Ductal carcinoma in situ (DCIS) may progress to ipsilateral invasive breast cancer (iIBC), but often never will. Because DCIS is treated as early breast cancer, many women with harmless DCIS face overtreatment. To identify features associated with progression, we developed an artificial intelligence-based DCIS morphometric analysis pipeline (AIDmap) on hematoxylin-eosin-stained (H&E) tissue sections. We analyzed 689 digitized H&Es of pure primary DCIS of which 226 were diagnosed with subsequent iIBC and 463 were not. The distribution of 15 duct morphological measurements was summarized in 55 morphometric variables. A ridge regression classifier with cross validation predicted 5-years-free of iIBC with an area-under the curve of 0.67 (95% CI 0.57–0.77). A combined clinical-morphometric signature, characterized by small-sized ducts, a low number of cells and a low DCIS/stroma ratio, was associated with outcome (HR = 0.56; 95% CI 0.28–0.78). AIDmap has potential to identify harmless DCIS that may not need treatment.

Abstract Background. Ductal carcinoma in situ (DCIS) is a frequently found precursor of invasive breast cancer (IBC). However, the majority of DCIS will never progress to IBC. As we cannot distinguish yet which DCIS will remain indolent (‘harmless’) from those that might progress in the future to IBC, almost all women with DCIS are intensively treated by surgery, often followed by radiotherapy. This brings an urgent clinical need to learn distinguishing harmless from potentially progressive DCIS to save many women with indolent DCIS the burden of unnecessary overtreatment. Aim. We aimed to investigate if the geometry and spatial configuration of DCIS ducts in Hematoxylin-Eosin (H&E) stained tissue sections are related to the risk of progression of DCIS to ipsilateral IBC (iIBC). Methods. We obtained data from a population-based cohort of women diagnosed with primary DCIS between 1989 and 2004 in the Netherlands, treated with breast conserving surgery (BCS) only and a median follow-up time of 12 years. A nested case-control study (n=689) was designed in which patients diagnosed with iIBC recurrences during follow-up were considered as “cases” (n= 226) and those with no subsequent iIBC as “controls” (n=463). The DCIS and stroma regions were digitally annotated on H&E-stained whole slide images (WSIs) by a pathologist as ground truth for the deep learning neural network of HALO AI module (IndicaLabs). We developed a computational pipeline to automatically detect and measure stroma areas, DCIS ducts, and the nucleus of their cells. We validated the accuracy of DCIS detection in H&Es WSIs from an external study, in which DCIS regions were digitally annotated by an independent pathologist (Translational Breast Cancer Research Consortium, TBCRC). We classified cases and controls according to morphological measurements using logistic ridge regression with double-loop cross-validation, followed by hierarchical clustering. The risk of subsequent iIBC after primary DCIS diagnosis was evaluated by multivariate Cox proportional hazards models. Results. The accuracy of DCIS detection performed by the computational pipeline was compared with pathologist annotations in 20 slides from the TBCRC study. Results showed a satisfactory DCIS overlap area agreement of 0.76 (0.68 – 0.83). We applied the DCIS computational pipeline on the case-control series. We obtained 15 morphological measurements for each DCIS duct, such as duct area, cell density, distance between ducts, average nucleus area, etc. We calculated 8 distribution parameters from each measurement in each WSI, including median and range. After leaving out redundant variables, 55 unique morphometric variables were obtained, representing the heterogeneity of DCIS ducts per WSI. The c lassifier revealed a median area-under the curve (AUC) of 0.66 (0.55-0.77) to predict 5-years free of iIBC, 0.59 (0.50-0.67) to predict 10-years and 0.60 (0.52-0.68) to predict 15-years. The 30 variables with the highest association with outcome were used to build four morphometric signatures. Signature number 1, which is characterized by lesions with small-sized ducts, a lower number of cells and a lower DCIS/stroma area ratio, showed a significant lower risk of developing iIBC compared to the other three signatures in a multivariate Cox regression model including grade, ER, COX-2 and HER2 expression: HR = 0.56 (0.28-0.78 95%CI). Conclusion. We developed a computational pipeline able to detect and measure DCIS ducts in H&E WSIs with high accuracy and reproducibility. DCIS lesions presenting the morphometric signature of small-sized DCIS ducts have a very low chance to progress to invasive breast cancer. After successful validation, our morphometric method will serve as a robust and easy to implement biomarker for de-escalation strategies in DCIS, and as such, could limit unnecessary overtreatment in the near future. Citation Format: Marcelo Sobral-Leite, Simon Castillo, Shiva Vonk, Xenia Melillo, Noomie Lam, Brandi de Bruijn, Yeman Hagos, Joyce Sanders, Mathilde Almekinders, Lindy Visser, Emilie Groen, Carolien Van der Borden, Petra Kristel, Ercan Caner, Leyla Azarang, Yinyin Yuan, Renee Menezes, Esther Lips, Jelle Wesseling, Grand Challenge PRECISION Consortium. Morphometric signature identifies ductal carcinoma in situ of the breast with low risk of progression to invasive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-07.

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision‐making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one‐third of patients with ERα‐positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα‐conversion. It remains unclear whether ERα‐converted cancers are biologically similar to bona fide ERα‐negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα‐positive, ERα‐converted and ERα‐negative cases. Our data revealed that the loss of ERα‐dependency in this metastatic site leads to only a partial switch to an ERα‐negative molecular phenotype, with preservation of a luminal‐like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα‐converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα‐dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.

Supplementary Materials and Methods

Ductal Carcinoma in Situ (DCIS) is a non-invasive non-obligate precursor of invasive breast cancer (IBC). DCIS is usually treated by surgery combined with radiotherapy, which can have a large impact on the life of patients. However, many of these DCIS lesions would never progress into IBC. To reduce the overtreatment of DCIS, but assure proper treatment for high risk DCIS, it is crucial to understand the biology underlying DCIS. To study the biology of DCIS we established Mouse INtraDuctal (MIND) patient-derived xenograft (PDX) models by intraductally injecting patient DCIS material into the mammary ducts of female immunocompromised mice. We engrafted 130 samples, which have been incubated in vivo for a period of 12 months. We obtain a take rate of 88% with 46% of our models showing invasive progression. Histology and molecular subtyping by PAM50 classification are well preserved in the MIND models compared to the primary counterpart, ensuring that our MIND models represent the patient disease well. For 102 primary samples we obtained RNAseq profiles as well as for 64 matched MIND-PDX models. In addition whole exome-/panel sequencing data is generated from the same primary DCIS samples together with 12 matched MIND-PDX WES profiles as well as 60 matched Copy Number Variation (CNV) MIND-PDX profiles. Together these data revealed multiple biomarkers related to invasive progression, including factors such as high grade, solid growth, a high copy number aberrations burden, HER2, PTK6 & MYC amplifications and a high Ki67. On top of this we used whole mount imaging of the injected mammary glands extracted from our MIND-PDX models, showing two distinct growth patterns correlated with invasion. And as this is all done in the context of the PRECISION consortium this allows us to confirm and validate our findings in larger sequencing and imaging efforts of human samples. We have also successfully passaged 42 MIND-PDX models which showed minimal changes in pheno- and genotype over time indicating invasive behavior is an intrinsic phenotype of DCIS with minimal evolution, supporting a multiclonal evolution model. Moreover, this provided a collection of 19 stable sequentially transplantable DCIS MIND models including Luminal A, Luminal B, ER+/HER2+ and ER-/HER2+ models. Ultimately these models can be used to validate the biomarkers found to be related to invasive progression, as an example we proved the direct role of HER2 overexpression in invasive progression by inhibiting the HER2 receptor or by overexpressing HER2. In conclusion all this data together enabled us to create a well-characterized biobank of DCIS models with the unique opportunity to follow the natural progression, sequentially transplant 42 models, find genomic and transcriptomic profiles related to high risk DCIS and manipulate gene expression to validate the role of genes in DCIS progression. Citation Format: Stefan J. Hutten, Roebi de Bruijn, Catrin Lutz, Madelon Badoux, Timo Eijkman, Xue Chao, Marta Ciwinska, Andrea Herencia-Ropero, Petra Kristel, Lennart Mulder, Joyce Sanders, Mathilde Almekinders, Alba Llop-Gueverra, Helen R. Davies, Fariba Behbod, Serena Nik-Zainal, Violeta Serra, Jacco van Rheenen, Esther H. Lips, Lodewyk F.A. Wessels, Jelle Wesseling, Colinda Scheele, Jos Jonkers. A living biobank of patient-derived ductal carcinoma in situ (DCIS) Mouse-INtraDuctal (MIND) xenografts identifies multiple risk factors of invasive progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR006.

Ductal carcinoma in situ (DCIS) is a non-invasive breast neoplasia that accounts for 25% of all screen-detected breast cancers diagnosed annually. Neoplastic cells in DCIS are confined to the ductal system of the breast, although they can escape and progress to invasive breast cancer in a subset of patients. A key concern of DCIS is overtreatment, as most patients screened for DCIS and in whom DCIS is diagnosed will not go on to exhibit symptoms or die of breast cancer, even if left untreated. However, differentiating low-risk, indolent DCIS from potentially progressive DCIS remains challenging. In this Review, we summarize our current knowledge of DCIS and explore open questions about the basic biology of DCIS, including those regarding how genomic events in neoplastic cells and the surrounding microenvironment contribute to the progression of DCIS to invasive breast cancer. Further, we discuss what information will be needed to prevent overtreatment of indolent DCIS lesions without compromising adequate treatment for high-risk patients.

Ductal Carcinoma in Situ (DCIS) is a starting lesion in the milk duct of the breast, which accounts for 25% of all ‘breast cancers’ detected. DCIS is usually treated by surgery combined with radiotherapy, which can have a large impact on the life of patients. However, there is little to no evidence that treatment of low and intermediate grade DCIS reduces mortality, while women diagnosed with DCIS do perceive their risk of dying the same as patients with invasive disease. To reduce the negative perception and the overtreatment of DCIS, but assure proper treatment for high risk DCIS, it is crucial to understand the biology underlying DCIS. In order to study the biology of DCIS we established patient-derived xenograft (PDX) models by intraductally injecting fresh patient DCIS material into the mammary ducts of female immunocompromised mice (Mouse Mammary Intraductal(MIND)). To date, 150 primary DCIS patient samples were engrafted and >100 have been incubated in vivo for a period of 12 months. Using this method we obtain a take rate of 89% with 36% of our models showing progression. Histology and molecular subtyping by PAM50 classification are well preserved in the outgrown PDX lesions compared to the original human counterpart, ensuring that our models are a good representation of the patient disease. For >80 primary samples we obtained RNAseq as well as >50 matched PDX profiles. In addition WES or WGS data is generated from the same primary DCIS samples together with 10 matched PDX WES profiles as well as >40 matched CNV PDX profiles. On top of this we used whole mount imaging, in order to create 3D sections of the injected mammary glands extracted from our PDX models, showing two distinct growth patterns correlated with invasion. And as this is all done in the context of the PRECISION consortium this allows us to confirm and validate our findings in larger sequencing and imaging efforts of human samples. Besides collecting large amounts of data which we can now link to DCIS progression we have successfully sequentially transplanted >25 of our PDX models in order to create models that can be shared with the scientific community and can be manipulated to validate the role of genes in DCIS progression. As an example we took HER2 overexpression, which in our models was found to be associated to progressive disease. On the one hand we treated three progressive HER2 overexpressing DCIS PDX models with a HER2-antibody Herceptin showing a massive decline in growth, while on the other hand we infected two indolent HER2 negative DCIS PDX models with a HER2-GFP lentivirus showing a transformation to HER2 positive progressive growth. In conclusion all this data together enables us to create a well-characterized biobank of DCIS models of which we can follow the progression, sequentially transplant >25 models, find mutational and expression profiles related to high risk DCIS and manipulate gene expression to validate the role of genes in DCIS progression. Citation Format: Jelle Wesseling, Stefan Hutten, Catrin Lutz, Colinda Scheele, Madelon Badoux, Timo Eijkman, Roebi de Bruijn, Mathilde Almekinders, Joyce Sanders, Esther Lips, Jacco van Rheenen, Jos Jonkers, Grand challenge PRECISION consortium. Intraductal patient-derived xenograft models of ductal carcinoma in situ (DCIS) to distinguish indolent DCIS from aggressive DCIS [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 P5-01-01.

Background Ductal carcinoma in situ (DCIS) is treated to prevent subsequent ipsilateral invasive breast cancer (iIBC). However, many DCIS lesions will never become invasive. To prevent overtreatment, we need to distinguish harmless from potentially hazardous DCIS. We investigated whether the immune microenvironment (IME) in DCIS correlates with transition to iIBC. Methods Patients were derived from a Dutch population-based cohort of 10,090 women with pure DCIS with a median follow-up time of 12 years. Density, composition and proximity to the closest DCIS cell of CD20 + B-cells, CD3 + CD8 + T-cells, CD3 + CD8 − T-cells, CD3 + FOXP3 + regulatory T-cells, CD68 + cells, and CD8 + Ki67 + T-cells was assessed with multiplex immunofluorescence (mIF) with digital whole-slide analysis and compared between primary DCIS lesions of 77 women with subsequent iIBC (cases) and 64 without (controls). Results Higher stromal density of analysed immune cell subsets was significantly associated with higher grade, ER negativity, HER-2 positivity, Ki67 ≥ 14%, periductal fibrosis and comedonecrosis ( P < 0.05). Density, composition and proximity to the closest DCIS cell of all analysed immune cell subsets did not differ between cases and controls. Conclusion IME features analysed by mIF in 141 patients from a well-annotated cohort of pure DCIS with long-term follow-up are no predictors of subsequent iIBC, but do correlate with other factors (grade, ER, HER2 status, Ki-67) known to be associated with invasive recurrences.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). Due to a lack of biomarkers able to distinguish high- from low-risk cases, DCIS is treated similar to early IBC even though the minority of untreated cases eventually become invasive. Here, we characterized 115 patient-derived mouse-intraductal (MIND) DCIS models reflecting the full spectrum of DCIS observed in patients. Utilizing the possibility to follow the natural progression of DCIS combined with omics and imaging data, we reveal multiple prognostic factors for high-risk DCIS including high grade, HER2 amplification, expansive 3D growth, and high burden of copy number aberrations. In addition, sequential transplantation of xenografts showed minimal phenotypic and genotypic changes over time, indicating that invasive behavior is an intrinsic phenotype of DCIS and supporting a multiclonal evolution model. Moreover, this study provides a collection of 19 distributable DCIS-MIND models spanning all molecular subtypes.

Background: Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer (IBC). However, the majority of DCIS will never progress to IBC if left untreated. As almost all DCIS are treated by surgery often supplemented by radiotherapy, overtreatment of indolent DCIS occurs. Consequently, there is an urgent need to identify biomarkers to distinguish harmless from aggressive DCIS. We hypothesised that morphometric features observed in Hematoxylin-Eosin (HE) sections of DCIS, such as number, geometry and density of DCIS ducts and cells could identify harmless DCIS lesions.

Although ductal carcinoma in situ (DCIS) is a non-obligate precursor to ipsilateral invasive breast cancer (iIBC), most DCIS lesions remain indolent. Hence, overdiagnosis and overtreatment of DCIS is a major concern. There is an urgent need for prognostic markers that can distinguish harmless from potentially hazardous DCIS. We hypothesised that features of the breast adipose tissue may be associated with risk of subsequent iIBC. We performed a case–control study nested in a population-based DCIS cohort, consisting of 2658 women diagnosed with primary DCIS between 1989 and 2005, uniformly treated with breast conserving surgery (BCS) alone. We assessed breast adipose features with digital pathology (HALO ® , Indica Labs) and related these to iIBC risk in 108 women that developed subsequent iIBC (cases) and 168 women who did not (controls) by conditional logistic regression, accounting for clinicopathological and immunohistochemistry variables. Large breast adipocyte size was significantly associated with iIBC risk (odds ratio (OR) 2.75, 95% confidence interval (95% CI) = 1.25–6.05). High cyclooxygenase (COX)-2 protein expression in the DCIS cells was also associated with subsequent iIBC (OR 3.70 (95% CI = 1.59–8.64). DCIS with both high COX-2 expression and large breast adipocytes was associated with a 12-fold higher risk (OR 12.0, 95% CI = 3.10–46.3, P < 0.001) for subsequent iIBC compared with women with smaller adipocyte size and low COX-2 expression. Large breast adipocytes combined with high COX-2 expression in DCIS is associated with a high risk of subsequent iIBC. Besides COX-2, adipocyte size has the potential to improve clinical management in patients diagnosed with primary DCIS.

OBJECTIVES We aimed to determine the presence, amount and origin of microchimerism in peripheral blood of pregnant and non-pregnant parous women with systemic lupus erythematosus (SLE) as compared to control subjects. METHODS We performed a comparative study in which peripheral blood was drawn from eleven female non-pregnant SLE-patients and 22 control subjects, and from six pregnant SLE-patients and eleven control subjects during gestation and up to six months postpartum. Quantitative PCR for insertion-deletion polymorphisms and null alleles was used to detect microchimerism in peripheral blood mononuclear cells and granulocytes. RESULTS Microchimerism was detected more often in non-pregnant SLE-patients than control subjects (54.4% vs. 13.6%, respectively; p=0.03). When present, the median total number of foetal chimeric cells was 5 gEq/106 in patients and 2.5gEq/106 in control subjects (p=0.048). Microchimerism was mostly foetal in origin; maternal microchimerism was detected in one patient and one control subject. In control subjects, microchimerism was always derived from only one source whereas in 50% of patients it originated from multiple sources. The pregnant patients had a significantly higher median number of foetal chimeric cells in the granulocyte fraction just after delivery than control subjects (7.5 gEq/106 vs. 0 gEq/106, respectively; p=0.02). CONCLUSIONS Just after delivery, SLE-patients had more microchimerism than control subjects. Three months post-partum, microchimerism was no longer detectable, only to reappear many years after the last pregnancy, more often and at higher levels in SLE-patients than in control subjects. This suggests that these chimeric cells may originate from non-circulating foetal chimeric stem cells.

Purpose For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. Methods Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff’s alpha (KA) and Gwet’s AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. Results Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05–6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35–5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34–10.23) were independently associated with a higher iIBC risk. Conclusions Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.

Abstract Background: The incidence of ductal carcinoma in situ (DCIS) has greatly increased since the introduction of mammographic screening. However, so far, it is still not possible to predict which patient with DCIS will further develop invasive breast cancer. Adipocyte hypertrophy and adipocyte inflammation has been associated with increased risk of developing breast cancer in postmenopausal women and with worse prognosis in breast cancer patients. In this study, we aimed 1) at evaluating the association between mammary adiposity, as assessed by digital pathology, with DCIS progression using a unique case-control series with long-term follow up and 2) study the interaction between DCIS cells and the surrounding adipocytes. Patients and methods: Mammary adipocyte size was measured in surgical specimens of 279 women with primary DCIS. These mammary adipocyte measurements were compared in 109 women with primary DCIS who subsequently developed ipsilateral invasive breast cancer (iIBC) (cases) and 170 women who did not (controls). Patients were matched for age and follow-up duration. Median follow-up time was 12.8 years. Post-menopausal patients were defined as >50 years at diagnosis and represented 220/279 (78,9%) of patients. All patients were treated with breast cancer-conserving surgery only. Intact adipocytes distant from the DCIS lesions were measured (largest diameter and area) using HALO™ image analysis software (Indica Labs, Corrales, NM) on H&E-stained whole slide images of primary DCIS. Given the specific interest in the larger adipocytes, we systematically considered the 75th percentile as unique value for each patient. An average of 4259 adipocytes (min:70, max:21107) was measured per patient. Tissue segmentation was used to measure the adipose area. Adipose triglyceride lipase (ATGL clone 2138, Cell Signaling) immunohistochemistry (IHC) was applied to study ATGL expression in DCIS cells. Her2, ER and COX-2 IHC and RNAseq of microdissected pure DCIS was already available for this series (LL. Visser et al. Clin Can Res 24 (15) 2018). Conditional logistic regression was applied to investigate differences in adipocyte hypertrophy between groups of patients (e.g., cases and controls). Results: Adipocytes were larger and the proportion of adipose tissue was higher in post-menopausal patients (both p<0.001). Mean adipocyte maximum diameter and mean adipocyte area of the 75th percentile ranged from 46.9-115.9 µm and 2016-11336 µm2 respectively. For every 1000 µm2 increase in mean adipocyte area a DCIS patient had a 1.18 increased risk for iIBC (95% CI 1.02-1.36, p= 0.028). DCIS patients with large adipocytes (above the 75th percentile) had a 2.1 increased risk for developing iIBC (95% CI: 1.18-3.72, p=0.011). This observation remained when restricting the analysis to post-menopausal patients (OR 1.19 per 1000 µm2 95% CI 1.01-1.40, p=0.038 and adipocytes above 75th percentile OR 2.2, 95% CI 1.22-3.99, p= 0.009). To further explore the potential interactions between the epithelial cells from DCIS with the adjacent adipocytes, we compared the size of the DCIS-adjacent adipocytes to those away from DCIS lesions. DCIS-adjacent adipocytes showed a reduction in size, suggesting delipidation. Adipose triglyceride lipase (ATGL) is a rate limiting lipase that can release stored free fatty acids (FFA) in DCIS cells for fatty acid ß-oxidation. In a pilot series strong ATGL expression was observed in a subset of DCIS lesions (4 of 25). Conclusion and perspective: This study is the first to demonstrate that mammary adipocyte hypertrophy is associated with an increased risk of progression for patients with DCIS. Our findings might help to distinguish potentially hazardous from harmless DCIS, enabling overtreatment of indolent DCIS. Adipocyte size will be correlated to immunohistochemical expression of ATGL, Her2, ER and COX2 in DCIS, and RNAseq data of pure microdissected DCIS. Citation Format: Mathilde Matthea Machteld Almekinders, Michael Schaapveld, Bram Thijssen, Ingrid Hofland, Marjolijn Mertz, Dennis Peters, Annegien Broeks, Lodewijk Wessels, Wilbert Zwart, Jos Jonkers, Esther Lips, Christine Desmedt, Jelle Wesseling, on behalf of the PRECISION Team. Impact of increased mammary adiposity on DCIS progression [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 P6-15-07.

Background. Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer (IBC). However, the natural course of a particular DCIS lesion is unknown, because almost all women with DCIS are treated. Furthermore, most studies are biased because these comprise DCIS adjacent to IBC, also known as synchronous DCIS and IBC, indicating that such DCIS lesions already have the capacity to progress to IBC. It is still unknown which proportion and type of subsequent ipsilateral IBCs (iIBC) are related to the initial primary "pure" DCIS lesion. Therefore, we performed an extensive molecular characterization of DCIS and matched subsequent iIBC, to better understand the natural course of DCIS.Patients and methods. We used a unique series of 78 women diagnosed with DCIS and treated by breast conserving surgery (BCS) alone, which subsequently developed iIBC. Mean time to iIBC event was 6.3 years (range 0.5-17.0). These 78 women are a representative sample of a case-control series, nested in a nation-wide, population-based cohort including all patients diagnosed with DCIS between 1989 and 2005 in the Netherlands (Visser, et al Clin Can Res 2018). Data on tumor location (ICD-10) was available for all lesions. DNA and RNA was simultaneously extracted for 78 DCIS lesions and 78 matched subsequent iIBC (DNA >20ng; RNA >100ng), and RNA sequencing (RNAseq) and low coverage whole genome sequencing (CNVseq) was performed. Panel sequencing (PanelSeq), using a custom panel of 53 breast cancer driver genes, was performed with the remaining DNA of 42 DCIS and iIBC matched pairs. We determined if the iIBC lesion and DCIS lesion were related, by comparing tumor location and genomic features.Results. Based on tumor location and histological grade, >95% of the subsequent iIBC reflected outgrowth of residual disease. Based on RNAseq data, 77% of all DCIS and IBC lesions classified into the same PAM50 subtypes. The CNVseq data showed that the DCIS lesions contained copy number aberrations on typical breast cancer-associated loci, such as 1q gain, 8q gain, 16q loss, 20q gain. However, when we compared DCIS with their matched iIBC, we observed in 41% of the cases very distinct copy number profiles, indicating either outgrow of a different tumor (minority) subclone or a second primary tumor. Analysis of PanelSeq mutation data supported this clonal or independent origin of the subsequent iIBC.Conclusion. To our knowledge, our study is the first to investigate if subsequent iIBC is likely to originate from the initial primary "pure" DCIS in a large series with long-term follow-up.Surprisingly, our CNVseq and PanelSeq results indicate that more than one third of the subsequent iIBCs after BCS alone treated primary DCIS are likely to be second primary tumors or represent selective outgrowth of a minority DCIS subclone.Citation Format: Lindy Visser, Marlous Hoogstraat, Tycho Bismeijer, Lotte Elshof, Koen van de Vijver, Emilie Groen, Mathilde Almekinders, Joyce Sanders, Carolien Bierman, Dennis Peters, Ingrid Hofland, Frank Nieboer, Michiel de Maaker, Petra Kristel, Lennart Mulder, Annegien Broeks, Michael Schaapveld, Marjanka Schmidt, Lodewyk Wessels, Esther Lips, Jelle Wesseling, On behalf of the PRECISION team. Ductal carcinoma in situ of the breast: Cancer precursor or not [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 751.

Ductal carcinoma in situ (DCIS) is considered a potential precursor of invasive breast carcinoma (IBC). Studies aiming to find markers involved in DCIS progression generally have compared characteristics of IBC lesions with those of adjacent synchronous DCIS lesions. The question remains whether synchronous DCIS and IBC comparisons are a good surrogate for primary DCIS and subsequent IBC. In this study, we compared both primary DCIS and synchronous DCIS with the associated IBC lesion, on the basis of immunohistochemical marker expression. Immunohistochemical analysis of ER, PR, HER2, p53, and cyclo-oxygenase 2 (COX-2) was performed for 143 primary DCIS and subsequent IBC lesions, including 81 IBC lesions with synchronous DCIS. Agreement between DCIS and IBC was assessed using kappa, and symmetry tests were performed to assess the pattern in marker conversion. The primary DCIS and subsequent IBC more often showed discordant marker expression than synchronous DCIS and IBC. Strikingly, 18 of 49 (36%) women with HER2-positive primary DCIS developed an HER2-negative IBC. Such a difference in HER2 expression was not observed when comparing synchronous DCIS and IBC. The frequency of discordant marker expression did not increase with longer time between primary DCIS and IBC. In conclusion, comparison of primary DCIS and subsequent IBC yields different results than a comparison of synchronous DCIS and IBC, in particular with regard to HER2 status. To gain more insight into the progression of DCIS to IBC, it is essential to focus on the relationship between primary DCIS and subsequent IBC, rather than comparing IBC with synchronous DCIS.

Abstract Background. Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer, because: DCIS often accompanies invasive breast cancer; its risk factors are similar to those of invasive breast cancer; and genetic markers found in DCIS are similar to the ones found in invasive breast cancer. However, clinical behavior of DCIS is still poorly understood, as there is only limited information on its long-term natural history. Altogether, this makes it difficult to understand the relatedness of DCIS and its subsequent ipsilateral invasive breast cancer (iIBC). Here, we set-up a comparison between primary DCIS and matched subsequent iIBC, by making use of pathological and molecular data. Patients and methods. For this study, we used a unique series of 155 DCIS cases which developed a subsequent iIBC during a median follow up period of 12.6 years. We assessed histological characteristics, tumor location, estrogen and progesterone receptor status, p16 expression, and HER2 and p53 overexpression. RNA sequencing and copy number sequencing was done on 78 DCIS lesions and 78 matched invasive breast cancer relapses. We determined if the iIBC lesion and DCIS lesion were related, with respect to tumor location, immunohistochemical (IHC) markers, and genomic features. Results. Based on tumor location and histological grade, >95% of the subsequent invasive breast cancers reflected outgrowth of residual disease. HER2 was the only IHC marker that showed a significant difference in expression between DCIS and matched iIBC: 40% of the HER2 positive DCIS was followed by a HER2 negative invasive recurrence. In addition, RNAseq data was used to classify DCIS and IBC lesions into PAM50 subtypes. 77% of the DCIS IBC pair belonged to the same subtype. The DCIS lesions showed copy number aberrations on typical breast cancer-associated loci. However, when we compared the DCIS with its matched iIBC, we saw in 41% of the cases very distinct copy number profiles, indicating either outgrow of a different tumor subclone or a second primary. Conclusion. This is the first time that a sound comparison could be made between primary DCIS and its subsequent invasive breast cancer with such a large patient group, integrating pathological and molecular data. Our results strongly suggest that many subsequent iIBCs after treatment of pure DCIS could be considered as second primary breast cancer lesions. To provide definite proof for this, in depth DNA sequencing and heterogeneity studies will be presented at SABCS 2018. Citation Format: Visser LL, Hoogstraat M, Elshof LE, van de Vijver K, Groen EJ, Almekinders MM, Bierman C, Nieboer F, de Maaker M, Kristel P, Mulder L, Schaapveld M, Schmidt MK, Lips E, Wesseling J. Approximately 40% of invasive recurrences after treatment of ductal carcinoma in situ is likely to be a second primary tumor [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 PD8-09.

Introduction: DCIS is a non-obligate precursor to invasive breast cancer (IBC). DCIS patients are treated similarly to breast cancer with surgery, often followed by radiotherapy and/or endocrine treatment. However, most DCIS lesions will never progress to IBC, indicating that overdiagnosis and overtreatment exists. DCIS lesions show variable amounts of immune cells, particularly in the periductal stroma. Immune escape might be a critical step for transition from DCIS to IBC. We aim to identify factors within the immune microenvironment of DCIS lesions that distinguish harmless from potentially hazardous DCIS.Methods: A case-control study is being conducted consisting of women with pure DCIS diagnosed between 1989-2005 with median follow-up of 12 years, treated with breast conserving surgery only. Cases are defined as women with DCIS developing subsequent ipsilateral breast cancer (iIBC), controls as women with DCIS without subsequent iIBC. Multispectral immunohistochemical imaging was performed on primary DCIS lesions, aiming at detection of CD20+ B-cells, CD8+ T-cells, CD3+ T-cells, CD3+Foxp3+ regulatory T-cells, and CD68+ macrophages. Density of immune cell subsets in cells/mm2, immune cell ratios and spatial relationships were calculated for 27 cases and 28 controls. These immune cell related factors were correlated to outcome and integrated with RNAseq data of pure microdissected DCIS. We performed gene set enrichment analysis on the correlation between DCIS gene expression and density of immune cell types with sample permutation (flexgsea R package).Results: Stromal lymphocyte, B-cell, CD8+ T-cell, regulatory T-cell and macrophage density did not significantly differ between cases and controls. Immune cell composition (CD8+ T-cell/lymphocyte, CD8+ T-cell/CD3+Foxp3+ regulatory T-cell and CD20+/lymphocyte ratio) and fraction of regulatory T-cells in close proximity of a CD8+ T-cell did not differ between cases and controls. We find a negative association between stromal B-cell density and DCIS gene expression of estrogen receptor (ESR1) targets. Higher stromal T-cell density was associated with proliferation and expression of genes characteristic for luminal B and basal-like subtypes. Furthermore, higher density of specific immune cell subsets within the DCIS compartment was associated with several immune and cancer pathways.Conclusion: A first set of analyzed DCIS cases (n=27) and controls (n=28) show no significant differences regarding immune cell density, composition and spatial relationships. Considering the entire group of DCIS patients (n=55), a negative association between stromal B-cell density and gene expression of ESR1 targets was found. Higher density of lymphocytes was associated with proliferation and expression of genes characteristic for luminal B and basal-like subtypes. The full set of 175 DCIS lesions will be presented at AACR Annual Meeting 2019.Citation Format: Mathilde M. Almekinders, Lindy Visser, Bram Thijssen, Rianne van der Linden, Charlotte van Rooijen, Petra Kristel, Annegien Broeks, Tycho Bismeijer, Lodewyk Wessels, Erik Hooijberg, Karin de Visser, Esther Lips, Jelle Wesseling, on behalf of the PRECISION team (PREvent ductal Carcinoma In Situ Invasive Overtreatment Now). Progression of ductal carcinoma in situ (DCIS), is it in the immune microenvironment [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 2806.

Abstract Introduction Since the introduction of population-based mammographic screening, the incidence of ductal carcinoma in situ (DCIS) increased manifold. DCIS lesions are non-obligate precursors to invasive breast cancer, because only a minority of DCIS patients later develops invasive breast cancer. DCIS patients are treated intensively with surgery, frequently supplemented by radiotherapy and/or endocrine treatment. However, treatment of DCIS lesions did not result in a decreased incidence of advanced stages of breast cancer, suggesting overdiagnosis and hence overtreatment exists. Because the immune microenvironment plays an important role in cancer progression, we performed a pilot study to assess the amount, composition and spatial distribution of immune cells aiming at the identification of biomarkers that distinguish aggressive from indolent DCIS. Methods A representative series of 32 paraffin-embedded DCIS lesions was studied with multispectral immunohistochemical imaging, providing simultaneous detection and quantification of CD20+ B-cells, CD8+ T-cells, CD4+ T-cells, CD4+Foxp3+ regulatory T-cells, CD68+ macrophages and pankeratin. Cellular density of immune cell subsets per tissue compartment and spatial distribution was analyzed by Inform software, SPSS and R. The number of CD4+FoxP3+ T-cells within 30µm of a CD8+ T-cell was assessed and expressed in a CD4+FoxP3+ T-cell per CD8+ T-cell ratio. Immune cell density and composition were correlated to grade and immunohistochemical ER, Her2 and p53 status. Results Multispectral immunohistochemical quantification showed a range of 30 to 2100 lymphocytes/mm2 in the stroma of DCIS lesions. High grade positively correlated with higher number of stromal lymphocytes/mm2 (p<0.01). Negative ER status, positive Her2 status and aberrantly expressed p53 was significantly associated with higher number of stromal lymphocytes/mm2, CD8+ T-cells/mm2, CD4+FoxP3+ regulatory T-cells/mm2 and CD20+ B-cells/mm2 (p<0.05). Within the DCIS-epithelium, the number of CD4+FoxP3+ regulatory T-cells positively correlated with negative ER-status (p=0.02) and positive Her2 status (p=0.03). The spatial distribution of the number of CD4+Foxp3+ T-cells within 30 μm of a CD8+ T-cell (expressed in a Treg per CD8+T-cell ratio) varied from 0 to 0.23 in the stromal compartment and from 0 to 0.60 in the DCIS compartment. Conclusions Within the immune microenvironment, CD20+ B-cells, CD8+ T-cells, CD4+ T-cells, CD4+Foxp3+ regulatory T-cells and CD68+ macrophages were successfully and simultaneously detected. Stromal lymphocyte density and CD8+ T-cell, CD4+ T-cell, CD4+FoxP3+ regulatory T-cell and CD20+ B-cell density positively correlated with negative ER status, positive Her2 status and aberrant expression of p53. The next step will be to analyze this multiplex panel in our nationwide DCIS cohort (1989-2005, median follow-up 12.0 years) for correlation with clinical outcome. Citation Format: Mathilde M. Almekinders, Lindy L. Visser, Bram Thijssen, Petra Kristel, Rianne van der Linden, Annegien Broeks, Erik Hooijberg, Karin de Visser, Esther H. Lips, Jelle Wesseling. Towards analysis of the immune microenvironment in ductal carcinoma in situ [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 3137.