Human Ductal Carcinoma In Situ Research Articles

Human Ductal Carcinoma In Situ: Advances and Future Perspectives.

Due to widespread adoption of screening mammography, there has been a significant increase in new diagnoses of ductal carcinoma in situ (DCIS). However, DCIS outcomes remain unclear. A large fraction of human DCIS (>50%) may not need the multimodality treatment options currently offered to all DCIS patients. More importantly, while we may be overtreating many, we cannot identify those most at risk of invasion or metastasis following a DCIS diagnosis. This review summarizes the studies that have furthered our understanding of DCIS pathology and mechanisms of invasive progression by using advanced technologies including spatial genomics, transcriptomics, and multiplex proteomics. This review also highlights a need for rethinking DCIS with a more focused view on epithelial states and programs and their cross talk with the microenvironment.

MYO10-filopodia support basement membranes at pre-invasive tumor boundaries.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs invivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion invitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.

Molecular signatures of in situ to invasive progression for basal-like breast cancers: An integrated mouse model and human DCIS study

Ductal carcinoma in situ (DCIS) of the breast is a non-obligate precursor of Invasive Ductal Carcinoma (IDC) and thus the identification of features that may predict DCIS progression would be of potential clinical value. Experimental mouse models can be used to address this challenge by studying DCIS-to-IDC biology. Here we utilize single cell RNA sequencing (scRNAseq) on the C3Tag genetically engineered mouse model that forms DCIS-like precursor lesions and for which many lesions progress into end-stage basal-like molecular subtype IDC. We also perform bulk RNAseq analysis on 10 human synchronous DCIS-IDC pairs comprised of estrogen receptor (ER) positive and ER-negative subsets and utilize 2 additional public human DCIS data sets for comparison to our mouse model. By identifying malignant cells using inferred DNA copy number changes from the murine C3Tag scRNAseq data, we show the existence of cancer cells within the C3Tag pre-DCIS, DCIS, and IDC-like tumor specimens. These cancer cells were further classified into proliferative, hypoxic, and inflammatory subpopulations, which change in frequency in DCIS versus IDC. The C3Tag tumor progression model was also associated with increase in Cancer-Associated Fibroblasts and decrease in activated T cells in IDC. Importantly, we translate the C3Tag murine genomic findings into human DCIS where we find common features only with human basal-like DCIS, suggesting there are intrinsic subtype unique DCIS features. This study identifies several tumor and microenvironmental features associated with DCIS progression and may also provide genomic signatures that can identify progression-prone DCIS within the context of human basal-like breast cancers.

Abstract P5-08-03: Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: Increased detection of ductal carcinoma in situ (DCIS) has been a consequence of mammographic screening. Currently there is a reappraisal of the necessity of aggressive treatment, with their attendant toxicities for a preneoplastic lesion with 15-20% chance of recurrence or progression. The recombinant immunotoxin, TFR(fv)PE40, binds to the abundantly expressed transferrin receptors on cancer cells, is internalized, processed and trafficked to the cytosol. The released Pseudomonas exotoxin (PE) catalyzes the transfer of ADP-ribose from NAD to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation, and cell death by apoptosis. However, TFR(fv)PE40 is toxic to liver cells. We hypothesized that, when administered via the opening at the mammary teat, the immunotoxin will destroy preneoplasias in the entire ductal tree. Because of the closed nature of the ductal system, the toxic effects of the TFR(fv)PE40 will be confined to the tumor cells alone, and systemic effects will be minimal or absent. The intraductal approach, we proposed, will provide a direct, safe and effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using two luc-tagged cell lines-SUM225-luc, derived from a human DCIS, and in MCF-7-luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS develop within one week in MIND models following intraductal (i.duc) injection of tumor cells (right and left 4th mammary gland) of ex-breeder NSG mice. DCIS progressed to invasive cancer in 2-3 weeks. At day 7, mice were administered vehicle, TFR mAb alone, or the HB21(fv)PE40 conjugate at two doses (0.15 or 1.5 ug per teat x 2) by the i.duc route, 3 times at weekly intervals, and observed for 1-3 months. Mice treated with the TFR-Ab showed no significant reduction in tumor size. Treated with HB21(fv)PE40 i.duc, by IVIS imaging, tumors were undetectable in both models (P<0.0001) within two weeks. No recurrence was observed during 3 months of followup. Mammary whole mount analysis and histopathology showed that i.duc toxin rendered more than 90% of the mice cancer-free. Immunohistochemical analysis of tumor sections for huTFR (CD71), Ki67, and CD31 supported the loss of TFR-expressing and proliferating cancer cells, and reduction of blood vessels. Pharmacokinetics studies showed that toxin was detectable by TFR-ELISA in blood 5, 30 and 60 min after i.p injection, but was undetectable in the blood of i.duc-treated mice. Conclusions: Overall, we have shown that i.duc HB21(fv)PE40 has remarkable antitumor activity in cell culture with an IC50 in the nM range, and in two MIND models of DCIS. Blood levels of the toxin after i.duc injection were undetectable, suggesting that the toxin remains inside the ductal system during its 30 min half-life, and its effect occurs during this time. This preclinical study provides a strong basis for conducting human trials. The toxicity of HB21(Fv)PE40 by the i. duc route of drug administration could be determined in dose escalation studies in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen, using a previously successful study design (PMID:2230751). This design will allow detailed examination of the local and systemic effects of HB21(fv)PE40 administered i.duc and reintroduce a very potent agent into DCIS treatment, and in the future, cancer prevention in women at high risk of developing breast cancer. Citation Format: Saraswati Sukumar, Guannan Wang, Alok Kumar, Wanjun Ding, Preethi Korangath, Priya Pai, Kathleen Gabrielson, Ira Pastan. Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-03.

Abstract P5-01-03: Mouse-intraductal (MIND): An in vivo model for studying the underlying mechanisms of DCIS malignancy

Abstract Background: 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. Methods: To address this gap, we developed an in vivo model, Mouse-INtraDuctal (MIND), by which patient-derived DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. The source of DCIS samples reflected clinical practice as predominantly high grade (70%), but also included intermediate grade (27%) and low grade (3%). Thirty-seven patient samples were injected into 202 mouse mammary glands and evaluated for invasive progression at a median duration of 9 months. The expression of clinically relevant biomarkers (ER, PR, Ki-67, HER2 and p53) on patient DCIS FFPE sections and xenografts’ extent of in vivo growth were evaluated for their utility in predicting DCIS invasive progression in the xenografts. Targeted DNA sequencing using Tempus XT oncology assay was used on patient DCIS in order to find a unique pattern of cancer related gene mutations that predicted DCIS invasiveness in the xenografts. Results: 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 noninvasive. Among the 20 samples that showed invasive progression in the MIND model, 9 patient samples injected into 54 xenografts exhibited a mixed pattern in which some xenografts showed invasive progression while others remained noninvasive. The mean duration of follow-up was not significantly different among the progressed, non-progressed or mixed groups (ANOVA; p-value=0.44). Among the clinically relevant biomarkers, only elevated progesterone receptor expression in patient DCIS and extent of in vivo growth in xenografts predicted an invasive outcome in the xenografts. Tempus XT oncology assay was used on 16 patient DCIS FFPE sections including eight patient DCIS that showed invasive progression (P), five patient DCIS that remained non-invasive (NP) and three patient DCIS that showed a mixed pattern (M) in the xenografts. Variant severity was called using SnpSift which is a program for identifying phenotype-relevant variants and predicts the severity of SNPs based on their effect on gene expression and function. COSMIC database was also used to identify mutations with pathogenic scores >0.5. Analysis of the frequency of cancer related pathogenic mutations showed no significant differences (P=27, NP=79, M=43, Kruskal-Wallis: P value=>0.05). There were also no differences in the frequency of low, moderate or high severity mutations (P= 25 highly severe, 120 moderately severe and 50 low-severity; NP=9 highly severe, 58 moderately severe and 28 low severity; M=3 highly severe, 33 moderately severe and 14 low severity; Kruskal-Wallis; P value >0.05). Conclusions: Highly severe and pathogenic variants in the patient’s DCIS were not associated with whether the DCIS developed into invasive lesions or remained non-invasive in the MIND models. These results are in agreement with previous studies that showed no significant differences in frequency of non-synonymous mutations and CNAs when comparing pure DCIS with synchronous IDC-DCIS. The MIND models are in immunocompromised mice, so the contribution of the immune system to DCIS progression may not recapitulate cancer progression in an immunocompetent state. However, the MIND model suggests that genetic changes in the DCIS are not the primary driver for the development of invasive disease. Citation Format: Aditi Rastogi, Jerome Lin, Yan Hong, Darlene Limback, Hanan S. Elsarraj, Haleigh Harper, Haley Haines, Hayley Hansford, Michael Ricci, Carolyn Kaufman, Emily Wedlock, Mingchu Xu, Jianhua Zhang, Lisa May, Terri Cusick, Marc Inciardi, Mark Redick, Jason Gatewood, Onalisa Winblad, Allison Aripoli, Ashley Huppe, Christa Balanoff, Jamie Wagner, Amanda Amin, Kelsey E. Larson, Lawrence Ricci, Ossama Tawfik, Hana Razek, Ruby O Meierotto, Rashna Madan, Andrew K. Godwin, Jeffrey Thompson, Susan G. Hilsenbeck, Andy Futreal, Alastair Thompson, E. Shelley Hwang, Fang Fan, Nicholas Navin, Fariba Behbod, Grand Challenge PRECISION Consortium. Mouse-intraductal (MIND): An in vivo model for studying the underlying mechanisms of DCIS malignancy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-03.

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.

Abstract PD5-09: Mouse-intraductal (MIND): The first in vivo model to recapitulate the full spectrum of human DCIS pathology

Abstract Introduction. Due to advances in imaging technology and an increase in mammographic screening, there has been a significant increase in the diagnosis rate of ductal carcinoma in situ (DCIS). At the present time, nearly all women undergo surgical removal of DCIS, often followed by adjuvant radiation and in some cases anti-hormonal therapy. Currently, there are no means by which to diagnose DCIS accurately, or to predict which patients benefit from aggressive therapy. Thus, the recommendation for surgery persists, despite studies which support that not all DCIS will subsequently progress to invasive disease. In this context, animal models can be particularly useful in studying DCIS progression. Here, we present the first in vivo model of DCIS, referred to as Mouse-INtraDuctal (MIND), in which patient-derived DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Methods. We performed intraductal injection of DCIS epithelial cells derived from 30 patient samples into 194 total glands. Of the 194, 146 xenografts showed in vivo growth, for a 75% take rate). Among the DCIS samples injected into mice, 18 (103 mouse mammary glands) were followed for a median of 9 months . Among those, 50% (9) showed invasive progression while 50% (9) remained non-invasive. DCIS invasive progression was evaluated by performing immunofluorescene staining using anti-smooth muscle actin (SMA) antibody and confirmed by the loss of SMA around the xenografted DCIS like lesions on 3 consequetive sections of FFPE tissues. Results. Progressed xenografts exhibited invasive progression, evident by the loss of SMA, as early as 6 months following transplantation. Similar to human DCIS, the cancer cells initially formed in situ lesions inside the mouse mammary ducts and mimicked all histologic subtypes including micropapillary, papillary, cribriform, solid and comedo. Among the biomarkers tested, including ER, PR, Ki67, HER2, p53, histology, nuclear and tumor grade, only low ER & PR expression and extent of DCIS growth in xenografts significantly correlated with invasive progression. A high depth targeted sequencing platform (T200) on DNA isolated from LCM captured DCIS of patient and xenograft pairs identified shared (i.e., EGFR) as well as unique (STK11, RUNX1, PIK3CA) mutations in patient/xenograft pairs. Notably, we also observed private mutations that were not shared within the same patient/xenograft pairs. These results indicate the presence of DCIS clonal heterogeneity and that DCIS xenografts may represent one or more clonal subpopulations of patient DCIS. Conclusion. The MIND model represents the first realistic in vivo model that recapitulates human DCIS progression in a manner that represents the inter- and intra-tumoral heterogeneity of human disease. These innovative mouse models will be invaluable for the discovery of molecular signatures of invasive DCIS by allowing comparison of xenografts with variable propensity for invasive progression. These models will enable the discovery of extrinsic factors that regulate DCIS malignancy as well as testing of pharmaceutical and natural compounds for prevention of DCIS progression to invasive disease. Citation Format: Fariba Behbod, Yan Hong, Darlene Limback, Hannan S Elsarraj, Haleigh Harper, Haley Haines, Hayley Hansford, Michael Ricci, Carolyn Kaufman, Mingchu Xu, Jianhua Zhang, Lisa May, Therese Cusick, Marc inciardi, Mark Redick, Jason Gatewood, Alison Aripoli, Ashley Huppe, Onalisa Winblad, Christa Balanoff, Jamie Wagner, Amanda L Amin, Kelsey E. Larson, Lawrence Ricci, Ossama Tawfik, Hana Razek, Ruby O Meierotto, Rashna Madan, Andrew K Godwin, Jeffrey Thompson, Andy Futreal, Alastair Thompson, Shelley Hwang, Fang Fan, On behalf of the Grand Challenge PRECISION consortium. Mouse-intraductal (MIND): The first in vivo model to recapitulate the full spectrum of human DCIS pathology [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD5-09.

Essential role of STAT5a in DCIS formation and invasion following estrogen treatment.

Ductal carcinoma in situ (DCIS) is one of the earliest stages of breast cancer (BCa). The mechanisms by which DCIS lesions progress to an invasive state while others remain indolent are yet to be fully characterized and both diagnosis and treatment of this pre-invasive disease could benefit from better understanding the pathways involved. While a decreased expression of Caveolin-1 (Cav-1) in the tumor microenvironment of patients with DCIS breast cancer was linked to progression to invasive breast cancer (IBC), the downstream effector(s) contributing to this process remain elusive. The current report shows elevated expression of Signal Transducer and Activator of Transcription 5a (STAT5a) within the DCIS-like lesions in Cav-1 KO mice following estrogen treatment and inhibition of STAT5a expression prevented the formation of these mammary lesions. In addition, STAT5a overexpression in a human DCIS cell line (MCF10DCIS.com) promoted their invasion, a process accelerated by estrogen treatment and associated with increased levels of the matrix metalloproteinase-9 (MMP-9) precursor. In sum, our results demonstrate a novel regulatory axis (Cav-1♦STAT5a♦MMP-9) in DCIS that is fully activated by the presence of estrogen. Our sudies suggest to further study phosphorylated STAT5a (Y694) as a potential biomarker to guide and predict outcome of DCIS patient population.

Abstract B021: Canine CAR T-cells therapy for mammary carcinoma in dogs

Abstract In women, ductal carcinoma in situ (DCIS) is an often-diagnosed breast disease that is widely considered to be a nonobligate precursor of invasive carcinoma. TNBC is the most aggressive and lethal form of breast cancer and predominantly occurs in women at high genetic risk including those with mutated BRCA1 genes. We provide for the first time an immunocompetent animal model for triple-negative DCIS (TN-DCIS) that will facilitate molecular analysis of preinvasive TNBC and will provide an invaluable resource for identifying and selecting targets for TNBC vaccine immunoprevention or immunotherapeutic intervention. Unlike most studied rodent models, dogs develop TN-DCIS spontaneously without genetic or chemical manipulation. We have shown that canine DCIS resembles human DCIS with shared histopathologic and molecular features and with similar imaging and behavioral characteristics. Dogs are much more outbred than laboratory rodents, yet certain breeds are at increased risk for developing mammary tumors. Indeed, we have found that 50% of randomly screened asymptomatic hound dogs have premalignant mammary lesions and that mammary TN-DCIS often progresses to invasive carcinoma within one year. Given the many common features of canine breast cancer and the high homology between the canine and human genome, the dog model offers an outstanding opportunity for exploiting TNBC immunoprevention and immunotherapeutic strategies. Moreover, the prevalence and rapid progression of canine TN-DCIS provides a much more rapid and cost-effective alternative to human trials for evaluation of the clinical effectiveness of cancer vaccine strategies. One immunotherapeutic strategy we are testing using our canine TN-DCIS model is chimeric antigen receptor (CAR) T-cells. CAR T-cells have shown promise in treating many malignancies, but in solid tumors have been hindered by many limitations. To overcome these limitations, we designed genetically engineered universal canine CAR T-cells that must be activated and targeted by a small-molecule adaptor before it can kill cancer cells. Our results showed that universal CAR T cells are functional and killed canine mammary tumor cell lines in vitro. Citation Format: Xavier E. Ramos-Cardona, Sulma I. Mohammed, Yong Gu Lee, Phillip Low. Canine CAR T-cells therapy for mammary carcinoma in dogs [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B021.

Abstract PD6-9: Molecular signatures of DCIS to invasive progression for basal-like breast cancers: An integrated mouse model and human tumor study

Abstract Invasive ductal cancers (IDC) have a variety of precursor lesions and molecular subtypes. Ductal Carcinoma in Situ (DCIS) is considered an important precursor stage immediately preceding IDC and has increased in its incidence in recent times. 20-40% of DCIS form IDC however, currently we lack robust molecular signatures to predict progression prone DCIS. Performing next generation sequencing in breast cancer DCIS precursors is likely to help elucidate their progression potential. Sequencing the progression path of IDC lesions in a mouse model, and in human samples of the same molecular subtype, should help to understand the drivers of transformation. We utilized the C3-TAG genetically engineered mouse model that forms DCIS like precursor lesions, and human breast DCIS-IDC pairs for genomic analyses. Through single cell RNA-sequencing, and bulk mRNA-seq, we identified disease stage specific, and cell population specific, signatures at three stages of breast cancer tumor progression in the C3-TAG mouse model. Through integrated analysis we subset cancer cells in the DCIS and tumor stage in C3-TAG and identify genes and gene signatures relevant in the IDC stage including HIF-1-alpha pathway (Fos proto-oncogene, lactate dehydrogenase A); glycolysis pathway (phosphoglycerate mutase 1, pyruvate kinase M1/2, aldolase, fructose-bisphosphate A, enolase 1); Metastasis associated protein (MTA3) complex (tubulin alpha 1c, tubulin alpha 4a) and endocrine resistance (cyclin dependent kinase inhibitor 2A, KRAS proto-oncogene, GTPase, heparin binding EGF like growth factor, cyclin D1). We also identified microenvironment signatures and detected gene changes in the immune, endothelial and fibroblast cells as DCIS transitions to IDC, including a sharp decline of CD8 T cell genes (CD274, C-C motif chemokine ligand 5, SLAM family member 7) from DCIS to IDC, highlighting an immune dysregulation as part of the transition from DCIS to IDC. Simultaneously, we retrieved a public microarray dataset of human DCIS/IDC pairs and performed Ribo-zero RNA-sequencing of 5 human basal-like DCIS/IDC and 5 Luminal A DCIS/IDC pairs. Utilizing gene set enrichment analysis, we show that our C3-TAG DCIS stage tumor cell signature was significantly enriched in the human basal-like DCIS, but not in Luminal A DCIS. These results highlight that the C3-TAG DCIS may recapitulate the biology of basal-like DCIS and that progression in basal-like may be different than in Luminal/ER+ cancers. Further interrogation of these DCIS and IDC stage specific signatures may lead to identification of a clinical marker of progression and/or a therapeutic target of early intervention. Citation Format: Aatish Thennavan, Xiaping He, Charles M Perou. Molecular signatures of DCIS to invasive progression for basal-like breast cancers: An integrated mouse model and human tumor study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD6-9.

Abstract LB-140: Myoepithelial-immune cell crosstalk in the transition from non-invasive to invasive breast cancer

Abstract The incidence of ductal carcinoma in situ (DCIS), a pre-invasive malignancy of the breast, has increased in the U.S. since the 1980s, from 3% to 20% of all breast cancer diagnoses. This increase is largely attributed to mammographic screening. Common treatments of DCIS are breast-conserving surgery followed by adjuvant radiation therapy or mastectomy. Further, despite efforts to detect and treat early stage cancer, the incidence of invasive breast cancer (IBC) has not declined, suggesting that many women with DCIS are over treated. Thus, prognostic markers that predict risk of DCIS progression are needed. Genomic studies have yet to find tumor intrinsic alterations that predict DCIS progression to IBC. In contrast, genetic changes in the myoepithelium are found at the transition from DCIS to IBC, suggesting myoepithelial cells may play a role in progression. To investigate the myoepithelium for potential biomarkers, we advanced a non-surgical intraductal tumor cell delivery model in mice using human DCIS.com cells. We reported a progressive loss in myoepithelial differentiation markers p63, calponin, and smooth muscle actin (SMA) prior to transition to IBC, observations that were confirmed in human DCIS. Thus, compromise in the myoepithelium may be an early warning for DCIS at high risk for progression. Here, we hypothesize that the myoepithelial differentiation state informs immune response, and that DCIS lesions with loss of myoepithelial Calponin and SMA will be associated with a pro-tumorigenic immune milieu. This hypothesis is based on observations by us and others showing lymphocyte infiltration in areas where the DCIS myoepithelium has been focally compromised. To address this, we analyzed the expression of 10 myoepithelial and lymphoid biomarkers using multiplex immunohistochemistry to analyze the relationship between myoepithelial cell integrity and immune cells (N=36 cases). DCIS in the background of IBC (mixed DCIS) is assumed to be high-risk DCIS. We find compromised myoepithelial calponin in mixed compared to pure DCIS. Mixed lesions also show reduced infiltration of immune and T-cells, consistent with reduced immune surveillance. However, calponin loss in pure DCIS cases was associated with a decrease in Foxp3+ T-regulatory cells and increase in CD8+PD-1+ T-cells. Next we found that DCIS areas with focal loss of the myoepithelium and micro-invasion showed an increase in immune infiltration, increased T-cells, and increased PD-1+CD8+ T-cells. These data suggest that early compromise in the myoepithelial layer may associate with activation of cytotoxic effectors, consistent with increased immune surveillance. However, since we find T-cell numbers are reduced in mixed DCIS, effective immune surveillance may be dampened with progression. These data support an unreported relationship between myoepithelial cell integrity and the activation state of local immune cells. Citation Format: Elizabeth Mitchell, Sonali Jindal, Tiffany Chan, Jayasri Narasimhan, Sheila Weinmann, Pepper Schedin. Myoepithelial-immune cell crosstalk in the transition from non-invasive to invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-140.

Abstract PD8-08: Development of humanized immune DCIS models using patient peripheral blood derived hematopoietic stem cells (CD34+)

Abstract Background: Ductal carcinoma in situ (DCIS) is the most common form of non-invasive breast cancer. To accurately study the natural progression of DCIS lesions in mice, we devised the mouse-intraductal (MIND) animal model, which involves intraductal injection of human DCIS epithelial cells into the mammary ducts of immunocompromised mice. To improve the translational application of the MIND model, we aimed to mimic the natural microenvironment of human DCIS with patient-derived immune cells and assess the role of engrafted immune cells on human DCIS progression. In order to achieve successful engraftment of the entire immune system in mice, we utilized MISTRG mice. These mice were developed by Rongvaux et al., on an immunodeficient (Rag2-/-IL2rγ-/-) background. The genes encoding human M-CSF (M), human IL-3 (I), SIRP1α (S), human thrombopoietin (TPO)(TR), and GM-CSF (G) were knocked into their respective mouse loci. As such, MISTRG mice are highly permissive for human hematopoiesis, supporting the development and function of lymphocytes, monocytes, and natural killer (NK) cells. In contrast, previous studies have used the humanized CD34+ NOD-SCID IL2rγ-/- mice (CD34+NSG), which are unable to support myeloid cell differentiation due to lack of expression of human-specific cytokines. Moreover, prior xenograft studies in the CD34+NSG mice have not used immune cells derived from the same patient as the tumor. Results: Human CD34+ cells derived from patients' peripheral blood were expanded ex vivo ˜100-fold using a novel formulation of culture medium. Transplantation of ex vivo expanded CD34+ cells via tail vein injection of MISTRG mice resulted in the successful engraftment of human immune cells as early as 4 weeks following injection. Successful engraftment was confirmed by flow cytometry using human specific antibodies that recognize human leukocytes (anti-CD45), T cells (anti-CD3), B cells (anti-CD20), and myeloid cells (anti-CD33) in spleen, bone marrow, and peripheral blood of MISTRG mice. Once engraftment was confirmed, DCIS epithelial cells from the same DCIS patients or DCIS cell lines were injected intraductally. Recruitment of patient-derived immune cells to the DCIS lesions was confirmed by immunofluorescence using human-specific antibodies that recognize neutrophils (anti-myeloperoxidase), macrophages (anti-CD68), M2-polarized macrophages (anti-c-MAF), natural killer cells (anti-CD56), dendritic cells (anti-CD21), T cells (anti-CD3) and B cells (anti-CD20). Conclusion: This model represents the first to enable the study of mechanisms of DCIS progression in a manner that fully represents the heterogeneity of human disease, including the influence of the patients' own immune cells on DCIS progression. Citation Format: Behbod F, Harper H, Hansford H, Limback D, Hong Y, Elsarraj H, Ricci LR, Fan F, Tawfik O, May L, Cusick T, Inciardi M, Redick M, Gatewood J, Winblad O, Fields TA, Fabian C, Godwin AK, Fields PE, Meierotto R, Perry J. Development of humanized immune DCIS models using patient peripheral blood derived hematopoietic stem cells (CD34+) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD8-08.

Abstract P6-05-09: Exosome microRNA contents are altered during breast cancer progression

Abstract Background: The progression of breast cancer involves the transformation of normal mammary epithelial cells to ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC). This process is initiated by genetic alterations and characterized by changes to gene expression programs and microenvironmental alterations. However, the specific drivers of DCIS progression to IBC are not well understood nor has an indicator of progression been identified. Exosomes are small secretory vesicles that can contribute to cancer progression by transferring oncogenic factors, such as microRNAs (miRNAs), to surrounding cells in the tumor microenvironment, and enter the circulation to act at distant sites. miRNAs are short noncoding RNAs that regulate the expression of a target messenger RNA (mRNA). Altered regulation by miRNAs is implicated in cancer progression. In this study, we sought to characterize the exosome miRNAs in the MCF10 isogenic model of breast cancer progression in order to identify potential drivers of breast cancer. Methods: Exosomes were isolated from the conditioned media of the MCF10 isogenic cell line model of breast cancer progression representing the following stages: normal, benign proliferative, carcinoma in situ, and invasive carcinoma. RNA was extracted from the exosomes and next generation RNA sequencing was performed. Exosome miRNA expression was validated in breast cancer cell lines and in plasma exosomes collected from a mouse-intraductal transplantation (MIND) model implanted with MCF10DCIS.com (DCIS) cells that can mimic human DCIS progression in vivo. Results: Comparisons were made between differentially expressed miRNAs among each condition (fold change >1.5; Kruskal-Wallis p<0.05,). Twenty-nine miRNAs were differentially expressed among invasive and DCIS exosomes. The expression of 5 oncogenic miRNAs (miR-30c-5p, -210, -182-5p, -200c-3p, and -200b-3p) were consistently increased, while 2 tumor suppressive miRNAs (miR-423-5p and -92b-3p) were consistently decreased with invasive progression. Exosome miRNA expression was confirmed in breast cancer cell lines and mouse plasma exosomes. Conclusion: This work demonstrates that the microRNA contents of exosomes change upon malignant transformation to invasive breast cancer and indicate that certain exosome microRNAs are consistently up- or down-regulated and may contribute to breast cancer progression. Citation Format: Hannafon BN, Gin AL, Behbod F, Ding W-Q. Exosome microRNA contents are altered during breast cancer progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-09.

IL-6-mediated cross-talk between human preadipocytes and ductal carcinoma in situ in breast cancer progression

BackgroundThe function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model.MethodsGFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody.ResultsIn the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition.ConclusionsOur findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Multiplex Immunohistochemistry Provides Insight Into Cross‐Talk Between Myoepithelial And Immune Cells In Ductal Carcinoma In Situ (DCIS) Progression

Every year in the U.S. ~50,000 women are diagnosed with Ductal Carcinoma in Situ (DCIS). Long term studies of untreated DCIS show ~30% of women progressed to invasive breast cancer (IBC), providing strong rational for treating DCIS similar to early stage IBC. Disease management entails breast‐conserving surgery (BCS) in ~70% of women with or without adjuvant radiation therapy (RT), or unilateral and/or bilateral mastectomy in about 30% of women. While RT and mastectomy reduce risk of IBC or DCIS ipsilateral recurrence, support of potential overtreatment is suggested by long term outcome studies demonstrating low mortality from DCIS (1–2% at 10 years) regardless of treatment received. Thus, prognostic markers that can distinguish DCIS at high versus low risk of progression are needed to personalize treatment decisions. The tumor microenvironment, including myoepithelial and immune cells, has been shown by us and others to play a pivotal role in tumor progression, and we have advanced murine models to study human DCIS progression. In an immune compromised model, the loss of myoepithelial cell calponin, a regulatory component of smooth muscle contraction, specifically corresponded with a ~130‐fold increased probability of adjacent human tumor cells expressing the poor prognostic tumor biomarker p63; implicating calponin as a tumor suppressor protein that acts on adjacent tumor cells. Using immune competent murine models, we find faster progression to invasive tumors, suggesting a supportive role for the immune system in DCIS to IBC progression. We hypothesized that pro‐tumorigenic immune cells will be found near DCIS lesions that have lost myoepithelial differentiation markers and/or tumor cells that have a poor‐prognostic basal phenotype. To test this hypothesis, we have developed novel computer‐assisted computational methods to characterize the integrity of the myoepithelial cell layer in DCIS lesions and to provide near‐neighbor analysis to identify proximity to immune cells. This method involves image segmentation to identify the myoepithelial cell layer using the combined IHC images of SMA, calponin, and CK18, followed by geometric quantification to determine relative expression as well as physical proximity between distinct biomarker (Figure 1). We validated the ability of this computational method to determine loss of myoepithelial cell‐differentiation markers by comparing results to a pathologist's visual annotation, and found a Pearson's‐correlation coefficient of R = 0.959. Utilizing FFPE breast tissues from a cohort of women diagnosed with DCIS (OHSU IRB #15514), we performed multiplex sequential immunohistochemical (IHC) staining for markers of myoepithelial differentiation state (p63, SMA, calponin), immune cells (lymphoid: CD45, CD8, CD3, Foxp3, Granzyme B, PD‐1) and prognostic biomarkers (cyclooxygenase‐2, p16, ki67, ER, PR, Her‐2, tenascin‐c, cytokeratin 5). Our results show that loss of the myoepithelial differentiation marker calponin is associated with a relative decrease in CD4+Foxp3+ T regulatory cells and a concurrent increase in CD8+PD‐1+ T cells. These data suggest that 1) during DCIS progression there may be activation of cytotoxic effectors and concomitant decrease in Tregs to support tumor suppression, and 2) myoepithelial cell integrity may facilitate the activation state of local immune cells. Future studies include assessment for the role of myeloid lineages in myoepithelial cell integrity and DCIS progression.Support or Funding InformationThis research work is supported by OHSU Knight Cancer Institute – Borchard Early Investigator Award to SJ, OHSU OCTRI ‐ NW Kaiser specimen retrieval opportunity to PS and SJ, Susan G. Komen Post‐Doctoral fellowship to EM (PDF17480342).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The PPARγ agonist efatutazone delays invasive progression and induces differentiation of ductal carcinoma in situ.

Ductal carcinoma in situ (DCIS) is a pre-invasive lesion of the breast considered a precursor of invasive ductal carcinoma. This study aimed to determine whether activated PPARγ acts as a tumor suppressor in human DCIS progression. We utilized the high-affinity PPARγ agonist, efatutazone, to activate endogenous PPARγ in a well-defined model for the progression of basal (triple negative) DCIS, MCFDCIS cells, cultured under 2D and 3D conditions. We studied the effects of activated PPARγ on DCIS progression in MCFDCIS xenograft and C3(1)/Tag transgenic mice treated with 30mg/kg of efatutazone. In vitro, efatutazone did not alter the MCFDCIS cell proliferation but induced phenotypic and gene expression changes, indicating that activated PPARγ is able to differentiate MCFDCIS cells into more luminal and lactational-like cells. In addition, MCFDCIS tumorsphere formation in 3D was reduced by PPARγ activation. In vivo, efatutazone-treated MCFDCIS tumors exhibited fat deposition along with upregulation of PPARγ responsive genes in both epithelial and stromal compartments, suggesting features of milk-producing mammary epithelial cell differentiation. The efatutazone-treated lesions were less invasive with fewer CD44+/p63+ basal progenitor cells. PPARγ activation downregulated Akt phosphorylation in these tumors, although the ERK pathway remained unchanged. Similar trends in gene expression changes consistent with lactational and luminal cell differentiation were observed in the C3(1)/Tag mouse model after efatutazone treatment. Our data suggest that activation of the PPARγ pathway differentiates DCIS lesions and may be a useful approach to delay DCIS progression.

Abstract CT140: PINC trial: Preventing invasive breast neoplasia with chloroquine

Abstract The PINC trial (NCT01023477) examined the dosage efficacy of oral chloroquine (CQ), an autophagy inhibitor, as a neoadjuvant therapy to reduce the volume, cause regression and decrease the recurrence of breast ductal carcinoma in situ (DCIS), for any grade or ER/PR/Her2 status. Study Objectives: Establish the safety of preventive doses of chloroquine in patients receiving external beam radiation. Elucidate functional molecular and cellular impacts of in vivo autophagy pathway treatment for DCIS. Study the impact of autophagy inhibitors on the MRI characteristics of DCIS lesions. Study the molecular cytogenetic profile of DCIS lesions before and after therapy. This trial implemented a general strategy to accelerate the pace of community-based translational research. Technology for providing immediate feedback on the therapeutic efficacy at the molecular level can be broadly extended to other trials. Methodology: 12 patients diagnosed with DCIS (any grade or ER/PR/Her2 status) were enrolled and randomly assigned to receive CQ at 250mg/week (n=5) or 500mg/week (n=7) for 4 weeks, followed by standard of care surgical therapy. MRI was performed before/after CQ treatment. DCIS spheroid forming cells were isolated and propagated from fresh human DCIS lesions. DCIS cells were characterized by organ culture, xenograft transplantation, molecular cytogenetics, and 59 cell signaling kinases were quantified by Reverse Phase Protein Microarrays. Results: 12 patients completed 4 weeks of CQ treatment prior to surgical excision of their DCIS lesion, with 1 yr follow-up information. CQ treatment reduced PCNA proliferation index in DCIS lesions compared to untreated controls (p=0.001) and inhibited autophagic flux (LC3B positive puncta by IHC). CQ reduced the number of mammospheres in organoid culture without altering copy number variation. Xenograft transplants in NOD/SCID mouse mammary fat pads failed to generate tumors (n=4). 7/12 patients exhibited a reduction in lesion diameter by MRI, 3/12 patients exhibited no measurable change, and 2/12 had a slight increase. Calcium export channel protein (PMCA2) co-localized with 3+ HER2 positive DCIS lesions. Tumor infiltrating macrophages migrated into DCIS ducts following CQ therapy compared to controls (p=0.006). Conclusion: Oral chloroquine, as anti-autophagy therapy, generates a measurable reduction in proliferation of DCIS lesions and enhances immune cell migration into the duct. Citation Format: Virginia A. Espina, Lance Liotta, Svetlana Rassulova, Holly Gallimore, Thalia Grant-Wisdom, Geetha Menezes, Hassan Nayer, Kirsten Edmiston. PINC trial: Preventing invasive breast neoplasia with chloroquine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT140. doi:10.1158/1538-7445.AM2017-CT140

Cytochrome c1 in ductal carcinoma in situ of breast associated with proliferation and comedo necrosis.

It is well known that comedo necrosis is closely associated with an aggressive phenotype of ductal carcinoma in situ (DCIS) of human breast, but its molecular mechanisms remain largely unclear. Therefore, in this study, we first examined the gene expression profile of comedo DCIS based on microarray data and identified CYC1 as a gene associated with comedo necrosis. Cytochrome c1 (CYC1) is a subunit of complex III in the mitochondrial oxidative phosphorylation that is involved in energy production. However, the significance of CYC1 has not yet been examined in DCIS. We therefore immunolocalized CYC1 in 47 DCIS cases. CYC1 immunoreactivity was detected in 40% of DCIS cases, and the immunohistochemical CYC1 status was significantly associated with tumor size, nuclear grade, comedo necrosis, van Nuys classification, and Ki‐67 labeling index. Subsequent in vitro studies indicated that CYC1 was significantly associated with mitochondrial membrane potential in MCF10DCIS.com DCIS cells. Moreover, CYC1 significantly promoted proliferation activity of MCF10DCIS.com cells and the cells transfected with CYC1 siRNA decreased pro‐apoptotic caspase 3 activity under hypoxic or anoxic conditions. Considering that the center of DCIS is poorly oxygenated, these results indicate that CYC1 plays important roles in cell proliferation and comedo necrosis through the elevated oxidative phosphorylation activity in human DCIS.

Biomarker discovery to improve prediction of breast cancer survival: using gene expression profiling, meta-analysis, and tissue validation.

PurposeBreast cancer is the leading cause of cancer death worldwide in women. The molecular mechanism for human breast cancer is unknown. Gene microarray has been widely used in breast cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis survival. So far, the valuable multigene signatures in clinical practice are unclear, and the biological importance of individual genes is difficult to detect, as the described signatures virtually do not overlap. Early prognosis of this disease, breast invasive ductal carcinoma (IDC) and breast ductal carcinoma in situ (DCIS), is vital in breast surgery.MethodsThus, this study reports gene expression profiling in large breast cancer cohorts from Gene Expression Omnibus, including GSE29044 (N=138) and GSE10780 (N=185) test series and four independent validation series GSE21653 (N=266), GSE20685 (N=327), GSE26971 (N=276), and GSE12776 (N=204). Significantly differentially expressed genes in human breast IDC and breast DCIS were detected by transcriptome microarray analysis.ResultsWe created a set of three genes (MAMDC2, TSHZ2, and CLDN11) that were significantly correlated with disease-free survival of breast cancer patients using a univariate Cox regression model (significance level P<0.01) in a meta-analysis. Based on the risk score of the three genes, the test series patients could be separated into low-risk and high-risk groups with significantly different survival times. This signature was validated in the other three cohorts. The prognostic value of this three-gene signature was confirmed in the internal validation series and another four independent breast cancer data sets. The prognostic impact of one of the three genes, CLDN11, was confirmed by immunohistochemistry. CLDN11 was significantly overexpressed in human breast IDC as compared with normal breast tissues and breast DCIS.ConclusionUsing novel gene expression profiling together with a meta-analysis validation approach, we have identified a three-gene signature with independent prognostic impact. Furthermore, CLDN11 may offer a biomarker to predict prognosis as well as a new target for prognostic and therapeutic intervention for human breast IDC.

Different Biological Action of Oleic Acid in ALDHhigh and ALDHlow Subpopulations Separated from Ductal Carcinoma In Situ of Breast Cancer.

The mechanisms underlying breast cancer progression of ductal carcinoma in situ (DCIS) associated with fatty acids are largely unknown. In the present study, we compared the action of oleic acid (OA) on two human DCIS cell lines, MCF10DCIS.COM (ER/PR/HER2-negative) and SUM225 (HER2 overexpressed). OA led to a significant increase in proliferation, migration, lipid accumulation and the expression of lipogenic proteins, such as SREBP-1, FAS and ACC-1, in MCF10DCIS.COM cells but not SUM225 cells. The ALDHhigh subpopulation analyzed by the ALDEFLUOR assay was approximately 39.2±5.3% of MCF10DCIS.COM cells but was small (3.11±0.9%) in SUM225 cells. We further investigated the different biological action of OA in the distinct ALDHlow and ALDHhigh subpopulations of MCF10DCIS.COM cells. OA led to an increase in the expression of ALDH1A1, ALDH1A2 and ALDH1A3 in MCF10DCIS.COM cells. SREBP-1 and ACC-1 were highly expressed in ALDHhigh cells relative to ALDHlow cells, whereas FAS was higher in ALDHlow cells. In the presence of OA, ALDHhigh cells were more likely to proliferate and migrate and displayed significantly high levels of SREBP-1 and FAS and strong phosphorylation of FAK and AKT relative to ALDHlow cells. This study suggests that OA could be a critical risk factor to promote the proliferation and migration of ALDHhigh cells in DCIS, leading to breast cancer progression.