University of Groningen
Targeting breast cancer cells and their microenvironment
Nienhuis, Hilje Harmina
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Lower PD-1 and PD-L1 Expression in
Male Compared to Female Breast
Cancer Samples
H.H. Nienhuis1, J.W.M. Martens2, H.Timmer-Bosscha1, S.J.H. Waaijer1, B. van der Vegt3, A. Niezink1,
A.E. van Leeuwen-Stok 4, G.H. de Bock 5, P.J. Van Diest 6, E.G.E. de Vries1, C.H.M. van Deurzen7, C.P.
Schröder1
1 Department of Medical Oncology, University Medical Centre Groningen, Groningen, The Netherlands
2 Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Centre, Rotterdam, The Netherlands
3 Department of Pathology, University Medical Centre Groningen, The Netherlands 4 Dutch Breast Cancer Research Group (BOOG) Study Centre, Amsterdam, The Netherlands 5 Department of Epidemiology, University of Groningen, University Medical Centre Groningen,
Groningen, The Netherlands.
6 Department of Pathology, University Medical Centre Utrecht, Utrecht, the Netherlands. 7 Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Centre,
Rotterdam, The Netherlands
Manuscript in preparation
ABSTRACT
Background: Male breast cancer is rare compared to female breast cancer. The density of
tumour infiltrating lymphocytes (TIL)s and expression of programmed cell death ligand (PD-L)1 and programmed cell death (PD)-1 is unknown in this disease. We aimed to evaluate these factors in male breast cancer and compare them to female breast cancer.
Methods: The EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program
was conducted as global effort to retrospectively assess tumour tissue of men diagnosed with breast cancer between 1989-2009. From the Dutch patients enrolled in this study, 803 paraffin embedded primary tumours were collected and compared with tumour tissue from 434 female breast cancer patients, consecutively collected between 1996 - 2005. Tissue microarrays were constructed with three cores per sample. Sections (4 μm) were analysed for TILs on haematoxylin & eosin staining and immunohistochemically for PD-1 and PD-L1 expression.
Results: Compared to breast cancer in female patients, tissue from male breast cancer showed
similar TIL score (moderate to high expression in 19% vs 23%, (NS), less PD-1 expression and PD-L1 expression (0.4% versus 4.8% (odds ratio (OR) 0.15; 95% confidence interval (CI) 0.06-0.40;
P < 0.001; 13.2% vs 18.5%, OR 0.67; 95%CI 0.48-0.94; P = 0.02). Oestrogen receptor (ER) absence
was observed less frequently in male than female breast cancer (9% vs 24%), and was related to higher tumour cell PD-L1 expression in both sexes. TIL score of male- and female BC was related to PD-L1 and PD-1 expression in univariate logistic regression analysis.
Conclusion: PD-1 and PD-L1 expression in male breast cancer is rare and less frequent than in
female breast cancer. In breast cancer of both sexes, ER is inversely related with PD-L1 presence on tumour cells. Therefore, the immune cell make up of breast cancer appears to be influenced by tumor- rather than environment characteristics (such as patient gender).
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INTRODUCTION
Male breast cancer accounts for less than 1% of all cancers in men and 1% of all breast cancers (1). Many differences exist between male and female breast cancer including stage at diagnosis and nodal involvement, disease occurrence, treatment response and outcome (2) and tumour receptor status (3). Compared to female breast cancer, male breast cancers generally have a lower grade, are more often oestrogen receptor (ER) positive, and less frequently human epidermal growth factor (HER)2 positive (3).
Men with breast cancer are treated according to therapy regimens optimised for female breast cancer patients. Despite the generally more favourable characteristics, outcome is worse compared to women (4), implying that therapeutic interventions need to be re-examined.
Next to the tumour cells the microenvironment might provide a harbour for specific targets, as the male and female breast have a distinct, gender specific, embedding framework. This is why differences in the microenvironment may modulate breast cancer behaviour gender specifically (5). An important component of the breast cancer microenvironment includes the interaction between the tumour cells and the immune system. The immune system attempts to control and eliminate tumour cells (6). In both sexes presence of tumour infiltrating lymphocytes (TILs) is correlated with better patient outcome, which is breast cancer subtype dependent (7-9). Cancer cells develop mechanisms to escape immune surveillance (10). This so-called immunoediting, includes the upregulation of immune checkpoint proteins on cancer cells as well as immune cells. Blocking of these proteins and their ligands, cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed death (PD)-1 and programmed dead ligand (PD-L)1, results in anti-tumour activity across different malignancies including although in a modest way in breast cancer (11, 12). In female breast cancer samples, expression of PD-L1 and PD-1 measured immunohistochemically is present in respectively 0-70% (PD-L1) and 16-60% (PD-1) of tumour and/or immune cells respectively (13-15). Expression of both PD-1 and PD-L1 is correlated with a more aggressive histology and worse patient outcome (13, 14). For male breast cancer, expression pattern of these factors and their relation with clinicopathological parameters is unknown which precluded until now comparison with female breast cancer.
Therefore, we aim to evaluate in this study the expression pattern of PD-L1 and PD-1 in relation to TILs and clinicopathological parameters in primary breast tumours of males. Furthermore, we compare the presence and expression patterns to female breast cancer samples.
MATERIALS AND METHODS
Patient material
The EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program was conducted as global effort to retrospectively assess tumour tissue of men diagnosed with breast cancer between 1989-2009. Male patients in the Netherlands were identified through the Dutch Cancer
Registry. Paraffin embedded male breast cancer tissue was retrospectively collected by the ‘Borstkanker onderzoeksgroep’ (BOOG). Tissue of the primary tumour was available in 803 of the 1,487 Dutch patients diagnosed in this period. Follow up was recorded until December 2013. Archival tissue of all patients was handled according to the Dutch Code for Proper Use of Human Tissue (www.federa.org).
Female breast cancer tumour tissue arrays (TMAs) from formerly published studies were used to compare with the male samples (16, 17). These TMAs comprise tumour tissue of 453 consecutive patients treated for primary operable invasive breast cancer at the University Medical Centre Groningen between January 1996-and December 2005.
Tissue microarray construction
From each paraffin embedded tumour, representative cores selected by a pathologist, were taken. For TMA construction three needle core biopsies of 0.6 mm in diameter from a paraffin embedded tissue block were allocated to a blank recipient block using an Automated Tissue Arrayer ATA-27 (Beecher Instruments Inc) (17). From these TMA blocks, slices of 4 µm were cut. The TMAs had previously been centrally reviewed (CHMvD) for histology and ER, progesterone receptor (PR) and HER2 expression (9). The TMA also contained tissues with known expression for PD-1 and PD-L1 as positive controls.
Female breast cancers TMAs were earlier prepared in an analogous way (16, 17). Tumour infiltrating lymphocytes
Haematoxylin and eosin (H&E) staining was used to quantify TILs. TMA slides were deparaffinised and stained. Slides were digitalised by using Digital Slide Scanner NanoZoomer and NDP software (Hamamatsu). The presence of TILs was quantified based on the recently validated International TILs Working Group 2014 recommendations (18-20). TILs were assessed by determining the percentage of the stromal area occupied by TILs. All mononuclear cells were included in the analysis, polynuclear cells were excluded. Scoring was performed by two independent observers (HHN and SJHW), blinded for the clinical characteristics. Random samples of each staining were validated by an expert breast pathologist (BvdV). The TIL density of the three cores was categorized into low density (≤ 1%), moderate (> 1 and < 30%) and high (≥ 30%). Based on the variations of TIL presence, the average TIL density (AvTIL) score of the three cores was determined and dichotomized (low AvTIL (average TIL density ≤ 1%) moderate/high AvTIL (average TIL density > 1%). The maximal TIL density (maxTIL) was also determined based on the maximal score of the categories of the three cores.
Immunohistochemistry
Immunohistochemical staining of PD-L1 and PD-1 was performed in one batch per factor. Heat induced antigen retrieval was performed in citrate buffer (pH 6.0) for PD-L1 and Tris-HCl buffer (pH 9.0) for PD-1. Endogenous peroxidase was blocked with 0.3% H202 in phosphate buffered saline (PBS; Cl2H3K2Na3O8P2, pH 7.4). Aspecific binding was blocked with human
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serum. Primary antibodies (PD-L1: clone SP142, Roche; PD-1: clone MRQ-22, Acris) were diluted in PBS supplemented with 1% bovine serum albumin. Envision anti-rabbit for PD-L1 and anti-mouse for PD-1 (DAKO) served as secondary antibodies. Staining was visualised using 3,3 0-diaminobenzidine and haematoxylin counterstaining. Positive and negative controls (immunoglobulin class-matched control sera) for each staining were included on positive control and female breast cancer tissue.
Slides were digitised with a Digital Slide Scanner NanoZoomer and NDP software (Hamamatsu). Patients were included for analysis when ≥2 cores with tumour and stromal cells were present. PD-1 and PD-L1 expression was determined for breast cancer and immune cells. For breast cancer cells, also localisation of expression was determined (nuclear, cytoplasmic, membrane). Scoring of the digitalised images was performed by two observers (HHN and SJHW), who were blinded for the clinicopathologic characteristics. All samples scored as positive, were validated by an expert breast pathologist (BvdV). If any of the cores were positive, the sample was considered positive. Statistical analysis
Overall survival (OS) was defined as the interval between the date of diagnosis and the documented date of death or last follow up alive, relapse-free survival (RFS) as the time between the date of diagnosis and the date of recurrence, death, or last follow-up alive. The relationship between clinicopathological patient characteristics and immunohistochemical expression of the microenvironmental factors was investigated using univariate logistic regression analysis. The prognostic value of the microenvironmental factor expression was determined using univariate and multivariate Cox regression analysis.
Clinicopathological patient characteristics and tumour microenvironmental factors were compared between male and female patients by logistic regression analysis. For logistic and cox regressions parameters showing a P-value < 0.05 or odds (OR) or hazard ratio (HR) of ≥ 3 in the univariate analysis were included in the stepwise multivariate analysis. All P-values were two-sided and P < 0.05 was considered statistically significant. Statistical analyses were performed with IBM SPSS version 24.0.
RESULTS
Patient characteristics
Out of 1487 male patients diagnosed within the Netherlands from 1989 to 2009, 803 patients could be included for analysis next to 434 female patients. The inclusion steps for the male breast cancer patients are illustrated in a flow diagram (supplemental Figure 1). Patient and tumour characteristics are presented in Table 1. The median age of the male patients was 67 years versus 58 years for female patients. In this set, 91% of male tumours expressed ER, and 5% showed HER2 overexpression. In female tumours, ER expression was present in 76% and 11% showed HER2 overexpression.
Table 1 | Patient characteristics at moment of primary tumour collection
Men n = 803 Women n = 434
Age at diagnosis, median (range) in years 67 (25-99) 58 58 (27-91)
OS, median (range) in months 76 0-299 48 48 (0-134)
RFS, median (range) in months 74 0-299 43 43 (0-134)
Tumour grade n (%) I 183 24% 109 25% II 395 53% 187 43% III 171 23% 135 31% Missing 54 3 T status n (%) T1 316 47% 254 60% T2 or higher 352 53% 171 40% Missing 135 9 N status n (%) N0 314 50% 224 53% N1 or higher 317 50% 196 47% Missing 172 14 ER n (%) Negative 72 9% 93 24% Positive 703 91% 288 76% Missing 28 53 PR n (%) Negative 28 4% 144 37% Positive 746 96% 242 73% Missing 29 48 HER2 n (%) Negative 703 95% 359 89% Positive 38 5% 44 11% Missing 62 Subtype n (%) ER-HER2- 62 9% 71 20% ER+HER2- 621 86% 247 69% ER+HER2+ 29 4% 26 7% ER-HER2+ 7 1% 15 4%
one of the receptor status unknown 143 75
Adjuvant treatment
chemotherapy 165 21% unknown
hormonal therapy 356 44% unknown
radiotherapy 302 38% unknown
Abbreviations: n: number, OS: overall survival, RFS: relapse free survival, T status: tumour status, N status: nodal-status, ER:
oestrogen receptor, PR: progesterone receptor, HER2: human epidermal growth factor receptor 2
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TILs, PD-1 and PD-L1 expression
MaxTIL was low in 28% of male tumours, moderate in 64% and intermediate in 9%. For female tumours, the maxTIL was low in 35%, moderate in 57% and intermediate in 8%. AvTIL was low in 81% of male tumours and in 77% of the female tumours (Figure 1).
PD-1 was expressed in tumours of male patients in only 0.4% by tumour cells and in 0.3% by immune cells. Only 4.8% tumours of female patients showed PD-1 expression, 0.5% by tumour cells and 4.3% by immune cells (Figure 1).
In tumours of male patients, cytoplasmic PD-L1 expression was present in 2.5% of tumour cells, in 12.1% of immune cells and in 13.2% of tumour and/or immune cells. In 8.8% of the tumours of male patients also nuclear tumour cell staining was observed. When nuclear staining was included, 20.3% of the tumours showed PD-L1 expression (Figure 1).
In tumours of female patients, cytoplasmic PD-L1 expression was present in 2.2% of tumour cells, in 18.2% of immune cells and in 18.5% of tumour and/or immune cells. In contrast to the tumours of the male patients, no nuclear staining was observed in female breast cancer tumours. PD-1 and PD-L1 related to adverse factors in male breast cancer, but not OS or RFS
Higher AvTIL density correlated with PD-L1 presence in male tumours (Figure 2, supplemental Table 1 and 2; data of female tumours in supplemental Table 4 and 5). The limited number of PD-1 positive male breast cancer tumours, precluded logistic regression. PD-L1 expression by tumour cells was correlated with adverse factors: higher tumour grade and absence of ER/PR, but not HER2 expression (Figure 2 and supplemental Table). PD-L1 expression in immune cells was also correlated with adverse factors: higher tumour grade and positive nodal stage. Correlation with adverse factors differed between L1 expression in tumour cells and immune cells. Higher PD-L1 expression by both tumour and immune cells was correlated with higher TIL score.
Age, tumour size, nodal status, ER-status and PR-status were associated with OS and RFS in male patients (Supplemental Table 3), which is in line with literature (21) and partly with findings in female patients (Supplemental Table 3). TIL density, PD-1 and PD-L1 expression were not associated with OS or RFS in male breast cancer patients.
PD-1 and PD-L1 expression is lower in male than female breast cancer TIL density, PD-1 and PD-L1 expression differed between tumours of male and female patients (Figure 3). Tumours of male patients had a higher MaxTIL density while PD-1 expression was less frequently present, cytoplasmic PD-L1 expression and PD-L1 expression in immune cells were less frequently present than in tumours of female breast cancer patients. PD-L1 expression in tumour cells was similar in male and female patients.
In line with previous analyses (9), the male patients were diagnosed at higher age, had larger tumours which expressed more frequently PR, less frequently HER2 and higher MaxTIL than female breast cancer patients.
PD-L1 positive PD-L1 negative PD-1 positive PD-1 negative PD-1 Male Female 0 1 2 3 4 5 % ex pr es si on in pa tie nt s w ith po si tiv e sc or e PD-L1 Male Female 0 5 10 15 20
25 tumour cells cytoplasmic
immune cells combined expression nuclear staining % ex pr es si on in pa tie nt s w ith po si tiv e sc or e a b e f c d g h TIL 0 TIL 1 TIL 2 TILmax Male Female 0 25 50 75 100 negative low high % TI L de ns ity TILav Male Female 0 25 50 75 100 negative positive % TI L de ns ity i j k l m n p o q r x200 x200 x200 x200 x400 x400 x400 x400
Figure 1| PD-L1, PD-1 expression and TIL score
Upper part of the fi gure shows samples of PD-L1 and PD-1 staining on left panels (a – h) and TIL density on right panels (i – n). Quantifi cation of the scoring is depicted in the lower fi gures (o - r). PD-L1 staining (a-d) and PD-1 staining (e-h) on upper panels (x200 magnifi cation) and lower panels (x400 magnifi cation). Positive PD-L1 staining (a, c) of tumour as well as stromal cells. A PD-L1 negative samples is also depicted (b, d). Positive PD-1 staining (e, g) of tumour and stromal cells. A PD-1 negative sample is also depicted (f, h). TIL density on upper panels (x200) and lower panels (x400). TIL score was scored as: 1 (0-1% stromal TILS) (I, k) , TIL score 2 (>1%- < 30% stromal TILs) (j, l) and TIL score 3 (≥ 30% stromal TILS) (m, n) are depicted. For data analysis the average TIL density (AvTIL) score of the three cores was determined and dichotomized (low AvTIL (average TIL density ≤ 1%) moderate/high AvTIL (average TIL density > 1%).
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Average TIL score PD-L1 tumour and immune cells PD-L1 tumour cells PD-L1 immune cells PD-1 tumour and immune cells 0. 01 0. 1 1 10 100 Odds ratio (log scale ) 0. 01 0. 1 1 10 100 H igher av er age T IL scor e PD -L1 posi tiv e H ER 2 posi tiv e PR p os itiv e E R p os itiv e N 1 or hi gher T2 or hi gher Tum our gr ade I II Tum our gr ade I I Age > 50 0. 01 0. 1 1 10 100 0. 01 0. 1 1 10 100 0. 01 0. 1 1 10 100 0. 01 0. 1 1 10 100 low er P D -L1 pr ev al en ce hi ghe r P D -L1 pr ev al en ce 0. 01 0. 1 1 10 100 low er P D -L1 pr ev al en ce hi ghe r P D -L1 pr ev al en ce 0. 01 0. 1 1 10 100 H igher av er age T IL scor e PD -L1 posi tiv e H ER 2 posi tiv e PR p os itiv e E R p os itiv e N 1 or hi gher T2 or hi gher Tum our gr ade I II Tum our gr ade I I Age > 50 low er TI L pr ev al en ce hi ghe r TI L pr ev al en ce 0. 01 0. 1 1 10 100 low er P D -L1 pr eva le nce hi ghe r P D -L1 pr eva le nce Figur e 2 |O dds ratio f
or patient and tumour charac
ter istics in r elation with PD -L1, PD -1 expr ession and TIL scor e O dd s r at io o f s eve ra l p ar am et er s i s d ep ic te d o f p at ie nt a nd t um ou r c ha ra ct er ist ic s i n r el at io n w ith t he s tu dy p ar am et er s i s s ho w ed w ith a pp ro pr ia te 9 5% C I c al cu la te d b y l og ist ic r eg re ss io n. Co rre la tio ns i n m al e p at ie nt s a re d ep ic te d o n t he u pp er s p an el s a nd l ow er p an el s r ep re se nt c or re la tio ns i n f em al e p at ie nt s. S ta tis tic al ly s ig ni fi c an t c or re la tio ns a re b ol d. L im ite d P D -1 p os iti ve m al e br ea st c an ce r t um ou rs , p re clu de d l og ist ic r eg re ss io n.
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Figure 3 | Odds ratio of patient and tumour characteristics and PD-1, PD-L1 and TIL score between
male and female breast cancer
Odds ratio of several parameters is depicted with appropriate 95% CI. Higher odds ratio implicates a higher prevalence of expression in tumours of male breast cancer patients.
DISCUSSION
This is the first study of TIL density, PD-1 and PD-L1 expression in male breast cancer. Here we show in male breast cancer samples that PD-L1 expression correlates with adverse factors such as lymph node positive disease and higher tumour grade. TIL density, PD-1 and PD-L1 expression correlated strongly with each other in male as well as female breast cancers. In male breast cancers PD-L1 expression was less frequently present than in the female breast cancers. This most likely coincides with the higher prevalence of ER positive breast cancers in males as in both sexes immune checkpoint presence was related to ER expression. Therefore, the immune cell make up of breast cancers seems to relate more to tumour- than gender environment characteristics.
While in female breast cancer PD-L1 expression of immune cells correlated with ER negative tumours, in male breast cancer this was the case for PD-L1 expression in tumour cells. PR absence in male breast cancer was even stronger correlated with combined PD-L1 expression in tumour and immune cells.
For other malignancies, such as non small cell lung cancer, tumours sensitive to checkpoint inhibition are being selected based on PD-L1 expression. However, the precise predictive role of PD-L1 as biomarker for PD-1/PD-L1 checkpoint inhibition antibody therapy is ongoing. Although PD-L1 expression correlates with clinical activity of anti PD-1 and anti PD-L1 antibodies, a recent meta-analysis showed that 17% of the patients without PD-L1 expression still responded (22). The PD-L1 staining we used, serves also as companion diagnostic in clinical studies and is FDA
Univariate comparison men and women
0.1 1 10
PD-L1 immune cellsPD-L1 tumour cells
PD-L1 immune and tumour cellsPD-1 immune and tumour cells
Maximal TIL scoreAverage TIL score
HER2 PR ER N status T status Tumour grade Age at diagnosis higher prevalence
in women higher prevalencein men
Odds ratio (log scale) chapter3
approved. However, the debate regarding the best suitable antibody for determining PD-L1 expression immunohistochemically is still continuing (23) and illustrated by reported range of 0-50% of PD-L1 positive tumours acros female breast cancer studies.
We did not observe a prognostic role for PD-L1, PD-1 expression or TIL density in the total group of patients. Some studies have found a prognostic role for PD-L1 expression in basal breast cancers (24-26), while other studies found no correlation with survival (25, 27). A possible explanation for the absence of a correlation in our study is the limited data regarding the survival of male as well as female patients.
PD-L1 expression in breast cancer, especially ER positive, is relatively low compared to other malignancies such as melanoma. The expression of prostate cancer, another relatively indolent, hormone sensitive malignancy has similar low expression of PD-L1 of approximately 10% in recent studies (28, 29). In men, breast cancer mainly consists of the genetically stable, luminal subtype. In women this subtype is related to reduced immunogenicity and lower PD-L1 compared to other subtypes. This does not exclude these patients from responding to immunotherapy as an objective response rate of 12% to PD-1 antibody pembrolizumab was observed in women with luminal breast cancer (30). Thus also genetically stable tumours can show immunogenicity, implying that male breast cancer patients cannot be excluded a priori for response on immunotherapy. Further research is needed to study mechanisms for selecting luminal (male) breast cancers or to activate the immune system to enhance response to immunotherapy.
Our study has some limitations. As we have used retrospective, archival samples there exists heterogeneity with regards to several aspects including treatment of patients. This makes the survival analyses of smaller significance compared to prospective studies. Next, due to the scarceness of the samples, we have used TMAs instead of whole slides. Although in breast cancer, tumour characteristics such as ER status on TMA correspond well with whole tissue sections (31), this may not be the case for microenvironment. Moreover, using ex vivo analysis for studying the immune system provides only a snapshot of information, as this system is highly dynamic. For future studies in vivo analyses by for example PET-imaging could provide more in depth whole body information at several moments in time.
In conclusion, our descriptive study shows that higher TIL density and PD-L1 expression in male breast cancer are correlated with adverse prognostic factors such as higher nodal status. Moreover, PD-1 and PD-L1 expression in tumours of males are lower compared to female breast cancer samples. This study improves the insights in differences between male and female breast cancer. Eventually this can support intervention strategies to optimize treatment for male breast cancer.
Acknowledgements
A.M. Timmermans (Rotterdam) for preparation of the TMAs. The International Male Breast Cancer Program and this work is supported by grants from the Breast Cancer Research Foundation, the Dutch Pink Ribbon Society, the Dutch Cancer Society, the European Breast Cancer Council, the Susan G. Komen for the Cure, the Swedish Breast Cancer Association and the Erasmus MC
Cancer Institute. The authors thank the registration of the Comprehensive Cancer centres for the collection of data for the Netherlands Cancer Registry and the scientific staff of the Netherlands Cancer Registry.
Funding
This work was supported by Dutch Cancer Society grant RUG 2010-4739 and 2010 Dutch Pink Ribbon Foundation grant Male Breast.
Disclosure
The authors have declared no conflicts of interest.
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30. Rugo H, Delord J, Im S et al. Preliminary efficacy and safety of pembrolizumab (MK-3475) in patients with PD-L1–positive, estrogen receptor-positive (ER+)/HER2-negative advanced breast cancer enrolled in KEYNOTE-028. Cancer Res 2016; 76: 4 Supplement: abstract S5-07.
31. Camp RL, Charette LA, Rimm DL. Validation of tissue microarray technology in breast carcinoma. Lab Invest 2000; 80: 1943-1949.
chapter3
SUPPLEMENTAL DATA
Association of TILs, PD-1 and PD-L1 expression with clinicopathological parameters in female patients
AvTIL score was higher in tumours of younger patients (OR 0.39 95%CI: 0.24 – 0.63 P < 0.001), tumours with higher differentiation grade (OR 6.45 95%CI 2.99 – 13.92 P < 0.001), ER negative tumours (OR 0.44 95% CI 0.26 - 0.74 P = 0.002) and HER2 overexpression (OR 2.64 95% CI 1.35 – 6.16 P = 0.004) in tumours of women. AvTIL was higher in tumours expressing PD-L1 and PD-1 (Supplemental Table 4). Multivariate analysis showed that AvTIL expression was higher in tumours of patients with younger age (OR 0.38; 95%CI 0.21 – 0.69; P = 0.002) and PD-L1 presence (OR 12.39; 95%CI 6.33 – 24.27; P < 0.001) (Supplemental Table 5).
Total PD-1 expression was correlated with younger patient’s age (OR 0.35 95%CI 0.14 – 0.87 P = 0.023). PD-1 expression was also correlated with higher TIL density.
Presence of cytoplasmic PD-L1 staining (in tumour cells and immune cells) was associated with higher tumour differentiation grade (OR 7.76; 95%CI 3.14 – 19.18 for grade III; P < 0.001), ER absence (OR 0.28; 95% CI 0.16 – 0.49; P < 0.001) and PR absence (OR 0.46; 95% CI 0.26 – 0.79 P = 0.005). Multivariate logistic regression showed that PD-L1 presence was correlated with higher tumour differentiation grade (OR 4.26; 95% CI 1.34 – 13.53; P = 0.014; for grade III) and ER absence (OR 0.37; 95%CI 0.19 – 0.73; P = 0.004) in tumours of women. PD-L1 expression was also correlated with higher AvTIL density. PD-L1 expression in tumour cells was not correlated with clinicopathological parameters, but was associated with higher TIL score.
Presence of PD-L1 expression in immune cells was correlated with higher tumour differentiation grade (OR 9.42 95% CI 3.56 – 24.90; P < 0.001 for grade III), ER absence (OR 0.28 95% CI 0.16 – 0.49 P < 0.001) and PR absence (OR 0.46 95% CI 0.26 – 0.79 P = 0.005). Multivariate analysis showed that PD-L1 expression in immune cells was associated with higher tumour grade (OR 4.26; 95%CI 1.35 – 13.51; P = 0.014) and ER absence (OR 0.35; 95%CI 0.18 – 0.68; P = 0.002). PD-L1 expression in immune cells was also higher in tumours with higher TIL density.
In female patients, tumour differentiation grade, tumour size and positive nodal-status were associated with OS (Supplemental Table 2). Presence of TILs, PD and PD-L1 expression in the total group of patients was not associated with OS. We did observe a trend towards worse survival of PD-L1 positive triple negative breast cancer compared to PD-L1 negative triple negative breast cancer patients (men: median OS 76 months for PD-L1 negative versus 20 months for PD-L1 positive; women: median OS 136 months for PD-L1 negative versus 90 months for PD-L1 positive). Multivariate analysis showed that higher tumour differentiation grade was predictive for worse OS (OR 2.94; 95%CI 1.34–6.43; P = 0.007; for grade III). Also larger tumour size was associated with lower OS (OR 1.68; 95%CI 1.02 – 2.75; P = 0.04).
Univariate Cox regression analysis of clinicopathological parameters and TIL density, PD-1 and PD-L1 expression in association with RFS showed a similar pattern as for OS (Supplemental Table 5). Presence of PD-L1 expression, PD-1 expression and TIL score were not associated with RFS.
Supplemental Figure 1
Inclusion of 803 patients with invasive breast cancer. Clinical data of 1487 patients Clinical data of 1487 patients
Exclusion
Male gender not confirmed 12 Non invasive breast tumour 142 No or too limited tissue available for analysis 530
803 male patients with invasive breast cancer 803 male patients with invasive breast cancer chapter3
Supplemen tal T able 1| A ssociation of TILs , PD -1 and PD -L1 expr
ession with clinicopatholog
ical paramet
ers in male patients
Pa tie nt c ha ra cte rist ic s Av er age T IL s core PD -L 1 i m m un e a nd t um ou r c el ls PD -L 1 t umo ur c el ls PD -L 1 i mm une c el ls OR 95 % C I P-va lu e OR 95 % C I P-va lu e OR 95 % C I P-va lu e OR 95 % C I P-va lu e A ge a t d ia gno si s n = 8 03 n = 6 80 n = 6 80 n = 6 80 ≤ 50 re f re f re f re f > 50 0.9 9 0. 55 1.7 8 0. 97 2.4 8 0. 88 6.9 9 0. 09 1.7 1 0. 22 13 .10 0. 61 2. 22 0. 78 6. 26 0.13 Tumo ur gr ad e n = 7 49 0. 80 n = 6 43 <0 .01 n = 6 43 <0 .01 n = 6 43 <0 .01 I re f re f re f re f II 1.15 0. 74 1.8 1 0. 53 1.3 5 0. 70 2. 62 0. 37 1.3 9 0.1 4 13 .4 4 0. 88 1.4 3 0.7 2 2. 83 0. 31 III 1.17 0. 69 1.9 9 0. 56 3. 28 1.6 6 6. 47 <0 .01 10 .5 5 1.3 3 83 .47 0. 03 3. 08 1.5 2 6. 24 <0 .01 T s ta tu s n = 6 68 n = 5 68 n = 5 68 n = 5 68 T1 re f re f re f re f T2 o r h ig her 1.12 0. 76 1.6 4 0. 56 1.0 0 0. 62 1.6 2 1.0 0 1.6 8 0. 50 5. 65 0.4 0 1.0 2 0. 62 1.6 8 0.9 5 N s ta tu s n = 6 31 n = 5 35 n = 5 35 n = 5 35 N0 re f re f re f re f N1 o r h ig her 1.4 7 0.9 9 2.19 0. 05 1. 70 1.0 2 2. 84 0. 04 1.5 8 0. 51 4. 88 0. 43 1. 78 1.0 4 3. 04 0. 03 ER n = 7 75 n = 6 64 n = 6 64 n = 6 64 ne gat iv e re f re f re f re f po sit iv e 0. 74 0. 42 1.3 9 0. 31 1.2 6 0. 52 3. 04 0. 61 0. 27 0. 09 0. 87 0. 03 1.8 1 0. 63 5.1 4 0. 27 PR n = 7 74 n = 6 70 n = 6 70 n = 6 70 ne gat iv e re f re f re f re f po sit iv e 0.9 0 0. 36 2. 26 0. 82 0. 49 0.1 6 1.5 3 0. 22 0.1 0 0. 03 0.4 0 <0 .01 0. 63 0.1 8 2. 25 0.4 8 HE R2 n = 7 41 n = 6 46 n = 6 46 n = 6 46 ne gat iv e re f re f re f re f po sit iv e 1.9 2 0.9 5 3. 91 0. 07 1.5 8 0. 63 3.9 8 0. 33 2. 76 0. 60 12 .6 4 0.19 1.0 7 0. 36 3.13 0. 91 PD -L 1 i m m un e c el ls an d t um or c el ls n = 6 80 ne gat iv e re f po sit iv e 4. 92 3. 08 7.8 6 <0 .01 Av er age T IL s core n = 6 80 n = 6 80 n = 680 low re f re f re f m od er ate /in te rm ed ia te 4. 92 3. 08 7.8 6 <0 .01 5. 86 2.19 15 .6 8 <0 .01 4. 74 2. 93 7.6 9 <0 .01 M ax im al T IL s core n = 6 80 to o l im ite d p os iti ve sa mp le s t o c al cu la te n = 6 80 low re f re f M od er at e/ hi gh 5.7 6 2. 47 13 .4 4 <0 .01 5. 11 2.19 11 .9 6 <0 .01 A bb re vi at ion s: n : n um ber , O R: o dd s r ati o, C I: c on fi d en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: hu m an e pi der m al g ro w th f ac to r r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th l ig an d 1 , T IL : t um ou r i nfi lt ra tin g l ym ph oc yt e
3
Supplemen tal T able 2| M ultivar iat e analysis of association of TILs , PD -1 and PD -L1 expr
ession with clinicopatholog
ical paramet ers in male br east cancer samples Pa tie nt c ha ra cte rist ic s Av er age T IL s core PD -L 1 i m m un e a nd t um ou r c el ls PD -L 1 t umo ur c el ls PD -L 1 i mm une c el ls OR 95 % C I P-va lu e OR 95 % C I P-va lu e OR 95 % C I P-va lu e OR 95 % C I P-va lu e A ge a t d ia gno si s ≤ 50 > 50 Tumo ur gr ad e <0 .01 <0 .01 <0 .01 I re f re f re f II 1.4 0 0. 63 3.13 0. 41 1.1 4 0.11 11 .5 4 0. 91 1.6 0 0. 70 3. 69 0. 29 III 4.0 0 1.7 7 9. 04 <0 .01 8. 59 1.0 5 70 .6 0 0. 05 3. 75 1.5 9 8. 84 <0 .01 N s ta tu s N0 N1 o r h ig her ER negat iv e po sit iv e PR negat iv e re f po sit iv e 0. 11 0. 02 0. 54 <0 .01 PD -L 1 i m m un e c el ls a nd tumo ur c el ls ne gat iv e re f po sit iv e 4. 92 * 3. 08 7.8 6 <0 .01 Av er age T IL s core low re f re f re f m od er ate /in te rm ed ia te 4. 43 2. 55 7.6 9 <0 .01 7. 72 2.4 0 24 .8 7 <0 .01 4. 37 2. 49 7.6 7 <0 .01 M ax im al T IL s core low Mod er at e/ hi gh A bb re vi at ion s: n : n um be r, O R: o dd s r at io , C I: c on fid en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: h um an e pi de rm al gr ow th f ac to r r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th li ga nd 1 , T IL : t um ou r i nfi ltr at in g l ym ph oc yt e * O R b as ed o n u ni va ria te a na ly se s a s o nl y o ne p ar am et er s l ef t chapter3
3
Supplemen tal T able 3 | univar iat e and multivar iat e co
x analysis of association bet
w een TILs , PD -1 and PD -L1 expr
ession and clinicopatholog
ical paramet
ers
with o
verall sur
vival in male and f
emale br
east cancer samples
Pa tie nt c ha ra cte rist ic s uni var ia te ana ly si s mu lt iv ar ia te ana ly si s uni var ia te ana ly si s mu lt iv ar ia te ana ly si s m en n = 8 03 n = 5 61 wo m en n = 4 34 n = 6 7 HR 95 % C I P-va lu e HR 95 % C I P-va lu e HR 95 % C I P-va lu e HR 95 % C I P-va lu e A ge a t d ia gno si s n = 4 48 n = 7 1 ≤ 50 re f re f re f > 50 2.40 1.6 0 3. 59 <0 .01 1. 99 1.2 4 3.1 8 <0 .01 0. 92 0. 56 1.51 0. 79 Tumo ur gr ad e n = 4 21 0. 80 n = 3 9 I re f re f <0 .01 re f <0 .01 II 1.0 5 0. 83 1.3 3 0. 66 6. 74 0. 88 51 .8 5 0. 07 1.5 7 0. 71 3. 48 0. 27 III 1.1 0 0. 83 1.4 5 0. 50 22. 44 3. 04 16 5. 46 <0 .01 2.9 4 1.3 4 6. 43 <0 .01 T s ta tu s n = 3 55 <0 .01 n = 7 1 T1 re f re f re f re f T2 o r h ig he r 1. 67 1.3 5 2. 07 <0 .01 1. 66 1.3 0 2.1 <0 .01 1. 92 1.2 0 3. 08 <0 .01 1. 68 1.0 2 2. 75 0. 04 N s ta tu s n = 6 30 n = 6 8 N0 re f re f N1 o r h ig her 1. 45 1.1 6 1.8 1 <0 .01 1. 71 1.0 6 2. 77 0. 03 ER n = 4 28 n = 5 8 ne gat iv e re f re f po sit iv e 0. 66 0. 49 0. 89 <0 .01 1.0 4 0. 58 1.8 9 0. 89 PR n = 4 32 n = 6 2 ne gat iv e re f re f re f po sit iv e 0. 22 0.1 4 0. 33 <0 .01 0.1 6 0.1 0 0. 28 <0 .01 1.5 6 0. 91 2. 69 0.11 HE R2 n = 4 04 n = 6 6 ne gat iv e re f re f po sit iv e 0. 91 0. 58 1.4 3 0. 69 2. 00 0.9 9 4. 07 0. 06 PD -L 1 i m m un e c el ls a nd tumo ur c el ls n = 3 82 n = 6 7 ne gat iv e re f re f po sit iv e 1.6 0 0. 79 1.4 3 0. 69 1.31 0. 74 3. 33 0. 36 PD -1 e xp re ssi on n = 3 82 n = 7 0 ne gat iv e re f re f po sit iv e 0.7 7 0. 25 2. 41 0. 66 0.9 8 0. 31 3.11 0. 97 Av er age T IL s core n = 4 48 n = 7 1 low re f re f m od er ate /in te rm ed ia te 0. 87 0. 68 1.1 0 0. 23 1.0 2 0. 59 1.7 5 0.9 6 A bb re vi at ion s: n : n um be r, O R: o dd s r at io , C I: c on fid en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: h um an e pi de rm al gr ow th f ac to r r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th li ga nd 1 , T IL : t um ou r i nfi ltr at in g l ym ph oc yt e Supplemen tal T able 4 | univar iat e and multivar iat e co
x analysis of association bet
w een TILs , PD -1 and PD -L1 expr
ession and clinicopatholog
ical paramet
ers
with r
elapse fr
ee sur
vival in male and f
emale br
east cancer samples
uni var ia te ana ly si s mu lt iv ar ia te ana ly si s uni var ia te ana ly si s mu lt iv ar ia te ana ly si s Pa tie nt c ha ra cte rist ic s m en n = 8 03 n = 5 27 wo m en n = 4 34 n = 6 4 HR 95 % C I P-va lu e HR 95 % C I P-va lu e HR 95 % C I P-va lu e HR 95 % C I P-va lu e A ge a t d ia gno si s n = 4 62 n = 7 1 ≤ 50 re f re f re f > 50 2. 30 1.5 5 3. 42 <0 .01 1. 86 1.1 6 2. 97 0. 01 0. 58 0. 36 0. 93 0. 02 Tumo ur gr ad e n = 4 33 0. 65 n = 7 1 I re f re f <0 .01 re f <0 .01 II 1.11 0. 88 1.4 0 0. 37 4. 20 1.4 8 11 .9 5 <0 .01 3.1 2 1.0 8 9. 00 0. 04 III 1.1 0 0. 84 1.4 5 0. 49 8. 45 3. 02 23. 68 <0 .01 7. 10 2. 50 20 .18 <0 .01 T s ta tu s n = 3 65 n = 7 1 T1 re f re f re f <0 .01 re f T2 o r h ig he r 1. 68 1.3 6 2. 07 <0 .01 1. 69 1.3 2 2.17 <0 .01 2. 51 1.5 5 4. 05 <0 .01 1. 90 1.1 4 3.1 6 0. 01 N s ta tu s n = 6 31 n = 6 8 N0 re f re f N1 o r h ig her 1. 49 1.2 0 1.8 5 <0 .01 2. 07 1.2 7 3. 39 <0 .01 ER n = 4 42 n = 5 8 ne gat iv e re f re f po sit iv e 0. 70 0. 52 0.9 4 0. 02 1.0 4 0. 57 1.9 0 0.9 0 PR n = 4 45 n = 6 0 ne gat iv e re f re f re f po sit iv e 0. 22 0. 22 0. 33 <0 .01 0.1 9 0.1 0 0. 35 <0 .01 0. 82 0. 49 1.3 7 0.4 6 HE R2 n = 4 17 n = 6 7 ne gat iv e re f re f po sit iv e 0. 93 0. 60 1.4 6 0.7 3 2. 21 1.15 4. 23 0. 02 PD -L 1 i m m un e c el ls a nd tumo ur c el ls n = 3 95 n = 6 7 ne gat iv e re f re f po sit iv e 1.0 4 0. 78 1.3 9 0. 80 1.2 5 0. 71 2. 23 0.4 4 PD -1 e xp re ssi on n = 3 95 n = 6 8 ne gat iv e re f re f po sit iv e 1.19 0. 38 3. 70 0.7 7 0. 62 0.15 2. 54 0. 51 Av er age T IL s core n = 4 62 n = 7 0 low re f re f m od er ate /in te rm ed ia te 0. 81 0. 64 1.0 3 0. 09 0. 80 0. 45 1.4 4 0.4 6 A bb re vi at ion s: n : n um be r, O R: o dd s r at io , C I: c on fid en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: h um an e pi de rm al gr ow th f ac to r r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th li ga nd 1 , T IL : t um ou r i nfi ltr at in g l ym ph oc yt e chapter3
3
with r
elapse fr
ee sur
vival in male and f
emale br
east cancer samples
uni var ia te ana ly si s mu lt iv ar ia te ana ly si s uni var ia te ana ly si s mu lt iv ar ia te ana ly si s Pa tie nt c ha ra cte rist ic s m en n = 8 03 n = 5 27 wo m en n = 4 34 n = 6 4 HR 95 % C I P-va lu e HR 95 % C I P-va lu e HR 95 % C I P-va lu e HR 95 % C I P-va lu e A ge a t d ia gno si s n = 4 62 n = 7 1 ≤ 50 re f re f re f > 50 2. 30 1.5 5 3. 42 <0 .01 1. 86 1.1 6 2. 97 0. 01 0. 58 0. 36 0. 93 0. 02 Tumo ur gr ad e n = 4 33 0. 65 n = 7 1 I re f re f <0 .01 re f <0 .01 II 1.11 0. 88 1.4 0 0. 37 4. 20 1.4 8 11 .9 5 <0 .01 3.1 2 1.0 8 9. 00 0. 04 III 1.1 0 0. 84 1.4 5 0. 49 8. 45 3. 02 23. 68 <0 .01 7. 10 2. 50 20 .18 <0 .01 T s ta tu s n = 3 65 n = 7 1 T1 re f re f re f <0 .01 re f T2 o r h ig he r 1. 68 1.3 6 2. 07 <0 .01 1. 69 1.3 2 2.17 <0 .01 2. 51 1.5 5 4. 05 <0 .01 1. 90 1.1 4 3.1 6 0. 01 N s ta tu s n = 6 31 n = 6 8 N0 re f re f N1 o r h ig her 1. 49 1.2 0 1.8 5 <0 .01 2. 07 1.2 7 3. 39 <0 .01 ER n = 4 42 n = 5 8 ne gat iv e re f re f po sit iv e 0. 70 0. 52 0.9 4 0. 02 1.0 4 0. 57 1.9 0 0.9 0 PR n = 4 45 n = 6 0 ne gat iv e re f re f re f po sit iv e 0. 22 0. 22 0. 33 <0 .01 0.1 9 0.1 0 0. 35 <0 .01 0. 82 0. 49 1.3 7 0.4 6 HE R2 n = 4 17 n = 6 7 ne gat iv e re f re f po sit iv e 0. 93 0. 60 1.4 6 0.7 3 2. 21 1.15 4. 23 0. 02 PD -L 1 i m m un e c el ls a nd tumo ur c el ls n = 3 95 n = 6 7 ne gat iv e re f re f po sit iv e 1.0 4 0. 78 1.3 9 0. 80 1.2 5 0. 71 2. 23 0.4 4 PD -1 e xp re ssi on n = 3 95 n = 6 8 ne gat iv e re f re f po sit iv e 1.19 0. 38 3. 70 0.7 7 0. 62 0.15 2. 54 0. 51 Av er age T IL s core n = 4 62 n = 7 0 low re f re f m od er ate /in te rm ed ia te 0. 81 0. 64 1.0 3 0. 09 0. 80 0. 45 1.4 4 0.4 6 A bb re vi at ion s: n : n um be r, O R: o dd s r at io , C I: c on fid en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: h um an e pi de rm al gr ow th f ac to r r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th li ga nd 1 , T IL : t um ou r i nfi ltr at in g l ym ph oc yt e tal T able 5 | A ssociation of TILs , PD -1 and PD -L1 expr
ession with clinicopatholog
ical paramet
ers in f
emale br
east cancer samples
tie nt c ha ra cte rist ic s Av er age T IL s core P D -L 1 i mm une a nd tumo ur c el ls PD -L 1 t umo ur c el ls PD -L 1 i mm une c el ls PD -1 OR 95% C I P-va lu e OR 95% C I P-va lu e OR 95% C I P-va lu e OR 95% C I P-va lu e OR 95% C I P-va lu e ge a t d ia gno sis n = 4 20 n = 4 06 n = 4 06 n = 4 06 n = 4 21 5 0 re f re f re f re f re f 0 0. 39 0. 24 0. 63 <0 .01 0. 76 0.4 4 1. 31 0. 32 0. 74 0. 43 1. 28 0. 28 1. 33 0. 27 6.4 8 0.7 3 0. 35 0.1 4 0. 87 0. 02 ur gr ad e n = 4 18 <0 .01 n = 4 03 <0 .01 n = 403 <0 .01 n = 4 03 0.11 n = 4 18 0. 82 re f re f re f re f re f 2. 72 1. 26 5. 88 0. 01 2. 58 1.0 2 6. 50 <0 .01 3.1 3 1.1 6 8. 43 0. 02 0. 27 0. 02 2.9 8 0. 28 1.15 0. 38 3. 46 0. 80 6. 45 2.9 9 13 .92 <0 .01 7. 76 3.1 4 19 .18 <0 .01 9. 42 3. 56 24 .9 0 <0 .01 2. 36 0.4 6 11 .93 0. 31 0. 81 0. 23 2. 86 0. 74 ta tu s n = 4 11 n = 3 97 n = 3 97 n = 3 97 n = 4 12 re f re f re f re f re f r h ig he r 1.4 3 0.9 0 2. 27 0.13 1.5 9 0.9 6 2. 66 0. 07 1.6 5 0.9 9 2. 75 0. 06 1.1 4 0. 30 4. 31 0. 85 0. 78 0. 31 2. 00 0. 61 ta tu s n = 4 08 n = 3 92 n = 3 92 n = 3 92 n = 4 08 re f re f re f re f re f r h ig he r 1.0 6 0. 67 1.6 8 0. 82 1. 23 0. 74 2. 04 0. 43 1. 27 0. 76 2.11 0. 36 0.1 6 0. 02 1. 27 0. 08 1.0 1 0.4 0 2. 55 0.9 8 n = 3 71 n = 3 56 n = 3 56 n = 3 56 n = 3 70 gat iv e re f re f re f re f re f sit iv e 0. 44 0. 26 0. 74 <0 .01 0. 28 0.1 6 0. 49 <0 .01 0. 28 0.1 6 0. 49 <0 .01 0. 56 0.13 2. 41 0.4 4 0.4 0 0.15 1.0 4 0. 06 n = 3 76 n = 3 60 n = 3 60 n = 3 60 n = 3 75 gat iv e re f re f re f re f re f sit iv e 0. 63 0. 39 1.0 2 0. 06 0. 46 0. 26 0. 79 <0 .01 0. 46 0. 26 0. 79 <0 .01 1.0 6 0. 25 4. 52 0.9 4 0. 59 0. 23 1.5 2 0. 27 R2 n = 3 89 n = 3 77 n = 3 77 n = 3 77 n = 3 91 gat iv e re f re f re f re f re f sit iv e 2.6 4 1. 35 5.1 6 <0 .01 2. 00 0.9 5 4. 25 0. 07 2. 00 0.9 5 4. 25 0. 07 1. 38 0.1 6 11 .71 0. 77 1.6 7 0.4 6 6.0 0 0.4 4 -L 1 i m m un e c el ls a nd t um or lls n = 3 94 n = 4 01 gat iv e re f re f sit iv e 10 .7 2 6. 07 18 .9 4 <0 .01 2.4 8 0.9 5 6.4 4 0. 06 ve ra ge T IL s core n = 394 n = 394 n = 394 n = 408 re f re f re f re f od er ate /in te rm ed ia te 10 .7 2 6. 07 18 .9 4 <0 .01 11 .1 9 6. 31 19. 87 <0 .01 27 .40 3. 38 222 .12 <0 .01 8. 71 3. 25 23. 36 <0 .01 ax im al T IL sc or e n = 394 n = 3 94 n = 394 n = 408 re f re f re f re f od er at e/ hi gh 7. 56 3.1 8 17. 93 <0 .01 9.1 4 3. 59 23 .28 <0 .01 4. 22 0. 52 34 .12 0.1 8 4. 97 1.1 4 21. 73 0. 03 bb re vi at ion s: n : n um be r, O R: o dd s r at io , C I: c on fid en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: h um an e pi de rm al ow th f ac to r r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th li ga nd 1 , T IL : t um ou r i nfi ltr at in g l ym ph oc yt e
3
Supplemen tal Table 6| M ultivar iat e analysis of association of TILs , PD -1 and PD -L1 expr ession with clinicopatholog ical paramet ers in female br east cancer samples Patie nt c ha ra cte rist ic s Av er age T IL s core PD -L 1 i m m un e a nd t um ou r c el ls PD -L 1 t umo ur c el ls PD -L 1 i mm une c el ls PD -1 OR 95% C I P-va lu e OR 95% C I P-va lu e OR 95% C I P-va lu e OR 95% C I P-va lu e OR 95% C I P-va lu e A ge a t d ia gno si s ≤ 50 re f > 50 0. 38 0. 21 0. 69 <0 .01 Tumo ur gr ad e <0 .01 <0 .01 I re f re f II 1.5 7 0.4 8 5.1 4 0. 54 1.6 6 0. 51 5. 39 0.4 0 III 4. 26 1.3 4 13 .5 3 0. 01 4. 26 1.3 5 13 .51 0. 01 N s ta tu s N0 N1 o r h ig her ER negat iv e re f re f po sit iv e 0. 37 0.19 0.7 3 <0 .01 0. 35 0.1 8 0. 68 <0 .01 PR negat iv e po sit iv e PD -L 1 i m m un e c el ls an d t um or c el ls ne gat iv e re f po sit iv e 12 .3 9 6. 33 24 .2 7 <0 .01 Av er age T IL s core low re f re f re f re f m od er ate /in te rm ed ia te 10 .7 5 5. 49 21 .0 5 <0 .01 10 .2 4 5. 27 19 .9 0 <0 .01 27 .40 3. 38 222 .11 <0 .01 8. 71 3. 25 23. 36 <0 .01 M ax im al T IL s core low Mod er at e/ hi gh A bb re vi at ion s: n : n um be r, O R: o dd s r at io , C I: c on fid en ce i nt er va l, T s ta tu s: t um ou r s ta tu s, N s ta tu s: n od al -s ta tu s, E R: o es tro ge n r ec ep to r, P R: p ro ge st er on e r ec ep to r, H ER 2: h um an e pi de rm al g ro w th fa ct or r ec ep to r 2 , P D -L 1: p ro gr am m ed d ea th li ga nd 1 , T IL : t um ou r i nfi ltr at in g l ym ph oc yt e chapter3
