71-year-old female with a history of breast carcinoma who was found to have an incidental small bowel mass on her follow-up. Clinically and radiologically, the mass was suspicious for either metastatic disease or gastrointestinal stromal tumor. The patient underwent open small bowel resection, and a 6.5 cm segment of the small bowel was sent to pathology. Grossly, a 2.0 cm tan-pink smooth round submucosal polyploid mass protruding into the lumen, mimicking a gastrointestinal stromal tumor, was identified. The tumor was hard and serially sectioned to reveal a white, calcified cut surface. Microscopically, the tumor appeared hypocellular and composed of scant spindle cells embedded in a dense, hyalinized and calcified collagenous stroma. Immunohistochemical stains for pan-cytokeratin, DOG1, desmin, S100, CD34, and MUC4 were negative, and a diagnosis of the calcifying fibrous tumor was rendered. This case provides a rare gross specimen image of calcifying fibrous tumor and highlights the importance of knowledge of rare entities in providing an accurate diagnosis for entities that can mimic other lesions.

Metastasis from tongue squamous cell carcinoma to the kidney.

Metastasis to the kidney from other primary sites is extremely rare. Previous studies reported the lung as the most common primary site. Distant metastasis from the tongue to the kidney is exceedingly rare. Herein, we describe a case of metastatic squamous cell carcinoma to the kidney in a 71-year-old male with a detailed discussion of differentiating it from potential mimickers. The patient underwent a total glossectomy and bilateral cervical lymph node dissection. A diagnosis of well-differentiated squamous cell carcinoma of the tongue was rendered and the tumor was staged pT3 pN3b. Within two years of initial presentation, the patient developed widely metastatic disease, including pulmonary nodules, renal masses, left adrenal mass, and pancreatic mass. Accurate diagnosis of a secondary involvement of the kidney by a metastatic tumor requires the appropriate correlation of clinical and imaging findings as well as morphologic and immunohistochemical clues.

BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

Calcifying fibrous tumor

ABSTRACT Calcifying fibrous tumor is a rare benign mesenchymal neoplasm. The etiology and pathogenesis of this tumor are uncertain. It has wide anatomical distribution. The tumor is most commonly found in the soft tissues of the extremities in younger individuals. However, in middle-aged patients, it tends to affect the visceral locations more commonly. In visceral location, it can mimic aggressive lesions clinically. The purpose of this report is to describe a case of calcifying fibrous tumor in a 71-year-old female with a history of breast carcinoma who was found to have an incidental small bowel mass on her follow-up. Clinically and radiologically, the mass was suspicious for either metastatic disease or gastrointestinal stromal tumor. The patient underwent open small bowel resection, and a 6.5 cm segment of the small bowel was sent to pathology. Grossly, a 2.0 cm tan-pink smooth round submucosal polyploid mass protruding into the lumen, mimicking a gastrointestinal stromal tumor, was identified. The tumor was hard and serially sectioned to reveal a white, calcified cut surface. Microscopically, the tumor appeared hypocellular and composed of scant spindle cells embedded in a dense, hyalinized and calcified collagenous stroma. Immunohistochemical stains for pan-cytokeratin, DOG1, desmin, S100, CD34, and MUC4 were negative, and a diagnosis of the calcifying fibrous tumor was rendered. This case provides a rare gross specimen image of calcifying fibrous tumor and highlights the importance of knowledge of rare entities in providing an accurate diagnosis for entities that can mimic other lesions.

NEMO, a Transcriptional Target of Estrogen and Progesterone, Is Linked to Tumor Suppressor PML in Breast Cancer.

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO, the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR.

Metastasis from tongue squamous cell carcinoma to the kidney

Metastasis to the kidney from other primary sites is extremely rare. Previous studies reported the lung as the most common primary site. Distant metastasis from the tongue to the kidney is exceedingly rare. Herein, we describe a case of metastatic squamous cell carcinoma to the kidney in a 71-year-old male with a detailed discussion of differentiating it from potential mimickers. The patient underwent a total glossectomy and bilateral cervical lymph node dissection. A diagnosis of well-differentiated squamous cell carcinoma of the tongue was rendered and the tumor was staged pT3 pN3b. Within two years of initial presentation, the patient developed widely metastatic disease, including pulmonary nodules, renal masses, left adrenal mass, and pancreatic mass. Accurate diagnosis of a secondary involvement of the kidney by a metastatic tumor requires the appropriate correlation of clinical and imaging findings as well as morphologic and immunohistochemical clues.

Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines.

The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2-4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec).