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Purpose: Ductal carcinoma in situ (DCIS) is treated to prevent progression to invasive breast cancer. Yet, most lesions will never progress, implying that overtreatment exists. Therefore, we aimed to identify factors distinguishing harmless from potentially hazardous DCIS using a nested case–control study.Experimental Design: We conducted a case–control study nested in a population-based cohort of patients with DCIS treated with breast-conserving surgery (BCS) alone (N = 2,658) between 1989 and 2005. We compared clinical, pathologic, and IHC DCIS characteristics of 200 women who subsequently developed ipsilateral invasive breast cancer (iIBC; cases) and 474 women who did not (controls), in a matched setting. Median follow-up time was 12.0 years (interquartile range, 9.0–15.3). Conditional logistic regression models were used to assess associations of various factors with subsequent iIBC risk after primary DCIS.Results: High COX-2 protein expression showed the strongest association with subsequent iIBC [OR = 2.97; 95% confidence interval (95% CI), 1.72–5.10]. In addition, HER2 overexpression (OR = 1.56; 95% CI, 1.05–2.31) and presence of periductal fibrosis (OR = 1.44; 95% CI, 1.01–2.06) were associated with subsequent iIBC risk. Patients with HER2+/COX-2high DCIS had a 4-fold higher risk of subsequent iIBC (vs. HER2−/COX-2low DCIS), and an estimated 22.8% cumulative risk of developing subsequent iIBC at 15 years.Conclusions: With this unbiased study design and representative group of patients with DCIS treated by BCS alone, COX-2, HER2, and periductal fibrosis were revealed as promising markers predicting progression of DCIS into iIBC. Validation will be done in independent datasets. Ultimately, this will aid individual risk stratification of women with primary DCIS. Clin Cancer Res; 24(15); 3593–601. ©2018 AACR.

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Supplementary Materials and Methods

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.

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 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.

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.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 CD20Higher 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.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 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.

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

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.

Purpose: Ductal carcinoma in situ (DCIS) is treated to prevent progression to invasive breast cancer. Yet, most lesions will never progress, implying that overtreatment exists. Therefore, we aimed to identify factors distinguishing harmless from potentially hazardous DCIS using a nested case–control study. Experimental Design: We conducted a case–control study nested in a population-based cohort of patients with DCIS treated with breast-conserving surgery (BCS) alone (N = 2,658) between 1989 and 2005. We compared clinical, pathologic, and IHC DCIS characteristics of 200 women who subsequently developed ipsilateral invasive breast cancer (iIBC; cases) and 474 women who did not (controls), in a matched setting. Median follow-up time was 12.0 years (interquartile range, 9.0–15.3). Conditional logistic regression models were used to assess associations of various factors with subsequent iIBC risk after primary DCIS. Results: High COX-2 protein expression showed the strongest association with subsequent iIBC [OR = 2.97; 95% confidence interval (95% CI), 1.72–5.10]. In addition, HER2 overexpression (OR = 1.56; 95% CI, 1.05–2.31) and presence of periductal fibrosis (OR = 1.44; 95% CI, 1.01–2.06) were associated with subsequent iIBC risk. Patients with HER2+/COX-2high DCIS had a 4-fold higher risk of subsequent iIBC (vs. HER2−/COX-2low DCIS), and an estimated 22.8% cumulative risk of developing subsequent iIBC at 15 years. Conclusions: With this unbiased study design and representative group of patients with DCIS treated by BCS alone, COX-2, HER2, and periductal fibrosis were revealed as promising markers predicting progression of DCIS into iIBC. Validation will be done in independent datasets. Ultimately, this will aid individual risk stratification of women with primary DCIS. Clin Cancer Res; 24(15); 3593–601. ©2018 AACR.

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 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 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.

INTRODUCTION. The wide spread adoption of population-based breast cancer screening has led to a substantial increase in diagnosis of ductal carcinoma in situ (DCIS). When detected, almost all DCIS is treated to prevent progression to invasive disease, even though the majority of DCIS will never progress. Yet, we are unable to discriminate harmless from potentially hazardous DCIS. Hence, there is an urgent need to find characteristics of DCIS and biomarkers that predict subsequent invasive tumor development. In this study, we compared primary DCIS lesions with their subsequent ipsilateral invasive breast cancer (iIBC), to explore how the initial DCIS lesion and its subsequent iIBC are related. PATIENTS AND METHODS. We used a population-based, nation-wide cohort consisting of 2,654 women who were treated for primary DCIS by breast conserving surgery (BCS) only. Within a median follow-up time of 10.7 years, 316 women developed a subsequent iIBC (12%). FFPE tissue blocks of both DCIS and subsequent iIBC could be collected for 158 of these 316 women. We assessed histology characteristics, tumor location, estrogen and progesterone receptor status, p16 expression, and HER2 and p53 overexpression. Additionally, DNA and RNA were simultaneously isolated from 100 DCIS and 100 matched subsequent iIBC specimens for extensive molecular profiling. RESULTS. More than 95% of the invasive recurrences were located at or near the site of the primary DCIS. Concordant histological grade was found in 94% of the matched pairs and identical immunohistochemical (IHC) marker expression in 58%. Of the 44 patients with HER2 positive DCIS, 36% developed HER2 negative iIBC. Furthermore, this change in HER2 status was also a main cause of a change in surrogate intrinsic subtype (based on ER, PR, HER2) between DCIS and iIBC, which was observed in 16% of the patients. These dissimilarities were not found when we compared invasive disease with synchronous DCIS (present in 83 of 158 patients). Molecular analyses is still ongoing. CONCLUSION. This is the first time that an unbiased comparison could be made between primary DCIS and its subsequent iIBC within such a large patient group, integrating clinical, histological and IHC data. Our results suggest that the majority of invasive breast cancers in our cohort reflect outgrowth of residual disease based on tumor location and histological grade. DCIS and iIBC are very similar when comparing histological grade, but frequently show differences on the IHC level. Remarkably, the dissimilarities in HER2 status and surrogate intrinsic subtype as seen in primary DCIS vs. iIBC are missing when comparing IBC with synchronous DCIS. As a next step, we will analyse the lesions on the molecular level, to verify these findings and to look for molecular characteristics of DCIS that could be associated with progression to invasive disease. Citation Format: Lindy L. Visser, Lotte L. Elshof, Koen van de Vijver, Emma J. Groen, Mathilde Almekinders, Marjanka K. Schmidt, Flora van Leeuwen, Emiel J. Rutgers, Michael Schaapveld, Esther H. Lips, Jelle Wesseling. Risk of ipsilateral invasive breast cancer after DCIS: a comparison of primary DCIS and subsequent invasive disease by morphological and immunohistochemical analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4738. doi:10.1158/1538-7445.AM2017-4738

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 these women that may forego treatment, we hypothesized that DCIS morphometric features relate to the risk of subsequent iIBC. We developed an artificial intelligence-based DCIS morphometric analysis pipeline (AIDmap) to detect DCIS as a pathologist and measure morphological structures in hematoxylin-eosin-stained (H&E) tissue sections. These were from a case-control study of patients diagnosed with primary DCIS, treated by breast-conserving surgery without radiotherapy. We analyzed 689 WSIs of DCIS of which 226 were diagnosed with subsequent iIBC (cases) and 463 were not (controls). The distribution of 15 duct morphological measurements in each H&E 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 0.65 (95% CI 0.55-0.76). A morphometric signature based on the 30 variables most associated with outcome, identified lesions containing small-sized ducts, low number of cells and low DCIS/stroma area ratio. This signature was associated with lower iIBC risk in a multivariate regression model including grade, ER, HER2 and COX-2 expression (HR = 0.56; 95% CI 0.28-0.78). AIDmap has potential to identify harmless DCIS that may not need treatment., Competing Interests: Conflict of Interests The authors declare that they have no conflicts of interest.