- Email: [email protected]
The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast
Annals of Oncology Advance Access published November 14, 2016
The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast.
G. Pruneri1,10,§, M. Lazzeroni2§, V. Bagnardi3,12, G.B. Tiburzio1, N. Rotmensz3, A. DeCensi2,11, A. Guerrieri-Gonzaga2, A. Vingiani1, G. Curigliano 4, S. Zurrida5, F. Bassi5, R. Salgado6, G. Van den Eynden7, S. Loi8, C. Denkert9, B. Bonanni2,†, G. Viale1,10,†.
Pathology, European Institute of Oncology, Milan, Italy.
2
Cancer Prevention and Genetics, European Institute of Oncology, Milan, Italy.
3
Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy.
4
Experimental Therapeutics, European Institute of Oncology, Milan, Italy.
5
Senology, European Institute of Oncology, Milan, Italy.
6
Department of Pathology, GZA, Breast Cancer Translational Research Group, Jules Bordet
Institute, Belgium. 7
Molecular Immunology Lab, Institut Jules Bordet, Université Libre de Bruxelles, 1000, Brussels,
Belgium. 8
Division of Research and Cancer Medicine, Peter MacCallum Cancer Centre, University of
Melbourne, East Melbourne, Victoria, Australia. 9
Institute of Pathology, Charité University Hospital, Berlin, Germany and German Cancer
Consortium, Berlin, Germany. 10
University of Milan, School of Medicine, Milan, Italy.
11
Division of Medical Oncology, E.O. Ospedali Galliera, Genoa, Italy.
12
University of Milan-Bicocca, Department of Statistics and Quantitative Methods, Milan, Italy.
§
These authors contributed equally to the study.
†
These authors contributed equally to the study as senior scientists.
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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1
Corresponding author: Dr. Matteo Lazzeroni, Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia (IEO), Via Ripamonti 435, 20141, Milan, Italy e-mail: [email protected]
T: 00390294372651
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Running head: Clinical value of tumor infiltrating lymphocytes in DCIS patients
ABSTRACT 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 1,488 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
variable. Levels of TILs were examined for their associations with ipsilateral breast event (IBE), whether in situ or invasive. Results: Of the 1,488 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-yr 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-years risk of IBE, our data suggest that immunotherapies might be considered in subsets of DCIS patients.
Key words: ductal carcinoma in situ; tumor infiltrating lymphocytes; Key message: 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
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Biomarker Working Group. TILs percentage was considered both as a continuous and categorical
Luminal A/B subtypes (1%) (p<0.0001). Our data confirm previous data on a putative immunogenic role of Her-2 in DCIS patients and provide evidence that a fraction of patients may have an immune response to DCIS cells.
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INTRODUCTION Most of ductal carcinoma in situ (DCIS) patients usually withstand a multimodality treatment including surgery, radiotherapy (RT) and/or hormone therapy (HT). RT following breast conserving surgery and HT protect DCIS patients from the risk of relapse, lowering the 10 years ipsilateral local recurrence rate by 15% [1] and 30% [2], respectively, but exert a negligible effect on overall mortality [2]. In this scenario, the painstaking search for identifying predictive and
expression signatures [4], and proliferation markers [5], but none of them however entered the clinical practice yet. Tumor-infiltrating lymphocytes (TILs) have been recently proposed as a reliable surrogate of the adaptive immune response, and proved to be associated with a better prognosis in invasive breast cancer [6], with particular reference to the triple negative subtype [7,8]. Detailed guidelines for evaluating TILs in invasive breast cancer have been recently issued by a panel of expert professionals worldwide - the International Immuno-Oncology Biomarker Working Group [9], in an effort to foster interobserver reproducibility, a prerequisite for a putative prognostic factor to be routinely used in the clinical practice. In this study, we aimed to investigate the distribution and clinical relevance of TILs in the DCIS setting. To this end, we firstly evaluated the prognostic value of TILs in a large and well annotated series of DCIS patients, diagnosed and treated in a single Institution. Because of the absence of previous studies in this setting, we also developed guidelines for evaluating TILs, involving members of the International Immuno-Oncology Biomarker Working Group.
PATIENTS AND METHODS
Study Population
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prognostic adjuncts yielded a number of candidates, including complex nomograms [3], gene
The study cohort consisted of all consecutive women who underwent primary breast surgery at the European Institute of Oncology (IEO) between January 1, 1997 and December 31, 2008, who were diagnosed with DCIS, and who were followed up by the IEO staff. Routine measurement of estrogen receptor (ER) and progesterone receptor (PgR) status began in January 1998. A dedicated database (the IEO Breast Cancer Database) was developed in order to collect prospectively all relevant clinical data. Exclusion criteria for the present cohort were: previous breast cancer (DCIS
axillary lymph nodes. For patients with multiple breast cancer samples episodes, only the first was considered in the analysis.
Treatment assignment Treatment assignments have been discussed by a multidisciplinary team on a weekly basis. A few differences in treatment proposal over the 10-year follow-up period were observed, as previously reported [5].
Pathology methods The criteria adopted for the evaluation of DCIS characteristics (histological type, grade, intrinsic subtype, stage, presence of necrosis and microcalcifications, ER, PgR, Ki-67 labeling index, and Her-2 expression) have been previously described [5]. ER/PgR and Her-2 immunoreactivity was assessed in line with the clinical practice procedures applicable at diagnosis. In line with the St. Gallen recommendations for invasive cancer [10], DCIS were classified as Luminal A-like (ER and PgR positive, absence of Her-2 overexpression and Ki-67 <14%), Luminal B-like (ER and PgR positive, absence of Her-2 overexpression and Ki-67 ≥14%), Luminal Blike/Her-2 positive (same as above plus Her-2 3+), Her-2 positive (ER and PgR <1%, Her-2 3+) and triple negative (ER and PgR <1% and absence of HER2 overexpression).
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or invasive), a history of extramammary primary tumor, any neoadjuvant treatment, and positive
TILs assessment in in-situ (CIS)-lesions of the breast: guidelines from the TILs Working Group. In view of the absence of previous reports on TILs in DCIS samples, detailed criteria for their evaluation were pre-planned after discussions with members of the International ImmunoOncology Biomarker Working Group, as described in supplementary table 1.
The percentage of TILs was considered in the analyses as both continuous or by predefined categorical groups (<1%, 1-49% and ≥50%), and its association with clinico-pathological characteristics was evaluated with the F-test (continuous data) or chi-square test (categorical data). The main outcome measure was the ipsilateral breast event (IBE) as first observed event, whether in situ or invasive. The cumulative incidence of IBE was defined as the time from the date of surgery to the time of recurrence. Other first events such as contralateral recurrences, other primary tumors or deaths for non-breast related causes were considered as competing events. In case of no events, the observation was censored at the last follow-up visit. The Gray’s test was used to test the difference in the cumulative incidence of IBE between subgroups of patients. Departure from linearity in the relationship between TILs (treated as a continuous variable) and the hazard of considered events was investigated fitting restricted cubic spline (RCS) models [11]. The Likelihood Ratio Test (LRT) was used to determine if the RCS model significantly increased the likelihood function when compared to a simpler model that assumed a linear relationship. The pvalue of the LRT was reported. A p-value less than 0.05 was considered statistically significant. A multivariable Cox proportional hazards regression model was used to adjust the effect of TILs (both as continuous and categorical) on IBE risk by clinico-pathological characteristics which were significantly associated with TILS and/or recurrence risk at the univariable analysis. The LRT was used to test the heterogeneity of the TILs effect among strata of clinico-pathologic features. The agreement between pathologists in TILs evaluation was measured by the intraclass correlation
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Statistical Methods
coefficient (ICC) [12]. All analyses were carried out with the SAS software (SAS Institute, Cary, NC) and the R (http://cran.r-project.org/) software. RESULTS Associations between TILs and clinico-pathological factors Table 1 summarizes the main baseline characteristics of the entire cohort. Preventive systemic treatment included any active pharmacological treatment: 474 of 601 (78.9%) patients
5 years. One-hundred and sixty patients received other preventive drugs, including the retinoid fenretinide (200 mg/day), or tamoxifen standard dose (20 mg/day), or the aromatase inhibitor anastrozole (1 mg/day) as part of phase II or III clinical trials. One hundred and seventy-nine patients received electron intraoperative radiotherapy (ELIOT). Overall, TILs were heterogeneously distributed across the cases analyzed: among the 1,488 DCIS under study, 523 (35.1%) had <1%, 868 (58.3%) 1-49% and 97 (6.5%) ≥50% peri-ductal stromal lymphocytes (Supplementary Figure 1). TILs assessment, as carried by four different experienced pathologists (RS, GVE, KD, GP) according to the pre-planned guidelines, showed an excellent interobserver agreement, with an interclass correlation coefficient of 0.96 (95% CI 0.95– 0.97). Factors significantly associated with TILs (p≤0.001) were intrinsic subtype, grade, necrosis, type of surgery, hormone and radiotherapy treatment (Table 1). Patients with higher levels of TILs were more likely to receive hormonal therapy, radiotherapy and have a mastectomy, likely due to the association of TILs with poor prognostic factors, including necrosis, higher grade and Her-2 immunoreactivity. Notably, tumors in the Her-2 positive category were significantly associated with TILs (p <0.0001) (Figure 1). More than 60% of DCIS with a prevalence of TILs ≥50% (n=60) had a Her-2 phenotype, as compared to the Luminal B-like/Her-2 positive (23%), Triple Negative (7%), Luminal B-like (4%) and Luminal A-like (3%) subtypes. Approximately 60% of the entire cohort (n=868) had a stromal lymphocytic infiltration between 1-49% (Luminal A-like 26.5%, Luminal B-
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treated with preventive HT received low dose tamoxifen (either 5 mg/day or 20 mg/week) for up to
like 25.8%, Luminal B-like/Her-2 positive 21%, Her-2 positive 21%, and triple negative 5%). More than half of our Luminal A-like DCIS (n=293) had a lymphocyte infiltration accounting for less than 1%. The median TILs level was <1%, <1%, 6%, 25%, and 8% or Luminal A-like, Luminal Blike, Luminal B-like/Her-2 positive, Her-2 positive and Triple Negative DCIS subtypes, respectively (Figure 1).
The pre-planned endpoint was ipsilateral breast event (in situ or invasive): after a median follow-up of 8.2 years, a total of 245 (16.5%) IBEs occurred, 109 (7.3%) of which were in situ and 136 (9.1%) invasive. Overall, 144 (9.7%) other events occurred (61 second primaries, 11 deaths for non-breast related causes as first event, 22 contralateral DCIS, and 50 contralateral invasive breast cancers). We did not find a significant association between the prevalence of stromal lymphocytic infiltration and the cumulative incidence of IBEs, whether in situ or invasive (Figure 2). The analysis of TILs as a continuous variable did not show any significant association between the increase of lymphocytes in the peri-DCIS stroma and the relative risk of IBE (p overall effect 0.44, p non-linear component: 0.30, Figure 2). Likewise, when patients were stratified by TILs percentage (<1%, n=523: 35%; between 1% and 49.9%, n=868: 58%; and ≥50%, n=97: 7%), no statistically significant associations were observed between TILs and the 10 years’ cumulative incidence of in situ and invasive IBEs (p=0.767) (Table 2). Results from crude models were not significantly different from multivariable models. The effect of TILs association on the hazard of IBE adjusted by age, intrinsic subtype, necrosis and therapy (surgery, RT, HT) was negligible (HR 0.92 comparing <1% vs. 1-49%, 95% CI 0.69-1.23; and 0.98 comparing <1% vs. ≥50%, 95% CI, 0.56-1.73) (Table 2). No association between TILs and contralateral breast cancers (supplementary figure 3) nor second primaries (data not shown) was observed. Finally, even after stratifying in accordance with intrinsic subtype or local treatment, we did not detect any significant association
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Association between TILs and ipsilateral breast cancer events (IBE)
between the cumulative incidence of IBEs and the presence of TILs (p-value testing for homogeneity of TILs effects among subtypes: 0.99; p-value testing for homogeneity of TILs effects among different local treatments: 0.36) (Figure 3 and supplementary figure 2). The same results were obtained with a subgroup analysis, excluding patients treated with mastectomy and patients who received intraoperative radiotherapy (Supplementary table 2). As expected from our previous studies [5,13], factors significantly associated with ipsilateral
surgery (data not shown).
DISCUSSION In the present report, we firstly present the prevalence and clinical relevance of TILs in the DCIS setting, investigating a large and well annotated series of patients diagnosed and treated in a single Institution. In view of the absence of established guidelines for TILs evaluation, we predefined detailed criteria for scoring stromal lymphocytes in the DCIS setting, involving members of the International Immuno-Oncology Biomarker Working Group. Their reproducibility was ascertained by a comparison of the readings generated by four experienced pathologists in 8.3% of the cases included in the present study. We observed an excellent inter-observer concordance rate, suggesting that this method may be broadly used for comparing data across different cohorts of patients. Her-2-positive (ER/PgR-negative, Her-2 3+ by IHC) DCIS showed a significantly higher prevalence of TILs than the other intrinsic subtypes, including the triple negative subtype: a substantial (23.6%) fraction of the Her-2-positive DCIS had 50% or more of peri-ductal stromal lymphocytes and could be therefore considered as lymphocyte-predominant DCIS (LPDCIS), mirroring the definition used in invasive breast cancer. Their prevalence in the Her-2 positive subtype doubled that observed in the triple negative subtype, somewhat different from reports in invasive breast tumors, where the triple negative cancers represent the lymphocyte-richest subtype
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recurrence at univariate analysis were menopausal status, microcalcifications, Ki-67 LI, and type of
[14]. Furthermore, within the Luminal B-like subtype, we found that the occurrence of Her-2 overexpression led to a significantly increase of TILs, with a shift in LPDCIS prevalence from 1% to 8.9%. A number of experimental evidences suggest an immunogenic role of Her-2 in DCIS patients [15-18]. Interestingly, the incidence of Her-2 positive DCIS is much higher (36% in our series) than that of invasive cancer (roughly 15%), suggesting that Her-2 positivity is negatively selected during progression to invasive disease. Taken together, these data suggest that Her-2
underlying mechanism is unknown. TILs have been repeatedly proven to be a robust prognostic marker in the triple negative invasive cancer subset, either in retrospective and randomized phase III clinical studies [8]. In our study, TILs were not associated with ipsilateral in situ or invasive tumor recurrence, irrespective of the treatment (surgery, hormonal therapy or radiotherapy), biological subtype (Luminal A and B-like, triple negative, Her-2 positive) or type of evaluation (continuous variable or 50% threshold). In view of the large series analyzed and the robustness of the scoring system used, it seems unlikely that it could be due to statistical or technical inconsistencies. Nevertheless, the absence of a prognostic value in DCIS in this dataset is not fully surprising, especially if one considers that to date TILs have been convincingly related to survival only in the subset of invasive breast cancer patients treated by chemotherapy, alone or in combination with Trastuzumab, but not in ER-positive patients receiving hormone therapy [14]. In this regard, our data in patients who did not receive chemotherapy, indirectly support an immunogenic effect of chemotherapy and Trastuzumab [9]. The absence of a prognostic value notwithstanding, it seems premature disregarding TILs evaluation in this setting of patients: firstly, our data need to be validated in independent cohorts; secondly, the lack of a prognostic signal should not deny the fact that a fraction of patients do reasonably have an immune response to DCIS cells, which could be sustained and exploited by immunotherapy. The cross-talk between leukocytes, tumoral microenvironment and tumor cells is complex. There is a multifaceted interplay between anti- or pro-tumoral leukocytes and mediators
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overexpression in DCIS cells represents a potent stimulator of the immune system, even if the
[19]. Accordingly, the relative density of specific subpopulations of lymphoid cells has been reported to specify the prognosis of breast cancer patients independent of the absolute TILs value: a high prevalence of T-suppressor/T-regulator lymphocytes [20] points towards favorable survival, while there is evidence of a poor clinical outcome in patients with a high content of CD68-positive tumor associated macrophages [21]. Of course, these leukocytes are almost indistinguishable in H&E routine slides. Hence, whilst evaluating TILs by H&E seems highly relevant for TNBC
therefore needed to understand the leukocytes subpopulations in DCIS, especially in the Her-2 positive and triple negative subtypes. This could be accomplished by highlighting leukocyte subpopulations using multispectral immunohistochemistry, gene expression or flow cytometry where fresh tissue is available. In conclusion, we provide for the first time a comprehensive evaluation of TILs in a large and well characterized cohort of DCIS patients. We observed a significantly higher prevalence of TILs across the Her-2 positive and, to a lesser extent, the Triple Negative DCIS subtypes. We also report a highly reproducible scoring method. Further studies characterizing the landscape and prognostic role of TILs subpopulations in DCIS are warranted and may provide important prognostic information.
Acknowledgments SL is funded by Cancer Council Victoria and the Breast Cancer Research Foundation, NY.
Funding Supported by the Ministry of Health, Clinical Health Care Research (Grant number: GR-201302355967).
Disclosure
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patients, it may not be appropriate in other cancer settings, including DCIS. Further studies are
The authors have declared no conflicts of interest.
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Figure 1. Boxplot of distribution of TILs as a continuous variable according to histological subtype. The median TILs level: Luminal A-like: <1%; Luminal B-like: <1%; Luminal B-like/Her2 positive: 6%; Her-2 positive: 25%; Triple Negative: 8%.
Figure 2. Cumulative incidence of ipsilateral in situ recurrences (panel A) and ipsilateral invasive recurrences (panel B), by stromal lymphocytic infiltration. Cumulative incidence of ipsilateral
infiltration, treated as a continuous variable, on the relative risk of ipsilateral event (panel D).
Figure 3. Cumulative incidence of ipsilateral recurrences by TILs, according to subtype (p-value testing homogeneity of TILs effect among subtypes: 0.99).
Supplementary Figure 1. Examples of TILs distribution in DCIS, in accordance with the scoring guidelines issued by the TILs working group. All the mononuclear inflammatory cells in the stromal compartment, defined as the area within the specialized stroma surrounding the CIS-lesions (within two high-power microscopic fields) were registered. Any type of circumferential TILsinfiltration was taken into account, including partial infiltration (arrows). TILs around normal lobules were not taken into account (arrowhead), while stromal lymphocytes surrounding areas of lobular cancerization were registered (asterisks).
Supplementary Figure 2. Cumulative incidence of ipsilateral recurrences by TILs, according to local treatment (p-value testing the homogeneity of TILs effect among different local treatments: 0.36).
Supplementary Figure 3. Cumulative incidence of contralateral breast cancers by TILs.
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recurrences by stromal lymphocytic infiltration (panel C). Effect of stromal lymphocytic
Table 1. Main baseline characteristics of the 1,488 DCIS patients included in the study. Stromal lymphocytic infiltration All patients
<1%
1%-49.9%
≥50%
No. 1,488
No. 523
No. 868
No. 97
30 (2)
10 (1.9)
19 (2.2)
1 (1)
Age <35
p-value 0.004
35-50
635 (42.7)
241 (46.1)
361 (41.6)
33 (34)
51-65
624 (41.9)
200 (38.2)
376 (43.3)
48 (49.5)
>65
199 (13.4)
72 (13.8)
112 (12.9)
15 (15.5)
Premenopausal
705 (47.4)
273 (52.2)
394 (45.4)
38 (39.2)
Postmenopausal BMI (kg/m2) median (IQR) (129 Missing) Grade
783 (52.6)
250 (47.8)
474 (54.6)
59 (60.8)
22.8 (20.8-25.6)
23.0 (21.0-26.3)
22.7 (20.6-25.3)
23.1 (20.7-25.4)
Low (G1)
251 (16.9)
154 (29.4)
95 (10.9)
2 (2.1)
Intermediate (G2)
802 (53.9)
320 (61.2)
460 (53)
22 (22.7)
High (G3)
435 (29.2)
49 (9.4)
313 (36.1)
73 (75.3)
573
219
319
35
< 2.5 cm
620 (67.8)
223 (73.4)
355 (64.7)
42 (67.7)
≥ 2.5 cm
295 (32.3)
81 (26.6)
194 (35.3)
20 (32.3)
Absent
577 (38.8)
553 (48.4)
297 (34.2)
30 (27.8)
Present
911 (61.2)
270 (51.6)
571 (65.8)
70 (72.2)
<0.001
With Microcalcifications
1162 (78.1)
414 (79.2)
673 (77.5)
75 (77.3)
0.811
With Multifocality
271 (18.2)
105 (20.1)
151 (17.4)
15 (15.5)
0.506
Menopausal status
0.052 <0.001
0.034
Necrosis
ER
Negative (0%)
318 (21.4)
18 (3.4)
233 (26.9)
60 (69.1)
Positive
1170 (78.6)
505 (96.6)
635 (73.1)
30 (30.9)
PgR (1 missing)
1
Negative
481 (32.4)
67 (12.8)
339
76 (78.4)
Positive
1006 (67.6)
456 (87.2)
529 (61.0)
21 (21.6)
5
2
3
<14%
594 (40.0)
320 (61.4)
270 (31.2)
4 (4.1)
≥ 14%
889 (60.0)
201 (38.6)
595 (68.8)
93 (95.9)
Negative/equivocal
976 (65.6)
460 (87.9)
502 (57.8)
14 (14.4)
Positive
512 (34.4)
63 (12.1)
366 (42.2)
83 (85.6)
526 (35.3)
293 (56)
230 (26.5)
3 (3.1)
Ki-67% (5 missing)
<0.001
1 <0.001
<0.001
HER2
Luminal A
Luminal B (Ki67≥14%)
387 (26)
159 (30.4)
224 (25.8)
4 (4.1)
Luminal B (Her2 positive)
258 (17.3)
53 (10.1)
182 (21)
23 (23.7)
Her2 positive
254 (17.1)
10 (1.9)
184 (21.2)
60 (61.9)
63 (4.2)
8 (1.5)
48 (5.5)
7 (7.2)
Triple Negative Local treatment
<0.001
<0.001
Subtype
<0.001
Mastectomy
364 (24.5)
107 (20.4)
228 (26.2)
29 (29.9)
Quadrantectomy with RT standard
417 (28.0)
108 (20.6)
271 (31.2)
38 (39.1)
Quadrantectomy with ELIOT
179 (12.0)
36 (6.8)
130 (14.9)
13 (13.4)
Quadrantectomy without RT
528 (35.5)
272 (52)
239 (27.5)
17 (17.5)
Hormone therapy
601 (40.4)
281 (53.7)
308 (35.5)
12 (12.4)
<0.001
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Tumor size (573 missing)
0.012
Table 2. Incidence of ipsilateral recurrences (all, in situ, invasive) by TILs. Association of TILs with the hazard of ipsilateral recurrence.
Incidence of ipsilateral recurrences (all, in situ, invasive) by TILs. All ipsilateral recurrence
Ipsilateral in situ recurrence
Ipsilateral invasive recurrence
10-yr cumulative incidence (95% CI)
Ev./N
10-yr cumulative incidence (95% CI)
Ev./N
10-yr cumulative incidence (95% CI)
<1%
90/523
19 (15.4-23.5)
33/523
6.7 (4.6-9.5)
57/523
12.4 (9.4-16.3)
1-49.9%
137/868
17.3 (14.7-20.4)
67/868
8.3 (6.5-10.6)
70/868
9.0 (7.0-11.5)
18/97
18.7 (12.1-29)
9/97
8.6 (4.4-16.8)
9/97
10.1 (5.4-19)
TILs
≥50% p-value
0.767
0.402
0.351
Association of TILs with the hazard of ipsilateral recurrence. TILs
Hazard Ratio* (95% CI) Univariable analysis
<1%
Reference
1%-49.9%
0.97 (0.74-1.26)
0.81
0.92 (0.69-1.23)
0.56
≥50%
1.14 (0.69-1.89)
0.61
0.98 (0.56-1.73)
0.98
+10% increase
1.02 (0.96-1.10)
0.51
1.01 (0.93-1.10)
0.78
p-value
Hazard Ratio* (95% CI) Multivariable analysis*
p-value
Reference
*Hazard ratios adjusted by menopausal status, subtype, necrosis and therapy (surgery, radiotherapy and hormone therapy) in a Cox proportional hazard model.
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Ev./N
185x166mm (150 x 150 DPI)
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Boxplot of distribution of TILs as a continuous variable according to histological subtype. The median TILs level: Luminal A-like: <1%; Luminal B-like: <1%; Luminal B-like/Her-2 positive: 6%; Her-2 positive: 25%; Triple Negative: 8%.
226x179mm (150 x 150 DPI)
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Cumulative incidence of ipsilateral in situ recurrences (panel A) and ipsilateral invasive recurrences (panel B), by stromal lymphocytic infiltration. Cumulative incidence of ipsilateral recurrences by stromal lymphocytic infiltration (panel C). Effect of stromal lymphocytic infiltration, treated as a continuous variable, on the relative risk of ipsilateral event (panel D).
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Cumulative incidence of ipsilateral recurrences by TILs, according to subtype (p-value testing homogeneity of TILs effect among subtypes: 0.99) 184x249mm (150 x 150 DPI)
The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast.
G. Pruneri1,10,§, M. Lazzeroni2§, V. Bagnardi3,12, G.B. Tiburzio1, N. Rotmensz3, A. DeCensi2,11, A. Guerrieri-Gonzaga2, A. Vingiani1, G. Curigliano 4, S. Zurrida5, F. Bassi5, R. Salgado6, G. Van den Eynden7, S. Loi8, C. Denkert9, B. Bonanni2,†, G. Viale1,10,†.
Pathology, European Institute of Oncology, Milan, Italy.
2
Cancer Prevention and Genetics, European Institute of Oncology, Milan, Italy.
3
Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy.
4
Experimental Therapeutics, European Institute of Oncology, Milan, Italy.
5
Senology, European Institute of Oncology, Milan, Italy.
6
Department of Pathology, GZA, Breast Cancer Translational Research Group, Jules Bordet
Institute, Belgium. 7
Molecular Immunology Lab, Institut Jules Bordet, Université Libre de Bruxelles, 1000, Brussels,
Belgium. 8
Division of Research and Cancer Medicine, Peter MacCallum Cancer Centre, University of
Melbourne, East Melbourne, Victoria, Australia. 9
Institute of Pathology, Charité University Hospital, Berlin, Germany and German Cancer
Consortium, Berlin, Germany. 10
University of Milan, School of Medicine, Milan, Italy.
11
Division of Medical Oncology, E.O. Ospedali Galliera, Genoa, Italy.
12
University of Milan-Bicocca, Department of Statistics and Quantitative Methods, Milan, Italy.
§
These authors contributed equally to the study.
†
These authors contributed equally to the study as senior scientists.
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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1
Corresponding author: Dr. Matteo Lazzeroni, Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia (IEO), Via Ripamonti 435, 20141, Milan, Italy e-mail: [email protected]
T: 00390294372651
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Running head: Clinical value of tumor infiltrating lymphocytes in DCIS patients
ABSTRACT 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 1,488 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
variable. Levels of TILs were examined for their associations with ipsilateral breast event (IBE), whether in situ or invasive. Results: Of the 1,488 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-yr 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-years risk of IBE, our data suggest that immunotherapies might be considered in subsets of DCIS patients.
Key words: ductal carcinoma in situ; tumor infiltrating lymphocytes; Key message: 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
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Biomarker Working Group. TILs percentage was considered both as a continuous and categorical
Luminal A/B subtypes (1%) (p<0.0001). Our data confirm previous data on a putative immunogenic role of Her-2 in DCIS patients and provide evidence that a fraction of patients may have an immune response to DCIS cells.
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INTRODUCTION Most of ductal carcinoma in situ (DCIS) patients usually withstand a multimodality treatment including surgery, radiotherapy (RT) and/or hormone therapy (HT). RT following breast conserving surgery and HT protect DCIS patients from the risk of relapse, lowering the 10 years ipsilateral local recurrence rate by 15% [1] and 30% [2], respectively, but exert a negligible effect on overall mortality [2]. In this scenario, the painstaking search for identifying predictive and
expression signatures [4], and proliferation markers [5], but none of them however entered the clinical practice yet. Tumor-infiltrating lymphocytes (TILs) have been recently proposed as a reliable surrogate of the adaptive immune response, and proved to be associated with a better prognosis in invasive breast cancer [6], with particular reference to the triple negative subtype [7,8]. Detailed guidelines for evaluating TILs in invasive breast cancer have been recently issued by a panel of expert professionals worldwide - the International Immuno-Oncology Biomarker Working Group [9], in an effort to foster interobserver reproducibility, a prerequisite for a putative prognostic factor to be routinely used in the clinical practice. In this study, we aimed to investigate the distribution and clinical relevance of TILs in the DCIS setting. To this end, we firstly evaluated the prognostic value of TILs in a large and well annotated series of DCIS patients, diagnosed and treated in a single Institution. Because of the absence of previous studies in this setting, we also developed guidelines for evaluating TILs, involving members of the International Immuno-Oncology Biomarker Working Group.
PATIENTS AND METHODS
Study Population
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prognostic adjuncts yielded a number of candidates, including complex nomograms [3], gene
The study cohort consisted of all consecutive women who underwent primary breast surgery at the European Institute of Oncology (IEO) between January 1, 1997 and December 31, 2008, who were diagnosed with DCIS, and who were followed up by the IEO staff. Routine measurement of estrogen receptor (ER) and progesterone receptor (PgR) status began in January 1998. A dedicated database (the IEO Breast Cancer Database) was developed in order to collect prospectively all relevant clinical data. Exclusion criteria for the present cohort were: previous breast cancer (DCIS
axillary lymph nodes. For patients with multiple breast cancer samples episodes, only the first was considered in the analysis.
Treatment assignment Treatment assignments have been discussed by a multidisciplinary team on a weekly basis. A few differences in treatment proposal over the 10-year follow-up period were observed, as previously reported [5].
Pathology methods The criteria adopted for the evaluation of DCIS characteristics (histological type, grade, intrinsic subtype, stage, presence of necrosis and microcalcifications, ER, PgR, Ki-67 labeling index, and Her-2 expression) have been previously described [5]. ER/PgR and Her-2 immunoreactivity was assessed in line with the clinical practice procedures applicable at diagnosis. In line with the St. Gallen recommendations for invasive cancer [10], DCIS were classified as Luminal A-like (ER and PgR positive, absence of Her-2 overexpression and Ki-67 <14%), Luminal B-like (ER and PgR positive, absence of Her-2 overexpression and Ki-67 ≥14%), Luminal Blike/Her-2 positive (same as above plus Her-2 3+), Her-2 positive (ER and PgR <1%, Her-2 3+) and triple negative (ER and PgR <1% and absence of HER2 overexpression).
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or invasive), a history of extramammary primary tumor, any neoadjuvant treatment, and positive
TILs assessment in in-situ (CIS)-lesions of the breast: guidelines from the TILs Working Group. In view of the absence of previous reports on TILs in DCIS samples, detailed criteria for their evaluation were pre-planned after discussions with members of the International ImmunoOncology Biomarker Working Group, as described in supplementary table 1.
The percentage of TILs was considered in the analyses as both continuous or by predefined categorical groups (<1%, 1-49% and ≥50%), and its association with clinico-pathological characteristics was evaluated with the F-test (continuous data) or chi-square test (categorical data). The main outcome measure was the ipsilateral breast event (IBE) as first observed event, whether in situ or invasive. The cumulative incidence of IBE was defined as the time from the date of surgery to the time of recurrence. Other first events such as contralateral recurrences, other primary tumors or deaths for non-breast related causes were considered as competing events. In case of no events, the observation was censored at the last follow-up visit. The Gray’s test was used to test the difference in the cumulative incidence of IBE between subgroups of patients. Departure from linearity in the relationship between TILs (treated as a continuous variable) and the hazard of considered events was investigated fitting restricted cubic spline (RCS) models [11]. The Likelihood Ratio Test (LRT) was used to determine if the RCS model significantly increased the likelihood function when compared to a simpler model that assumed a linear relationship. The pvalue of the LRT was reported. A p-value less than 0.05 was considered statistically significant. A multivariable Cox proportional hazards regression model was used to adjust the effect of TILs (both as continuous and categorical) on IBE risk by clinico-pathological characteristics which were significantly associated with TILS and/or recurrence risk at the univariable analysis. The LRT was used to test the heterogeneity of the TILs effect among strata of clinico-pathologic features. The agreement between pathologists in TILs evaluation was measured by the intraclass correlation
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Statistical Methods
coefficient (ICC) [12]. All analyses were carried out with the SAS software (SAS Institute, Cary, NC) and the R (http://cran.r-project.org/) software. RESULTS Associations between TILs and clinico-pathological factors Table 1 summarizes the main baseline characteristics of the entire cohort. Preventive systemic treatment included any active pharmacological treatment: 474 of 601 (78.9%) patients
5 years. One-hundred and sixty patients received other preventive drugs, including the retinoid fenretinide (200 mg/day), or tamoxifen standard dose (20 mg/day), or the aromatase inhibitor anastrozole (1 mg/day) as part of phase II or III clinical trials. One hundred and seventy-nine patients received electron intraoperative radiotherapy (ELIOT). Overall, TILs were heterogeneously distributed across the cases analyzed: among the 1,488 DCIS under study, 523 (35.1%) had <1%, 868 (58.3%) 1-49% and 97 (6.5%) ≥50% peri-ductal stromal lymphocytes (Supplementary Figure 1). TILs assessment, as carried by four different experienced pathologists (RS, GVE, KD, GP) according to the pre-planned guidelines, showed an excellent interobserver agreement, with an interclass correlation coefficient of 0.96 (95% CI 0.95– 0.97). Factors significantly associated with TILs (p≤0.001) were intrinsic subtype, grade, necrosis, type of surgery, hormone and radiotherapy treatment (Table 1). Patients with higher levels of TILs were more likely to receive hormonal therapy, radiotherapy and have a mastectomy, likely due to the association of TILs with poor prognostic factors, including necrosis, higher grade and Her-2 immunoreactivity. Notably, tumors in the Her-2 positive category were significantly associated with TILs (p <0.0001) (Figure 1). More than 60% of DCIS with a prevalence of TILs ≥50% (n=60) had a Her-2 phenotype, as compared to the Luminal B-like/Her-2 positive (23%), Triple Negative (7%), Luminal B-like (4%) and Luminal A-like (3%) subtypes. Approximately 60% of the entire cohort (n=868) had a stromal lymphocytic infiltration between 1-49% (Luminal A-like 26.5%, Luminal B-
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treated with preventive HT received low dose tamoxifen (either 5 mg/day or 20 mg/week) for up to
like 25.8%, Luminal B-like/Her-2 positive 21%, Her-2 positive 21%, and triple negative 5%). More than half of our Luminal A-like DCIS (n=293) had a lymphocyte infiltration accounting for less than 1%. The median TILs level was <1%, <1%, 6%, 25%, and 8% or Luminal A-like, Luminal Blike, Luminal B-like/Her-2 positive, Her-2 positive and Triple Negative DCIS subtypes, respectively (Figure 1).
The pre-planned endpoint was ipsilateral breast event (in situ or invasive): after a median follow-up of 8.2 years, a total of 245 (16.5%) IBEs occurred, 109 (7.3%) of which were in situ and 136 (9.1%) invasive. Overall, 144 (9.7%) other events occurred (61 second primaries, 11 deaths for non-breast related causes as first event, 22 contralateral DCIS, and 50 contralateral invasive breast cancers). We did not find a significant association between the prevalence of stromal lymphocytic infiltration and the cumulative incidence of IBEs, whether in situ or invasive (Figure 2). The analysis of TILs as a continuous variable did not show any significant association between the increase of lymphocytes in the peri-DCIS stroma and the relative risk of IBE (p overall effect 0.44, p non-linear component: 0.30, Figure 2). Likewise, when patients were stratified by TILs percentage (<1%, n=523: 35%; between 1% and 49.9%, n=868: 58%; and ≥50%, n=97: 7%), no statistically significant associations were observed between TILs and the 10 years’ cumulative incidence of in situ and invasive IBEs (p=0.767) (Table 2). Results from crude models were not significantly different from multivariable models. The effect of TILs association on the hazard of IBE adjusted by age, intrinsic subtype, necrosis and therapy (surgery, RT, HT) was negligible (HR 0.92 comparing <1% vs. 1-49%, 95% CI 0.69-1.23; and 0.98 comparing <1% vs. ≥50%, 95% CI, 0.56-1.73) (Table 2). No association between TILs and contralateral breast cancers (supplementary figure 3) nor second primaries (data not shown) was observed. Finally, even after stratifying in accordance with intrinsic subtype or local treatment, we did not detect any significant association
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Association between TILs and ipsilateral breast cancer events (IBE)
between the cumulative incidence of IBEs and the presence of TILs (p-value testing for homogeneity of TILs effects among subtypes: 0.99; p-value testing for homogeneity of TILs effects among different local treatments: 0.36) (Figure 3 and supplementary figure 2). The same results were obtained with a subgroup analysis, excluding patients treated with mastectomy and patients who received intraoperative radiotherapy (Supplementary table 2). As expected from our previous studies [5,13], factors significantly associated with ipsilateral
surgery (data not shown).
DISCUSSION In the present report, we firstly present the prevalence and clinical relevance of TILs in the DCIS setting, investigating a large and well annotated series of patients diagnosed and treated in a single Institution. In view of the absence of established guidelines for TILs evaluation, we predefined detailed criteria for scoring stromal lymphocytes in the DCIS setting, involving members of the International Immuno-Oncology Biomarker Working Group. Their reproducibility was ascertained by a comparison of the readings generated by four experienced pathologists in 8.3% of the cases included in the present study. We observed an excellent inter-observer concordance rate, suggesting that this method may be broadly used for comparing data across different cohorts of patients. Her-2-positive (ER/PgR-negative, Her-2 3+ by IHC) DCIS showed a significantly higher prevalence of TILs than the other intrinsic subtypes, including the triple negative subtype: a substantial (23.6%) fraction of the Her-2-positive DCIS had 50% or more of peri-ductal stromal lymphocytes and could be therefore considered as lymphocyte-predominant DCIS (LPDCIS), mirroring the definition used in invasive breast cancer. Their prevalence in the Her-2 positive subtype doubled that observed in the triple negative subtype, somewhat different from reports in invasive breast tumors, where the triple negative cancers represent the lymphocyte-richest subtype
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recurrence at univariate analysis were menopausal status, microcalcifications, Ki-67 LI, and type of
[14]. Furthermore, within the Luminal B-like subtype, we found that the occurrence of Her-2 overexpression led to a significantly increase of TILs, with a shift in LPDCIS prevalence from 1% to 8.9%. A number of experimental evidences suggest an immunogenic role of Her-2 in DCIS patients [15-18]. Interestingly, the incidence of Her-2 positive DCIS is much higher (36% in our series) than that of invasive cancer (roughly 15%), suggesting that Her-2 positivity is negatively selected during progression to invasive disease. Taken together, these data suggest that Her-2
underlying mechanism is unknown. TILs have been repeatedly proven to be a robust prognostic marker in the triple negative invasive cancer subset, either in retrospective and randomized phase III clinical studies [8]. In our study, TILs were not associated with ipsilateral in situ or invasive tumor recurrence, irrespective of the treatment (surgery, hormonal therapy or radiotherapy), biological subtype (Luminal A and B-like, triple negative, Her-2 positive) or type of evaluation (continuous variable or 50% threshold). In view of the large series analyzed and the robustness of the scoring system used, it seems unlikely that it could be due to statistical or technical inconsistencies. Nevertheless, the absence of a prognostic value in DCIS in this dataset is not fully surprising, especially if one considers that to date TILs have been convincingly related to survival only in the subset of invasive breast cancer patients treated by chemotherapy, alone or in combination with Trastuzumab, but not in ER-positive patients receiving hormone therapy [14]. In this regard, our data in patients who did not receive chemotherapy, indirectly support an immunogenic effect of chemotherapy and Trastuzumab [9]. The absence of a prognostic value notwithstanding, it seems premature disregarding TILs evaluation in this setting of patients: firstly, our data need to be validated in independent cohorts; secondly, the lack of a prognostic signal should not deny the fact that a fraction of patients do reasonably have an immune response to DCIS cells, which could be sustained and exploited by immunotherapy. The cross-talk between leukocytes, tumoral microenvironment and tumor cells is complex. There is a multifaceted interplay between anti- or pro-tumoral leukocytes and mediators
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overexpression in DCIS cells represents a potent stimulator of the immune system, even if the
[19]. Accordingly, the relative density of specific subpopulations of lymphoid cells has been reported to specify the prognosis of breast cancer patients independent of the absolute TILs value: a high prevalence of T-suppressor/T-regulator lymphocytes [20] points towards favorable survival, while there is evidence of a poor clinical outcome in patients with a high content of CD68-positive tumor associated macrophages [21]. Of course, these leukocytes are almost indistinguishable in H&E routine slides. Hence, whilst evaluating TILs by H&E seems highly relevant for TNBC
therefore needed to understand the leukocytes subpopulations in DCIS, especially in the Her-2 positive and triple negative subtypes. This could be accomplished by highlighting leukocyte subpopulations using multispectral immunohistochemistry, gene expression or flow cytometry where fresh tissue is available. In conclusion, we provide for the first time a comprehensive evaluation of TILs in a large and well characterized cohort of DCIS patients. We observed a significantly higher prevalence of TILs across the Her-2 positive and, to a lesser extent, the Triple Negative DCIS subtypes. We also report a highly reproducible scoring method. Further studies characterizing the landscape and prognostic role of TILs subpopulations in DCIS are warranted and may provide important prognostic information.
Acknowledgments SL is funded by Cancer Council Victoria and the Breast Cancer Research Foundation, NY.
Funding Supported by the Ministry of Health, Clinical Health Care Research (Grant number: GR-201302355967).
Disclosure
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patients, it may not be appropriate in other cancer settings, including DCIS. Further studies are
The authors have declared no conflicts of interest.
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Figure 1. Boxplot of distribution of TILs as a continuous variable according to histological subtype. The median TILs level: Luminal A-like: <1%; Luminal B-like: <1%; Luminal B-like/Her2 positive: 6%; Her-2 positive: 25%; Triple Negative: 8%.
Figure 2. Cumulative incidence of ipsilateral in situ recurrences (panel A) and ipsilateral invasive recurrences (panel B), by stromal lymphocytic infiltration. Cumulative incidence of ipsilateral
infiltration, treated as a continuous variable, on the relative risk of ipsilateral event (panel D).
Figure 3. Cumulative incidence of ipsilateral recurrences by TILs, according to subtype (p-value testing homogeneity of TILs effect among subtypes: 0.99).
Supplementary Figure 1. Examples of TILs distribution in DCIS, in accordance with the scoring guidelines issued by the TILs working group. All the mononuclear inflammatory cells in the stromal compartment, defined as the area within the specialized stroma surrounding the CIS-lesions (within two high-power microscopic fields) were registered. Any type of circumferential TILsinfiltration was taken into account, including partial infiltration (arrows). TILs around normal lobules were not taken into account (arrowhead), while stromal lymphocytes surrounding areas of lobular cancerization were registered (asterisks).
Supplementary Figure 2. Cumulative incidence of ipsilateral recurrences by TILs, according to local treatment (p-value testing the homogeneity of TILs effect among different local treatments: 0.36).
Supplementary Figure 3. Cumulative incidence of contralateral breast cancers by TILs.
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recurrences by stromal lymphocytic infiltration (panel C). Effect of stromal lymphocytic
Table 1. Main baseline characteristics of the 1,488 DCIS patients included in the study. Stromal lymphocytic infiltration All patients
<1%
1%-49.9%
≥50%
No. 1,488
No. 523
No. 868
No. 97
30 (2)
10 (1.9)
19 (2.2)
1 (1)
Age <35
p-value 0.004
35-50
635 (42.7)
241 (46.1)
361 (41.6)
33 (34)
51-65
624 (41.9)
200 (38.2)
376 (43.3)
48 (49.5)
>65
199 (13.4)
72 (13.8)
112 (12.9)
15 (15.5)
Premenopausal
705 (47.4)
273 (52.2)
394 (45.4)
38 (39.2)
Postmenopausal BMI (kg/m2) median (IQR) (129 Missing) Grade
783 (52.6)
250 (47.8)
474 (54.6)
59 (60.8)
22.8 (20.8-25.6)
23.0 (21.0-26.3)
22.7 (20.6-25.3)
23.1 (20.7-25.4)
Low (G1)
251 (16.9)
154 (29.4)
95 (10.9)
2 (2.1)
Intermediate (G2)
802 (53.9)
320 (61.2)
460 (53)
22 (22.7)
High (G3)
435 (29.2)
49 (9.4)
313 (36.1)
73 (75.3)
573
219
319
35
< 2.5 cm
620 (67.8)
223 (73.4)
355 (64.7)
42 (67.7)
≥ 2.5 cm
295 (32.3)
81 (26.6)
194 (35.3)
20 (32.3)
Absent
577 (38.8)
553 (48.4)
297 (34.2)
30 (27.8)
Present
911 (61.2)
270 (51.6)
571 (65.8)
70 (72.2)
<0.001
With Microcalcifications
1162 (78.1)
414 (79.2)
673 (77.5)
75 (77.3)
0.811
With Multifocality
271 (18.2)
105 (20.1)
151 (17.4)
15 (15.5)
0.506
Menopausal status
0.052 <0.001
0.034
Necrosis
ER
Negative (0%)
318 (21.4)
18 (3.4)
233 (26.9)
60 (69.1)
Positive
1170 (78.6)
505 (96.6)
635 (73.1)
30 (30.9)
PgR (1 missing)
1
Negative
481 (32.4)
67 (12.8)
339
76 (78.4)
Positive
1006 (67.6)
456 (87.2)
529 (61.0)
21 (21.6)
5
2
3
<14%
594 (40.0)
320 (61.4)
270 (31.2)
4 (4.1)
≥ 14%
889 (60.0)
201 (38.6)
595 (68.8)
93 (95.9)
Negative/equivocal
976 (65.6)
460 (87.9)
502 (57.8)
14 (14.4)
Positive
512 (34.4)
63 (12.1)
366 (42.2)
83 (85.6)
526 (35.3)
293 (56)
230 (26.5)
3 (3.1)
Ki-67% (5 missing)
<0.001
1 <0.001
<0.001
HER2
Luminal A
Luminal B (Ki67≥14%)
387 (26)
159 (30.4)
224 (25.8)
4 (4.1)
Luminal B (Her2 positive)
258 (17.3)
53 (10.1)
182 (21)
23 (23.7)
Her2 positive
254 (17.1)
10 (1.9)
184 (21.2)
60 (61.9)
63 (4.2)
8 (1.5)
48 (5.5)
7 (7.2)
Triple Negative Local treatment
<0.001
<0.001
Subtype
<0.001
Mastectomy
364 (24.5)
107 (20.4)
228 (26.2)
29 (29.9)
Quadrantectomy with RT standard
417 (28.0)
108 (20.6)
271 (31.2)
38 (39.1)
Quadrantectomy with ELIOT
179 (12.0)
36 (6.8)
130 (14.9)
13 (13.4)
Quadrantectomy without RT
528 (35.5)
272 (52)
239 (27.5)
17 (17.5)
Hormone therapy
601 (40.4)
281 (53.7)
308 (35.5)
12 (12.4)
<0.001
Downloaded from http://annonc.oxfordjournals.org/ at Main Library of Gazi University on December 17, 2016
Tumor size (573 missing)
0.012
Table 2. Incidence of ipsilateral recurrences (all, in situ, invasive) by TILs. Association of TILs with the hazard of ipsilateral recurrence.
Incidence of ipsilateral recurrences (all, in situ, invasive) by TILs. All ipsilateral recurrence
Ipsilateral in situ recurrence
Ipsilateral invasive recurrence
10-yr cumulative incidence (95% CI)
Ev./N
10-yr cumulative incidence (95% CI)
Ev./N
10-yr cumulative incidence (95% CI)
<1%
90/523
19 (15.4-23.5)
33/523
6.7 (4.6-9.5)
57/523
12.4 (9.4-16.3)
1-49.9%
137/868
17.3 (14.7-20.4)
67/868
8.3 (6.5-10.6)
70/868
9.0 (7.0-11.5)
18/97
18.7 (12.1-29)
9/97
8.6 (4.4-16.8)
9/97
10.1 (5.4-19)
TILs
≥50% p-value
0.767
0.402
0.351
Association of TILs with the hazard of ipsilateral recurrence. TILs
Hazard Ratio* (95% CI) Univariable analysis
<1%
Reference
1%-49.9%
0.97 (0.74-1.26)
0.81
0.92 (0.69-1.23)
0.56
≥50%
1.14 (0.69-1.89)
0.61
0.98 (0.56-1.73)
0.98
+10% increase
1.02 (0.96-1.10)
0.51
1.01 (0.93-1.10)
0.78
p-value
Hazard Ratio* (95% CI) Multivariable analysis*
p-value
Reference
*Hazard ratios adjusted by menopausal status, subtype, necrosis and therapy (surgery, radiotherapy and hormone therapy) in a Cox proportional hazard model.
Downloaded from http://annonc.oxfordjournals.org/ at Main Library of Gazi University on December 17, 2016
Ev./N
185x166mm (150 x 150 DPI)
Downloaded from http://annonc.oxfordjournals.org/ at Main Library of Gazi University on December 17, 2016
Boxplot of distribution of TILs as a continuous variable according to histological subtype. The median TILs level: Luminal A-like: <1%; Luminal B-like: <1%; Luminal B-like/Her-2 positive: 6%; Her-2 positive: 25%; Triple Negative: 8%.
226x179mm (150 x 150 DPI)
Downloaded from http://annonc.oxfordjournals.org/ at Main Library of Gazi University on December 17, 2016
Cumulative incidence of ipsilateral in situ recurrences (panel A) and ipsilateral invasive recurrences (panel B), by stromal lymphocytic infiltration. Cumulative incidence of ipsilateral recurrences by stromal lymphocytic infiltration (panel C). Effect of stromal lymphocytic infiltration, treated as a continuous variable, on the relative risk of ipsilateral event (panel D).
Downloaded from http://annonc.oxfordjournals.org/ at Main Library of Gazi University on December 17, 2016
Cumulative incidence of ipsilateral recurrences by TILs, according to subtype (p-value testing homogeneity of TILs effect among subtypes: 0.99) 184x249mm (150 x 150 DPI)