DCIS Lesions Research Articles

Abstract 2176: Mutational profiling of premalignant breast microbiopsies

Abstract Ductal carcinoma in situ (DCIS) comprises ~20% of all breast cancer diagnoses in the United States. Nearly all DCIS are treated to prevent progression to potentially lethal invasive cancer, yet it is estimated about half of DCIS progress within 10 years making the systematic treatment controversial. This highlights an urgent need for reliable molecular markers to stratify patients by risk. Small, degraded biopsies have made molecular profiling historically challenging; furthermore, a single biopsy can have several regions varying in nuclear grade, hormone receptor status, and histological architecture requiring laser-capture microdissection. Recent advances in whole-exome sequencing now enable mutational profiling from limited quantities (<10ng) of damaged, formalin-fixed, paraffin-embedded (FFPE) DNA, and present an opportunity to sequence individual regions and relate the phenotypic heterogeneity with genetic alterations. Here, we apply these advances to obtain mutational profiles from pure DCIS microbiopsies, enabling a comprehensive molecular profile of pure DCIS, direct measurement of genetic heterogeneity, and associations between molecular and histopathological features. Whole-exome sequencing was performed on 59 regions across 31 pure DCIS cases varying in nuclear grade (including 19% low-grade), and histological architecture (52% cribriform, 39% solid). Driver analysis revealed breast cancer-like driver alterations in 30/31 cases, and evidence for multiple putative drivers in 26/31, even in low-grade DCIS. The most frequently mutated genes across patients were common breast cancer driver genes: PIK3CA (37%), TP53 (22%), and GATA3 (11%). Phylogenetic trees constructed on multi-region biopsies revealed that regions of the same biopsy shared 12-336 alterations, and all pathogenic driver point mutations. Nevertheless, 1-22 region-specific alterations were found in each biopsy, some of which were likely driver CNA. We identified several histological associations with genetic alterations including high grade (p=2x10-4) and solid DCIS (p=3x10-4) associated with high copy number burden, and a non-significant increase in GATA3 (13% vs 0%) and PIK3CA (50% vs 28%) pathogenic mutations in cribriform vs solid architecture. Lastly, the most genetically diverse DCIS were HER2+/ER- (p=0.07) and those displaying necrosis (p=0.006). Overall, using novel methodology we performed detailed multi-region mutational profiling on difficult to sequence precancer lesions, revealing varying genetic diversity within a biopsy and novel associations between histology and underlying genetic landscape. We illustrate multiple driver genetic alterations and diversity present even in low-grade, pure DCIS lesions, providing key insight into early breast carcinogenesis and representing a critical step towards the development of prognostic markers of progression. Citation Format: Daniela Nachmanson, Mark F. Evans, Joseph Steward, Adam Officer, Huazhen Yao, Thomas J. O'Keefe, Farnaz Hasteh, Gary S. Stein, Kristen Jepsen, Donald L. Weaver, Gillian L. Hirst, Brian L. Sprague, Laura J. Esserman, Alexander Borowsky, Janet L. Stein, Olivier Harismendy. Mutational profiling of premalignant breast microbiopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2176.

Multi-modal imaging of high-risk ductal carcinoma in situ of the breast using C2Am: a targeted cell death imaging agent

BackgroundDuctal carcinoma in situ (DCIS) is a non-invasive form of early breast cancer, with a poorly understood natural history of invasive transformation. Necrosis is a well-recognized adverse prognostic feature of DCIS, and non-invasive detection of its presence and spatial extent could provide information not obtainable by biopsy. We describe here imaging of the distribution and extent of comedo-type necrosis in a model of human DCIS using C2Am, an imaging agent that binds to the phosphatidylserine exposed by necrotic cells.MethodsWe used an established xenograft model of human DCIS that mimics the histopathological features of the disease. Planar near-infrared and optoacoustic imaging, using fluorescently labeled C2Am, were used to image non-invasively the presence and extent of lesion necrosis.ResultsC2Am showed specific and sensitive binding to necrotic areas in DCIS tissue, detectable both in vivo and ex vivo. The imaging signal generated in vivo using near-infrared (NIR) fluorescence imaging was up to 6-fold higher in DCIS lesions than in surrounding fat pad or skin tissue. There was a correlation between the C2Am NIR fluorescence (Pearson R = 0.783, P = 0.0125) and optoacoustic signals (R > 0.875, P < 0.022) in the DCIS lesions in vivo and the corresponding levels of cell death detected histologically.ConclusionsC2Am is a targeted multi-modal imaging agent that could complement current anatomical imaging methods for detecting DCIS. Imaging the presence and spatial extent of necrosis may give better prognostic information than that obtained by biopsy alone.

Abstract 1583: The tumor immune microenvironment in early breast cancer progression

Abstract Ductal carcinoma in situ (DCIS), comprising 20% of all breast cancer diagnoses, is a neoplastic proliferation of epithelial cells confined to the luminal compartment of mammary ducts, which precedes invasive ductal carcinoma (IDC) formation. Although not all DCIS progresses to IDC, there remains no reliable method to determine which DCIS lesions are most likely to become invasive. Recent studies on paired HER2+ and triple negative DCIS and IDC, indicate biological modulation of the microenvironment as a possible mechanism of progression. In particular, the immune microenvironment plays a crucial role in modulating cancer cell behavior and invasion potential. The role of the immune microenvironment in luminal (ER/PR+, HER2+/-) DCIS progression to IDC has not been well studied, however it is an area of keen interest given that this subtype accounts for 50-65% of all diagnosed breast cancers. To address this gap in knowledge, we employed imaging mass cytometry (IMC) to evaluate the tumor immune microenvironment of co-existing luminal DCIS and IDC in patient tumor samples. IMC allows for the comprehensive analysis of up to 35 different metal-tagged antibodies simultaneously, by coupling laser ablation of the tissue with mass cytometry, while maintaining spatial integrity. Using a panel of epithelial, immune and signaling markers, we characterized the tumor microenvironment of DCIS and IDC tumor components across multiple patient samples, investigating the differences between IDC, DCIS adjacent to IDC, and distant DCIS that is further away from the IDC component, while also controlling for inter-individual heterogeneity. A segmentation mask for each image was generated using a combination of Ilastik, CellProfiler, and ImageJ open source platforms. Single-cell information was extracted and utilized to categorize cells by phenotypes and reconstruct spatial organization maps. Immune phenotype composition, cell-cell interactions, and tumor infiltrating lymphocytes (TILs) were evaluated, and nearest neighbor analysis was performed. In addition to a significant increase in TILs within IDC tumors, we observe a switch towards an immunosuppressed microenvironment between DCIS (both adjacent and distant) and IDC. This is evidenced by an increase in T regulatory cell (Treg) infiltration and Treg-CD8 T cell interactions, as well as a decrease in the proportion of CD8 T cells observed in IDC tumors. Furthermore, there is an increase in T cell infiltration in adjacent DCIS, when compared to both IDC and distant DCIS. This may indicate a more activated immune microenvironment phenotype in these tumors. Results from this novel study will give insight into how the overall immune landscape is reprogrammed during DCIS progression, and may contribute to efforts to better predict early-stage breast cancer progression. Citation Format: Alyssa Victoria Francis, Luke McCaffrey. The tumor immune microenvironment in early breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1583.

Abstract 2502: Genetic and functional heterogeneity of DCIS as predictors of invasive cancer

Abstract Genetic diversity both between and within individual tumors constitutes a challenge to personalized cancer medicine. Intra-tumor heterogeneity provides the genetic fuel for natural selection in clonal evolution and cancer progression. Tumors with high levels of genetic heterogeneity are hypothesized to be more likely to demonstrate aggressive behavior and progress to invasion and metastasis. We analyzed the mutational loads from separate areas of pure DCIS and compared this to genetic heterogeneity in DCIS lesions found adjacent to invasive and metastatic cancer. Two spatially distinct areas of DCIS from each case were macro-dissected and the DNA extracted from FFPE samples. To analyze the data, we developed new bioinformatics methods that allowed analysis of small amounts of degraded DNA extracted from FFPE samples across multiple regions. Our bioinformatics pipeline was optimized on a series of 28 independent technical replicates of the same DNA sample sequenced twice, as training tools to find the best filtering parameters. Whole exome sequencing was performed on each of the two geospatially separated samples for each case. Minimum coverage for inclusion in this study was 40X over at least 50% of the exome. We used the ratio of private mutations (only in 1 area) to public (found in both areas) mutations as a measure of intra-tumor heterogeneity. We present an approach to measure clonal heterogeneity using a bulk sequencing strategy applied to geospatially distinct foci of DCIS. We found statistically significant difference between DCIS adjacent to invasive disease and metastatic patients' genetic divergence (t-test, p=0.013). Our findings suggest that genetic and functional heterogeneity may play an important evolutionary role as a driver for invasive progression. Citation Format: Angelo Fortunato, Diego Mallo, Lorraine King, Timothy Hardman, Allison Hall, Jeffrey R. Marks, Shelley Shelley Hwang, Carlo C. Maley. Genetic and functional heterogeneity of DCIS as predictors of invasive cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2502.

Breast intraductal nanoformulations for treating ductal carcinoma in situ I: Exploring metal-ion complexation to slow ciclopirox release, enhance mammary persistence and efficacy

IntroductionDuctal Carcinoma In Situ (DCIS) represents a significant fraction (~20–25%) of all newly diagnosed breast cancer cases and, if left untreated, a significant fraction of patients will progress to invasive disease. Surgery is the only treatment option. Ciclopirox (CPX), an FDA-approved antifungal drug, has exhibited promising antitumor activity by down-regulating the expression of vital antiapoptotic cellular proteins and inhibiting the genetic expression of several oncogenic pathways. In this study, the feasibility of using nanoscale delivery systems to control release and prolong mammary tissue persistence of a lipophilic metal complex of CPX and Zinc (CPXZn) after intraductal administration was investigated. MethodsCPX and CPX-Zn nanosuspensions (NSs) were prepared using an evaporative nanoprecipitation-ultra-sonication method. Flash nanoprecipitation was used to prepare PLGA nanoparticles (NPs) loaded with CPXZn. Our established orthotopic DCIS rat model was used to evaluate efficacy. Briefly, two days after 13762 Mat B III cell intraductal inoculation, rats were divided into treatment groups and a single intraductal injection of CPX NS, CPX-Zn NS or CPX-Zn NPs was administered. In the first study arm, the efficacy of CPX NS (1, 3, 5 mg/duct) was evaluated. In the second arm, the in vivo efficacy of CPX NS, CPX-Zn NS and CPX-Zn loaded NPs was evaluated and compared at equivalent CPX doses. The mammary persistence of CPX from CPX NS, CPX-Zn NS, and CPX-Zn PLGA NPs was also assessed. ResultsCPX-Zn complex was successfully synthesized and characterized by several spectral analyses. CPX release was slowed from the CPX-Zn NS and further slowed by incorporating CPX-Zn into PLGA NPs as compared to the CPX NS with release half times following the order: CPX NS < CPX-Zn NS << CPX-Zn NP. Intraductal CPX NS administration was dose and time dependent in suppressing tumor initiation suggesting prolonged mammary exposure may improve efficacy. In the second arm, mammary tissue persistence of CPX followed the rank order CPX NS < CPX-Zn NS << CPX-Zn NP at 6 h and 48 h post-administration. Prolonged mammary CPX exposure was highly correlated to improved efficacy. Prolonged CPX tissue persistence, attributed to slower release from the zinc complex and the PLGA NPs, resulted in a 5-fold dose reduction compared to the CPX NS. ConclusionsThe current results demonstrate that slowing drug release in the mammary duct after intraductal administration overcomes the rapid ductal clearance of CPX, prolongs mammary tissue persistence, improves efficacy against DCIS lesions in vivo, and requires 5-fold less CPX to achieve equivalent efficacy. The studies also provide a strategic path forward for developing a locally administered drug delivery system for treating DCIS, for which no primary chemotherapy option is available.

Abstract P1-20-20: Intraoperative ultrasound localization (IOUS) for non-palpable breast cancer

Abstract Background: With the introduction of breast cancer screening the number of non-palpable breast cancers and so the number of breast conserving surgery (BCS) has increased. Wire-guided localization (WGL) is used as invasive standard method for localization. IOUS is an alternative non-invasive, time-sparing and safe localization method with many cost benefits. Patient and methods: A retrospective observational study was performed in a single breast unit where IOUS was used as localization method by two surgeons in the period June 2013 to October2016. The surgeons did not have any special experience with Ultrasound (US) apart from one week of bed-side training. Results: During a 41 months period the two surgeons treated 452 with BCS. Of them 126 were non-palpable and visible by US 28%. The 126 cancers were identified with IOUS at surgery and removed successfully by the surgeons. In the period a total of 156 had a BCS with WGL. These cases had a nonpalpable cancer which were non-visible on US with either calcifications, DCIS or non-specific lesions. In the study we also found that the re-lumpectomy rate for BCS with IOUS was lower than without IOUS. In the entire breast unit, the number of BCS were 848 and number of BCS+WGL were 320 in the same period. Of them 263 (22.5%) had a re-lumpectomy. The number BCS with IOUS were 452 and the number of BCS+WGL with IOUS were 156. Of them 72 (11.8%) had a re-lumpectomy. Conclusion: In our breast unit, IOUS was used after a short bed-side training, with 100% success rate, to remove non-palpable cancers if visible on US. IOUS is easy to learn and easy to use and offers many financial and time-sparing benefits. IOUS has a reasonable learning curve and can reduce the work load on the Radiology Department. Citation Format: Shideh Pour Zahed, Thor Knudsen Andersen. Intraoperative ultrasound localization (IOUS) for non-palpable breast cancer [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 P1-20-20.

Establishment of a 3D co-culture model to investigate the role of primary fibroblasts in the development of an invasive phenotype of DCIS lesions

Establishment of a 3D co-culture model to investigate the role of primary fibroblasts in the development of an invasive phenotype of DCIS lesions

Abstract P3-06-12: Genetic heterogeneity of DCIS is a predictor of invasive cancer

Abstract Background: Heterogeneity is a hallmark of human cancers that is apparent both between and within individual tumors. Intra-tumor heterogeneity provides the genetic fuel for natural selection in clonal evolution and cancer progression. Tumors with high levels of genetic heterogeneity are hypothesized to be more likely to progress to invasion and metastasis Methods: We measured the mutational loads from separate areas of pure DCIS and compared this to genetic heterogeneity in DCIS lesions that co-exist with invasive cancer, as a surrogate for progression. Cases of pure DCIS and DCIS diagnosed concurrent with invasive cancer were identified. Two areas of DCIS from each case a minimum of 0.8 cm apart and control tissues were macro-dissected and the DNA extracted from FFPE samples. To analyze the data, we developed new bioinformatics methods that allowed analysis of small amounts of degraded DNA extracted from FFPE samples across multiple regions. Our bioinformatics pipeline was optimized on a series of 28 independent technical replicates of the same DNA sample sequenced twice, as training tools to find the best filtering parameters. Results: Whole exome sequencing was performed on two geospatially separated blocks for each case (41 pure DCIS and 30 DCIS adjacent to invasive disease). Minimum coverage for inclusion in this study was 40X over at least 50% of the exome. We used the ratio of private mutations (only in 1 area) to public (found in both areas) mutations as a measure of intra-tumor heterogeneity. Overall mutational load of DCIS was not a significant predictor to progression; however notably, DCIS adjacent to invasive disease patients had a higher ratio of private/public mutations (heterogeneity) in coding domains (Mann-Whitney p=0.016. Functional analysis of mutated coding genes (DAVID 6.8) shows a statistically significant enrichment in signal transduction, olfactory receptors (G-protein-coupled receptors) and cell-matrix interactions in both tumor types, after FDR correction. DCIS adjacent to invasive disease had an enrichment of mutated genes involved in additional cellular functions such as microtubule activity (fold enrichment =7.6, FDR=0.002), protein-protein interactions (fold enrichment =3.65, FDR=5.11E-04) and extracellular matrix remodeling (fold enrichment =8.3, FDR=0.02). Conclusion: We present an approach to measure clonal heterogeneity using a bulk sequencing strategy applied to geospatially distinct foci of DCIS. Our findings suggest that functional heterogeneity may play an important evolutionary role as a driver for invasive progression. Citation Format: Fortunato A, Mallo D, King L, Hardman T, Hall A, Marks JR, Hwang S, Maley CC. Genetic heterogeneity of DCIS is a predictor of invasive cancer [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 P3-06-12.

Abstract P6-16-03: The impact of ductal carcinoma in situ of the breast on health services utilization and general health and well-being of women

Abstract Background: The impact of DCIS diagnosis on the development of significant anxiety and depression and on the utilization of breast imaging and breast biopsy procedures, which may reflect fears of breast cancer recurrence, is unknown. We established a population cohort of women with DCIS and examined the impact of the DCIS diagnosis on health services utilization (physician, emergency room visits) before and after DCIS, including those related to anxiety or depression, and all breast-related interventions, compared to similar women without DCIS. Methods: From hospital claims we identified a population-based cohort of women with a first diagnosis of DCIS between 2010-2015. Cases with prior history of cancer were excluded. We identified treatment (mastectomy, breast-conserving surgery (BCS) +/- radiation therapy (RT) by linkage of administrative databases. Using DCIS diagnosis date for each case, we matched 5 unaffected controls (with no history of DCIS) by age, mammography history, socioeconomic status, and comorbidity at same date. For all cases and controls we extracted the following from physician billing claims: breast imaging (mammography, ultrasound, MRI), breast biopsies, breast surgery, all physician visits, and all hospital claims (including those with a diagnosis of anxiety or depression), for five years prior to and following the index date. We excluded claims from the year prior to and subsequent to the index date to remove the effect of services utilization surrounding the diagnosis and management of the index DCIS lesion. We then computed the rate of each service per 100 person-years for 5 years before and after the index date. Cases who developed local recurrence (LR) (and their matched controls) were censored 3 months prior to LR date. We used negative binomial regression to test differences in rates of service utilization before and after DCIS diagnosis and between cases and unaffected controls. Results: The cohort includes 4,977 women treated for DCIS were identified, each matched to 5 controls. 911/4,977 (18.3%) of cases were &amp;lt; 50 years old at diagnosis; 1,006/4,977 (20.2%) were treated by BCS alone, 2,995/4,977 (60.2%) by BCS+RT, and 976 (19.6%) by mastectomy. For cases diagnosed with visits with a breast diagnosis code, utilization of breast imaging procedures and the use of breast surgery was significantly greater relative to unaffected controls irrespective of age at diagnosis and treatment (all p values &amp;lt;0.0001). Primary care visits were higher among cases (RR=1.10 (95% CI 1.06, 1.14), p&amp;lt;0.0001), including those with a breast diagnosis code (RR=3.69 (95%CI 3.29, 4.14)). However, there was no increase in relative rates of physician or hospital visits for anxiety or depression (RR 1.13 (95% CI 0.97, 1.32 p=0.11), psychiatry visits (RR 1.07 (95% CI 0.82, 1.40) p=0.6), non-breast surgical procedures (RR 1.10 (95% CI 0.88, 1.37) p=0.4), or emergency department visits (unrelated to breast) in cases with DCIS compared to unaffected controls. Conclusion: DCIS is associated with more visits and procedures related to the breast compared to women without a diagnosis of DCIS but overall health services utilization and visits related to anxiety and depression were not increased. Citation Format: Rakovitch E, Sutradhar R, Zhou L, Nofech-Mozes S, Hanna W, Paszat L. The impact of ductal carcinoma in situ of the breast on health services utilization and general health and well-being of women [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 P6-16-03.

Abstract P4-15-04: Neoadjuvant endocrine therapy for ER+ DCIS can lead to disease regression and allows BCS in up to a third of patients with disease &amp;gt;40mm at diagnosis

Abstract Background: The role of neoadjuvant endocrine therapy (NET) for ER+ DCIS is an area of evolving study. It may allow down-sizing prior to surgery, converting DCIS requiring mastectomy to disease suitable for breast conservation surgery (BCS). Here we report the results from the first European single-institution series of its type. Methods: Data were prospectively collected from patients diagnosed with ER+ DCIS and treated with NET prior to surgery, at a single unit between 2009 -2015. The size of the tumour on initial imaging (mammography) was compared to the size of the tumour on final imaging and pathology using RECIST criteria to determine disease progression. Blocks from initial core biopsy and final pathology are being interrogated by immunohistochemistry and DNA and RNA comparisons. Results: 42 patients diagnosed with ER+ DCIS received NET with median age at diagnosis of 63y (range 37-94y). 7/42 premenopausal women were treated with tamoxifen, 35/42 post-menopausal women were treated with letrozole. 36/42 (85.7%) patients underwent surgery with 18/36 (50%) requiring mastectomy and 18/36 (50%) treated by BCS. 3/18 (16.7%) of the BCS patients required re-excision for positive margins. The median time to operation was 72d (range 15-308d). In total 12/42 (28.6%) had invasive disease on final pathology. 2/36 (5.6%) patients had a pathological complete response (PCR), 14/36 (38.9%) had a partial response (PR), 17/36 (47.2%) had stable disease and 3/36 (8.3%) had larger disease on pathology than imaging; this is a common feature of many lower grade DCIS lesions. 26/42 (61.9%) patients initially had DCIS &amp;gt;40mm (largest 240mm) and yet 9/26 (34.6%) of these patients still underwent successful BCS. There was a significant correlation between length of endocrine therapy and reduction in size of disease. Immunohistochemical and molecular analyses are ongoing. Conclusions: •NET is an effective treatment for ER+ DCIS. It reduces the rate of re-excision to 16.7% in this series - substantially lower than the national (UK) figures for DCIS at 30%. •It produces path CRs (5.6%) and high response rates that relate to the duration of treatment. •This unique study shows that the optimal duration of NET is of the order of 6 months, achieving high rates of conversion for mastectomy to BCS. Citation Format: Cartlidge CW, Johns N, Hackney RJ, Turnbull AK, Dixon JM. Neoadjuvant endocrine therapy for ER+ DCIS can lead to disease regression and allows BCS in up to a third of patients with disease &amp;gt;40mm at diagnosis [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 P4-15-04.

Abstract OT1-04-01: A phase IIB pre-surgical trial of oral tamoxifen (TAM) versus transdermal 4-hydroxytamoxifen (4-OHT) in women with DCIS of the breast

Abstract Background Ductal carcinoma in situ (DCIS) is diagnosed in 60,000 women annually in the US. TAM is proven to reduce risk of local recurrence and new primary breast cancer in women with estrogen receptor (ER) positive DCIS. However, acceptance of TAM has been low, primarily because of toxicity related to systemic exposure. Local delivery to the breast is an attractive alternative since low systemic levels could minimize toxicity. 4-OHT is an active metabolite of TAM. When formulated as a gel and applied to the breast skin, it is well tolerated, and results in 4-OHT breast tissue drug levels comparable oral TAM. In small pilot studies, its anti-proliferative effects on invasive breast tumors and DCIS are also similar to oral TAM [Lee O, et al. PMID 25028506]. The goal of our study is to validate these results in preparation for a Phase III trial of 4-OHT gel in comparison to oral TAM. Methods We are conducting a randomized, double-blinded, placebo-controlled, Phase IIB pre-surgical trial to demonstrate that daily application of 4-OHT gel will result in a reduction in the Ki-67 labeling index of DCIS lesions that is not inferior to that seen in women receiving daily oral TAM 20 mg daily. Ki-67 of the base-line diagnostic core needle biopsy will be compared to that of the therapeutic surgical excision sample after oral TAM or 4-OHT gel for 8 ± 2 weeks. Secondary endpoints include changes in Oncotype DCIS-Score, IHC markers (CD68, COX2, p16), hormone levels, coagulation markers, drug concentration in the plasma and breast tissue, the fraction of women with no residual DCIS in the surgical sample, and experienced symptoms. 100 women (assuming 20% non-evaluable samples or compliance issues) with DCIS (10% ER-positive) will be enrolled across six institutions into two intervention arms: oral TAM 20 mg daily, placebo gel and 4-OHT gel 4mg daily (2mg/breast), placebo capsule. All participants will be evaluable for toxicity from their first dose. All samples from all participants who receive drug will be evaluated and included in the primary analysis, which will be based on intent to treat principle. To date 15 of 100 participants have been enrolled across six institutions including: Northwestern University in Chicago, IL, St. Elizabeth Healthcare in Edgewood, KY, Duke University Medical Center in Durham, NC, Cleveland Clinic in Cleveland, OH, Memorial Sloan Kettering Cancer Center in New York, NY, and Mayo Clinic in Rochester, MN. Since study open, 69 potential participants have been contacted, 52 did not consent for screening, 17consented for screening, 2 are pending consent, and 15 have started study intervention. The most common reasons potential participants chose not to consent are wanting to schedule surgery as soon as possible, attitudes toward medical research, and current use of a prohibited concomitant medication such as a potent inhibitor of tamoxifen metabolism or exogenous sex steroid. Funding Source: NCI Contract # HHSN2612201200035I. Citation Format: Benante KA, Xu Y, Tull MB, Segura AJ, Alber KM, Kalinichenko K, Hou L, Jovanovic B, Perloff M, Heckman-Stoddard B, Dimond E, Khan SA. A phase IIB pre-surgical trial of oral tamoxifen (TAM) versus transdermal 4-hydroxytamoxifen (4-OHT) in women with DCIS of the breast [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 OT1-04-01.

Abstract P3-08-08: SOX11 is a potential prognostic marker of high-risk breast ductal carcinoma in situ

Abstract Ductal carcinoma in situ (DCIS) comprises 20-25% of screen-detected breast cancers and, like invasive ductal carcinoma (IDC), is heterogenous in terms of the underlying biology, presentation, and outcome. While there are limited potential biomarkers of outcome for DCIS, estrogen receptor (ER)- positive, progesterone receptor (PR)- positive, and HER2- negative DCIS appears to have a better prognosis compared to ER- negative, PR- negative, and HER2- positive DCIS. The aim of this study was to identify additional clinically relevant markers to stratify DCIS according to risk of relapse or progression to invasive disease. In order to determine the driver genes involved in DCIS evolution, we utilized transcriptional data sets (GSE788, GSE16873), containing data from both normal mammary glands (NMG) and DCIS. Upon performing class comparison (NMG vs DCIS), we identified 297 over-expressed genes and 187 under-expressed genes. The over-expressed genes represented mitotic and proliferative features annotated as mitotic spindle and condensed chromosomes, while the under-expressed genes were associated with loss of epithelial features annotated as epithelial cell differentiation and development. The 484 differentially expressed genes were further correlated with recurrence events using Kessler's breast cancer data set to identify genes contributing to the aggressive features across IDC and subsequently associated with DCIS. Genes correlating with recurrence events were selected. Of the 484 genes, 99 genes were found to be significantly associated with recurrence events of IDC (with P&amp;lt;0.003). Among these 99 genes, component genes of the Oncotype DCIS score and genes reported as relevant to DCIS biology were included for Nanostring transcriptomic analysis. The final number of genes-of-interest were 58, including 5 housekeeping genes. 40 DCIS lesions and 8 NMG tissue were macro- dissected from formalin- fix paraffin- embedded blocks (FFPE) and extracted transcripts were subjected for Nanostring analysis. Gene expression data was clustered in an unsupervised manner using R software. Two sample clusters were identified: an ER/PR- negative cluster and an ER/PR- positive cluster. Over-expression of transcription factor SOX11, along with HER2, was exclusively seen in the ER/PR- negative cluster. This cluster was further categorized into HER2-low/SOX11+ and HER2-high/SOX11+ groups. These RNA expression findings are undergoing confirmation by immunohistochemistry (IHC) of the FFPE tumor sections. An independent series of 15 DCIS cases that have recurred as DCIS or progressed to IDC were analyzed by IHC, revealing SOX11 expression only present in cases displaying a high proportion of HER2+ expression. SOX11 is exclusively expressed in ER/PR-negative DCIS and is a candidate clinical marker for recurrence of DCIS or progression to IDC. Citation Format: Ladha FA, Kai K, Lu W, Yang F, Tang X, Wistuba II, Sen S, Thompson A. SOX11 is a potential prognostic marker of high-risk breast ductal carcinoma in situ [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 P3-08-08.

Abstract B14: Sprouty4 regulates the transition to invasive breast ductal carcinoma through ERK/MAPK signaling

Abstract Breast cancer is one of the most common oncologic developments in U.S. women, with approximately 25% of these cases being in situ disease (DCIS). For many years DCIS lesions have been treated using surgery and radiotherapy with the goal of decreasing the amount of late-stage disease. Unfortunately, a number of reports have since shown that early intervention is not sufficient and have highlighted the likelihood of overtreatment. This is especially true for triple-negative breast cancers, which are more strongly associated with distant recurrence, metastasis, and death compared to other types of invasive breast ductal carcinoma (IDC). These factors have generated a substantial clinical need for reliable biomarkers or molecular determinants to assess disease progression. Sprouty proteins are recognized as important regulators of ERK/MAPK signaling, and have been studied in various cancers. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling, and its loss or reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry we found that Sprouty4 expression was reduced in IDC patient tissue samples compared to normal or DCIS tissues, and that ERK/MAPK activity had an inverse relationship to Sprouty4 expression. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient overexpression of Sprouty4 reduced both ERK/MAPK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments revealed data consistent with the relocation of E-cadherin back to the cell surface and the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug produced the restoration of an epithelial-like phenotype similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 may act to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions. Citation Format: Ethan J. Brock, Ryan Jackson, Julie L. Boerner, Quanwen Li, Bonnie F. Sloane, Raymond R. Mattingly. Sprouty4 regulates the transition to invasive breast ductal carcinoma through ERK/MAPK signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B14.

PO-069 Clinical and histological risk factors for subsequent in situ lesions after a primary diagnosis of ductal carcinoma in situ

IntroductionDuctal carcinoma in situ (DCIS) is considered a potential precursor lesion of invasive breast cancer and is treated to prevent the development of invasive disease. After DCIS treatment, three scenarios emerge: i) the DCIS lesion has been removed completely, ii) a remnant or new DCIS lesion will progress into invasive breast cancer, or iii) a remnant or new DCIS lesion will be diagnosed as a subsequent DCIS lesion. In the latter case a risk of developing invasive breast cancer still or again exists. Therefore, our aim was to investigate risk factors for subsequent DCIS lesions.Material and methodsAll patients diagnosed with DCIS between 1989 and 2004 in the Netherlands were selected by the Netherlands Cancer Registry (NCR). Pathology reports were made available by the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA) with follow-up to 2016. We selected for patients treated by breast conserving surgery with or without radiotherapy, who subsequently presented with a second DCIS lesion according to the pathology report. Afterwards, we linked the datasets of NCR and PALGA to analyse clinical and pathological risk factors for subsequent DCIS lesions.Results and discussionsAfter a median follow-up time of 13 years, 450 of 5584 (8.1%) women with primary DCIS presented with a subsequent DCIS lesion in the ipsilateral breast. The average time to a subsequent ipsilateral DCIS lesion was 4.1 years. Histological factors such as size, grade, margins and clinical factors such as age, radiotherapy yes or no, time of follow-up will be analysed. Results will be compared with the risk factors for developing invasive breast cancer after a primary diagnosis with DCIS (Elshof et al, BCRT 2016).ConclusionAccording to our results, the risk of a subsequent ipsilateral in situ lesion after primary DCIS is 8.1% after breast conserving surgery. Analysis of clinical variables available from the Netherlands Cancer Registry, histological variables available from the pathology reports, and comparison with risk factors for developing invasive disease is ongoing. Molecular analysis of subsequent in situ lesions will be performed as a follow-up project.

PO-070 Identification of risk factors for subsequent invasive breast cancer after primary DCIS by transcriptomic profiling

IntroductionDuctal carcinoma in situ (DCIS) is a pre-invasive breast lesion, frequently detected by breast cancer screening, with thousands of new cases each year. While DCIS is not life threatening, it does increase a women’s risk of developing invasive breast cancer; this in turn can lead to breast-cancer specific mortality. Therefore, almost all DCIS lesions are treated to prevent progression to invasive disease. However, many DCIS lesion will never develop into invasive breast cancer if left untreated, indicating that many women are overtreated. In this study, we performed RNA sequencing (RNAseq) on a large set of primary DCIS lesions, comparing lesions with a subsequent invasive breast cancer recurrence (IBCR) to those without, to identify factors distinguishing harmless from potentially hazardous DCIS.Material and methodsWe made use of a DCIS case-control series, nested in a nation-wide population-based cohort of Dutch women diagnosed with primary DCIS and treated with breast conserving surgery alone (standard clinical practice) between 1989–2005. Mean follow up time was 12.0 years. RNA was extracted from FFPE laser-microdissected tissue fragments of 183 primary DCIS lesions: 100 associated with subsequent IBCR and 83 which remained free of invasive recurrence. RNAseq was performed to identify differentially expressed genes between DCIS with and without IBCR. Subsequently, gene set enrichment analysis (GSEA) was performed. Finally, findings were correlated with patients’ clinical and histopathological characteristics.Results and discussionsDCIS lesions were categorised into PAM50 intrinsic subtypes: Luminal A 25%, Luminal B 38%, Her2 22%, Basal 10%, Normal 5%. Within each PAM50 subtype, 1 to 64 genes were statistically significant (p<0.05) differentially expressed between DCIS lesions with and without IBCR. GSEA revealed these genes were enriched in immune-related pathways. In Luminal A DCIS the pathways interferon alpha response and interferon gamma response and were associated with subsequent IBCR. Correlation of these findings with clinical and histopathological characteristics is ongoing.ConclusionTo our knowledge, we performed the first large-scale transcriptome analysis of primary DCIS lesions with and without subsequent IBCR and long-term follow-up. Our data suggest that immune-related pathways are involved in DCIS progression. The results of our study add to the current understanding of DCIS and to risk stratification of DCIS in the near future.

Abstract 171: Acid-induced collagen remodeling promotes cancer progress as a result of niche engineering

Abstract Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells in tumors adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to a disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, niche construction and remodeling promoted by adaptation to acidosis, which itself is a metabolic adaptation to the early harsh environment in intraductal neoplasms. Early carcinogenesis is an avascular and hypoxic disease leading to extracellular acidosis. Hence, we investigated how acid-adapted tumor cells engineer their niche to support their growth, proliferation, and invasion. We show that relatively rare collagens are produced by acid-adapted cancer cells to create an environment that contributes to their survival. We found collagen and other fibrillar structure in the periluminal region of DCIS lesions using Second Harmonic Generation Microscopy (SHG). We compared the microarray of DCIS samples to normal tissue and observed that Col10a1 and Col11a1 were significantly increased. We have shown previously that DCIS lesions are acidic. To test if these were related, we adapted low grade MCF7 and DCIS-10AT breast cancer cells to growth in acidic conditions and probed them for collagen production using qPCR, western blotting and Immunofluorescence. We found acid adaptation increases number of collagens including Col10a1, Col11a1, and different subtypes of Col4. Further, we developed a window chamber model to study the collagen remodeling real time through intra-vital microscopy. SNARF and collagen imaging simultaneously showed the collagen structural modifications in acidic regions. Further proteomic and secretome analysis of acid adapted cells versus non-adapted cells identified additional enzymes involved in collagen remodeling such as TGM2 and LOXL2 that have been shown to play major role in matrix remodeling. These findings were also validated by western blot and ICC. To test the role of collagens in viability of cancer cells we grew acid adapted and non-adapted MCF7 cells as 3D spheroids and treat them with FAK inhibitors. FAK inhibitors dramatically affected the acid adapted spheroids. The specific role of each of collagen genes in survival and invasion is still under study. We discuss that adaptation to acidosis induces cancer cells to adopt strategies that assist them to be more independent, particularly in harsh conditions such periluminal areas of DCIS that they don't have access to stroma cells; they produce the collagen they need and will remodel it by the enzymes produced by themselves to be able to survive, grow, and invade. Finally these adaptation mechanisms may present a vulnerability to design new therapeutics against the emerging aggressive phenotypes during cancer cell evolution. Citation Format: Ava Niazi, Johnson J. Joseph, Robert J. Gillies, Mehdi Damaghi. Acid-induced collagen remodeling promotes cancer progress as a result of niche engineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 171.

Abstract P4-15-13: When is cancer not really cancer? The PREvent ductal carcinoma in situ invasive overtreatment now (PRECISION)* initiative

Abstract Background Ductal carcinoma in situ (DCIS) now represents 20-25% of all breast neoplasia due to large-scale detection by widely adopted population-based breast cancer screening programs. As a result, thousands of women are confronted with DCIS each year: more than 8,000 in the UK, 2,500 in the Netherlands, and some 50,000 in the US. Conventional management includes surgery, supplemented by radiotherapy and/or endocrine therapy, but overtreats the majority of DCIS as ˜1% recur annually and breast cancer mortality is ˜3% at 20 years. Uncertainty as to which DCIS lesions will progress to invasive cancer or, after excision, which will return with recurrent DCIS or invasive breast cancer drives this overtreatment. This urges us to learn how to distinguish DCIS that may progress to invasive breast cancer from the majority of indolent DCIS. Such distinction may be best achieved by synergistic international collaboration between leading global experts from various disciplines, driven by the essential input from patient voices as full members of the research team. Aim PRECISION (PREvent ductal Carcinoma In Situ Invasive Overtreatment Now) aims to save thousands of women with low risk DCIS the burden of intensive inappropriate treatment of DCIS (surgery, radiation therapy, hormonal therapies) through the discovery of new data and development of novel tests that promote informed and shared decision-making between patients and clinicians, without compromising the excellent outcomes for DCIS management presently achieved. Methods First, three large DCIS cohorts and supplementary resources will be collected enabling in depth molecular studies. Second, extensive genomic characterization, immune profiling and imaging analysis will be performed. In vivo and in vitro modeling will be performed to study the biology of DCIS in detail. Finally, all clinical, immune, and molecular data will be incorporated into a clinical risk prediction model. This risk prediction model will be validated in three prospective randomized DCIS trials in the US (COMET trial), UK (LORIS trial), and mainland Europe (LORD trial). How the results of this research will be used The discoveries from our laboratory studies, including a risk stratification model, will be cross-validated in three prospective trials of DCIS active surveillance versus conventional treatment (the COMET, LORIS and LORD trials). As such, the main result of this study will be that we can identify a group of women for which active surveillance for DCIS could be a safer alternative to intensive treatment. Ultimately, this may also contribute to a more reassuring perception of risk regarding non-life threatening precancerous lesions in general, reducing anxiety and preserving quality of life. * The PRECISION Team is a Cancer Research UK Grand Challenge Award 2017 winning team and will be jointly funded by Cancer Research UK and the Dutch Cancer Society. Citation Format: Wesseling J, Thompson A, Nik-Zainal S, Futreal A, Hwang S, Jonkers J, Lips E, Rea D, On Behalf of the PRECISION Team. When is cancer not really cancer? The PREvent ductal carcinoma in situ invasive overtreatment now (PRECISION)* initiative [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-15-13.

Stereotactic vacuum-assisted breast biopsy: Comparison between 11- and 8-gauge needles

PurposeThe 11-gauge (11G) stereotactic vacuum-assisted breast biopsy (VABB) showed a better profile than 14G-VABB in terms of feasibility, safety, microcalcification sampling, and accuracy. Underestimation rates were significantly lower with 11G-VABB than with 14G-VABB. Thus, the introduction of an even larger needle at the VABB procedure could reduce this rate further. The purpose of this study was to compare the overall performance of stereotactic VABB with 8G and 11G needles. Materials and methodsFour hundred and three VABBs performed between July 2012 and February 2015 at the Breast Diagnostic Unit of Careggi Hospital in Florence were retrospectively analyzed; 197 were performed with 11G-VABB and 206 with 8G-VABB. Lesions were classified according to mammographical patterns in microcalcifications, architectural distortions, or opacities, and all biopsy targets were classified according to BIRADS classification as BIRADS III, IV or V. Data were collected on radiological classification of targets, imaging presentation, procedure time, number of specimens per procedure, and microcalcification retrieval on histological findings. Surgery was always performed when high-risk or malignant lesions (B3 or B5) were detected; the final diagnosis was made on surgical pathology. ResultsCompared to VABB with an 11G needle, 8G-VABB allows a reduction in the time needed to complete the procedure (20.6 versus 27.4, P < 0.00001) and the number of specimens collected per lesion (21.6 versus 12.2, P < 0.00001). Moreover, 8G-VABB resulted in the same diagnostic accuracy, and the underestimation rates were comparable between the two groups for both B3 and DCIS lesions. ConclusionsThe 8G needle should be considered as a valid alternative option in VABB for breast lesions.

Abstract P4-06-01: Patient derived DCIS mouse-intraductal (MIND) models recapitulate the full spectrum of human patient pathology and histologic features including progression to invasion in a subset of cases

Abstract Introduction: The 20-year breast cancer mortality rate following a DCIS diagnosis is ∼3%. Radiation and anti-hormonal therapy of DCIS has not resulted in improved overall survival, which argues against the non-selective use of such therapies in DCIS management. However, some DCIS cases do progress to invasive cancer, and these patients may benefit from treatment. A study of 80 DCIS patients followed for up to 30 years reported a 43% progression rate. Notably, progression was not predicted by grade, as 39% of even low-grade DCIS ultimately progressed to invasive cancer. Clearly, there is a need to identify which DCIS lesions are likely to progress. We have developed a novel mouse xenograft model (mouse-intraductal; MIND) to study the molecular basis of DCIS progression and enable identification of suitable biomarkers that predict invasive progression. Methods: MIND involves injection of epithelial cells derived from patient breast lesions into the mammary ducts of immunocompromised mice. Serial sections of mouse mammary glands containing DCIS xenograft lesions were examined at time intervals of 3-14 months post-engraftment by histology using hematoxylin and eosin (H&amp;E) and immunohistochemistry using anti-human cytokeratin 5/19, smooth muscle actin, ER, PR, p53, Ki67 and HER-2. Results: Intraductal injection of cells derived from breast lesions of 28 patients into 133 mice resulted in a successful engraftment rate of 60%. Among these, 12 pure DCIS samples were injected into 35 different mice to create MIND xenografts. As early as three months post-engraftment, the DCIS xenograft cells showed multilayered in situ growth consisting of atypical neoplastic cells with prominent and vesicular nuclei. DCIS MIND xenografts exhibited the full spectrum of human DCIS histologic features, including similar biomarker expression (ER, PR, Ki67, HER-2 and p53) at long-term follow-up after engraftment (up to 12 months). Most remarkably, a subset of xenografts representing 5 patients (5/12; 42%) showed progression to invasion 6-12 months post-engraftment in the absence of any external genetic manipulations. This rate is very similar to that reported for human DCIS progression in untreated patients. MIND DCIS xenograft lesions that progressed showed disruption of basement membrane and myoepithelial layer by the invasive cells, retraction of basement membrane, and micro-invasion. MIND DCIS lesions were enriched in small capillaries, and in some cases clusters of invasive cells appeared inside nearby blood vessels. Conclusion: The MIND xenograft is a viable model for human DCIS progression that recapitulates histologic features of human DCIS, as well as reported rates of progression to invasion. The availability of this innovative model provides a valuable tool for the discovery of new biomarkers to identify DCIS with invasive potential. The identification of high risk DCIS will ultimately help patients and clinicians choose the best course of therapy and avoid the morbidity and costs associated with unnecessary treatment. Citation Format: Behbod F, Limback D, Hong Y, Elsarraj H, Berger J, Heddens E, Valdez K, Smith WP, Inciardi M, Reddick M, Gatewood J, Darrah J, Winblad O, Meierotto R, Ricci L, Wagner J, Amin A, May L, Cusick T, Mammen J, Fields T, Godwin AK, Fabian C, Kaufman C, Tawfik O, Fan F. Patient derived DCIS mouse-intraductal (MIND) models recapitulate the full spectrum of human patient pathology and histologic features including progression to invasion in a subset of cases [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-01.

Easily recognizable sonographic patterns of ductal carcinoma in situ of the breast

BackgroundDuctal carcinoma in situ (DCIS) is a malignant proliferation of ductal epithelium confined by the basement membrane of the involved breast ducts. The aim of this study was to categorize positive findings of DCIS of the breast on sonography. MethodsFrom 2007 to 2011, 100 pathologically proven DCIS lesions were evaluated. Four sonographic patterns used to identify DCIS have been characterized as cumulus-type, coral-type, pipe-type, and miscellaneous lesions. ResultsThe lesion numbers of nonhigh-grade and high-grade DCIS were 44 and 56, respectively. The coral type (42%) was the most commonly found lesion, followed by cumulus-type (38%), pipe-type (17%), and miscellaneous (3%) lesions. There was no significant difference between the sonographic pattern and nuclear grades. However, the coral-type group was composed of significantly more high-grade DCIS cases than the other three types (p < 0.05). ConclusionCoral-, cumulus-, and pipe-type lesions are three easily recognizable sonographic findings of DCIS. Improving the breast ultrasound technique to better demonstrate the sonographic pattern is necessary to facilitate breast lesion interpretation.