Breast Cancer : Th1/ Th2 balance impairments ART 1

 

subject: Survival: However, a FOXP3+/CD4+ ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033)/Raoul Droeser/Inti Zlobec/ Ergin Kilic / Uwe Güth/ Michael Heberer/ Giulio Spagnoli/ Daniel Oertli1 / Coya Tapia/
object_opposite: CD4+ infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers.
misc: In univariate but not in multivariate analysis CD4+ infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3+/CD4+ ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033)./Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers Raoul Droeser12, Inti Zlobec34, Ergin Kilic5, Uwe Güth6, Michael Heberer2, Giulio Spagnoli2, Daniel Oertli1 and Coya Tapia34*author_year: Raoul Droeser/Inti Zlobec/ Ergin Kilic / Uwe Güth/ Michael Heberer/ Giulio Spagnoli/ Daniel Oertli1 / Coya Tapia/2012journal_volume_page: BMC Cancer / 10.1186/1471-2407-12-134

 

 

Differential pattern and prognostic significance of CD4⁺, FOXP3⁺ and IL-17⁺ tumor infiltrating lymphocytes in ductal and lobular breast cancers

Raoul Droeser/   Inti Zlobec/ Ergin Kilic/ Uwe Güth/ Michael Heberer/ Giulio Spagnoli/Daniel Oertli/ Coya Tapia/2012

 

Background

Tumor-infiltrating lymphocytes (TILs) are frequently considered to reflect host immune response against malignant tumors [1]. TILs have been shown to infiltrate a variety of tumors of diverse histological origin [2,3]. Their exquisite tumor specificity has been demonstrated in a number of cases and it has led to the characterization of tumor associated antigens. Although resident TILs have frequently been reported to be in a functionally "anergic" state [4,5], importantly, following "ex vivo" culture, TILs have been used to treat different types of cancers [6]. In line with these data, tumor infiltration by T lymphocytes has been shown to be associated with favorable prognosis, particularly in melanoma and colorectal cancers [2,7]. On the other hand, tumor infiltration by T-lymphocytes subsets endowed with immuno-regulatory or suppressive potential, e.g. CD4⁺ T-cells expressing FOXP3 transcription factor, has been suggested to be associated with tumor progression and unfavorable prognosis [8]. More recently, a CD4⁺ T-cell subset producing IL-17 has been implicated in the pathogenesis of several autoimmune diseases [9]. However, the role of the so-called Th17 in antitumor immunity is still debated [10-13]

In normal breast tissue small numbers of lymphocytes representing the mucosa-associated lymphoid tissue can be detected [14]. In contrast, increased numbers of lymphocytes are frequently detectable around and within breast cancers [15-18]. The clinical significance of TILs in breast cancer is still controversial. In some studies, TILs were associated with unfavorable characteristics such as high grade tumors, estrogen receptor negativity, basal-like molecular subtype as well as her2/neu positive tumors [19,20]. High CD4⁺ and CD8⁺ lymphocytic infiltration has been associated with positive lymph node status as well as worse overall survival [21]. Furthermore, in early stage breast cancer, CD8⁺ lymphocytic infiltration has been suggested to correlate with lymph node involvement [22]. Other groups, however, have shown that breast cancers with increased TIL number display a better prognosis in comparison with breast cancers with lesser lymphocyte infiltration [23], as also confirmed by data from our institution for CD8⁺ TILs in the ER negative subgroup [24]. Additionally, high TIL counts may represent an independent predictor of response to neo-adjuvant chemotherapy [25]. Notably, infiltration by FOXP3⁺ lymphocytes in breast cancer has been proposed to represent an independent unfavorable prognostic factor, especially in the nodal positive subgroup [26] and to correlate with tumor invasiveness [27]. In contrast, a complete clinical response has been suggested to be associated with disappearance of tumor infiltrating FOXP3⁺ T-cells during treatment [28].

While the clinical significance of TILs is controversial, their distribution within stromal and intratumoral compartments in breast cancers is largely unknown. Furthermore, T-cell infiltration in different histological subtypes as well as the occurrence of IL-17⁺ lymphocytes in breast cancer tissue has not been reported to date. Here, we addressed these issues by using a tissue microarray (TMA) including a large number (> 1000) of breast cancers stratified according to ductal and lobular histological subtypes. By taking advantage of a comprehensive clinical follow-up database, numbers of CD4⁺, FOXP3⁺ and IL-17⁺ TILs and their occurrence in different tumor compartments was correlated with clinico-pathological features and survival data.

Discussion

The adaptive immune system is known to play a major role in the control of tumor progression in different types of cancer. Indeed, tumor infiltration by CD8⁺ T-cells has been shown to represent an important prognostic factor in melanoma [7], and, more recently, in colorectal cancers [2,33].

Early studies have suggested a favorable prognostic effect of lymphocyte infiltration in breast cancers [34]. More recently, infiltration by CD3⁺ T-cells has been suggested to predict responsiveness to neoadjuvant treatment in these tumors [25]. Furthermore, a predictive effect of breast cancer infiltration by FOXP3⁺ cells has also been reported [35]. Breast cancers, however, comprise histologically different tumor entities characterized by molecular specificities and differential prognosis [36,37]. The expression of hormone receptors and her2/neu also represent important factors in the biology of breast cancers and in its prognosis [36]. Most importantly, phenotypes and location of tumor infiltrating lymphocytes are emerging as important issues in cancer immunobiology [7]. Therefore, a thorough assessment of the immunobiological relevance of lymphocyte infiltration in breast cancer needs to accurately take into account these parameters.

Our data, deriving from the study of a large cohort of cases, including > 1000 specimens, are consistent with a significantly different pattern of lymphocyte infiltration in ductal and lobular breast cancers. In the lobular histological type, there is a lower lymphocytic infiltration than in ductal cancers, particularly regarding CD4⁺ and FOXP3⁺ cells. Furthermore, in lobular breast cancers, lymphocyte infiltration is not correlated with tumor grade and expression of hormonal receptors and it has no prognostic relevance.

In contrast, in ductal cancers, increased infiltration by CD4⁺ or FOXP3⁺ lymphocytes correlates significantly with histological grade, and ER loss. Her2/neu over-expression in ductal cancers is also significantly associated with increased numbers of FOXP3⁺ infiltration. Puzzlingly, loss of PR expression appears to be associated with a decrease of CD4⁺ infiltrate. In agreement with previous reports [27,38], we also found that FOXP3⁺ infiltration is significantly increased in triple negative ductal, but not in lobular breast cancers.

Whereas the molecular background underlying these effects is unclear, they do impact the clinical course of the disease, since in ductal cancers, high infiltration by CD4⁺ T-cells is associated with a significantly more severe prognosis, albeit only in univariate analysis.

On the other hand, in both ductal and lobular cancers only a modest infiltration by IL-17 producing cells was detectable.

Ductal breast cancers are more compactly growing tumors, sometimes characterized by a broad and pushing border, whereas lobular carcinomas mainly show indian file pattern, smoothly infiltrating the surrounding tissue. Interestingly, the detection of increasing numbers of TILs in high grade tumors with pushing borders was described earlier in medullary breast cancers [23]. In our study, higher counts of TILs are significantly (p < 0.001) associated with more aggressive tumor features such as loss of estrogen receptor, higher tumor grade (G3), or her2/neu over-expression in ductal breast cancers. More aggressive tumors are growing faster and may therefore present more necrotic areas while producing stroma damage possibly related to local hypoxia [39-42]. Indeed, increased numbers of CD4⁺ and FOXP3⁺ cells under hypoxic condition could be shown in several studies [43]. Therefore, the growth dynamics of the tumor could play a role in inducing lymphocyte infiltration.

FOXP3 represents a typical, although not exclusive, marker of regulatory CD4⁺ T-cells. Tumor infiltration by T-cells expressing this marker has been associated to severe prognosis in different tumors, including ovarian and lung cancers [8]. However, in colorectal cancers FOXP3⁺ T-cell infiltration has been found to correlate with significantly improved prognosis by us and others [44,45]. Our data clearly document that, although numbers of FOXP3⁺ lymphocyte infiltration in ductal breast cancer are significantly associated with unfavourable clinico-pathological features, this marker alone does not appear to represent a prognostic marker. However, a high ratio (> 1) of total FOXP3⁺/CD4⁺ TILs was independently associated with a better overall survival, thereby suggesting that FOXP3⁺ cells other than CD4⁺ T lymphocytes could be involved in the elicitation of the favorable prognostic effects. It is of note, that although FOXP3 still represents one of the most reliable Treg markers, it is known to be expressed by activated T-cells as well [8]. Indeed, FOXP3 has been shown to be expressed, albeit transiently, in activated CD8⁺ T cells [46], in tonsillar suppressive CD8⁺ T cells [47], and even in tumor cells [48].

Contrasting data have been reported regarding FOXP3 expression in tumor cells. In a mouse model FOXP3 could be identified as a X-linked tumor suppressor gene in breast cancer [49], but others have failed to detect expression of this gene in non hematopoietic tissues [50]. FOXP3 expression has also been reported in human breast cancer cells [26,51]. Other groups however, did not confirm these findings and only detected FOXP3 expression in breast cancer infiltrating lymphocytes [35,52]. Furthermore evidence has been reported that localization and activation status of FOXP3 positive cells might play a prognostic role in breast cancer [53,54]. Still unclear is the background underlying these discrepancies, possibly related to the use of different reagents and staining protocols. In our studies however, in keeping with previous results [55] expression of FOXP3 in breast cancer cells was found to be negligible while it was usually detectable as nuclear staining of TIL.

Accordingly, CD3 and CD163 specific staining data indicate that tumor infiltration by FOXP3⁺ cells is highly correlated with infiltration by CD3⁺ cells. Further studies are warranted to clarify nature, origin and functions of FOXP3⁺ cells associated with improved survival in ductal breast cancer. Nevertheless, our data suggest that FOXP3 expression might reflect an activated state of specific T cell subsets.

Our study indicates that ductal and lobular breast cancers are characterized by a significantly different pattern of lymphocyte infiltration. Notably, in ductal cancers, total and, in particular, intratumoral lymphocyte infiltration is significantly associated with higher histological grade and severe prognosis, although not independently from known prognostic factors.

Further research is warranted to clarify whether these features are related to differential growth patterns of these tumor types, or to a differential immunogenicity of these tumors. Alternatively, variable tumor microenvironments might differentially favour lymphocyte chemoattraction and expansion.

1. Takagi S, Chen K, Schwarz R, Iwatsuki S, Herberman RB, Whiteside TL: Functional and phenotypic analysis of tumor-infiltrating lymphocytes isolated from human primary and metastatic liver tumors and cultured in recombinant interleukin-2.
   

   Cancer 1989, 63:102-111. 

   
   
2. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, et al.: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.
   

   Science 2006, 313:1960-1964.

   
   
3. Su X, Ye J, Hsueh EC, Zhang Y, Hoft DF, Peng G: Tumor microenvironments direct the recruitment and expansion of human Th17 cells.
   

   J Immunol 2010, 184:1630-1641

   
   
4. Luscher U, Filgueira L, Juretic A, Zuber M, Luscher NJ, Heberer M, et al.: The pattern of cytokine gene expression in freshly excised human metastatic melanoma suggests a state of reversible anergy of tumor-infiltrating lymphocytes.
5. Int J Cancer 1994, 57:612-619. 
6. Zippelius A, Batard P, Rubio-Godoy V, Bioley G, Lienard D, Lejeune F, et al.: Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.
   

   Cancer Res 2004, 64:2865-2873.

   
   
7. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME: Adoptive cell transfer: a clinical path to effective cancer immunotherapy.
   

   Nat Rev Cancer 2008, 8:299-308. 

   
   
8. Oble DA, Loewe R, Yu P, Mihm MC Jr: Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human melanoma.
   

   Cancer Immun 2009, 9:3.

   
   
9. Curiel TJ: Regulatory T cells and treatment of cancer.
   

   Curr Opin Immunol 2008, 20:241-246. 

   
   
10. Bettelli E, Oukka M, Kuchroo VK: T(H)-17 cells in the circle of immunity and autoimmunity.
11. Nat Immunol 2007, 8:345-350.
12. Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A, et al.: Tumor-specific Th17-polarized cells eradicate large established melanoma.
    

    Blood 2008, 112:362-373.

    
    
13. Tartour E, Fossiez F, Joyeux I, Galinha A, Gey A, Claret E, et al.: Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice.
    

    Cancer Res 1999, 59:3698-3704.

    
    
14. Liu J, Duan Y, Cheng X, Chen X, Xie W, Long H, et al.: IL-17 is associated with poor prognosis and promotes angiogenesis via stimulating VEGF production of cancer cells in colorectal carcinoma.
    

    Biochem Biophys Res Commun 2011, 407:348-354.

    
    
15. Wilke CM, Kryczek I, Wei S, Zhao E, Wu K, Wang G, et al.: Th17 cells in cancer: help or hindrance?
    

    Carcinogenesis 2011, 32:643-649

    
    
16. Ferguson DJ: Intraepithelial lymphocytes and macrophages in the normal breast17.
    

    Virchows Arch A Pathol Anat Histopathol 1985, 407:369-378.

    
    
17. Kuroda H, Tamaru J, Sakamoto G, Ohnisi K, Itoyama S: Immunophenotype of lymphocytic infiltration in medullary carcinoma of the breast.
18. Virchows Arch 2005, 446:10-14.
19. Demaria S, Volm MD, Shapiro RL, Yee HT, Oratz R, Formenti SC, et al.: Development of tumor-infiltrating lymphocytes in breast cancer after neoadjuvant paclitaxel chemotherapy.
    

    Clin Cancer Res 2001, 7:3025-3030

    
    
20. Chin Y, Janseens J, Vandepitte J, Vandenbrande J, Opdebeek L, Raus J: Phenotypic analysis of tumor-infiltrating lymphocytes from human breast cancer.
    

    Anticancer Res 1992, 12:1463-1466
21. Murta EF, de Andrade JM, Falcao RP, Bighetti S: Lymphocyte subpopulations in patients with advanced breast cancer submitted to neoadjuvant chemotherapy. Tumori 2000, :403-407.
22. Ghebeh H, Barhoush E, Tulbah A, Elkum N, Al Tweigeri T, Dermime S: FOXP3+ Tregs and B7-H1+/PD-1+ T lymphocytes co-infiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy. BMC Cancer 2008, 8:57.
23. kim KH, Telisinghe PU, Abdullah MS, Ramasamy R: Possible significance of differences in proportions of cytotoxic T cells and B-lineage cells in the tumour-infiltrating lymphocytes of typical and atypical medullary carcinomas of the breast. Cancer Immun 2010, 10:3.
24. Matkowski R, Gisterek I, Halon A, Lacko A, Szewczyk K, Staszek U, et al.: The prognostic role of tumor-infiltrating CD4 and CD8 T lymphocytes in breast cancer. Anticancer Res 2009, 29:2445-2451.
25. Macchetti AH, Marana HR, Silva JS, de Andrade JM, Ribeiro-Silva A, Bighetti S: Tumor-infiltrating CD4+ T lymphocytes in early breast cancer reflect lymph node involvement. Clinics (Sao Paulo) 2006, 61:203-208.

     26. Rakha EA, Aleskandarany M, El Sayed ME, Blamey RW, Elston CW, Ellis IO, et al.: The prognostic significance of inflammation and medullary histological type in invasive carcinoma of the breast. Eur J Cancer 2009, 45:1780-1787.Baker K, Lachapelle J, Zlobec I, Bismar TA, Terracciano L, Foulkes WD: Prognostic significance of CD8+ T lymphocytes in breast cancer depends upon both oestrogen receptor status and histological grade. Histopathology 2011, 8:1107-1116.Denkert C, Loibl S, Noske A, Roller M, Muller BM, Komor M, et al.: Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010, 28:105-113.

1. Merlo A, Casalini P, Carcangiu ML, Malventano C, Triulzi T, Menard S, et al.: FOXP3 expression and overall survival in breast cancer.
2. J Clin Oncol 2009, 27:1746-1752
3. Bates GJ, Fox SB, Han C, Leek RD, Garcia JF, Harris AL, et al.: Quantification of regulatory T cells enables the identification of high-risk breast cancer patients and those at risk of late relapse.
   

   J Clin Oncol 2006, 24:5373-5380
4. Ladoire S, Arnould L, Apetoh L, Coudert B, Martin F, Chauffert B, et al.: Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating foxp3+ regulatory T cells.
   

   Clin Cancer Res 2008, 14:2413-2420.
5. Tapia C, Zlobec I, Schneider S, Kilic E, Guth U, Bubendorf L, et al.: Deletion of the inhibitor of growth 4 (ING4) tumor suppressor gene is prevalent in human epidermal growth factor 2 (HER2)-positive breast cancer.
   

   Hum Pathol 2011, 42:983-990. 
6. Guth U, Huang DJ, Schotzau A, Dirnhofer S, Wight E, Singer G: Breast cancer with non-inflammatory skin involvement: current data on an underreported entity and its problematic classification.
   

   Breast 2010, 19:59-64. 
7. Sauter G, Simon R, Hillan K: Tissue microarrays in drug discovery.
8. Nat Rev Drug Discov 2003, 2:962-972. 
9. Elston CW, Ellis IO: Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 1991, 19:403-410. 
10. Pages F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R, et al.: Effector memory T cells, early metastasis, and survival in colorectal cancer.
11. N Engl J Med 2005, 353:2654-2666.
12. Menard S, Tomasic G, Casalini P, Balsari A, Pilotti S, Cascinelli N, et al.: Lymphoid infiltration as a prognostic variable for early-onset breast carcinomas.
    

    Clin Cancer Res 1997, 3:817-819. 
13. de Kruijf EM, van Nes JG, Sajet A, Tummers QR, Putter H, Osanto S, et al.: The predictive value of HLA class I tumor cell expression and presence of intratumoral Tregs for chemotherapy in patients with early breast cancer.
    

    Clin Cancer Res 2010, 16:1272-1280.
14. Colleoni M, Viale G, Goldhirsch A: Lessons on responsiveness to adjuvant systemic therapies learned from the neoadjuvant setting.
    

    Breast 2009, 18:(Suppl 3):S137-S140.

    
    
15. Cowin P, Rowlands TM, Hatsell SJ: Cadherins and catenins in breast cancer.
    

    Curr Opin Cell Biol 2005, 17:499-508
16. Bohling SD, Allison KH: Immunosuppressive regulatory T cells are associated with aggressive breast cancer phenotypes: a potential therapeutic target.
    

    Mod Pathol 2008, 21:1527-1532. 
17. Minervini A, Di Cristofano C, Gacci M, Serni S, Menicagli M, Lanciotti M, et al.: Prognostic role of histological necrosis for nonmetastatic clear cell renal cell carcinoma: correlation with pathological features and molecular markers.
    

    J Urol 2008, 180:1284-1289.
18. Finger EC, Giaccia AJ: Hypoxia, inflammation, and the tumor microenvironment in metastatic disease.
    

    Cancer Metastasis Rev 2010, 29:285-293
19. Osinsky S, Zavelevich M, Vaupel P: Tumor hypoxia and malignant progression.
    

    Exp Oncol 2009, 31:80-86.
20. De Schutter H, Landuyt W, Verbeken E, Goethals L, Hermans R, Nuyts S: The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy +/- chemotherapy.
    

    BMC Cancer 2005, 5:42. 
21. Ben Shoshan J, Maysel-Auslender S, Mor A, Keren G, George J: Hypoxia controls CD4+ CD25+ regulatory T-cell homeostasis via hypoxia-inducible factor-1alpha.
    

    Eur J Immunol 2008, 38:2412-2418.
22. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, et al.: Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer.
    

    J Clin Oncol 2009, 27:186-192.
23. Frey DM, Droeser RA, Viehl CT, Zlobec I, Lugli A, Zingg U, et al.: High frequency of tumor-infiltrating FOXP3(+) regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients.
    

    Int J Cancer 2010, 126:2635-2643.
24. Roncarolo MG, Gregori S: Is FOXP3 a bona fide marker for human regulatory T cells?
    

    Eur J Immunol 2008, 38:925-927
25. Siegmund K, Ruckert B, Ouaked N, Burgler S, Speiser A, Akdis CA, et al.: Unique phenotype of human tonsillar and in vitro-induced FOXP3+CD8+ T cells.
    

    J Immunol 2009, 182:2124-2130.

    
    
26. Hinz S, Pagerols-Raluy L, Oberg HH, Ammerpohl O, Grussel S, Sipos B, et al.: Foxp3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer.
    

    Cancer Res 2007, 67:8344-8350.

    
    
27. Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W, et al.: FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene.
    

    Cell 2007, 129:1275-1286. 
28. Kim J, Lahl K, Hori S, Loddenkemper C, Chaudhry A, deRoos P, et al.: Cutting edge: depletion of Foxp3+ cells leads to induction of autoimmunity by specific ablation of regulatory T cells in genetically targeted mice.
    

    J Immunol 2009, 183:7631-7634.
29. Ladoire S, Arnould L, Mignot G, Coudert B, Rebe C, Chalmin F, et al.: Presence of Foxp3 expression in tumor cells predicts better survival in HER2-overexpressing breast cancer patients treated with neoadjuvant chemotherapy.
    

    Breast Cancer Res Treat 2011, 125:65-72
30. Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Lee AH, Ellis IO, et al.: An evaluation of the clinical significance of FOXP3(+) infiltrating cells in human breast cancer.
    

    Breast cancer Res Treat 2011, 127:99-108. 
31. Gobert M, Treilleux I, driss-Vermare N, Bachelot T, Goddard-Leon S, Arfi V, et al.: Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome.
    

    Cancer Res 2009, 69:2000-2009.

    Menetrier-Caux C, Gobert M, Caux C: Differences in tumor regulatory T-cell localization and activation status impact patient outcome.
32. Cancer Res 2009, 69:7895-7898. 
33. Wolf D, Wolf AM, Tzankov A: Comment on "Cutting edge: depletion of Foxp3+ cells leads to induction of autoimmunity by specific ablation of regulatory T cells in genetically targeted mice". J Immunol 2010, 184:4051.