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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.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies.

Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5–41% of breast cancers. Here, we investigated the genetic differences between HER2‐positive and HER2‐negative admixed breast cancer components. We performed an in‐depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin‐fixed, paraffin‐embedded breast cancer tissue of ten patients with at least one HER2‐negative and at least one HER2‐positive component was microdissected. Targeted next‐generation sequencing was performed using a customized 53‐gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2‐negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2‐negative tumour component. No common alternative drivers were identified in the HER2‐negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere ‘passenger’ molecular anomalies., This in‐depth analysis of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile: some genetic aberrations will be crucial for cancer progression, and others will be mere ‘passenger’ molecular anomalies.

Introduction: Ductal carcinoma in situ (DCIS) is considered a non-obligate precursor of invasive ductal carcinoma. With the aim of preventing a subsequent invasive cancer, all DCIS lesions are currently treated with surgical excision often supplemented with radiotherapy (RT). To prevent DCIS over- or undertreatment, a reliable marker of DCIS invasiveness risk is urgently needed. Methods: We studied two large DCIS cohorts: the Sloane cohort, a prospective breast screening cohort from the UK (median follow-up of 11 years), and a Dutch population-based cohort (NKI, median follow-up of 13 years). FFPE tissue specimens from patients with pure primary DCIS after breast-conserving surgery (BCS) +/- RT that did develop a subsequent ipsilateral event (DCIS or invasive) were considered as cases, whereas patients that did not develop any form of recurrence up to the last follow-up or death were considered as controls. We performed copy number analysis (CNA) and RNAseq analysis on 229 cases (80 DCIS only recurrences) and 344 controls. Results: DCIS was classified into the PAM50 subtypes using RNAseq data which revealed an enrichment of luminal A phenotype in DCIS that did not recur (P = 0.01, Fisher Exact test). No single copy number aberration was more common in cases compared to controls. RNAseq data did not reveal any genes significantly over/under-expressed in cases versus controls after FDR correction. However, by limiting the analysis to samples that had not had RT and excluding pure DCIS recurrences, we could develop a penalized Cox model from RNAseq data. The model was trained on weighted samples (to correct for the biased sampling of the case-control dataset) from the NKI series with double loop cross-validation. The genes were selected using the Elastic net framework of penalization. Using this predicted hazard ratio, the samples were split into high, medium, and low-risk quantiles, with a recurrence risk of 23%, 7% and 2%, respectively at 5 years (p = 10-10, Wald test). The NKI-trained predictor was independently validated in the Sloane No RT no DCIS recurrence cohort (p = 0.02, Wald test). GSEA analysis revealed proliferation hallmarks enriched in the recurrence predictor (FDR = 0.058). The RNAseq predictor was more predictive of recurrence than PAM50, clinical features (Grade, Her2 and ER) and the 12-gene Oncotype DCIS score (p < 0.001, permutation test using the Wald statistic) in both the NKI and Sloane series. Conclusion: Genomic profiling of two independent series of DCIS with outcome data did not reveal any clear associations with recurrence until analysis was limited to a set of samples who had not had radiotherapy and DCIS recurrences were excluded. We then identified an RNAseq-based classifier that could differentiate primary DCIS in low-, medium-, and high-risk groups, and validated it in an independent cohort. This classifier, if validated in other datasets, will allow us to identify women who do not need intensive treatment for their DCIS. Citation Format: Maria Roman Escorza, Michael Sheinman, Tycho Bismeijer, Ahmed A. Ahmed, Vandna Shah, Jeffrey R. Marks, Lorraine M. King, Anargyros Megalios, Lindy L. Visser, Marlous Hoogstrat, Helen R. Davies, Tapsi Kumar, Deborah Collyar, Hilary Stobart, Sarah Pinder, Nicholas N. Navin, Andrew Futreal, Serena Nik-Zainal, E. Shelley Hwang, Esther H. Lips, Alastair Thompson, Lodewyk F.A. Wessels, Jelle Wesseling, Elinor J. Sawyer. Genomic predictor can discriminate between high- and low-risk DCIS [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 PR002.

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 the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

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.

With the widespread adoption of breast cancer screening the incidence of pure ductal carcinoma in situ (DCIS) has increased. As DCIS is considered a non-obligate precursor of invasive ductal carcinoma most women with pure DCIS are treated with breast conserving surgery (BCS) +/- radiotherapy. However, for many this is likely to be overtreatment as only a minority will develop a subsequent ipsilateral recurrence. Studies also show that only ~60% of these ipsilateral recurrences are invasive disease with the remainder being pure DCIS. To predict which women are most likely to benefit from interventions, there is a need to identify biomarkers that are associated with invasive recurrence. Our aim was to assess whether copy number aberrations (CNAs) could be used to identify DCIS that was likely to recur as invasive disease or remain recurrence-free during long-time follow up. We performed somatic copy number profiling on 309 pure DCIS samples that had not developed an ipsilateral event (controls), 198 that had developed subsequent ipsilateral invasive disease (INV-cases) and 58 that had developed subsequent ipsilateral pure DCIS (DCIS-cases). The samples were obtained from two large nation-wide cohorts: the Sloane cohort, a prospective breast screening cohort from the UK with a median follow up of 12.5 years and a Dutch population based cohort, with a median follow up of 13 years. CNAs were assessed using the CytoSNP array or low pass whole genome sequencing and analyzed using GISTIC. Integrative cluster (IntClust) subtyping revealed that only 5 subtypes were well represented in DCIS compared to 10 in invasive disease and the distribution of clusters between INV-cases and controls was similar with the exception of IntClust 4, which was significantly more common in controls (P= 0.025, Fishers exact test). IntClust 4 is characterized to have low levels of genomic instability and a CNA-devoid. INV-cases were globally more aberrant than controls (P = 0.006, Wilcoxon test) as assessed by the chromosomal instability index (CIN) score. GISTIC identified 17 recurrent amplifications, 21 recurrent gains and 22 recurrent losses in the whole cohort. Six of these regions were more common in INV-cases compared to controls: amplifications at 17q24.1 and 8p11.23, losses at 1p36.13 and 11q23.2 ​and gains at 17q21.33​ and 16p (Nominal P < 0.05 and FDR < 0.1, Fishers exact test). Subgroup analysis of ER+, Her2- INV-cases versus controls revealed an additional differential CNA, amplification at 11q13.3 more common in cases. DCIS-cases had similar CNAs to INV-cases and were more aberrant than controls in terms of CIN score (P < 0.037, Wilcoxon test) but not as aberrant as INV-cases. In conclusion, we have identified potential CNAs that are associated with invasive recurrence. Further analysis will integrate gene expression with copy number data to identify which genes are being targeted by these CNAs in order to identify pathways important in progression of DCIS. Citation Format: Ahmed A. Ahmed, Maria Roman-Escorza, Tycho Bismeijer, Michael Sheinman, Vandna Shah, Rana Shami, Jeffrey R. Marks, Lorraine M. King, Anargyros Megalios, Lindy L. Visser, Marlous Hoogstraat, Helen R. Davies, Tapsi Kumar, Deborah Collyar, Hilary Stobart, Sarah Pinder, Nicholas N. Navin, Andrew Futreal, Serena Nik-Zainal, E. Shelley Hwang, Lodewyk F. Wessels, Esther H. Lips, Alastair Thompson, Jelle Wesseling, Elinor J. Sawyer. Copy number analysis of pure DCIS and association with recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5108.

Pure ductal carcinoma in situ (DCIS) is being diagnosed more frequently through breast screening programmes and is associated with an increased risk of developing invasive breast cancer. We assessed the clonal relatedness of 143 cases of pure DCIS and their subsequent events using a combination of whole exome, targeted and copy number sequencing, supplemented by single cell analysis. Unexpectedly, 18% of all invasive events after DCIS were clonally unrelated to the primary DCIS. Single cell sequencing of selected pairs confirmed our findings. In contrast, synchronous DCIS and invasive disease (n=44) were almost always (93%) clonally related. This challenges the dogma that almost all invasive events after DCIS represent invasive transformation of the initial DCIS and suggests that DCIS could be an independent risk factor for developing invasive disease as well as a precursor lesion. Our findings support a paradigm shift that confirms a more complex role for DCIS than previously recognized, and that the future management of DCIS should take into account both the precursor and risk factor implications of this diagnosis.

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: 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.

With the widespread adoption of population-based breast cancer screening, ductal carcinoma in situ (DCIS) has come to represent 20–25% of all breast neoplastic lesions diagnosed. Current treatment aims at preventing invasive breast cancer, but the majority of DCIS lesions will never progress to invasive disease. Still, DCIS is treated by surgical excision, followed by radiotherapy as part of breast conserving treatment, and/or endocrine therapy. This implies over-treatment of the majority of DCIS, as less than 1% of DCIS patients will go on to develop invasive breast cancer annually. If we are able to identify which DCIS is likely to progress or recur as invasive breast cancer and which DCIS would remain indolent, we can treat the first group intensively, while sparing the second group from such unnecessary treatment (surgery, radiotherapy, endocrine therapy) preserving the quality of life of these women. This review summarizes our current knowledge on DCIS and the risks involved regarding progression into invasive breast cancer. It also shows current knowledge gaps, areas where profound research is highly necessary for women with DCIS to prevent their over-treatment in case of a harmless DCIS, but provide optimal treatment for potentially hazardous DCIS.

Background. The incidence of DCIS has increased since the introduction of population-based screening. This has not resulted in a decrease in invasive breast cancer incidence, implying overdiagnosis exists. All women with DCIS are still intensively treated, by surgery, radiotherapy, and/or hormonal treatment, although only a minority will develop a subsequent invasive breast cancer. As we cannot discriminate such hazardous from harmless DCIS lesions, accurate prognostic biomarkers are urgently needed. In the current study we aim to identify molecular markers for DCIS aggressiveness, using a large population-based cohort. Patients and methods. We used a population-based, nation-wide cohort consisting of 10,090 women treated for primary DCIS between 1989 and 2004 with a median follow-up time of 10.7 years. Within this cohort, a case-control study was set up to analyse which markers are associated with progression to invasive breast cancer. Formalin-fixed paraffin embedded (FFPE) tissue blocks were retrieved from 1580 DCIS patients who were treated by breast conserving surgery without radiotherapy (316 DCIS patients with a subsequent ipsilateral invasive breast cancer (iiBC): i.e. the "cases"; and 1264 DCIS patients without subsequent invasive breast cancer: i.e. the "controls"). A first study using this population-based cohort will involve immunohistochemistry (IHC) on 200 "cases" and 500 "controls" for an 8-marker IHC panel (ER, PR, HER2, Ki67, p16, p53, COX-2, and Annexin A1). Molecular subtypes of the DCIS and invasive breast cancer lesions will be determined and intra-individual heterogeneity will be assessed. IHC marker expression will be both compared between "cases" and " controls" as well as between DCIS lesions and its subsequent invasive breast cancer. In a second study, DNA and RNA will be isolated from these specimens, using laser microdissection, and extensive molecular profiling will be performed. Results. We have collected FFPE tissue blocks of 287 "cases" and 1149 "controls" (86% of requested material) from 56 participating hospitals. At present, the specimens of 223 "cases" (matched DCIS and iiBC specimen) and 103 "controls" have been centrally revised for extensive morphological characteristics. Only a small part (14%) of the specimens had to be excluded from the study population. IHC staining of the tissue specimens, using the 8-marker IHC panel is ongoing. Conclusion. Within a nation-wide cohort of 10,090 patients diagnosed with primary DCIS, we were able to collect tissue material of a representative case-control series of 200 "cases" with subsequent invasive breast cancer and 500 invasive breast cancer-free "controls". This is the first time such a large unique, unbiased DCIS series, with long-term follow-up is analysed integrating clinical, histological, and immunohistochemical data. The results will be presented at SABCS 2015. Citation Format: Visser L, Elshof L, Groen E, van de Vijver K, Lips E, de Maaker M, Nieboer F, Schaapveld M, Rutgers E, Wesseling J. Biomarkers to distinguish hazardous from harmless ductal carcinoma in situ (DCIS) of the breast. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-17-09.

We performed a systematic review with meta-analyses to summarize current knowledge on prognostic factors for invasive disease after a diagnosis of ductal carcinoma in situ (DCIS). Eligible studies assessed risk of invasive recurrence in women primarily diagnosed and treated for DCIS and included at least 10 ipsilateral-invasive breast cancer events and 1 year of follow-up. Quality in Prognosis Studies tool was used for risk of bias assessment. Meta-analyses were performed to estimate the average effect size of the prognostic factors. Of 1,781 articles reviewed, 40 articles met the inclusion criteria. Highest risk of bias was attributable to insufficient handling of confounders and poorly described study groups. Six prognostic factors were statistically significant in the meta-analyses: African-American race [pooled estimate (ES), 1.43; 95% confidence interval (CI), 1.15–1.79], premenopausal status (ES, 1.59; 95% CI, 1.20–2.11), detection by palpation (ES, 1.84; 95% CI, 1.47–2.29), involved margins (ES, 1.63; 95% CI, 1.14–2.32), high histologic grade (ES, 1.36; 95% CI, 1.04–1.77), and high p16 expression (ES, 1.51; 95% CI, 1.04–2.19). Six prognostic factors associated with invasive recurrence were identified, whereas many other factors need confirmation in well-designed studies on large patient numbers. Furthermore, we identified frequently occurring biases in studies on invasive recurrence after DCIS. Avoiding these common methodological pitfalls can improve future study designs.

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.

Resident memory T cells sound the alarm Immunological memory protects against reinfection. Resident memory T cells (T RM ) are long-lived and remain in the tissues where they first encountered a pathogen (see the Perspective by Carbone and Gebhardt). Schenkel et al. and Ariotti et al. found that CD8 + T RM cells act like first responders in the female reproductive tissue or the skin of mice upon antigen reencounter. By secreting inflammatory proteins, T RM cells rapidly activated local immune cells to respond, so much so that they protected against infection with an unrelated pathogen. Iijima and Iwasaki found that CD4 + T RM cells protected mice against reinfection with intravaginal herpes simplex virus 2. Science , this issue p. 98 , p. 101 , p. 93 ; see also p. 40

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 Ductal 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 methods We 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 discussions DCIS 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 Conclusion To 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.

Introduction Ductal 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 methods All 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 discussions After 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). Conclusion According 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.

The CD34+ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8+ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8+ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.

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

Harvesting the potential of the immune system in order to eradicate (residual) acute myeloid leukemia (AML) cells is the long pursued goal of immunotherapy in AML. Strategies using apoptotic tumor cell vaccines have been explored for many years, without significant clinical improvements. In recent years insight has been gained into the mechanisms activating and interfering with tumor-directed immunity. With the arrival of novel immune-modulating agents allowing for the interference with regulatory molecules and interaction with immune-propelling mechanisms, new doors are opening for increasing vaccination efficacy. Combined with advances in the design of apoptotic tumor-based vaccines, we are on the verge of creating an effective AML vaccine strategy, offering a much needed novel therapeutic option for this devastating disease.

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.