Mixed Ductal Carcinoma In Situ Research Articles

Abstract PR02: Inferring the evolutionary dynamics of ductal carcinoma in situ through multi-regional sequencing and mathematical modeling

Abstract Introduction. The natural history of preinvasive breast cancer, or ductal carcinoma in situ (DCIS) remains poorly understood. Overcoming this gap would allow risk-appropriate treatment for patients diagnosed with DCIS. We used a multiregional sequencing approach in combination with mathematical modeling to characterize the evolutionary dynamics of DCIS initiation and progression. Methods. We analyzed a cohort of 18 patients diagnosed with DCIS, either with (n=9) or without (n=9) synchronous invasive cancer. Based on whole exome sequencing, tumor-specific mutation panels were constructed, each targeting 29-75 mutations (median: 60). From each tumor, and using selective ultraviolet radiation fractionation (SURF), we microdissected small spots (encompassing 1-3 duct cross-sections) from 3-4 spatially separated microscope sections (mean slide separation: 1.25cm, range: 0.34-6.0cm). Spots were spatially registered and genotyped based on targeted sequencing of the tumor-specific mutation panels. For each tumor, we performed unsupervised clonal deconvolution of the spot genotypes (CloneFinder) and constructed phylogenetic subclone trees. To quantify the spatial patterns of subclonal mutations, we introduced a dispersion index (DI), ranging from low (DI=0%) to high (DI=100%). To provide a spatio-temporal context for the heterogeneity patterns we developed a family of stochastic mathematical models of DCIS initiation and progression. Thereby, we embedded the evolutionary dynamics of tumor cell expansion in the branching topology of mammary ductal trees. Results. A total of 485 microdissected spots (median per tumor: 23, range: 10-50) were spatially registered and sequenced (median depth: 9,000x). All tumors were multiclonal, containing a median of 5 subclones (range: 2-14). Surprisingly, the correlation between spatial and genomic distances of spots was low. Individual subclones were diffusely dispersed across tumors. DCIS with synchronous DCIS and invasive cancer (mixed DCIS) had a higher mutation dispersion (DI=84.7%) than those without (pure DCIS, DI=70.5%; p=0.03, Wilcoxon rank-sum test). Mixed DCIS also had a higher fraction of spots containing more than one subclone than pure DCIS (median: 30.4% vs 0%, p=0.03). Among 7 mixed DCIS with invasive spots, 5 showed evidence of multiclonal invasion, that is more than one invading subclones were found in both in situ and invasive regions of the tumor. Mathematical modeling analyses show that the observed spatial patterns of genetic heterogeneity are consistent with a single expansion of mixing subclones across the ductal tree architecture. Conclusions. Our findings provide novel insights into the early growth and invasion dynamics of DCIS lesions. Further, we identified potential evolutionary markers for the delineation between indolent (pure) and aggressive (mixed) DCIS. This constitutes an important step towards identification of patients with low-risk DCIS who could be appropriately managed with less aggressive treatment. Citation Format: Marc D. Ryser, Inmaculada C. Sorribes, Matthew Greenwald, Ethan Wu, Allison Hall, Diego Mallo, Lorraine M. King, Timothy Hardman, Lunden Simpson, Carlo C. Maley, Jeffrey R. Marks, Darryl Shibata, E. Shelley Hwang. Inferring the evolutionary dynamics of ductal carcinoma in situ through multi-regional sequencing and mathematical modeling [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR02.

Abstract 2683: Ductal carcinoma in situ is a multiclonal disease

Abstract Introduction. Ductal carcinoma in situ (DCIS) of the breast has a limited propensity to progress to invasive breast cancer. However, due to a lack of markers that predict the risk of progression to invasive cancer, many DCIS patients may receive unnecessary surgery. Here, we explore whether the multiclonal intratumoral heterogeneity commonly observed in invasive breast cancers is also present in the precursor DCIS, and whether it differs between pure DCIS (without concurrent invasion) and mixed DCIS (with concurrent invasion). Methods. A total of 19 tumors (10 pure, 9 mixed) were analyzed. Based on whole exome sequencing, tumor-specific mutation panels were identified and contained a median of 60 point mutations per tumor (range: 35-80). From each tumor, 3-4 histologic sections were obtained, with a mean distance between slides of 1.26cm (range: 0.34-6.0cm). Individual DCIS ducts were microdissected from the sections using selective ultraviolet radiation fractionation (SURF); median number of ducts per tumor was 23 (range: 10-50). For each duct, the tumor specific mutation panel was assayed using targeted deep sequencing (median depth: 9,000x). Clonal deconvolution within each tumor was performed using the software package CloneFinder. Individual glands were classified as multiclonal if they contained 2 or more tumor subclones. In addition, the following evolutionary measures were computed for each tumor: (i) clone diversity as the mean pairwise Hamming distance between all ducts; (ii) clonal mixing as the number of clones that were present in more than one section. Comparisons between pure and mixed cases were performed using a two-sided Wilcoxon rank sum test. Results. All DCIS lesions were multiclonal. The number of subclones per tumor ranged from 2 to 9 overall, with a median of 5 and 6 subclones in pure and mixed DCIS, respectively (p-value for difference=.1). The median fraction of multiclonal glands per tumor was higher in mixed vs. pure DCIS (10% vs. 0%; p=.03). Median subclone diversity was higher in mixed vs. pure DCIS (1.48 vs. 1.04; p=.03), and the fraction of mixing subclones trended higher in mixed DCIS (median: 63%, range: 38-100%) compared to pure DCIS (median: 37%, range: 0-80%; p=.1). Subclones were spatially extensive: 18/19 tumors had at least one subclone that spanned across the analyzed sections, covering a mean distance of 2.5cm. Conclusions. All analyzed DCIS cases were multiclonal and individual subclones were found to span large regions of the tumor. Despite small sample sizes we found differences between pure and mixed DCIS. In particular, mixed DCIS harbored a higher fraction of multiclonal glands and a higher subclone diversity between individual ducts. Our findings characterize the evolutionary dynamics of breast cancer initiation and may provide evolutionary markers that distinguish indolent (pure) and progressive (mixed) disease. As such, this line of work has the potential to risk-stratify DCIS lesions. Citation Format: Marc D. Ryser, Inmaculada C. Sorribes, Matthew Greenwald, Allison Hall, Diego Mallo, Lorraine M. King, Timothy Hardman, Lunden Simpson, Carlo C. Maley, Jeffrey R. Marks, Darryl Shibata, E. Shelley Hwang. Ductal carcinoma in situ is a multiclonal disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2683.

Loss of myoepithelial calponin-1 characterizes high-risk ductal carcinoma in situ cases, which are further stratified by T cell composition.

A hallmark of ductal carcinoma in situ (DCIS) progression is a loss of the surrounding ductal myoepithelium. However, whether compromise in myoepithelial differentiation, rather than overt cellular loss, can be used to predict the risk of DCIS progression is unknown. Here we address this question utilizing pure and mixed DCIS cases (N = 30) as surrogates for DCIS at low and high risk for progression, respectively. We used multiplex immunohistochemical staining to evaluate the relationship between myoepithelial cell differentiation and lymphoid immune cell types associated with poor prognostic DCIS. Our results show that myoepithelial calponin‐1 discriminates between pure and mixed DCIS lesions better than histological subtype, presence of necrosis, or nuclear grade. Additionally, focal loss of myoepithelial cells associated with increased PD‐1+CD8+ T cells, which suggests a link between the myoepithelium and immune surveillance. To identify associations between calponin‐1 expression and immune response, we performed unsupervised hierarchical clustering of myoepithelial and immune cell biomarkers on 219 DCIS lesions from 30 cases. Notably, the majority of pure (low‐risk) DCIS lesions clustered in a high calponin‐1, T cell low group, whereas the majority of mixed (high‐risk) DCIS lesions clustered in a low calponin‐1, T cell high group, specifically with CD8+ and PD‐1+CD8+ T cells. However, a subset of pure DCIS lesions had a similar calponin‐1 and immune signature as the majority of mixed DCIS lesions, which have low calponin‐1 and T cell enrichment—raising the possibility that these pure DCIS lesions might be at a high risk for progression.

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

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

The clinical and biological significance of HER2 over-expression in breast ductal carcinoma in situ: a large study from a single institution

BackgroundPrevious studies have reported up to 50% of ductal carcinoma in situ (DCIS), is HER2 positive, but the frequency of HER2-positive invasive breast cancer (IBC) is lower. The aim of this study is to characterise HER2 status in DCIS and assess its prognostic value.MethodsHER2 status was evaluated in a large series of DCIS (n = 868), including pure DCIS and DCIS associated with IBC, prepared as tissue microarrays (TMAs). HER2 status was assessed using immunohistochemistry (IHC) and chromogenic in situ hybridisation (CISH).ResultsIn pure DCIS, HER2 protein was over-expressed in 9% of DCIS (3+), whereas 15% were HER2 equivocal (2+). Using CISH, the final HER2 status was positive in 20%. In mixed DCIS, HER2 amplification of the DCIS component was detected in 15% with amplification in the invasive component of only 12%. HER2-positive DCIS was associated with features of aggressiveness (p < 0.0001) and more frequent local recurrence (p = 0.03). On multivariate analysis, combined HER2+/Ki67+ profile was an independent predictor of local recurrence (p = 0.006).ConclusionsThe frequency of HER2 positivity in DCIS is comparable to IBC- and HER2-positive DCIS is associated with features of poor prognosis. The majority of HER2 over-expression in DCIS is driven by gene amplification.

Prolyl-4-hydroxylase \u0391 subunit 2 (P4HA2) expression is a predictor of poor outcome in breast ductal carcinoma in situ (DCIS)

BackgroundExtracellular matrix (ECM) plays a crucial role in tumour behaviour. Prolyl-4-hydroxlase-A2 (P4HA2) is a key enzyme in ECM remodelling. This study aims to evaluate the prognostic significance of P4HA2 in breast ductal carcinoma in situ (DCIS).MethodsP4HA2 expression was assessed immunohistochemically in malignant cells and surrounding stroma of a large DCIS cohort comprising 481 pure DCIS and 196 mixed DCIS and invasive carcinomas. Outcome analysis was evaluated using local recurrence free interval (LRFI).ResultsHigh P4HA2 expression was detected in malignant cells of half of pure DCIS whereas its expression in stroma was seen in 25% of cases. Higher P4HA2 expression was observed in mixed DCIS cases compared to pure DCIS both in tumour cells and in stroma. High P4HA2 was associated with features of high risk DCIS including younger age, higher grade, comedo necrosis, triple negative and HER2-positive phenotypes. Interaction between P4HA2 and radiotherapy was also observed regarding the outcome. High P4HA2 expression was an independent prognostic factor in predicting shorter LRFI.ConclusionP4HA2 plays a role in DCIS progression and can potentially be used to predict DCIS outcome. Incorporation of P4HA2 with other clinicopathological parameters could refine DCIS risk stratification that can potentially guide management decisions.

Prognostic significance of tumour infiltrating B lymphocytes in breast ductal carcinoma in situ

Tumour-infiltrating lymphocytes (TILs) are an important component of the immune response to cancer and have a prognostic value in breast cancer. Although several studies have investigated the role of T lymphocytes in breast cancer, the role of B lymphocytes (TIL-Bs) in ductal carcinoma in situ (DCIS) remains uncertain. This study aimed to assess the role of TIL-Bs in DCIS. Eighty DCIS cases (36 pure DCIS and 44 mixed with invasive cancer) were stained immunohistochemically for B lineage markers CD19, CD20 and the plasma cell marker CD138. TIL-Bs density and localization were assessed, including relation to the in-situ and invasive components. An association with clinicopathological data and patient outcome was performed. Pure DCIS showed a higher number of TIL-Bs and lymphoid aggregates than DCIS associated with invasion. In pure DCIS, a higher number of peri- and paratumoral TIL-Bs was associated significantly with large tumour size (P = 0.016), hormone receptor (ER/PR) negative (P = 0.008) and HER2+ status (P = 0.010). In tumours with mixed DCIS and invasive components, cases with high-density B lymphocytes, irrespective of their location or topographic distribution, were associated significantly with variables of poor prognosis, including larger size, high grade, lymphovascular invasion, lymph node metastases, ER/PR-negative and HER2+ status. Outcome analysis showed that pure DCIS associated with higher numbers of B lymphocytes had shorter recurrence-free interval (P = 0.04); however, the association was not significant with the CD138+ plasma cell count (P = 0.07). Assessment of TIL-B cells based on location and topographic distribution can provide prognostic information. Validation in a larger cohort is warranted.

Validation of an oligo-gene signature for the prognostic stratification of ductal carcinoma in situ (DCIS).

Current evidence suggests that the majority of DCIS lesions do not progress to invasive carcinoma, and overtreatment of DCIS is a significant problem. We previously reported an 8-gene signature that differentiated microdissected low-grade (LG) DCIS lesions with and without associated stromal invasion, based on differential DNA copy number changes detected by quantitative (q) PCR. The current study was undertaken to validate our candidate breast cancer invasion gene panel in a larger series of non-microdissected LG DCIS cases, and to investigate its potential utility in intermediate-grade (IG) and high-grade (HG) DCIS. Representative paraffin blocks were selected from 267 resected DCIS cases with 5-15years of follow-up (139 pure DCIS ["PD"] and 128 mixed DCIS with associated invasion ["MD"]). These included 171 LG, 46 IG and 50 HG DCIS cases. Gene copy number changes were determined by qPCR, and their differential distribution in the PD and MD subgroups was evaluated. As an alternate platform, we employed immunohistochemistry (IHC). Novel IHC assays were developed for all eight candidate genes, and increased or reduced protein expression was manually scored. Separate multi-gene models were developed for qPCR and IHC to distinguish progressing and non-progressing DCIS lesions. By qPCR analysis, a panel of six genes, as well as CELSR1 alone (a potential invasion suppressor), differentiated PD and MD cases in LG and IG, but not in HG DCIS. By IHC, a panel of three genes, as well as GRAP2 alone (a potential invasion promoter), also distinguished PD and MD cases in LG and IG, but not in HG DCIS. The combination of CELSR1 (by qPCR) and GRAP2 (by IHC) had the best discriminatory power (p=0.00004). Assays testing either or both of these genes have the potential to become important adjuncts for choosing appropriate treatment for LG/IG DCIS patients.

Methylation profiling of ductal carcinoma in situ and its relationship to histopathological features.

IntroductionDNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material.MethodsPromoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction.ResultsMethylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification.ConclusionsDNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0423-9) contains supplementary material, which is available to authorized users.

A Multigene Expression Assay to Predict Local Recurrence Risk for Ductal Carcinoma In Situ

A Multigene Expression Assay to Predict Local Recurrence Risk for Ductal Carcinoma In Situ

Differential copy number aberrations in novel candidate genes associated with progression from in situ to invasive ductal carcinoma of the breast

Only a minority of intraductal carcinomas of the breast give rise to stromally invasive disease. We microdissected 206 paraffin blocks representing 116 different cases of low-grade ductal carcinoma in situ (DCIS). Fifty-five were pure DCIS (PD) cases without progression to invasive carcinoma. Sixty-one cases had a small invasive component. DNA was extracted from microdissected sections and hybridized to high-density bacterial artificial chromosome arrays. Array comparative genomic hybridization analysis of 118 hybridized DNA samples yielded data on 69 samples that were suitable for further statistical analysis. This cohort included 20 pure DCIS cases, 25 mixed DCIS (MD), and 24 mixed invasive carcinoma samples. PD cases had a higher frequency of DNA copy number changes than MD cases, and the latter had similar DNA profiles compared to paired invasive carcinomas. Copy number changes on 13 chromosomal arms occurred at different rates in PD versus MD lesions. Eight of 19 candidate genes residing at those loci were confirmed to have differential copy number changes by quantitative PCR. NCOR2/SMRT and NR4A1 (both on 12q), DYNLRB2 (16q), CELSR1, UPK3A, and ST13 (all on 22q) were more frequently amplified in PD. Moreover, NCOR2, NR4A1, and DYNLRB2 showed more frequent copy number losses in MD. GRAP2 (22q) was more often amplified in MD, whereas TAF1C (16q) was more commonly deleted in PD. A multigene model comprising these candidate genes discriminated between PD and MD lesions with high accuracy. These findings suggest that the propensity to invade the stroma may be encoded in the genome of intraductal carcinomas.

Recurrent Cancer After Breast-Conserving Surgery with Radiation Therapy for Ductal Carcinoma in Situ: Mammographic Features, Method of Detection, and Stage of Recurrence

The purpose of our study was to determine the mammographic appearance, detection method, and stage of ipsilateral breast tumor recurrence in women treated with breast-conserving surgery and whole-breast radiation therapy for ductal carcinoma in situ (DCIS). Following institutional review board approval, records of women treated with breast-conserving surgery and radiation therapy for DCIS who developed an ipsilateral breast tumor recurrence from 1981 to 2003 were reviewed retrospectively. Multiinstitutional database records showed 513 women were treated, of whom 42 (8.2%) developed local recurrence. Study criteria were fulfilled and complete records were available for 32 women. Mean age at initial diagnosis was 49 years (range, 26-73 years). Of the 32 patients included in our study, 31 (97%) recurrences were mammographically apparent. Twenty-nine (91%) of 32 were diagnosed exclusively by mammography. Mammographic findings at recurrence were calcifications in 24 (75%) of 32, mass in six (19%) of 32, and distortion in one (3%) of 32. The mean time to recurrence was 4.5 years. Twelve (40%) of 30 had the recurrence in a remote quadrant from the original cancer. Recurrences were DCIS in 17 (53%) of 32, DCIS with microinvasion in six (19%) of 32, invasive ductal cancer in three (9%) of 32, invasive lobular cancer in two (6%) of 32, and mixed DCIS and invasive cancer in four (13%) of 32. Six (67%) of nine patients with invasive cancer (excluding microinvasion) had tumors smaller than 1 cm. Ninety-one percent of recurrences were minimal cancers. All recurrences were stage 0 or 1. Mammography successfully detected ipsilateral breast tumor recurrence, predominantly as calcifications or masses, after breast-conserving surgery with radiation therapy for DCIS in 97% of cases. The recurrences were located at variable distances from the lumpectomy site. Ninety-one percent of recurrences were minimal cancers and all were early stage, connoting excellent prognosis.