Stromal lymphocytes are associated with upgrade of B3 breast lesions.

Various histopathological, clinical and imaging parameters have been evaluated to identify a subset of women diagnosed with lesions with uncertain malignant potential (B3 or BIRADS 3/4A lesions) who could safely be observed rather than being treated with surgical excision, with little impact on clinical practice. The primary reason for surgery is to rule out an upgrade to either ductal carcinoma in situ or invasive breast cancer, which occurs in up to 30% of patients. We hypothesised that the stromal immune microenvironment could indicate the presence of carcinoma associated with a ductal B3 lesion and that this could be detected in biopsies by counting lymphocytes as a predictive biomarker for upgrade. A higher number of lymphocytes in the surrounding specialised stroma was observed in upgraded ductal and papillary B3 lesions than non-upgraded (p < 0.01, negative binomial model, n = 307). We developed a model using lymphocytes combined with age and the type of lesion, which was predictive of upgrade with an area under the curve of 0.82 [95% confidence interval 0.77-0.87]. The model can identify some patients at risk of upgrade with high sensitivity, but with limited specificity. Assessing the tumour microenvironment including stromal lymphocytes may contribute to reducing unnecessary surgeries in the clinic, but additional predictive features are needed.

Evaluation of tumor infiltrating lymphocytes as a prognostic biomarker in patients with ductal carcinoma in situ of the breast.

PURPOSE: To assess the association between tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ (DCIS) samples and disease recurrence. METHODS: This retrospective cohort study included women aged 18 years and older who underwent treatment between January 2007 and December 2020. Male patients, individuals diagnosed with invasive or microinvasive disease based on anatomopathological examination of surgical specimens, and those with a personal history of any other cancers were excluded. Additionally, the presence of "touching TILs" (lymphocytes in direct contact with tumor cells) and periductal desmoplasia were evaluated as complementary methods to represent the immunological microenvironment. The primary outcome was relapse-free survival based on TIL quantification adjusted for potential confounders. Pathologists assessed TILs in the sample with the highest tumor representation and quantified them as a percentage. Survival was evaluated using Kaplan‒Meier curves, log-rank tests, and Cox regression models. RESULTS: A total of 191 patients met the eligibility criteria. The mean follow-up duration was 77.2 months, with a recurrence rate of 9.2%. Patients with TILs ≥ 17% had a greater risk of recurrence (HR 2.97, 95% CI 1.17-7.51; p = 0.02). Additionally, focal necrosis (HR 6.4, 95% CI 1.39-34.71; p = 0.018) or comedonecrosis (HR 4.53, 95% CI 1.34-15.28; p = 0.015) were associated with increased recurrence risk. According to the multivariate model, comedonecrosis and TILs ≥ 17% were significantly associated with recurrence (p = 0.034 and p = 0.035, respectively). Regarding the evaluations of "touching TILs" and periductal desmoplasia, no statistical significance was found when assessing their association with disease recurrence. CONCLUSION: In our cohort, a high percentage of TILs (≥ 17%) and the presence of comedonecrosis were independently associated with DCIS recurrence.

Single-cell transcriptome analysis reveals immune microenvironment changes and insights into the transition from DCIS to IDC with associated prognostic genes.

BACKGROUND: Ductal carcinoma in situ (DCIS) of the breast is an early stage of breast cancer, and preventing its progression to invasive ductal carcinoma (IDC) is crucial for the early detection and treatment of breast cancer. Although single-cell transcriptome analysis technology has been widely used in breast cancer research, the biological mechanisms underlying the transition from DCIS to IDC remain poorly understood. RESULTS: We identified eight cell types through cell annotation, finding significant differences in T cell proportions between DCIS and IDC. Using this as a basis, we performed pseudotime analysis on T cell subpopulations, revealing that differentially expressed genes primarily regulate immune cell migration and modulation. By intersecting WGCNA results of T cells highly correlated with the subtypes and the differentially expressed genes, we identified six key genes: FGFBP2, GNLY, KLRD1, TYROBP, PRF1, and NKG7. Excluding PRF1, the other five genes were significantly associated with overall survival in breast cancer, highlighting their potential as prognostic biomarkers. CONCLUSIONS: We identified immune cells that may play a role in the progression from DCIS to IDC and uncovered five key genes that can serve as prognostic markers for breast cancer. These findings provide insights into the mechanisms underlying the transition from DCIS to IDC, offering valuable perspectives for future research. Additionally, our results contribute to a better understanding of the biological processes involved in breast cancer progression.

Immune microenvironment in ductal carcinoma in situ: a comparison with invasive carcinoma of the breast.

BACKGROUND: The immune microenvironment in ductal carcinoma in situ (DCIS) and its significance are not well established. This study was conducted to evaluate the immune microenvironment of DCIS including the composition of tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells and to compare it with that of invasive breast cancer. MATERIALS AND METHODS: A total of 671 cases including three different disease groups of pure DCIS, DCIS with microinvasion (DCIS-M), and invasive carcinoma were included in this study. CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ immune cells were detected with immunohistochemistry using tissue microarrays and were analyzed in relation to clinicopathologic characteristics and different disease groups. RESULTS: In pure DCIS, high infiltrations of CD4+, CD8+, and FOXP3+ T cells and the presence of PD-L1+ immune cells were associated with high nuclear grade, comedo-type necrosis, hormone receptor (HR) negativity, and high Ki-67 proliferation index. All immune cell infiltrations were higher in invasive carcinoma than in pure DCIS regardless of the HR status. While CD4+ T cells were more abundant than CD8+ T cells in pure DCIS, CD8+ T cells were dominant in invasive carcinoma, especially in HR-negative tumors. Within individual cases of invasive carcinoma with DCIS component, all immune cell subset infiltration was higher in the invasive component than in the DCIS component; however, CD4+ TIL infiltration did not differ between the two components in HR-negative tumors. Comparing pure DCIS, DCIS-M, and DCIS associated with invasive carcinoma (DCIS-INV), CD4+ TIL infiltration revealed a gradual increase from pure DCIS to DCIS-M and DCIS-INV in the HR-negative group, whereas FOXP3+ TIL infiltration was significantly increased in DCIS-INV than in pure DCIS in the HR-positive group. The high infiltration of FOXP3+ TIL and the presence of PD-L1+ immune cells were associated with tumor recurrence in patients with pure DCIS. CONCLUSIONS: Our study showed that the immune microenvironment differs significantly not only between DCIS and invasive carcinoma but also between pure DCIS, DCIS-M, and DCIS-INV depending on the HR status.

The prognostic significance of immune microenvironment in breast ductal carcinoma in situ.

BACKGROUND: The role of different subtypes of tumour infiltrating lymphocytes (TILs) in breast ductal carcinoma in situ (DCIS) is still poorly defined. This study aimed to assess the prognostic significance of B and T lymphocytes and immune checkpoint proteins expression in DCIS. METHODS: A well characterised DCIS cohort (n = 700) with long-term follow-up comprising pure DCIS (n = 508) and DCIS mixed with invasive carcinoma (IBC; n = 192) were stained immunohistochemically for CD20, CD3, CD4, CD8, FOXP3, PD1 and PDL1. Copy number variation and TP53 mutation status were assessed in a subset of cases (n = 58). RESULTS: CD3+ lymphocytes were the predominant cell subtype in the pure DCIS cohort, while FOXP3 showed the lowest levels. PDL1 expression was mainly seen in the stromal TILs. Higher abundance of TILs subtypes was associated with higher tumour grade, hormone receptor negativity and HER2 positivity. Mutant TP53 variants were associated with higher levels of stromal CD3+, CD4+ and FOXP3+ cells. DCIS coexisting with invasive carcinoma harboured denser stromal infiltrates of all immune cells and checkpoint proteins apart from CD4+ cells. Stromal PD1 was the most differentially expressed protein between DCIS and invasive carcinoma (Z = 5.8, p < 0.0001). Dense TILs, stromal FOXP3 and PDL1 were poor prognostic factors for DCIS recurrence, while dense TILs were independently associated with poor outcome for all recurrences (HR = 7.0; p = 0.024), and invasive recurrence (HR = 2.1; p = 0.029). CONCLUSIONS: Immunosuppressive proteins are potential markers for high risk DCIS and disease progression. Different stromal and intratumoural lymphocyte composition between pure DCIS, DCIS associated with IBC and invasive carcinoma play a potential role in their prognostic significance and related to the underlying genomic instability. Assessment of overall TILs provides a promising tool for evaluation of the DCIS immune microenvironment.

Ductal carcinoma in situ of the breast: immune cell composition according to subtype.

Ductal carcinoma in situ of the breast includes several subtypes with a divergent biological behavior. Data regarding the composition of ductal carcinoma in situ-associated immune cells and their potential role in progression is limited. We studied ductal carcinoma in situ-associated immune response by characterizing immune cell subsets according to ductal carcinoma in situ subtypes. Ductal carcinoma in situ-associated tumor infiltrating lymphocyte (TIL) density was evaluated based on hematoxylin and eosin (H&E)-stained sections from 473 patients. Cases were subtyped based on ER, PR, and HER2. Patients were categorized as TIL-high or low. Ductal carcinoma in situ-associated immune cells of TIL-high cases were immunostained on whole slides with CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 and SP263). In total, 131/473 patients (28.0%) were considered as TIL-high. The percentage of TIL-high cases was significantly higher in HER2+ and triple-negative ductal carcinoma in situ (P < 0.0001). Overall, no statistical difference in immune cell composition according to subtypes was found. However, individual subtype comparison showed that ER+ HER2+ cases had a significantly higher proportion of CD8+ T cells compared with triple-negative cases (P = 0.047). In TIL-high cases, PD-L1-SP142 expression on tumor cells was associated with subtype (P = 0.037); the lowest number of positive cases was observed in the HER2+ subtype (independent of ER). However, in TIL-high ductal carcinoma in situ, PD-L1 expression by both clones was limited. In conclusion, high numbers of TILs are predominantly observed in HER+ and triple negative ductal carcinoma in situ. The ER+ HER2+ subtype seems to attract a higher proportion of CD8+ T cells compared with the triple negative subtype. Among TIL-high cases, the HER2+ subgroup had the lowest PD-L1-SP142 expression on tumor cells. This suggests a more pronounced antitumor immunity in HER2+ ductal carcinoma in situ, which could play a role in its biological behavior.

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.

Higher densities of tumour-infiltrating lymphocytes and CD4+ T cells predict recurrence and progression of ductal carcinoma in situ of the breast.

AIMS: Host immunity influences cancer progression and therapeutic response. We investigated the potential of tumour-infiltrating lymphocytes (TILs) around ductal carcinoma in situ (DCIS) in predicting recurrence and progression. METHODS AND RESULTS: CD4, CD8, programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression in DCIS from 198 patients was determined by immunohistochemistry. We correlated disease-free survival (DFS), clinicopathological parameters and biomarker expression with TIL density and CD4/CD8 ratio. High TIL density was associated with high nuclear grade (P < 0.001), DCIS PD-L1 expression (P = 0.008), TIL PD-L1 expression (P < 0.001), oestrogen (ER) negativity (P < 0.001), progesterone (PR) negativity (P < 0.001), human epidermal growth factor receptor 2 (HER2) positivity (P = 0.002) and triple negativity (P = 0.001). TIL PD-L1 expression was associated with triple-negative DCIS (P = 0.028). TIL density was associated with molecular subtypes (P < 0.001). High CD4+ T cell density was associated with high nuclear grade (P = 0.001), microinvasion (P = 0.037), ER negativity (P < 0.001), PR negativity (P = 0.001), HER2 positivity (P = 0.004), triple negativity (P = 0.023) and PD-L1 expression in TILs (P < 0.011). High CD4/CD8 ratio was associated with PD-L1 expression in DCIS (P = 0.035) and TILs (P < 0.001). DCIS with higher TIL density disclosed worse DFS (P = 0.012) and was affirmed with multivariate analysis [95% confidence interval (CI) = 1.109-2.554, hazard ratio (HR) = 1.683, P = 0.014]. Poorer DFS for ipsilateral invasive recurrence was found for DCIS with higher CD4+ T cell density (P = 0.006) or CD4/CD8 ratio (P = 0.02), confirmed by multivariate analysis for the former (95% CI = 1.369-10.196, HR = 3.736, P = 0.01) and latter (95% CI = 1.311-7.935, HR = 3.225, P = 0.011). CONCLUSION: DCIS with higher TIL density was associated with poorer prognostic parameters and predicted recurrence, while both CD4+ T cell density and CD4/CD8 ratio were associated with both recurrence and ipsilateral invasive recurrence.

Tumour-infiltrating lymphocytes in ductal carcinoma in situ (DCIS)-assessment with three different methodologies and correlation with Oncotype DX DCIS Score.

AIMS: Tumour-infiltrating lymphocytes (TILs) are prognostic in invasive breast cancer; however, their prognostic significance in ductal carcinoma in situ (DCIS) has not been established. The Oncotype DX (ODX) Breast DCIS Score test is a genomic assay used to predict the local recurrence risk. The aims of this study were to quantify TILs in DCIS by the use of three methodologies, and correlate them with the ODX DCIS Score. METHODS AND RESULTS: We studied 97 DCIS cases, all with an ODX DCIS Score. Cases with a low ODX DCIS Score were considered as one group, and those with an intermediate/high ODX Score were considered together. TILs were quantified on haematoxylin and eosin-stained slides. The methodologies used to quantify TILS included assessment of stromal TILs, assessment of touching TILs, and assessment of circumferential TILS. In cases with >5% stromal TILS, the percentage of stromal TILS was considered to be high. In cases with a mean number of more than five touching TILs per DCIS duct, TILs were considered to be present. The ODX DCIS Score was intermediate/high in 27 (28%) cases and low in 70 (72%) cases. There were >5% stromal TILs in 33 (34%) cases, and more than five touching TILs per DCIS duct in 15 (15%) cases; circumferential TILs were present in nine (9%) cases. In univariate analysis, a low ODX DCIS Score showed significant associations with absent touching TILS (P = 0.027), stromal TILs < 5% (P = 0.031), and absent circumferential TILs (P = 0.002). In logistic regression analysis adjusted for necrosis and nuclear grade, touching TILs and circumferential TILs showed significant associations with the ODX DCIS Score, whereas stromal TILs did not. CONCLUSIONS: Our results suggest that both the presence of TILs and the spatial arrangement of TILs or close proximity of TILs to DCIS, and TILs touching or encircling DCIS, may be predictive of recurrence.

Prognostic role of immune infiltrates in breast ductal carcinoma in situ.

PURPOSE: Ductal carcinoma in situ (DCIS) of the breast is often regarded as a non-obligate precursor to invasive breast carcinoma but current diagnostic tools are unable to accurately predict the invasive potential of DCIS. Infiltration of immune cells into the tumour and its microenvironment is often an early event at the site of tumourigenesis. These immune infiltrates may be potential predictive and/or prognostic biomarkers for DCIS. This review aims to discuss recent findings pertaining to the potential prognostic significance of immune infiltrates as well as their evaluation in DCIS. METHODS: A literature search on PubMed was conducted up to 28th January 2019. Search terms used were "DCIS", "ductal carcinoma in situ", "immune", "immunology", "TIL", "TIL assessment", and "tumour-infiltrating lymphocyte". Search filters for "Most Recent" and "English" were applied. Information from published papers related to the research topic were synthesised and summarised for this review. RESULTS: Studies have revealed that immune infiltrates play a role in the biology and microenvironment of DCIS, as well as treatment response. There is currently no consensus on the evaluation of TILs in DCIS for clinical application. CONCLUSIONS: This review highlights the recent findings on the potential influence and prognostic value of immunological processes on DCIS progression, as well as the evaluation of TILs in DCIS. Further characterisation of the immune milieu of DCIS is recommended to better understand the immune response in DCIS progression and recurrence.

Tumor-Infiltrating Lymphocytes in a Contemporary Cohort of Women with Ductal Carcinoma In Situ (DCIS).

BACKGROUND: Growing evidence suggests that the tumor immune microenvironment influences breast cancer development and prognosis. Density of tumor-infiltrating lymphocytes (TILs) within invasive breast cancer is correlated with response to therapy, especially in triple-negative disease. The clinical relevance and outcomes of TILs within ductal carcinoma in situ (DCIS) are less understood. METHODS: Our institutional database of 668 patients with pure DCIS from 2010 to 2018 was queried. TILs were evaluated by International TILs Working Group guidelines. Percentage of TILs was assessed from the densest focus (hotspot) in one high-power field of stroma touching the basement membrane. Statistical methods included cluster analyses (to define sparse versus dense TILs), logistic, and Cox regression models. RESULTS: Sixty-nine patients with DCIS and TILs were evaluated, of whom 54 (78%) were treated by breast-conserving surgery. Thirteen (19%) patients had ipsilateral recurrence. Each recurrence (n = 13) was matched to four controls (n = 56) based on date of surgery. Median follow-up was 6.7 years. TILs were defined as sparse (< 45%) or dense (≥ 45%). Dense TILs were associated with younger age (p = 0.045), larger tumor size (p < 0.001), high nuclear grade (p = 0.010), comedo histology (p = 0.033), necrosis (p = 0.027), estrogen receptor (ER) negativity (p = 0.037), and ipsilateral recurrence (p = 0.001). Nine patients with dense TILs had mean time to recurrence of 73.5 months compared with four patients with sparse TILs with mean time to recurrence of 97.9 months (p = 0.003). CONCLUSIONS: Dense TILs were significantly associated with age, tumor size, nuclear grade, comedo histology, necrosis, and ER status and was a significant predictor of recurrence in patients with pure DCIS.

Tumor-infiltrating lymphocytes and ductal carcinoma in situ of the breast: friends or foes?

In the past three decades, the detection rate of ductal carcinoma in situ of the breast has dramatically increased due to breast screening programs. As a consequence, about 20% of all breast cancer cases are detected in this early in situ stage. Some ductal carcinoma in situ cases will progress to invasive breast cancer, while other cases are likely to have an indolent biological behavior. The presence of tumor-infiltrating lymphocytes is seen as a promising prognostic and predictive marker in invasive breast cancer, mainly in HER2-positive and triple-negative subtypes. Here, we summarize the current understanding regarding immune infiltrates in invasive breast cancer and highlight recent observations regarding the presence and potential clinical significance of such immune infiltrates in patients with ductal carcinoma in situ. The presence of tumor-infiltrating lymphocytes, their numbers, composition, and potential relationship with genomic status will be discussed. Finally, we propose that a combination of genetic and immune markers may better stratify ductal carcinoma in situ subtypes with respect to tumor evolution.

Prognostic significance of tumor-infiltrating lymphocytes in ductal carcinoma in situ of the breast.

Tumor-infiltrating lymphocytes (TILs) provide prognostic value in invasive breast cancer and guidelines for their assessment have been published. This study aims to evaluate: (a) methods of TILs assessment, and (b) their prognostic significance in breast ductal carcinoma in situ (DCIS). Hematoxylin and eosin sections from two clinically annotated DCIS cohorts; a training set (n = 150 pure DCIS) and a validation set (n = 666 comprising 534 pure DCIS and 132 cases wherein DCIS and invasive breast carcinoma were co-existent) were assessed. Seven different scoring methods were applied to the training set to identify the most optimal reproducible method associated with strongest prognostic value. Among different methods, TILs touching ducts' basement membrane or away from it by one lymphocyte cell thickness provided the strongest significant association with outcome and highest concordance rate [inter-cluster correlation coefficient = 0.95]. Assessment of periductal TILs at increasing distances from DCIS (0.2 , 0.5 , and 1 mm) as well as percent of stromal TILs were practically challenging and showed lower concordance rates than touching TILs. TILs hotspots and lymphoid follicles did not show prognostic significance. Within the pure DCIS validation set, dense TILs were associated with younger age, symptomatic presentation, larger size, higher nuclear grade, comedo necrosis and estrogen receptor negativity as well as shorter recurrence-free interval (p = 0.002). In multivariate survival analysis, dense TILs were independent predictor of shorter recurrence-free interval (p = 0.002) in patients treated with breast conservation. DCIS associated with invasive carcinoma showed denser TILs than pure DCIS (p = 9.0 × 10-13). Dense TILs is an independent prognostic variable in DCIS. Touching TILs provides a reproducible method for their assessment that can potentially be used to guide management.

Analysis of tumour-infiltrating lymphocytes reveals two new biologically different subgroups of breast ductal carcinoma in situ.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) have been demonstrated to significantly influence prognosis and response to therapy of invasive breast cancer (IBC). Thus, it has been suggested that TIL density or/and immunophenotype could serve as biomarkers for selection of IBC patients for immunotherapy. However, much less is known about significance of TILs in breast ductal carcinoma in situ (DCIS). METHODS: We retrospectively investigated TIL density and immunophenotype in 96 pure DCIS and 35 microinvasive carcinomas (miCa). TIL density was assessed on H&E-stained breast biopsy sections as the percentage of tumour stromal area occupied by TILs, and classified into 4 grades: 0 (0%-9%), 1 (10-29%), 2 (30-49%) and 3 (50%-100%). TIL immunophenotype was assessed by immunohistochemistry for CD8, CD4, FoxP3, CD38 or CD20. RESULTS: Compared to pure DCIS, miCa contained significantly more cases with TIL density grade 3 (p = 0.028). Concordantly, CD8+, CD4+ and CD38+ cells were more numerous in miCa than in pure DCIS. In the pure DCIS subgroup with TIL density grades 2 and 3, all TIL subpopulations were more numerous than in the pure DCIS with TIL density grades 0 and 1, however the ratio between T-lymphocytes (CD8+ and CD4+) and B-lymphocytes (CD20+) was significantly lower (p = 0.029). On the other side, this ratio was significantly higher in miCa, in comparison with pure DCIS having TIL density grades 2 and 3 (p = 0.017). By cluster analysis of tumour cell pathobiological features we demonstrated similarity between miCa and the pure DCIS with TIL density grades 2 and 3. The only significant difference between those two categories was in the ratio of T- to B-TILs, higher in miCa. CONCLUSION: Results indicate that TIL density level can distinguish 2 biologically different DCIS subgroups, one of which (DCIS with ≥30% TILs, the TIL-rich DCIS) is like miCa. Similarity of TIL-rich pure DCIS and miCa as well as the role of B-lymphocytes in DCIS invasiveness are worth further investigating with regards to the potential development of immunotherapy-based prevention of DCIS progression.

The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast.

Background: Tumor-infiltrating lymphocytes (TILs) are a robust prognostic adjunct in invasive breast cancer, but their clinical role in ductal carcinoma in situ (DCIS) has not been ascertained. Patients and methods: We evaluated the prevalence and clinical relevance of TILs in a well annotated series of 1488 consecutive DCIS women with a median follow-up of 8.2 years. Detailed criteria for TILs evaluation were pre-defined involving the International Immuno-Oncology Biomarker Working Group. TILs percentage was considered both as a continuous and categorical variable. Levels of TILs were examined for their associations with ipsilateral breast event (IBE), whether in situ or invasive. Results: Of the 1488 patients with DCIS under study, 35.1% had <1%, 58.3% 1-49% and 6.5% ≥50% peri-ductal stromal lymphocytes. The interobserver agreement in TILs evaluation, measured by the intraclass correlation coefficient (ICC) was 0.96 (95% CI 0.95-0.97). At univariable analysis, clinical factors significantly associated with TILs (P ≤0.001) were intrinsic subtype, grade, necrosis, type of surgery. Her-2 positive DCIS were more frequently associated with TILs (24% of patients with TILs ≥50%), followed by the triple negative (11%), Luminal B/Her-2 positive (9%) and Luminal A/B subtypes (1%) (P < 0.0001). We did not find any association between TILs as a continuous variable and the risk of IBEs. Likewise, when patients were stratified by TILs percentage (<1%, between 1% and 49.9%, and ≥50%), no statistically significant association was observed (10-year cumulative incidence of IBEs: 19%, 17.3%, and 18.7% respectively, P = 0.767). Conclusion: TILs occur more frequently in the Her-2 positive DCIS. Although we did not find a significant association between TILs and the 10-year risk of IBE, our data suggest that immunotherapies might be considered in subsets of DCIS patients.

Characterizing the immune microenvironment in high-risk ductal carcinoma in situ of the breast.

PURPOSE: The recent increase in the incidence of ductal carcinoma in situ (DCIS) has sparked debate over the classification and treatment of this disease. Although DCIS is considered a precursor lesion to invasive breast cancer, some DCIS may have more or less risk than is realized. In this study, we characterized the immune microenvironment in DCIS to determine if immune infiltrates are predictive of recurrence. METHODS: Fifty-two cases of high-grade DCIS (HG-DCIS), enriched for large lesions and a history of recurrence, were age matched with 65 cases of non-high-grade DCIS (nHG-DCIS). Immune infiltrates were characterized by single- or dual-color staining of FFPE sections for the following antigens: CD4, CD8, CD20, FoxP3, CD68, CD115, Mac387, MRC1, HLA-DR, and PCNA. Nuance multispectral imaging software was used for image acquisition. Protocols for automated image analysis were developed using CellProfiler. Immune cell populations associated with risk of recurrence were identified using classification and regression tree analysis. RESULTS: HG-DCIS had significantly higher percentages of FoxP3+ cells, CD68+ and CD68+PCNA+ macrophages, HLA-DR+ cells, CD4+ T cells, CD20+ B cells, and total tumor infiltrating lymphocytes compared to nHG-DCIS. A classification tree, generated from 16 immune cell populations and 8 clinical parameters, identified three immune cell populations associated with risk of recurrence: CD8+HLADR+ T cells, CD8+HLADR- T cells, and CD115+ cells. CONCLUSION: These findings suggest that the tumor immune microenvironment is an important factor in identifying DCIS cases with the highest risk for recurrence and that manipulating the immune microenvironment may be an efficacious strategy to alter or prevent disease progression.

Loss of Anti-HER-3 CD4+ T-Helper Type 1 Immunity Occurs in Breast Tumorigenesis and is Negatively Associated with Outcomes.

BACKGROUND: We previously demonstrated a progressive loss of the anti-human epidermal growth factor receptor 2 (HER2) CD4+ T-helper type 1 (Th1) response during HER2pos breast tumorigenesis. This loss is associated with residual disease following neoadjuvant therapy and increased risk of recurrence. In this study, we assessed the fate of anti-HER3 Th1 immunity during breast tumorigenesis. METHODS: Peripheral blood from 131 subjects, including healthy donors (HDs), patients with benign breast disease (BD), ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC), was collected. Interferon (IFN)-γpos immune responses to four HER3-derived major histocompatibility complex (MHC) class II promiscuous peptides were tested via enzyme-linked immunosorbent (ELISPOT) assays, and three immune response parameters were compared: anti-HER3 (i) responsivity, or proportion of subjects responding to at least one peptide; (ii) repertoire, or number of responding peptides; and (iii) cumulative response, or summed peptide response. RESULTS: A significant decline in anti-HER3 Th1 response was observed going from HDs to IBC patients; patients with triple-negative breast cancer (TNBC) demonstrated the lowest responses. HDs had significantly higher Th1 responses versus estrogen receptor (ER)pos IBC and TNBC patients across all three immune parameters; HER2pos IBC patients displayed responses similar to HDs and BDs. Patients with recurrent breast cancer and residual disease following neoadjuvant therapy demonstrated significantly lower anti-HER3 Th1 immunity compared with patients without recurrence or with a pathologic complete response to neoadjuvant therapy. CONCLUSIONS: Anti-HER3 CD4+ Th1 responses decline during breast tumorigenesis, particularly in TNBC. Attempts to immunologically restore depressed responses in vulnerable subgroups may help mitigate recurrence.

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

AIMS: 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. METHODS AND RESULTS: 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). CONCLUSION: Assessment of TIL-B cells based on location and topographic distribution can provide prognostic information. Validation in a larger cohort is warranted.

The immune microenvironment of breast ductal carcinoma in situ.

The host immune response has a key role in breast cancer progression and response to therapy. However, relative to primary invasive breast cancers, the immune milieu of breast ductal carcinoma in situ (DCIS) is less understood. Here, we profile tumor infiltrating lymphocytes and expression of the immune checkpoint ligand programmed death ligand 1 (PD-L1) in 27 cases of DCIS with known estrogen receptor (ER), progesterone receptor, and human epidermal growth factor 2 (HER-2) expression using tissue microarrays. Twenty-four cases were pure DCIS and three had associated invasive ductal carcinoma. Tumors were stained by immunohistochemistry for PD-L1, as well as the lymphocyte markers CD3, CD4, CD8, FoxP3, and CD20. The expression of PD-L1 by DCIS carcinoma cells and tumor infiltrating lymphocytes was determined, and the average tumor infiltrating lymphocytes per high power field were manually scored. None of the DCIS cells expressed PD-L1, but 81% of DCIS lesions contained PD-L1+ tumor infiltrating lymphocytes. DCIS with moderate-diffuse tumor infiltrating lymphocytes was more likely to have PD-L1+ tumor infiltrating lymphocytes (P=0.004). Tumor infiltrating lymphocytes with high levels of PD-L1 expression (>50% cells) were seen only in triple-negative DCIS (P=0.0008), and PD-L1-tumor infiltrating lymphocytes were seen only in ER+/HER-2-DCIS (P=0.12). The presence of PD-L1+ tumor infiltrating lymphocytes was associated with a younger mean patient age (P=0.01). Further characterization of the DCIS immune microenvironment may identify useful targets for immune-based therapy and breast cancer prevention.

CD4+ T cell-dominant insulitis in acute-onset Type 1 diabetes mellitus associated with intraductal papillary mucinous adenoma.

The loss of insulin-producing pancreatic ß-cells in Type 1 diabetes mellitus (DM) is presumably the result of a T cell-mediated process. In general, CD8+ T cells are the predominant lymphocytes in the insulitis lesions, and CD4+ T cell-dominant insulitis is very rare. We present a case of a 72-year-old woman presented with excessive thirst and a 3-month history of weight loss. She was in a state of ketosis, and her plasma glucose concentration and HbA1c value were elevated. Moreover, anti-islet autoantibodies were positive, thus acute-onset Type 1 DM was diagnosed. At the time of diagnosis, a tumour was detected in the pancreas; total pancreatectomy was carried out 2 months later. The pathological diagnosis was intraductal papillary mucinous adenoma. Immunohistochemical staining of a sample of non-tumorous pancreatic tissue revealed 13 insulitis lesions infiltrated by both CD4+ and CD8+ T cells, and interestingly there were more CD4+ T cells than CD8+ T cells in the lesions. Moreover, B cells and macrophages had also infiltrated the lesions, and these two cell frequencies were both positively correlated with CD4+ as well as CD8+ T cell frequencies. This was a rare case with acute-onset Type 1 DM characterized by CD4+ T cell-dominant insulitis. Proinflammatory cytokines that can promote ß-cell apoptosis or CD8+ T cell function are reported to be secreted from CD4+ T cells. Thus, together with B cells and macrophages, CD4+ T cell-associated immune responses may have, directly and/or indirectly, played a role in the pathogenesis of the Type 1 DM in this patient.