The Immune Microenvironment of Breast Ductal Carcinoma in Situ

Elizabeth Thompson; Janis M Taube; Hillary Elwood; Rajni Sharma; Alan Meeker; Hind Nassar Warzecha; Pedram Argani; Ashley Cimino-Mathews; Leisha A Emens

Disclosures

Mod Pathol. 2016;29(3):249-258. 

In This Article

Results

Clinicopathologic Features of DCIS Cases

The clinicopathologic features of 27 evaluable patients with DCIS are detailed in Table 1. The median patient age was 38 years (mean 41 years, range 18–74), with 52% white, 26% black, 7% Asian, and 4% Hispanic patients. The DCIS phenotype of 26 evaluable patients included 62% luminal A, 15% luminal B, 12% HER-2+, and 12% triple negative, for a total of 77% ER+ and 23% ER cases. The ER/PR/HER-2 status of one case could not be determined, and the case was not included in analyses that subdivided by DCIS phenotype. Most cases were nuclear grade 2 (52%) or grade 3 (44%). The average tumor size was 2.4 cm; 37% were multifocal. Twenty-four cases were pure DCIS (pathologic stage pTis), without any associated invasive carcinoma. Three cases had associated infiltrating ductal carcinoma, which were also present on the tissue microarray cores. Two of these cases were triple-negative DCIS and one was luminal A DCIS. The mean and median follow-up time were 82 months and 80 months (approximately 7 years), respectively. Two (7%) DCIS patients had an ipsilateral recurrence, both of which were nuclear grade 3; one patient with triple-negative DCIS and concurrent infiltrating ductal carcinoma developed recurrent infiltrating ductal carcinoma in the ipsilateral chest wall 1 year after initial diagnosis, and one patient with HER-2+ DCIS experienced recurrent ipsilateral DCIS 3 years after initial diagnosis. No patients received neoadjuvant therapy. All patients received adjuvant therapy for pure DCIS or infiltrating carcinoma as per the standard of care.

Quantification of Tumor Infiltrating Lymphocytes in DCIS

We first evaluated tumor infiltrating lymphocytes associated with DCIS by histopathologic density scoring on a scale from 0 to 3. All cases had tumor infiltrating lymphocytes, with 78% showing either moderate (score 2) or diffuse (score 3) tumor infiltrating lymphocyte density (Table 2). Only two cases (7%) contained diffuse tumor infiltrating lymphocytes. ER+ DCIS and DCIS in older patients tended to have lower tumor infiltrating lymphocyte density scores than ER DCIS or DCIS in younger patients (P=0.28 and 0.32, respectively). There was only one case of grade 1 DCIS, precluding group wide comparison, and there was no discernible difference in tumor lymphocytic infiltration between the grade 2 DCIS and grade 3 DCIS.

We quantified subsets of tumor infiltrating lymphocytes using CD3, CD4, CD8, CD20, and FoxP3 staining. CD3+ T cells predominated across all DCIS subtypes at all ages, with slightly more CD4+ T cells than CD8+ T cells on average. CD20+ B cells were the next most common tumor infiltrating lymphocytes, followed by FoxP3+ Treg (Table 2). On average, ER DCIS contained higher numbers of all tumor infiltrating lymphocyte subsets than ER+ DCIS, and ER+ DCIS was more likely to have to have a high CD8/FoxP3 ratio (≥4) than ER DCIS. DCIS in young women also contained higher numbers of all tumor infiltrating lymphocyte subsets relative to older women; however, these differences were not statistically significant. There was no difference in tumor infiltrating lymphocyte subset distribution between the nuclear grade 2 DCIS and grade 3 DCIS.

Although the case numbers are small and results should be interpreted with caution, differences in tumor infiltrating lymphocytes were also seen in DCIS with concurrent infiltrating ductal carcinoma (n=3), and in DCIS that later recurred (n=2). The two cases of DCIS with diffuse tumor infiltrating lymphocytes (score 3) were DCIS with concurrent infiltrating ductal carcinoma (P=0.009). DCIS with concurrent infiltrating ductal carcinoma showed higher levels of infiltrating CD8+ T cells and CD20+ B cells than cases with only in situ disease, but this was not statistically significant. DCIS that recurred had higher numbers of all tumor infiltrating lymphocyte subsets relative to DCIS that did not recur. DCIS that recurred also had the greatest number of CD8+ T cells of any subset of DCIS cases examined. However, this DCIS phenotype also showed a lower CD8/FoxP3 ratio, indicating a commensurate increase in regulatory T cells in these two cases.

PD-L1 Expression in DCIS and Tumor Infiltrating Lymphocytes

We next examined the cell surface expression of PD-L1 by DCIS tumor cells and associated tumor infiltrating lymphocytes. There was no expression of PD-L1 on DCIS tumor cells in any case, although non-specific PD-L1 staining was seen within the necrotic debris of central comedonecrosis. In DCIS with concurrent infiltrating ductal carcinoma (n=3), the PD-L1 status was concordant (negative) between in situ and invasive components, as we have previously reported.[24] Although DCIS tumor cells were PD-L1, 81% of the DCIS cases showed PD-L1 expression by tumor infiltrating lymphocytes (Figure 1a and b and Table 2). All DCIS cases with PD-L1 tumor infiltrating lymphocytes were of the ER+ luminal A phenotype (P=0.12) (Figure 1c and d), while all ER cases had PD-L1+ tumor infiltrating lymphocytes (Table 3). PD-L1 expression was also scored as low (≤50% of tumor infiltrating lymphocytes) or high (>50% of tumor infiltrating lymphocytes). All luminal A, luminal B, and HER-2+ DCIS with PD-L1+ tumor infiltrating lymphocytes showed low PD-L1 expression on tumor infiltrating lymphocytes (Table 3). In contrast, 100% of triple-negative DCIS had high expression of PD-L1 on tumor infiltrating lymphocytes (P=0.0008). In addition, all cases with high expression of PD-L1 on tumor infiltrating lymphocytes were nuclear grade 3 DCIS compared with nuclear grade 2 (P=0.07) (Table 3). The average age of DCIS patients with PD-L1+ and PD-L1 tumor infiltrating lymphocytes was 38 and 54 years (P=0.01), respectively, with 80% of DCIS containing PD-L1 tumor infiltrating lymphocytes occurred in older patients (P=0.02). All high-risk DCIS (concurrent infiltrating ductal carcinoma and recurrent DCIS) had PD-L1+ tumor infiltrating lymphocytes, though this was not statistically significant.

Figure 1.

Programmed death ligand 1 (PD-L1) staining patterns differ in triple-negative and estrogen receptor (ER)+ ductal carcinoma in situ (DCIS). All cases of triple-negative DCIS (a, hematoxylin and eosin, H&E) contain tumor infiltrating lymphocytes with high PD-L1+ expression (>50% cells) (b). ER+ DCIS (c, H&E) shows low (<50% cells) or absent PD-L1 tumor infiltrating lymphocyte staining (d).

DCIS with low tumor infiltrating lymphocyte density scores (score 1) was more likely to be PD-L1 than PD-L1+ (P=0.004). In contrast, cases of DCIS with moderate or diffuse tumor infiltrating lymphocyte density scores (scores 2–3) were more likely to be PD-L1+ than PD-L1 (Figure 2 and Table 2). DCIS with PD-L1+ tumor infiltrating lymphocytes had higher numbers of tumor infiltrating lymphocytes across all subsets examined when compared to tumors with PD-L1 tumor infiltrating lymphocytes, though this was not statistically significant (Figure 2 and Table 2). Both PD-L1+ and PD-L1 DCIS had similar CD8/FoxP3 ratios.

Figure 2.

Immunologic features of the ductal carcinoma in situ (DCIS) tumor microenvironment. Most DCIS cases (81%) (panel 1; a, hematoxylin and eosin, H&E) display programmed death ligand 1 (PD-L1)+ tumor infiltrating lymphocytes (b). PD-L1+ tumor infiltrating lymphocytes are associated with greater numbers of all tumor infiltrating lymphocyte subsets including CD4 helper T cells (c), CD8+ cytotoxic T cells (d), CD20+ B cells (e), and FoxP3+ Tregs (f) relative to DCIS with PD-L1 tumor infiltrating lymphocytes (panel 2). Importantly, no DCIS carcinoma cell displayed cell surface PD-L1 staining.

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