Ductal Carcinoma In Situ Subtypes Research Articles

Abstract PO4-25-10: Towards personalized medicine for DCIS - the role of hormone receptors, HER2, and Ki67 status in high-grade DCIS

Abstract Objective: This study aimed to investigate the molecular details in breast ductal carcinoma in situ (DCIS). Expression status of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 were investigated among luminal A (LumA), luminal B HER2-negative (LumB HER2-), luminal B HER2-positive (LumB HER2+), HER2-enriched, and triple-negative (TPN), subtypes of DCIS. Materials & Methods: The study comprised formalin-fixed paraffin-embedded (FFPE) specimens of 357 DCIS grade 3 cases diagnosed between 1996 – 2018. Routine diagnostic immunohistochemical (IHC) staining were performed. DCIS cases were classified as LumA, LumB HER2-, LumB HER2+, HER2-enriched or TPN, according to the 2013 St. Gallen guidelines, which is used for molecular subtyping of invasive breast carcinoma (IBC). Each subtype was sorted into three subcategories: “pure” meaning those without an invasive component; “w/invasive” meaning those with an invasive component; and “all” meaning the entire group of the given subtype. Furthermore, ER and PR-receptor expression was registered for LumA, LumB HER2- and LumB HER2+ cases as intervals. The distribution of Ki67 was analyzed within each subtype. For some analyses, we combined the LumA and LumB HER2- subtypes into one entire group. The inter-observer variability of Ki67 was calculated by setting a cut-off value of 20% (2013 St. Gallen). A cohort (n = 47) of DCIS cases with a median Ki67 value of 18% was selected. Ki67 was calculated by analyzing 200 DCIS cells in two separate hotspots. Cohen's Kappa coefficient was calculated based on these data. HER2 (IHC) was scored based on ASCO/CAP guidelines established for routine diagnostic work-up for IBC. 16 equivocal cases (2+) were further investigated using dual SISH. Results: 98% of “all” cases of the LumA subtype showed an ER ≥ 50%. PR expression ≥ 50% was found in 91% of cases in this subtype. The incidence of ER-receptor at a cut-off ≥ 50% in the LumA subtype was significantly higher than that in the LumB HER2- and LumB HER2+ subtypes (p < 0.0001, Chi-square). In contrast, there was a statistically significant reduction in ER-receptor expression at a cut-off < 10% in LumA compared to the latter subtypes. The proportion of cases with PR-receptors with a cut-off of < 20% showed significant differences between LumA, LumB HER2- and LumB HER2+ subtypes (1.6%; 47% and 37%, respectively). There was also a significantly higher proportion of PR-receptor ≥ 50% cases among the LumA subtype. We found a significant association between PR < 20% and HER2 (3+) in luminal subtypes (p < 0.0004, Fisher's exact). There was no significant difference in ER/PR expression in “pure” cases of luminal subtypes of DCIS versus those with an invasive component (p = 0.1831, Chi-square). The Ki67 in the entire population (n = 357) varied from < 1% to > 80%. The mean and median were around 20% in those subtypes whose classifications were not depended on Ki67. There was a significant difference in the distribution of Ki67 when cases of LumA “all” and those of LumB HER2- “all” were combined as one entire group and were compared to LumB HER2+ “all” and HER2-enriched “all” (p-adjusted < 0.0001, Kruskal Wallis). The assessment of Ki67 among different observers showed a Cohen's kappa coefficient of 0.29 – 0.31 (fair agreement). We compared the HER2 (IHC) 0, 1+ and 2+ score among LumA and LumB HER2- subtypes and did not find a statically significant difference, when the “pure” and “w/invasive” were compared (p = 0.603, Chi-square). Conclusions: Ki67 was highly variable in DCIS grade 3. Inter-observer agreement was (as expected) suboptimal, and the cut-off at 20% defined by the 2013 St Gallen guidelines for IBC is not reliable for the distinction of LumA and LumB HER2 subtypes of DCIS. The LumB subtype of DCIS is heterogeneous with considerable variability among the four IHC markers used in the present study. A low PR is strongly associated with HER2 (3+), in luminal subtypes (p < 0.0004, Fisher's exact). Citation Format: Hossein Schandiz, Lorant Farkas, Daehoon Park, Solveig Norheim Andersen, Jürgen Geisler, Torill Sauer. Towards personalized medicine for DCIS - the role of hormone receptors, HER2, and Ki67 status in high-grade DCIS [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-10.

Abstract PO1-24-11: Distinct patterns of MHC class I, PD-L1 expression, and T-cell infiltration in different subtypes of ductal carcinoma in situ of the breast

Abstract Background: Ductal carcinoma in situ (DCIS) of the breast encompasses various subtypes with distinct behaviors. Immune cell infiltration within the tumor microenvironment is thought to influence DCIS progression. Tumor-infiltrating lymphocytes (TILs) serve as a surrogate marker for the adaptive immune response. The interaction between programmed death 1 (PD-1) and its ligand PD-L1 suppresses effector T-cell function. Major histocompatibility complex (MHC) class I molecules play a critical role in presenting tumor antigens to cytotoxic T cells. Despite the significance of PD-L1 and MHC class I in immune evasion, limited data exist on their correlation with T-cell infiltration in DCIS subtypes. Methods: Using immunohistochemistry, we examined MHC class I and PD-L1 expression, along with CD3+ and CD8+ T lymphocytes, in 131 DCIS cases through tissue microarrays. DCIS subtypes included hormone receptor-positive (HR+)/human epidermal growth factor 2-negative (HER2-), HR+/HER2+, HR-/HER2+, and triple-negative (TN) subtypes. Results: Among the 128 interpretable cases, the distribution was as follows: HR+/HER2- (50.8%), HR+/HER2+ (14.1%), HR-/HER2+ (26.5%), and TN (8.6%). HER2+ subtypes (HR+/HER2+ and HR-/HER2+) exhibited higher stromal TILs, CD3+ T cells, and CD8+ T cells compared to HR+/HER2- subtype. MHC class I loss was observed in 16.4% (21/128) of cases, with the highest frequency in the HR+/HER2- subtype (29.2%), followed by HR-/HER2+ (5.9%), HR+/HER2+ (0%), and TN (0%) subtypes. DCIS cases lacking MHC class I expression displayed lower stromal CD8+ T cell infiltration than those with intact MHC class I. PD-L1 expression in immune cells (≥1%) was detected in 18.8% (24/128) of cases and correlated with increased stromal TILs, CD3+ T cells, and CD8+ T cells. PD-L1 expression was more prevalent in HER2+ subtypes than in HR+/HER2- subtype. PD-L1-positive DCIS cases mostly retained MHC class I expression (95.8%), except for one case (4.2%) in the HR-/HER2+ subtype. The highest numbers of stromal CD3+ and CD8+ T cells were observed in DCIS cases with intact MHC class I and positive PD-L1 expression. The correlation between MHC class I and PD-L1 expression and infiltration of CD3+ and CD8+ T cells was maintained in HR+/HER2- and HR-/HER2+ subtypes. Notably, tumoral CD3+ and CD8+ T cells showed no significant association with MHC class I or PD-L1 expression. Furthermore, MHC class I and PD-L1 expression did not predict tumor recurrence. Conclusion: We observed distinct patterns of MHC class I and PD-L1 expression and T-cell infiltration among DCIS subtypes. Enhancing our understanding of MHC class I, PD-L1, and immune subsets, including CD8+ T cells, may contribute to the development of immune modulating therapies for DCIS, particularly in the HER2+ subtype. Citation Format: Nah Ihm Kim, Min Ho Park, Ji Shin Lee. Distinct patterns of MHC class I, PD-L1 expression, and T-cell infiltration in different subtypes of ductal carcinoma in situ of the breast [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-11.

Subtypes of high-grade breast ductal carcinoma in situ (DCIS): incidence and potential clinical impact

ObjectiveThe purpose of this study was to investigate and classify the molecular subtypes of high-grade ductal carcinoma in situ (DCIS) and identify possible high-risk subtypes. The heterogenicity of DCIS with variable clinical and histopathological presentations has been recognized. Nevertheless, only histopathological grading and diameter are currently implemented in clinical decision-making following the diagnosis of DCIS. The molecular subtypes of DCIS and their IHC surrogate markers have not been defined in conventional treatment guidelines and recommendations. We applied the definitions of molecular subtypes according to the IHC surrogate markers defined for IBC and subclassified high-grade DCIS, accordingly.MethodsHistopathological specimens were collected, revised, and regraded from 494 patients diagnosed with DCIS between 1996 and 2018. Other in situ and papillary lesions observed in breast biopsies were excluded from this study. 357 high-grade DCIS cases were submitted to IHC analysis. The markers investigated were ER, PR, HER2, and Ki67.Results45 cases were classified as grade 1, 19 as grade 2, and 430 as grade 3. Sixty patients with high-grade DCIS had an additional invasive component in the surgical specimen. Thirty-three patients were diagnosed with recurrent DCIS or invasive cancer (minimum one year after their primary DCIS diagnosis). The proportions of luminal A and luminal B HER2-negative subtypes varied depending on whether 2011 or 2013 St. Gallen Consensus Conference guidelines were adopted. Luminal A was the most prevalent subtype, according to both classifications. The luminal B HER2-positive subtype was found in 22.1% of cases, HER2-enriched subtype in 21.8%, and TPN subtype in 5.6%. There were strong indications that HER2-enriched subtype was significantly more frequent among DCIS with invasive component (p = 0.0169).ConclusionsHigh-grade DCIS exhibits all the molecular subtypes previously identified in IBC, but with a somewhat different distribution in our cohort. HER2-enriched subtype is substantially related to the presence of an invasive component in DCIS; consequently, it is regarded as a high-risk entity.

MCQ corner

1. Selenocysteine is coded by UGA UAG UAA AUG Ans: A. UGA Explanation Selenocysteine (SeCys/SeC) is the 21st amino acid and is found in thioredoxin reductase, glutathione peroxidase, 5’-deiodinase, and selenoprotein P. It is an exceptional case where a stop codon codes for an amino acid. The other stop codons are UAA and UAG. The ability of the protein synthetic apparatus to identify a selenocysteine-specific UGA codon involves the selenocysteine insertion element, a stem-loop structure in the untranslated region of the mRNA.[1] 2. A child with diarrhea for 2 days came to your out-patient department (OPD). On examination, the signs of dehydration were marked and the child was treated with oral rehydration solution (ORS). Glucose absorption was facilitated by which receptors? SGLT1 SGLT2 GLUT4 Sodium–hydrogen antiporter Ans: A. SGLT1 Explanation Absorption of glucose from the intestinal lumen to intestinal cells is by the cotransport mechanism. It is an active transport system and can transport against the concentration gradient.[2] 3. Skeletal derivative of the second pharyngeal arch is Malleus Incus Stapes Maxilla Ans: C. Stapes Explanation Maxilla, malleus, and incus are derived from the first arch.[3] 4. Blastocyst makes contact with a uterus on <3 days 5–7 days 8–11 days 15–16 days Ans: B. 5–7 days Explanation Contact of the blastocyst with endometrium occurs at the time of implantation. Implantation occurs at around 5-7 days.[4] 5. The father of antiseptic surgery is Louis Pasteur Robert Koch Julius Petri Joseph Lister Ans. D. Joseph Lister Explanation Louis Pasteur – the father of microbiology, Robert Koch – the father of microbiology (with Louis Pasteur), and the father of medical bacteriology, Julius Petri – invented the Petri dish.[5] 6. Following were the findings in a Glucose tolerance test (GTT). Fasting blood glucose <75 mg/dl; all blood samples show low blood glucose; urine glucose – nil. The possible diagnosis is Alimentary glycosuria Diabetes mellitus Hypopituitarism Diabetes insipidus Ans. C. Hypopituitarism Explanation In alimentary glycosuria, there will be a peak in the first sample after glucose load and sugar will appear in urine. In diabetes mellitus, fasting and the next two samples will show higher values than the normal reference range and the sample does not return to baseline at the end of the test. In hypopituitarism, there is hypoglycemia and the patient feels fatigued and stressed. In diabetes insipidus, GTT is mostly unaffected.[6] 7. A 10-year-old boy with a history of atopy presented with seasonal recurrent bilateral conjunctivitis and complained of blurred vision for the past 1 week. On lid eversion, giant papillae were seen. All of the following could be seen by slit-lamp examination except: Vascular pannus and punctate epithelial erosions involving the superior cornea. An oval epithelial ulceration with underlying stromal opacification in the central cornea Limbal follicles Conjunctival symblepharon Ans. D. Conjunctival symblepharon Explanation A conjunctival symblepharon is seen in the case of autoimmune disorders such as Sjogren’s syndrome, sarcoidosis, lichen planus, granulomatosis with polyangiitis, etc.[7] 8. Which of the following breast lesion is a noninvasive malignancy? Tubular carcinoma and mucinous carcinoma Intraductal carcinoma of the comedo type Infiltrating ductal carcinoma and lobular carcinoma Medullary carcinoma, including atypical medullary lesions Ans. B. Intraductal carcinoma of the comedo type Explanation A comedo-type ductal carcinoma in situ (DCIS), also known as comedocarcinoma insitu, is the high-grade subtype of DCIS. It completely fills and dilates the ducts and lobules in terminal duct lobular units with plugs of high-grade tumor cells with central necrosis “comedonecrosis.”[8] 9. In patients who develop a documented episode of deep-vein thrombosis, the most significant long-term sequelae are: Claudication Recurrent foot infections Development of stasis ulcer Pulmonary embolization Ans. C-Development of stasis ulcer Explanation Chronic symptoms may occur after 6 weeks or beyond. Pulmonary embolization is the most common complication but is seen as an acute complication.[9] 10. Identify the test from the diagram. Rothera’s test Benedict’s test Barfoed’s test Seliwanoff’s test Ans. A. Rothera’s testFigure 1: Purple permanganate coloured ring [Ferro-penta cyanine complex] at the junction of two fluidsExplanation Rothera’s test was done to identify the presence of ketone bodies in a urine sample. Benedict’s test will contain a green/yellow/orange/brick red precipitate at the bottom. Barfoed’s test has a red scum at the top of the fluid and a little red precipitate at the bottom. Seliwanoff’s test will have a cherry red color.[10] Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Towards the development of DCIS risk prediction models.

Ductal carcinoma in situ (DCIS) has a variable natural history, with a poorly understood biology. In this issue of Cancer Cell, Strand et. al. use two well-annotated DCIS repositories to identify an 812-gene classifier that associates with high risk of DCIS recurrence and define novel DCIS subtypes that informs future studies.

Abstract A017: Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer

Abstract Background: Screening mammography has dramatically increased incidental detection of Ductal Carcinoma in situ (DCIS), a non-obligate precursor of breast cancer. Approximately 10-50% of DCIS progress to the invasive disease and the risk of developing recurrence or progression after surgery is still not well defined based on clinical and pathological characteristics. Our study investigated the genetic and epigenetic mechanisms associated with recurrence and progression of DCIS to invasive cancer. Methods: To identify genes associated with recurrence and progression of DCIS to invasive cancer, we performed Nanostring nCounter analysis (MDACC, n = 40) and RNA-sequencing (Sloane study, n = 183) in two independent prospective primary DCIS cohorts. Mammary epithelial-specific Pten knockout mouse model developed in our lab was used to interrogate the genetic pathway deregulated during development of mammary intraepithelial neoplasia to adenocarcinoma. ATAC-seq and ChIP-qPCR were performed to assess chromatin accessibility in the genome and validate binding of histone-modifying enzymes on target promoters. Histopathological analyses were performed to determine DCIS grades, subtypes, and protein expression profiles. Results: RNA expression profiles of normal mammary and primary DCIS tissues (n = 40) identified 10 gene transcripts that were significantly and differentially expressed between normal and high-grade DCIS. Only SOX11 expression was significantly and progressively correlated with KI67 and the DCIS recurrence score as described by Solin (JNCI 2013). Mammary intraepithelial neoplasia in Pten knockout mice and 21T DCIS to IDC progression cell line model also revealed SOX11 upregulation compared to controls. Since PTEN/PI3K/AKT pathway is known to regulate global active histone marks and open accessible chromatin, we investigated the role of AKT on SOX11 expression. Results demonstrated that inhibition of AKT altered enrichment of histone-modifying enzymes KDM5A and EZH2 on the promoter accompanying downregulated SOX11 expression in DCIS cell lines. SOX11 and EZH2 expression profiles revealed significant correlation in DCIS tissue samples. In two independent DCIS cohorts, SOX11 was found significantly upregulated in HER2+ and basal-like DCIS subtypes (P &amp;lt; 0.001). RNA sequencing in 183 longitudinal DCIS cases of the Sloane study (median follow-up of 96 months) revealed significantly shorter recurrence-free survival in SOX11+ than SOX11- patients (P &amp;lt; 0.02 in all subtypes; P = 0.002 in HER2+ subtype). Based on COX proportional hazards model, SOX11 alone was significantly associated with the risk of developing recurrence and invasive breast cancer (HR = 1.7, 95% CI = 1.1 to 2.6; P &amp;lt; 0.02). Conclusions: Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer. SOX11 and its epigenetic regulators can be developed as prognostic markers for DCIS patients with recurrence risk independent of traditional clinical and pathologic characteristics. Citation Format: Warapen Treekitkarnmongkol, Vandna Shah, Kazuharu Kai, Hiroshi Katayama, Justin W. Wong, Farah A Ladha, Brian Menegaz, Sarah E. Pinder, Wei Lu, Fei Yang, Ximing Tang, Savitri Krishnamurthy, Ignacio I. Wistuba, Elinor J. Sawyer, Alastair M. Thompson, Subrata Sen. Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer [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 A017.

Is loss of p53 a driver of ductal carcinoma in situ progression?

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive carcinoma. Multiple studies have shown that DCIS lesions typically possess a driver mutation associated with cancer development. Mutation in the TP53 tumour suppressor gene is present in 15-30% of pure DCIS lesions and in ~30% of invasive breast cancers. Mutations in TP53 are significantly associated with high-grade DCIS, the most likely form of DCIS to progress to invasive carcinoma. In this review, we summarise published evidence on the prevalence of mutant TP53 in DCIS (including all DCIS subtypes), discuss the availability of mouse models for the study of DCIS and highlight the need for functional studies of the role of TP53 in the development of DCIS and progression from DCIS to invasive disease.

Abstract LB165: Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer

Abstract Background: There is an unmet need for the development of safer and more effective therapies for the prevention of human invasive breast cancer, for which ductal carcinoma in situ (DCIS) is a precursor. Currently, anti-hormonal therapies are the only option in individuals previously diagnosed with hormone receptor positive breast cancer. However, these agents are associated with many side effects. Furthermore, there are currently no preventive options for hormone receptor negative breast cancers. To study DCIS progression, we developed a model referred to as MIND (Mouse-INtraDuctal) in which patient-derived DCIS cells and cell lines are injected intraductally and studied as they progress to invasive ductal carcinoma (IDC). Previously we provided strong evidence for the role of B Cell Lymphoma-9 (BCL9) as a potential driver of DCIS malignancy. We chose to target BCL9 using RE40CA and its active ingredient CA previously identified in a screen for inhibitors of β-catenin binding to BCL9. Experimental Strategy: CA and RE40CA were administered to DCIS.COM MIND animal models for four weeks by oral gavage or mixed in a powder diet formulation. Drug plasma levels were evaluated by LC-MS. Four weeks following drug administration, mammary glands were excised and evaluated for efficacy and expression of biomarkers of aggressive DCIS, nuclear β-catenin and BCL9. For efficacy studies, mammary glands containing DCIS like lesions were processed and subjected to immunofluorescence staining using anti-human CK-5, anti-SMA, Dapi, β-catenin and BCL9. Extent of DCIS in vivo growth, number of invasive lesions, tumor number and tumor volume were evaluated for each mammary gland. Results: Efficacy studies showed a significant reduction in the number of invasive lesions by administration of CA by oral gavage (CA OG:10mg/kg), CA at 200mg/kg diet (CA200), CA400 and RE40CA 500. A significant reduction in extent of DCIS in vivo growth was achieved by CA OG and RE40CA 500. A significant reduction in the number of tumors was achieved by CA OG, RE40CA OG (25mg/kg equivalent to CA 10mg/kg), RE40CA 100, RE40CA 500 and RE40CA 1000. A significant reduction in tumor volume was achieved by CA OG, RE40CA OG, RE40CA 100, RE40CA 500 and RE40CA 1000. Linear regression modelling was used to compare statistical differences among the groups and differences that reached p-value &amp;lt;0.05 were considered significant. Administration of CA or RE40CA by oral gavage was also associated with a significant loss of nuclear BCL9/β-catenin expression in DCIS epithelial cells. Dietary administration of CA and RE40CA as OG or mixed in powder diet showed an increasing trend in plasma concentrations as the dose was increased. Evaluation of HE stained sections of animal organs by histopathology showed no evidence in any tissue of necrosis, degeneration, inflammation or neoplasia. Additionally, no consistent lesions attributable to treatment were observed in any of the animals or groups. Conclusion: Administration of CA and RE40CA are safe and effective in prevention of DCIS malignancy. Future studies are aimed at testing the efficacy of CA/RE40CA using DCIS cell lines representing other DCIS subtypes (ER+/PR+ and HER2 overexpressing) as well as patient derived DCIS MIND animal models. Supported by NCI PREVENT Cancer Preclinical Drug Development Program, 75N91019D00016/75N91020F00001. Citation Format: Yan Hong, Aditi Rastogi, Aryamitra Banerjee, Brandon L. Plattner, Matthew Lindeblad, Alexander V. Lyubimov, Timothy Fields, Seema A. Khan, Altaf Mohammed, Jeremy J. Johnson, Fariba Behbod. Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer [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 LB165.

Abstract P3-18-11: Meeting the challenge of successful one-stage lumpectomy for DCIS

Abstract Introduction: Positive lumpectomy margins are associated with an increased risk for ipsilateral tumor recurrence, and necessitate additional procedures to achieve acceptable margins. Re-excision rates are higher in patients with ductal carcinoma in situ (DCIS) versus those with invasive carcinoma. The purpose of this study was to investigate the clinicopathologic characteristics, surgical techniques, and other outcomes associated with re-excision over time following primary breast conserving surgery for DCIS. Methods: Our institutional Breast Cancer Database was queried for patients treated with lumpectomy as the primary procedure for pure DCIS from 2010-2021. Cases of microinvasive disease were excluded. The primary endpoint was the rate of re-excision following lumpectomy. Variables of interest included patient demographics and clinicopathologic characteristics. As a secondary analysis, adjusted odds ratios for the association of MarginProbe® with re-excisions were calculated using a multivariable logistic regression model controlling for age, tumor size, breast density and DCIS subtype. Individual comparisons of continuous variables were based on t-tests, group proportions compared with one-way ANOVA, and categorical variables compared using Chi Square tests. Results: Out of a total of 695 patients in our cohort, 221 (32%) had re-excisions. There was a statistically significant 2% decline per year in the number of re-excisions (p=0.005). Patients who underwent re-excision had larger size of DCIS (2.37 cm vs 1.37 cm, p&amp;lt;0.001). Papillary-type DCIS was associated with an increased rate of re-excision surgery (Table 1). Prior to the introduction of the MarginProbe® for intraoperative margin assessment in 2014, approximately 42% of patients with DCIS underwent re-excisions. This decreased to 24.9% in the post-2013 time frame (p=0.02). In an unadjusted estimate for the odds ratio for association, patients who underwent lumpectomy with MarginProbe® were 81% less likely to require re-excision (OR = 0.19, 95% CI = 0.12, 0.31, p&amp;lt;0.0001). In a logistic regression model controlling for potential confounders with re-excision as the dependent variable and MarginProbe® as the primary explanatory variable, patients for whom the MarginProbe® was used were 66% less likely to require re-excision when controlling for age, tumor size, menopausal status, breast density, and pathology (OR = 0.34, 95% CI 0.16, 0.69). Of patients who required re-excision, 20% went on to mastectomies as their second procedure. After each unsuccessful re-excision, a larger proportion of patients converted to mastectomy. In total, 26% of patients who had unsuccessful primary surgery went on to mastectomies. Of patients who proceeded to mastectomy as their second procedures, 13% were found to carry BRCA1 or 2 mutations. Conclusions: In our study of patients diagnosed with pure DCIS and treated with primary breast conserving surgery, larger size of DCIS and papillary subtype were associated with an increased rate of re-excision procedures. A proportion of patients with unsuccessful primary lumpectomies required multiple procedures to complete their surgical treatment and 26% went on to mastectomy. Patients whose primary procedures included intraoperative margin assessment with the MarginProbe® were significantly less likely to require re-excision. Intraoperative margin assessment is an important tool that can support breast conserving surgery in the challenging population of patients with DCIS. Table 1.Clinicopathologic CharacteristicsVariableNo Re-excision (n=313)Re-Excision (n=100)P-ValueAge; mean (SD)61.48 (12.01)58.04 (13.39)0.016Age of Menopause; mean (SD)50.22 (5.18)48.13 (5.45)0.004Size of DCIS; mean (SD)1.37 (1.25)2.37 (2.00)&amp;lt;0.001Breast Density (%)0.149N/A6 (1.9)2 (2.0)Entirely Fatty10 (3.2)3 (3.0)Extremely Dense19 (6.1)13 (13.0)Heterogeneously Dense165 (52.7)42 (42.0)Scattered Fibroglandular113 (36.1)40 (4.0)DCIS SubtypePapillary93 (29.7)48 (48.0)0.001Cribiform206 (65.8)69 (69.0)0.641Solid190 (60.7)60 (60.0)0.994Comedo54 (17.3)25 (25.0)0.117Surgical Margins (%)&amp;lt;0.001Close (&amp;gt;0 and &amp;lt;1mm)3 (1.0)60 (60.0)Negative (≥1 mm)310 (99.0)13 (13.0)Positive (≤0 mm)0 (0.0)27 (27.0) Citation Format: Joshua A Feinberg, Charles DiMaggio, Nakisa Pourkey, Jennifer Chun Kim, Jenny Goodgal, Amber Guth, Deborah Axelrod, Freya Schnabel. Meeting the challenge of successful one-stage lumpectomy for DCIS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-18-11.

Abstract OT1-09-01: A randomized study comparing surgical excision versus NeOadjuvant Radiotherapy followed by delayed surgical excision of Ductal carcinoma In Situ (NORDIS)

Abstract Lumpectomy and breast radiotherapy (RT) achieve good longterm control of disease in patients diagnosed with ductal carcinoma in situ (DCIS). In the combined analysis of NSABP B-17/B-24 DCIS trials, these treatments were shown to reduce invasive ipsilateral breast tumor recurrences by 52% compared to lumpectomy alone. RT has been consistently used as an adjuvant to surgery and therefore, little is known of its activity on intact/untreated DCIS. Elucidating the effectiveness of radiation therapy may help optimize the treatment of DCIS and avoid over-treatment.Trial Design The objective of the ongoing phase 2 pilot trial is to evaluate the ablative and treatment effects of neoadjuvant RT (neoRT) on DCIS. Patients diagnosed with DCIS on core needle biopsy are randomized to upfront lumpectomy (Arm 1) followed by partial breast irradiation (PBI) or neoRT PBI followed by delayed surgical excision (Arm 2). Arm 1 serves as a reference group for pathological-radiological correlations as well as to ascertain the rate of upstaging to invasive cancer. The neoRT regimen consists of 6 Gy daily x 5 (consecutive or non-consecutive days) to the intact tumor with a 0.5 cm margin. Surgical excision is delayed 12-16 weeks, allowing for the ablative effects of neoRT to occur. Specific Aims •To determine if neoRT can completely ablate 30% of DCIS cases•To determine if DCIS subtypes exhibit differential sensitivity to neoRT•To evaluate microscopic treatment effects; wound complication rates; post-RT breast imaging changes; and invasive carcinoma rate.Eligibility Women 18 years of age or older diagnosed with DCIS by core needle biopsy and intending to receive breast conserving surgery are eligible. Tumors must be ≤4 cm, discovered as mammographic microcalcifications and/or MRI non-mass enhancement measuring with evidence of residual imaging abnormality after diagnostic needle biopsy. Statistics A total of 50 patients will be recruited. Upstaging to invasive cancer is anticipated to be approximately 10%. With 25 subjects in each arm, we will have 80% power to detect a 30% or higher improvement in the DCIS complete response rate following neoadjuvant PBI. Accrual 10 (open 2019) Contact wapnir@stanford.edu. Wapnir IL, Dignam JJ, Fisher B, et al. Long-term outcomes of invasive ipsilateral breast tumor recurrences after lumpectomy in NSABP B-17 and B-24 randomized clinical trials for DCIS. J Natl Cancer Inst. 2011;103(6):478-488. doi:10.1093/jnci/djr027.McCormick B, Winter K, Hudis C, et al. RTOG 9804: a prospective randomized trial for good-risk ductal carcinoma in situ comparing radiotherapy with observation. J Clin Oncol. 2015;33(7):709-715. doi:10.1200/JCO.2014.57.9029. Citation Format: Irene Wapnir, Carol Marquez, Kimberly Stone, Kimberly Allison, Wendy DeMartini, Catherine Salem, Jacqueline Tsai, Robert West, Alex McMillan, Melinda Telli, Kathleen Horst. A randomized study comparing surgical excision versus NeOadjuvant Radiotherapy followed by delayed surgical excision of Ductal carcinoma In Situ (NORDIS) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-09-01.

Relationship of the breast ductal carcinoma in situ (DCIS) immune microenvironment with clinicopathological features: An institutional experience.

e12565 Background: DCIS is usually treated with resection followed by 5 years of adjuvant endocrine therapy for hormone receptor (HR) + DCIS. Endocrine therapy is not used in HR- DCIS. Although DCIS is considered a precursor lesion to invasive breast cancer, the different molecular subtypes confer variable clinical outcomes. The host immune response plays a key role in breast cancer progression and response to therapy. However, relative to invasive breast cancer, the immune milieu of DCIS is less understood. This retrospective study compares the clinical outcomes and tumor microenvironment of HR+ and HR- DCIS in order to identify clinical and immunological features in HR- DCIS that may predict an increased risk of recurrence or progression to invasive breast cancer. Methods: A single institution retrospective chart review was performed to identify patients diagnosed with DCIS between 2012 and 2017. A clinico-pathologic data set, as well as the PD-L1 expression of the DCIS and TILs were collected and correlated with various outcomes. Results: Our cohort consisted of 20 cases of HR- DCIS and 50 cases of HR+ DCIS. Overall, 56% were Caucasian, 20% Asian, 18% Hispanic, and 6% African American. Of the HR- patients, 70% were Caucasian, 15% Hispanic, and 15% Asian. Of the 17 HR- patients with available HER2 data, 76% had HER2+, and 24% triple negative (TN) DCIS. 18% of the HR+ patients and 38% of the HR- patients were PD-L1+. 25% of the HR-/HER2+ patients, and 75% of the TN patients were PD-L1+. 6% of the HR+ patients developed recurrent disease, 2 with DCIS and 1 with invasive ductal carcinoma. 20% of the HR- patients had recurrent disease, all of whom were HER2+. Of the HR- patients that recurred, 2 recurred with metastatic disease, 1 with ipsilateral invasive ductal carcinoma, and 1 with DCIS. All 7 patients that recurred had original DCIS pathology showing a high nuclear grade. Our future results at the time of the meeting will expand on this cohort. Conclusions: This retrospective analysis showed that HR- DCIS conferred higher rates of local and distant recurrence. Therefore, there is a need for treatments to reduce the recurrence rates of HR- DCIS. There are ongoing clinical trials for the high risk, HR-/HER2+ DCIS subtype. TN DCIS is also an aggressive phenotype. Given the high rate of PD-L1 positivity we detected in TN DCIS, immune-based therapy may be useful in the adjuvant setting to reduce the risk of recurrence in this cohort of patients.

Immune response and stromal changes in ductal carcinoma in situ of the breast are subtype dependent.

Ductal carcinoma in situ (DCIS) associated stromal changes and influx of immune cells might be mediators of progression to invasive breast cancer. We studied the interaction between DCIS-associated stromal changes, and immune cell distribution and composition in a well-characterized patient cohort. We included 472 patients with DCIS. The presence of stromal changes, signs of regression, and DCIS-associated immune cell position were determined on hematoxylin and eosin-stained slides. Immune cell composition was characterized by immunohistochemistry (CD4, CD8, CD20, CD68, and FOXP3). The number of intraductal immune cells was quantified per mm2. The interaction between stromal changes, signs of DCIS regression, immune cell composition and location was explored. Stromal changes and signs of DCIS regression were identified in 30 and 7% of the patients, respectively. Intraductal immune cells mainly comprised CD68+ macrophages and CD8+ T cells. Patients with stromal changes had significantly less influx of immune cells within the duct. DCIS regression was associated with an increased number of intraductal FOXP3+ T cells. The highest number of intraductal CD8+ T cells was seen in the ER+ HER2+ subtype. We suggest that DCIS-associated stromal changes prevent the interaction between immune cells and DCIS cells. However, in case of DCIS regression, we surmise a direct interaction between DCIS cells and immune cells, in particular FOXP3+ cells. Furthermore, the increased number of intraductal CD8+ T cells in the ER+ HER2+ DCIS subtype suggests a subtype-specific immune response, which is likely to play a role in the distinct biological behavior of different DCIS subtypes.

In Situ DESI-MSI Lipidomic Profiles of Breast Cancer Molecular Subtypes and Precursor Lesions.

Clinically meaningful molecular subtypes for classification of breast cancers have been established, however, initiation and progression of these subtypes remain poorly understood. The recent development of desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) facilitates the convergence of analytical chemistry and traditional pathology, allowing chemical profiling with minimal tissue pretreatment in frozen samples. Here, we characterized the chemical composition of molecular subtypes of breast cancer with DESI-MSI. Regions of interest were identified, including invasive breast cancer (IBC), ductal carcinoma in situ (DCIS), and adjacent benign tissue (ABT), and metabolomic profiles at 200 μm elaborated using Biomap software and the Lasso method. Top ions identified in IBC regions included polyunsaturated fatty acids, deprotonated glycerophospholipids, and sphingolipids. Highly saturated lipids, as well as antioxidant molecules [taurine (m/z 124.0068), uric acid (m/z 167.0210), ascorbic acid (m/z 175.0241), and glutathione (m/z 306.0765)], were able to distinguish IBC from ABT. Moreover, luminal B and triple-negative subtypes showed more complex lipid profiles compared with luminal A and HER2 subtypes. DCIS and IBC were distinguished on the basis of cell signaling and apoptosis-related ions [fatty acids (341.2100 and 382.3736 m/z) and glycerophospholipids (PE (P-16:0/22:6, m/z 746.5099, and PS (38:3), m/z 812.5440)]. In summary, DESI-MSI identified distinct lipid composition between DCIS and IBC and across molecular subtypes of breast cancer, with potential implications for breast cancer pathogenesis. SIGNIFICANCE: These findings present the first in situ metabolomic findings of the four molecular subtypes of breast cancer, DCIS, and normal tissue, and add to the understanding of their pathogenesis.

Abstract P4-05-05: A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions

Abstract Background: The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but has not translated into increased survival rates. There are currently no reliable biomarkers to stratify DCIS into those which would not develop into an aggressive invasive cancer from those that do, and avoid over-treatment. The interaction with the DCIS microenvironment has likely been under-appreciated or not studied in the adequate clinical context. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Here, we present an approach combining laser capture microdissection (LCM) with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Methods: Targeted DNA sequencing (whole exome and cancer gene panel) was performed on limited dilutions (200, 50, 10,3 ng) of a test DNA from archived breast tissue, or validation DNA from 10 LCM regions from 3 DCIS specimens. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), or alternate single-strand (SS) or blunt-end (BE) adapter ligations. The expression profiling was performed using SMART-3SEQ protocol on 15 LCM regions from 8 DCIS specimen. Results: The use of an FFPE-optimized hybrid-capture AT protocol resulted in poor coverage performance (7% of targeted base pairs covered at ≥20x) from test DNA inputs lower than 50 ng and suggested the ligation of the sequencing adapter as the initial bottleneck. Alternatively steps using SS or BE adapter ligation from 3 ng of test DNA (~500 cells) increased the library complexity, reducing the fraction of duplicate reads and increasing the fraction of targeted base pairs covered at 20x to 49% and 100% respectively. Artifactual cytosine to tyrosine substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (&amp;lt;5%). As a result, the use of molecular barcodes, ensemble variant calling and heuristic variant filtering, resulted in 98% precision and 96% recall in variant calling accuracy from 3ng input, when compared to 200 ng input from a fresh frozen mirrored specimen. Similarly, the presence of an ERBB2 amplification was validated in all tested conditions. The validation of the BE approach on 10 LCM regions from 3 DCIS showed decreasing levels of clonal relationships when comparing regions from adjacent sections, then from matching distinct regions, then from distinct specimen. The heterogeneity of TP53 loss of heterozygosity within a specimen was consistent with high-grade. Furthermore, gene expression profiling of 15 regions from 8 archived DCIS specimen differentiated expression profiles of stromal and epithelial regions, within and between specimen, highlighting sets of genes associated with DCIS subtype and the immune make-up of the micro-environment. Conclusion: The proposed approach - from coordinated specimen preparation to mutational and expression profiling - was therefore validated to support the spatial characterization of pre-cancerous lesions and the microenvironment in which they evolve. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome. Citation Format: Daniela Nachamanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Farnaz Hasteh, Kristen Jepsen, Laura J. Esserman, Alexander Borowsky, Olivier Harismendy. A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions [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 P4-05-05.

Abstract P1-16-04: How patient advocates and researchers work together in PRECISION* to identify low-risk ductal carcinoma in situ (DCIS) that may not need aggressive treatment

Abstract BACKGROUND: Ductal carcinoma in situ (DCIS) was virtually unknown before breast screening and now accounts for up to 25% of all ‘breast cancers’ detected. Over 50,000 women are diagnosed annually with DCIS in the US alone; most receive treatments intended for women with invasive breast cancer which may offer no benefit and are associated with physical and psychological complications, and financial burdens. Observational studies show a majority of DCIS never becomes invasive, even if left untreated. Lack of prospective data and concerns about “overtreatment” are now being addressed by PRECISION - an international consortium of clinicians, scientists and patient advocates - collaborating to understand the biological nature of DCIS and how it evolves. Our goal is to develop tests that distinguish between women with DCIS who will likely develop breast cancer and should be treated, and those who can safely avoid treatment. Patient advocates are fully integrated into PRECISION as well as 3 prospective randomized clinical trials - LORD (EU), LORIS (UK) and COMET (US) – aiming to produce evidence to show whether or not Active Surveillance has equivalent outcomes to surgery for low-risk DCIS. These trials are critical because PRECISION uses biospecimens from each trial, in addition to other DCIS cohorts around the world. PATIENT ADVOCATE AIMS: • Help foster teams that contribute to better outcomes for women with DCIS. • Assist in designing effective informational tools for providers, organizations, and the public. • Understand international differences in health services, medical culture, and patient expectations, and recommend improvements. • Measure the value of patient involvement in PRECISION research. • Raise awareness and communicate about ongoing research and (over)treatment of DCIS. METHODS: The PRECISION Patient Advocate Involvement Panel (PAIP) includes representatives from each country: NL, UK, and US. PAIP members provide encouragement that helps to shape ongoing projects into a cohesive team, while encouraging new synergistic projects that can potentially improve clinical practice. The PAIP actively participates in the Steering Group, all Work Packages (WPs), and a variety of internal and external communications. This includes making posters and presentations at international conferences, preparing for PRECISION reviews, creating website and newsletter content, giving interviews, co-authoring publications, and developing social media campaigns to raise awareness. RESULTS: The PAIP has: a) helped build collaborations between institutions; b) developed public-facing information about PRECISION and the 3 clinical trials; c) offered patient-oriented ideas and feedback to all WPs; d) helped resolve international contract barriers; e) helped identify additional DCIS cohorts; f) suggested ideas to improve recruitment for the clinical trials; g) given feedback on surveys and co-authored publications; h) built relationships with postdocs and PhD students; and i) engaged public and clinician audiences on DCIS dilemmas in each country. Additional ongoing PAIP work includes engaging in research to reduce pathology report DCIS grade discordance within and between countries (a critical issue for women diagnosed with DCIS). CONCLUSIONS: There is a need to better understand DCIS subtypes, stratify associated risks for invasive cancer, synergize accurate communication about DCIS, and improve pathology standards within and between countries. The PAIP will continue to work closely with researchers and communicate evidence that answers important questions related to risk factors for women who may or may not need treatment for DCIS. *PRECISION is sponsored by a Cancer Research UK Grand Challenge Award, which is also funded by the Dutch Cancer Society. Citation Format: Deborah Collyar, Donna Pinto, Hilary Stobart, Ellen Verschuur, Marja van Oirsouw, on behalf of the PRECISION Project. How patient advocates and researchers work together in PRECISION* to identify low-risk ductal carcinoma in situ (DCIS) that may not need aggressive treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-16-04.

Recurrence Outcomes After Nipple-Sparing Mastectomy and Immediate Breast Reconstruction in Patients with Pure Ductal Carcinoma In Situ.

Nipple-sparing mastectomy (NSM) has become increasingly prevalent for patients with ductal carcinoma in situ (DCIS) requiring mastectomy. However, few data regarding recurrence outcomes after NSM are available for this patient population. This study evaluated the locoregional recurrence (LRR) rate for patients with pure DCIS who underwent NSM followed by immediate breast reconstruction without adjuvant radiotherapy and investigated potential risk factors for LRR and/or nipple-areola complex recurrence (NR). A retrospective chart review was performed for 199 consecutive patients with pure DCIS who underwent NSM and immediate breast reconstruction between March 2003 and December 2015. Risk factors for LRR and NR were analyzed using univariate (Chi square test) and multivariate (Cox model) methods. The median follow-up duration after surgery was 97months (range, 39-186months). At 10years, the LRR rate was 4.5%, and the NR rate was 3%. The univariate analysis showed that high nuclear grade, negative receptor status, positive human epidermal growth factor receptor 2 (HER2) status, and negative hormone receptor/positive HER2 subtype were associated with increased risk for NR. The multivariate analysis demonstrated that negative progesterone receptor status was an independent risk factor for LRR. However, margin status and tumor-to-nipple distance (TND) were not associated with increased risk for either LRR or NR. The study findings suggest that NSM can be a feasible surgical option even for DCIS with a TND of 1cm or less if the retroareolar resection margin is negative for malignancy. Determining the molecular subtype of DCIS might be helpful in identifying patients at high risk for recurrence.

Molecular Subtypes of Ductal Carcinoma In Situ (DCIS) Identified by Expression of ER, PR, HER2, and Ki-67

Objective: The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 are used to classify invasive breast cancer into various subtypes. In the case of ductal carcinoma in situ (DCIS), however, the information relating to subtypes using these markers is still limited. Methods: The pathological specimens of 267 patients diagnosed with DCIS at Siriraj Hospital were analyzed. By using the expressions of ER, PR, HER2, and Ki-67, breast cancer patients were classified into the five molecular subtypes: luminal A, luminal B/HER2 negative, luminal B/HER2 positive, HER2 overexpression, and triple-negative. Based on the specific molecular subtypes, age, clinical presentation, tumor size, and tumor grade were analyzed separately using univariate analysis. Results: 135 (50.6%), 1 (0.4%), 58 (21.7%), 59 (22.1%), and 14 (5.2%) DCIS were luminal A, luminal B/HER2 negative, luminal B/HER2 positive, HER2 overexpression, and triple-negative, respectively. Patients with luminal A DCIS significantly presented with mass, compared to the other subtypes that displayed with mass and microcalcification in combination (p = 0.008). The luminal A subtype was also associated with tumors of a smaller size (less than 2 cm; p = 0.007) and a lower nuclear grade (p = 0.000) than the HER2 overexpression subtype. Conclusion: Although luminal A DCIS was the most common DCIS subtype of Thai women, HER2 overexpression DCIS was significantly correlated with poor clinicopathological features, including a large tumor size and a high nuclear grade, which are recognized prognostic factors for tumor recurrence.

Abstract 2704: Ductal carcinoma in situ of the breast: Immune cell subset composition according to subtype

Abstract Introduction: Ductal carcinoma in situ (DCIS) of the breast includes several subtypes with a divergent biological behavior. HER2+ DCIS is often detected at the in situ stage, while ER- HER2- DCIS is relatively rare. Besides, HER2+ DCIS has a more extensive growth pattern compared to other subtypes. Data regarding the composition of DCIS-associated immune cells and their potential role in DCIS progression is limited. We studied DCIS-associated immune response by characterizing immune cell subsets according to DCIS subtypes. Methods: We evaluated DCIS-associated tumor infiltrating lymphocyte (TIL) density and distribution based on H&amp;E stained sections of excision specimens from 473 patients with DCIS. These cases were subtyped based on ER, PR and HER2. Patients were categorized as TIL-high or TIL-low, whereby TIL-high was defined as high TILs density (&amp;gt;50% of the DCIS-associated stroma occupied by TILs) with a patchy or diffuse distribution. The DCIS-associated immune cells of TIL-high cases were immunostained on consecutive whole slides with CD4 (T-helper cells), CD8 (cytotoxic T-cells), CD20 (B-cells), CD68 (macrophages), FOXP3 (regulatory T-cells), PD-L1 (immune checkpoint ligand, clones SP142 and SP263). The percentage of CD4+, CD8+, CD20+ and CD68+ immune subsets was assessed relatively to one another, with a combined score of 100%. The percentage of FOXP3+ and PD-L1+ immune cells was determined as a proportion of all immune cells. PD-L1+ DCIS cells were scored using the histo-score (H-score). The immune cell composition according to DCIS subtypes was analyzed using a Kruskal Wallis or Mann-Whitney U test. Results: DCIS was subtyped as ER+PR+/-HER2- (n=225), ER+PR+/-HER2+ (n=80), ER-PR-HER2+ (n=85), triple negative (TN; n=22) or missing (n=31). In total, 131/473 patients (27.7%) were considered as TIL-high and the percentage of TIL-high cases was significantly associated with DCIS subtype (11.4% of ER+PR+/-HER2-, 38.8% of ER+PR+/-HER2+, 61.2% of ER-PR-HER2+ and 63.6% of the TN subtype, P&amp;lt;0.0001). There was no statistical difference in the immune cell composition according to DCIS subtypes. However, individual DCIS subtype comparison showed that the ER+PR+/-HER2+ subtype was associated with a significantly higher proportion of CD8+ T-cells compared to the TN subtype (P=0.047). The ER-PR-HER2+ subtype was associated with a higher proportion of CD4+ T cells compared to the TN subtype, though significance was not reached (P=0.061). PD-L1 expression by both clones was low (range: 0-10% of immune cells, H-score 0-54 of DCIS cells). However, the mean value of PD-L1 SP263 was higher compared to PD-L1 SP142, for both TILs and tumor cells (P&amp;lt;0.0001). Conclusion: High numbers of TILs are mainly observed in HER+ and TN DCIS subtypes. The ER+ HER2+ DCIS subtype attracts more CD8+ T-cells compared to the TN subtype. This suggests a more pronounced anti-tumor immunity in HER2+ DCIS, which could play a role in its biological behavior. Citation Format: Marie Colombe Agahozo, Mieke R. van Bockstal, Floris H. Groenendijk, Thierry P. van den Bosch, Pieter J. Westenend, Carolien H. van Deurzen. Ductal carcinoma in situ of the breast: Immune cell subset composition according to subtype [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 2704.

Biological Aggressiveness of Subclinical No-Mass Ductal Carcinoma In Situ (DCIS) Can Be Reflected by the Expression Profiles of Epithelial-Mesenchymal Transition Triggers.

Epithelial-mesenchymal transitions (EMTs) have been recently implicated in the process of cancer progression. The aim of this study was to assess how the preoperative expression patterns of EMT biomarkers correlate with the risk of postoperative invasion in ductal carcinoma in situ (DCIS) found on stereotactic breast biopsies. N-cadherin, Snail1, and secreted protein acidic and rich in cysteine (SPARC) immunoreactivity was observed in 8%, 62%, and 38% of tumors, respectively. Snail1 and SPARC expressions were significantly related to N-cadherin expression and to each other. The postoperative upgrading rate was associated with a positive preoperative expression of all biomarkers. Significance of Snail1 and SPARC persisted in multivariate analysis, but the impact of SPARC on invasion was more significant. When these two EMT triggers were considered together, the risk of invasion did not significantly differ between the subtypes of DCIS with single positive expression (SPARC−/Snail1+ vs. SPARC+/Snail1−). However, it was significantly lower in single-positive DCIS when compared to lesions of a double-positive profile (SPARC+/Snail1+). Moreover, there were no cases in the double-negative DCIS (SPARC−/Snail1−), with foci of infiltrating cancer found postoperatively in residual postbiopsy lesions. In contrast, DCIS with a combined high SPARC and Snail1 expression (intermediate or strong) had an invasive component in 66–100% of tumors.

Triple-negative and HER2 positive ductal carcinoma in situ of the breast: characteristics, behavior, and biomarker profile.

We compared the characteristics, clinical behavior, and biomarker profile between HER2 positive (HER2+) and triple-negative (TN) ductal carcinoma in situ (DCIS) which are considered more aggressive than other DCIS subtypes. In addition, we explored the impact of these features on its potential of progression to invasive breast carcinomas. Cases of DCIS diagnosed at the Department of Pathology, Singapore General Hospital from 1994 to 2010 were identified. TN and HER2+ DCIS cases formed the study cohort. Immunohistochemistry (IHC) was performed for ER, PR, HER2, CK14, EGFR, and p53. Comparisons of clinicopathological features, IHC results, and clinical outcomes were performed between the two groups. We evaluated 145 HER2+ and 85 TN DCIS cases. HER2 positive DCIS had significantly higher nuclear grade (p < 0.001) and more frequent necrosis (p < 0.001) than TN DCIS. HER2 positive DCIS also harbored significantly higher rates of nuclear p53 immunoreactivity (p = 0.002) than TN DCIS. Younger patients (age < 40) with HER2+ and TN DCIS demonstrated statistically significant worse invasive DFS than older women (p < 0.001). Multivariate cox regression analysis (HR 15.08, 95% CI 12.79-81.45, p = 0.002) also confirmed these findings. In addition, younger patients (age < 40) with HER2+ DCIS experienced significantly poorer prognosis when p53 was also positive (p = 0.033). HER2+ DCIS had more aggressive pathological characteristics compared to TN DCIS; accumulation of mutant p53 could possibly be contributory. Age was an independent predictor of aggressive biological behavior of HER2+ and TN DCIS. We demonstrated that younger patients with p53 positive HER2+ DCIS had significantly adverse clinical outcome.