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Year : 2021  |  Volume : 64  |  Issue : 1  |  Page : 28-37
Exploring the role CD163-labeled TAMs and FOXP3-labeled Tregs in different types of breast cancer: Reflections and putative benefits

1 Department of Surgical Pathology, Faculty of Medicine, Ain Shams University, Egypt
2 Department of Medical Oncology, Faculty of Medicine, Ain Shams University, Egypt

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Date of Submission07-Mar-2020
Date of Decision06-Apr-2020
Date of Acceptance01-Jun-2020
Date of Web Publication8-Jan-2021


Context: Tumor immune microenvironment (TIME) is heterogeneous and dynamic. It exerts bimodal pro and antitumor effects. Among the TIME contributors, TAMs and Tregs are condemned as cancer cells allies rather than enemies; however, such contribution is not universally equal in all tumors.
Aims: We aimed to explore and compare TAMs and Tregs in various breast cancers and link such findings to pathologic prognostic indices. Settings and Design: This was a retrospective study. Methods and Materials: Archival blocks of 108 breast cancers were immunohistochemically studied for CD163 and FOXP3 in tumor stroma (TS) and specialized DCIS periductal stroma. FOXP3 was additionally evaluated in tumor cells. CD163 and FOXP3 expressions were compared with different histopathological prognostic categories for statistical analysis. Statistical Analysis Used: Analysis of data was done using the Chi-Square test. Results: Both CD163+ TAM and FOXP3+ Tregs. showed statistically significant association with high tumor grade, T stage, multifocality and hormone negativity. Synchronous expression was consistent for both markers in almost all compared parameters, dual high expression of both CD163 and FOXP3 yielded additional statistically significant association with lymphovascular invasion (LVI). Periductal stromal CD163 and FOXP3 high expression showed statistically significant association with DCIS. FOXP3 tumor cells expression was similar to TS FOXP3 but additionally showed significant association with LVI and N stage; moreover, Her-2 over-expressing breast cancer was significantly associated with low FOXP3+ tumor cells. Conclusions: Breast cancer TS TAMs and Tregs. abundance reflects unfavorable prognosis in various breast cancers particularly hormone negative cancers.

Keywords: Breast cancer, TAM, Tregs, tumor immunity

How to cite this article:
Shash LS, M. Kamal KA, Abd Raboh NM. Exploring the role CD163-labeled TAMs and FOXP3-labeled Tregs in different types of breast cancer: Reflections and putative benefits. Indian J Pathol Microbiol 2021;64:28-37

How to cite this URL:
Shash LS, M. Kamal KA, Abd Raboh NM. Exploring the role CD163-labeled TAMs and FOXP3-labeled Tregs in different types of breast cancer: Reflections and putative benefits. Indian J Pathol Microbiol [serial online] 2021 [cited 2022 May 16];64:28-37. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/28/306499

   Introduction Top

Breast cancer (BC) is the most common cancer in females worldwide accounting for the second cause of cancer death after the lung cancer.[1] Although advanced therapeutic modalities had tremendously reduced cancer morbidity and improved overall survival, however, further research work is yet needed to unravel the mechanisms enabling BC cells to survive, relapse and metastasize despite of therapy.[2]

Tumor microenvironment provides innumerable interactive cross talks that influence cancer cell longevity and behavior.[3],[4] The tumor immune microenvironment (TIME) homes a dynamic battlefield between pro-tumor or anti-tumor effectors.[5],[6] This bimodal contribution of TIME has been intensively investigated to identify its functional and phenotypic diversity.[7],[8] Immune cells in tumor stroma (TS) rather than those of tumor nests (TN) were proved to reflect poor prognostic outcome.[9] The historic dogma of BC being poorly immunogenic has been recently wrecked; many BCs, particularly of the hormone negative group harbor an enriched mutational signature with a corresponding pronounced antigenic immune provocative signature.[2] Accumulating evidence emphasize the prognostic and potentially predictive value of tumor infiltrative lymphocytes population (TILs) particularly in hormone negative BC.[10],[11] Regulatory T lymphocytes (Tregs) are known to represent the immunosuppressive party of TILs population that help cancer cells evade anti-tumor immunity.[12],[13] FOXP3 is reported as the most specific immunohistochemical marker for labeling Treg[14],[15] its staining was also noted in tumor cells in several studies. Interpretation of FOXP3 expression was quite controversial in these studies.[16]

Another pivotal contributor in TIME is tumor-associated macrophages (TAM) inducing a tumor-promoting effect via two major attributions: promoting hypoxia induced angiogenesis and maintaining an immunosuppressive niche allowing cancer cells to evade immune surveillance.[17],[18] CD163 is currently regarded as a highly specific macrophage marker for polarized M2 subset exerting these effects.[19],[20]

Immunotherapy had achieved sounding success in several tumors; particularly, anti-PDL1.[21] Such premises encouraged researchers to explore new stations of the tumor immune cycle to alternatively or synergistically target.[2],[22],[23]

The primary objective of this study is to investigate and compare TAMs and Tregs in various BCs. A secondary objective was linking such findings to pathological prognostic indices for building up better knowledge about their differential role in BC subtypes thus inspiring putative selective immunotherapeutic targets.

   Subjects and Methods Top

Cohort and sample selection

This retrospective study included an initial cohort of 120-candidate primary BC cases that were diagnosed and treated in Ain Shams University Hospitals.

Her2-neu cases with IHC equivocal score of 2+ were not included in the initial selection due to absence of intra institutional facility for further work up by ISH techniques, pure ductal carcinoma in situ (DCIS) cases were also excluded. After initial selection, data concerning patients' clinical radiologic background were retrieved from our institutional archives anonymously through a numerically coded database; corresponding coded blocks and slides were then extracted. For cases receiving neoadjuvant therapy, the pre-treatment biopsy was extracted for pathological and immunohistochemical study. Recommendations of the 'International Immuno-Oncology Biomarkers Working Group' were considered; selection included only cases with available tissue to serve at least one section of 4–5 μm at a magnification of 200–400 x for each of the tested markers.[24] Final cohort of this study included 108 cases. Schematic presentation of sample selection process is shown in the following illustration.

Patient archival clinical/radiologic data:

Age of the patient was recorded from archival files. Data concerning size of the tumor, multifocality, axillary lymph node status were obtained from pathology reports in case of modified radical mastectomy specimens. Excision biopsies accounted for 81 cases; of which 70 were modified radical mastectomies and 11 were wide local excisions, on the other hand, 27 cases were represented as CNBs (not followed by intra-institutional excision). Cases having pretreatment CNB followed by post-treatment excision (MRM or WLE) were counted with the excision cases albeit tested on their CNB material to assure originality of their IHC reactions/results. Any case presented by more than one CNB (for multifocality or nodal involvement testing) was only counted once. For all CNB cases, data concerning tumor size, nodal status and focality status (unifocal versus multifocal) were recorded from their original (pre-treatment) radiology reports, provided actual involvement was confirmed on pathological biopsy basis (99 cases). Presence of associated ductal carcinoma in situ (DCIS) was only considered on pathologic proof basis (5 cases). Cases missing these aforementioned prerequisites were not included in statistical analysis for such parameters.

Pathological and immunohistochemical studies

Hematoxylin and Eosin stained slides of tumor sections were revised for selection of corresponding blocks comprising sufficient cancer and stromal tissue. Lymphovascular invasion was reported in 39 cases. ER, PR and Her2-neu stained slides were revised according to the updated ASCO-CAP scoring systems.

Four μm thick tissue sections were cut, dried, deparaffinized, and rehydrated following standard procedures. The sections were subjected to heat-induced antigen retrieval. Immunohistochemical staining was performed with monoclonal ready to use anti-FOXP3 (catalog no.: API 3164 AA-Biocompare-medical) and monoclonal concentrated anti CD163 (ED2-sc-58965-Santa Cruz with a 1:100 dilution) according to the manufacturer's instructions.

Immunohistochemical staining of each of FOXP3 and CD163 was evaluated according to a three tier semi quantitative score regarding density of stained cells in tumor stroma (TS). The percentage of positively stained cells was executed as an overall percentage of examined slides and not only in hot spots.[24]

Scoring of FOXP3 staining was modified from Takenaka et al., where the extent of TILs showing FOXP3+ nuclei was screened in TS compartment. TILs FOXP3 staining was scored 0 in absence of positive staining, 1+ when 1%-25% of positively stained TILs was observed, 2+ with a 26%-50% positive cells, and 3+ when 51%-100% positive lymphocytes were observed. Moreover, FOXP3+ tumor cells were assessed for nuclear or cytoplasmic staining, which was reported as absent (0), low (1+), intermediate (2+) or strong (3+). Since FOXP3 expression in tumor cells emphasizes another connotation, scoring of FOXP3 in tumor cells was separately assessed and analyzed.[20]

CD163 was recorded as positive when observed in the membrane or cytoplasm of macrophages. Positive staining was scored according to Medrek et al., in a nearly similar protocol to that of FOXP3 evaluation in TILs.[9]

Both FOXP3 and CD163 (TS) scores were further dichotomized into two categories of low expression (staining in =50%: corresponding to scores 0,1+ and 2+) and high expression (expression in >50%: corresponding to score 3+). This dichotomization was applied for easier comparisons and more reproducible statistical analysis.

In cases associated with DCIS, periductal stromal FOXP3+ and CD163 + infiltrates were semi-quantitatively assessed independent of the associated invasive carcinoma component TS infiltrate evaluation.[25]

Ethical statement

  • This retrospective study followed the revised tenets of the Declaration of Helsinki
  • The patient's blocks and slides were anonymously recruited. This is guaranteed by data and specimens coded retrieval protocol of Ain Shams University Hospital Pathology Lab policy statement in abidance to the local institutional Ethics Committee.

Statistical analysis

Analysis of data was done using SPSS (Statistical Package for Social Science) program version 23. Qualitative data were presented as count and percentage.

Chi-Square test was used to compare qualitative data between different groups. P-value represented the level of significance of relations. P-value =0.05 was considered not significant (NS), P-value <0.05 was considered significant (S) and <0.001 was considered highly significant (HS).

   Results Top

The mean age of patients in this study was 49.60 ±13 years. The invasive duct carcinoma (NOS) subtype accounted for the majority of our cases (61.1%), followed by classic invasive lobular carcinoma (19.4%), other histologic subtypes included metaplastic carcinoma, medullary subtype, mucinous subtype, pleomorphic lobular as well as others. Clinical and pathological profiles of cases are displayed in [Table 1].
Table 1: Clinical and pathologic profile of cases

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I. Immunohistochemical staining results for CD163 and FOXP3:

Spatial localization of FOXP3 and CD163 stained cells in TN and TS revealed considerable balanced distribution of both markers in both compartments for each examined tumor. Also, the distribution of TAMs and Tregs was remarkably more dispersed in the TN compartment compared to a rather clustered distribution in TS compartment in most of cases [Figure 1]a and [Figure 2]a. Additionally, lymphoid follicles suggestive of tertiary lymphoid structures were only encountered in TNBC; these cases showed both high FOXP3 expression and CD163 expression, such observation despite interesting was too rare to earn validation.
Figure 1: CD163 expression patterns in TS. (a) CD163 high balanced expression in TN (arrow heads) and TS (star) compartments in an IDC grade III (×200). (b) CD163 high expression in metaplastic carcinoma (×100). (c) CD163 high expression in medullary carcinoma (×200). (d) CD163 high expression in TS of mucinous carcinoma (×100). (e) CD163 low expression in grade I IDC (×100)

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Figure 2: FOXP3 expression: (a) High FOXP3 clustered TS in an IDC with papillary pattern grade II. DCIS periductal stromal high expression (arrow heads) (×100). (b) FOXP3 high expression in metaplastic carcinoma. Tertiary lymphoid structures (arrow head) (× 200). (c) Low TS-high FOXP3 expression in tumor cells of mucinous carcinoma (×400). (d) FOXP3 high expression in TS and tumor cells in IDC grade II (×400). (e) FOXP3 high expression in TS and in tumor cells in medullary carcinoma (×200). (f) FOXP3 high expression in TS and tumor cells in IDC grade III (×400)

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CD163 showed low TS expression in 51 (47.2%) of the cases and high expression in 57 (52.8%) of the cases. On the other hand, FOXP3 TS expression was high in 52 (48.1%) and low in 56 (51.8%). In addition to TILs staining, FOXP3 showed positive staining in tumor cells in 87 (80.5%) of the cases, 38.9% of which showed strong (3+) staining score. Details of FOXP3 and CD163 staining results are illustrated in [Table 2].
Table 2: Immunohistochemical staining results for CD163 and FOXP3

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II. Relation between CD163 TS expression and other tumor characteristics:

The relation between TAM infiltration as measured by TS CD163 staining (low/high) and various clinicopathologic features of the tumors are shown in [Table 3]. A statistically significant correlation was found between TS CD163 expression and each of tumor type, grade, T stage, focality and tumor biology (P = 0.001, <0.001, 0.01, 0.02 and 0.01, respectively). However, neither N stage nor lymphovascular invasion parameters showed a statistical correlation to CD163-labeled TS TAM.
Table 3: Relation between CD163 TS expression and tumor characteristics

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The highest level of TAMs infiltration (high TS CD163) was a hallmark of all metaplastic [Figure 1]b, medullary [Figure 1]c and mucinous carcinoma subtypes of breast carcinoma [Figure 1]d, followed by classic ILC cases showing high expression in 57.1%. IDC NOS cases showed a nearly equal tendency towards low CD163 expression (51.5%) and high expression (48.5%), on the other hand, pleomorphic lobular carcinomas showed low CD163.

Grade III tumors were exclusively associated with TS high CD163 expression (100%) in contrast to grade I tumors which were invariably associated with TS low CD163 [Figure 1]e. Regarding grade II tumors; 55.2% showed TS low CD163 expression and the remaining 44.8% cases showed high expression.

The T stage was associated with a statistically significant correlation with TS CD163 (P = 0.01); most of T1 cases had low expression (65.4%), while most of T2 cases had high expression (64.2%), however, this trend was not preserved in T3 cases, which showed a decline in TAM infiltration in the majority of cases with low TS CD163 expression in 75%. Meanwhile, the N stage designating lymph node status did not meet any statistically significant correlation (P = 0. 51). As regards tumor focality, a significant correlation was found between TS CD163 and tumor multifocality (P = 0.02) where 75% of multifocal tumors showed high TS expression.

Regarding tumor biology, a statistically significant TS high CD163 expression was observed in TNBC (75%), meanwhile, TPBC showed equal proportions of TS high and low CD163expression patterns, Her2+-EBC and HP cases revealed a slight trend for CD163 low expression as demonstrated in 62.5% of and 58.3% of these biologic groups, respectively.

III. Relation between FOXP3 TS expression and other tumor characteristics:

A significant correlation was found between TS FOXP3 expression in TILs and most of the studied tumor characteristics including tumor type, grade, T parameter of the stage, tumor focality and biologic state (P = 0.001.<0.001, 0.01,0.02, 0.01, respectively) [Table 4].
Table 4: Relation between FOX TS expression and other tumor characteristics

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In accordance to CD163 expression, all metaplastic and medullary carcinomas were associated with TS high FOXP3 expression [Figure 2]b, and in contrast to CD163 expression, FOXP3 expression was exclusively low in all cases of mucinous carcinoma [Figure 2]c. Likewise, all pleomorphic lobular carcinoma showed low expression, however, classic ILC showed predominant TS high FOXP3 (71.4%), meanwhile, IDC-NOS showed minor predilection to TS FOXP3 high expression (56.1%). TS FOXP3 expression also showed a statistical significant correlation with tumor grade since it was low in all grade 1 tumors; the difference in FOXP3 expression was minor among grade II tumors [Figure 2]d but markedly higher in grade III tumors reflecting a more consistent trend towards increased Treg. population in higher tumor grades. The majority of TNBC was associated with TS high FOXP3 expression (86.1%), whereas TPBC showed an equal distribution among low and high FOXP3 expressing tumors, the Her2+ -EBC tumors showed a higher tendency for low FOXP3 expression pattern (66.7%) and the majority of the HP group revealed low FOXP3 expression (83.3%).

IV. Relation between FOXP3 expression in tumor cells and other tumor characteristics:

FOXP3 expression in tumor cells shows a significant correlation with all the studied tumor characteristics [Table 5].
Table 5: Relation between tumor cells FOXP3 expression and tumor characteristics

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Moderate to strong expression was associated with all metaplastic, mucinous [Figure 2]c and medullary carcinomas [Figure 2]e, while negative expression was associated with all pleomorphic lobular cases (P < 0.001). Grade I tumors were all negative and grade III tumors showed strong positive expression in 50% of the cases (P = 0.03) [Figure 2]f. All cases positive for LVI expressed FOX3 staining in tumor cells with variable intensities (P < 0.001). TNBC and HP cases were mostly associated with moderate to strong expression, Her 2+- EBC was associated with negative to low expression while TPBC cases showed negative expression in 50% of cases and strong positive expression in 50% (P < 0.001).

V. Relation between FOXP3/CD163 dual expression and other tumor characteristics:

Correlating the dual TS expression pattern of FOXP3/ CD163 with tumor characteristics showed a statistically significant correlation between dual TS high expression and each of: metaplastic and medullary type tumors, high tumor grade, T2 stage tumors, presence of LVI and TNBC (P = 0.01, <0.001, <0.001,0.01,0.01, <0.001 respectively).

In contrast, the TS dual low expression of both markers was associated with pleomorphic lobular carcinoma, grade I, TPBC and HP tumors (P = 0.01, <0.001, 0.03, respectively).

Dual expression was also noted in the periductal specialized stroma of all DCIS components accompanying five of our cases regardless of their nuclear grade [Figure 2]a.

   Discussion Top

Studying TIME is an on growing research field motivated by the need to explore new oncotherapeutic modalities.[23] Successive breakthrough achievements like checkpoint inhibitors had raised hope in the utility of immune modulation as an effective and safer therapeutic approach.[26] Nevertheless, the immune modulatory effect of a particular immune cell type considerably differs from one tumor to the other, as such; investigating the differential immune infiltrate in different tumors is of paramount value in characterizing their contributions and accordingly tailoring competent targeted immunotherapies.[2],[27]

The forkhead box protein 3 (FOXP3) transcription factor is highly expressed in tumor cells as well as in regulatory T cells (Tregs), it plays a tumor-enhancing role in Tregs and suppresses carcinogenesis as a potent repressor of several oncogenes. The clinical prognostic value of FOXP3 expression has not yet been elucidated, few studies reported the absence of statistically significant association between FOX3p + cancer cells and tumor prognostic indices.[16]

Moreover, TAMs labeled by CD163 had been reported to exert an immunosuppressive effect thus promoting cancer cells survival and progression.[2],[23] These data are not restricted to breast cancer, for example, Cheng et al. demonstrated that CD163 is a novel predictor to evaluate gastric cancer immune status and tumor prognosis.[28]

This study was motivated by investigating possible functional diversities of these two contributors in various BC subtypes. Hormone negative BCs have limited sometimes poorly effective therapeutic options, nevertheless, they're frequently privileged by an immune rich stroma; therefore, it can be anticipated that immunotherapy would present a promising redeemer for this less fortunate group.[29]

Accordingly, in our study, we evaluated TAMs (by CD163 expression) and Tregs. (by FOXP3 expression) in different types of invasive BC with a variety of histologic subtypes, biologic categories of TNBC, TPBC, HPBC as well as HER2+ EBC, different pathologic tumor stages T1, 2 and 3 as well as pathologic nodal stages N0, 1 and 2, unifocal and multifocal tumors were included. Noninvasive (DCIS) tumors were also presented, however, in a biased selection where an associated invasive component was always present.

Considering tumor histopathologic type, both TAMs and Tregs., contributed abundantly in all metaplastic carcinomas and all medullary carcinomas as reflected by their statistically significant association with high CD163 and FOXP3 tumor stromal scores (TS). Such observations are quite expected in the metaplastic and medullary cancer subtypes since they're usually TNBCs; a group frequently reported to show high TAM populations.[30],[31] indeed our cases of metaplastic and medullary cancers were all in the TNBC group. High TS CD163 expression pattern extended to the rest of TNBC cases of our study with a statistically significant association, meanwhile the TPBC group, the Her2 +EBC and the HP groups failed to meet such statistically significant association. The TS high expression FOXP3 was also statistically significant in the TNBC only, but a statistically significant low expression was observed in the HP group, whereas FOXP3 expression pattern didn't mount to any statistical significance in either of TPBC group or the Her2+-EBC group. In agreement with our findings, high FOXP3 expression in TNBC TILs has been documented by many researchers.[32],[33],[34]] Tian et al. 2016, reported FOXP3 TILs to represent an independent poor prognostic parameter in TNBC, moreover, luminal tumors known by their consistent hormone-positive expression are regarded to show low FOXP3 expression profile.[35],[36]As regards the relation between Her2 neu status and Tregs. population; Jeong et al., also reported absence of statistical significant association,[37] but the opposite was reported by Takenaka et al., in this respect.[20] Mendoza and Mentor documented in their review that TIME of Her2 positive BCs comprise Tregs. among other TILs, neutrophils, dendritic cells and mast cells but they don't seem to project prognostic implications.[38] Nonetheless, the association of FOXP3 expression with TNBC seemed to be an agreement point.[20],[35],[37]

Many researchers agreed with us about the simultaneous TAMs and TILs profusion reporting strong association of high TS expression of both CD163 and FOXP3 with grade III tumors.[39] The importance of the subsets of T-cells in TILs as a prognostic value has been investigated by many, especially in TNBC, also location of these TILs, albeit their location has given less significance.[40] Moreover, Tekenaka et al., specifically studied FOXP3 Tregs. and reported similar results, in their study, multivariate analysis demonstrated that FOXP3 expression in TILs, unlike that in tumor cells, was an independent prognostic factor for overall survival.[20] TILs association with grade III tumors was also reported in Miyoshi et al. research however without specification of the subtype.[41] In our work, grade I tumors revealed statistically significant low TS CD163 and FOXP3, thus, further emphasizing a likely influential role of TAM and Tregs. on this prognostic index.

Classic ILC as well as the overall IDC-NOS subtype generally expressed CD163 in a rather balanced trend with an approximating ratio of low and high expression patterns despite of a slight tendency to high expression; whereas in Tregs., FOXP3 displayed predominant high expression in classic ILC cases (71.4%), inversely, pleomorphic lobular carcinoma expressed a statistically significant association with concomitant low TS CD163 and FOXP3. Du et al. mentioned dominance of tumor-infiltrating natural killer cells and Tregs. in classic ILC compared to IDC-NOS.[42] Additionally, in contrast to a statistically significant TAMs population in mucinous carcinoma as evidenced by high TS CD163, there was a contrasting paucity of Tregs in this subtype reflected by a statistically significant association of this histopathological type with TS low FOXP3 expression. Mucinous carcinomas in our study belonged to the HP group, having T stage of 2 and 3 and an N stage 2, these parameters suggest that the TAMs abundance might have played a role in dictating a rather aggressive behavior (manifested by high pTN) despite of a histologically and biologically privileged subtype, this assumption albeit noteworthy yet couldn't be reproducible in such limited number of cases.

It's worth mentioning that all the above-described relations concerning histologic subtypes of BCs (metaplastic, medullary, mucinous and pleomorphic lobular carcinomas) were tested on extremely small groups and yielded a statistically significance only based on the Fisher's exact test, thus they're far from validation, nonetheless, they're worth mentioning hypothetical deductions that may be inspiring for larger scale specified studies in this area.

Both CD163 and FOXP3 TS scores expression showed a statistical significant association with tumor stage in terms of T identifier (tumor size) but neither of them independently nor as dual high expression mounted to any statistically significant association when the N identifier (lymph nodal status) was considered. These findings were in agreement with Jeong et al. investigating TAMs in BC.[37]

Other tested poor prognostic indicators included lymphovascular invasion, which proved to show a statistically significant association with high TS FOXP3 but not CD163 TS. Multifocality showed a statistically significant association with both high TS FOXP3 and CD163 expressions.

The consistent parallel TS expression patterns observed in CD163 and FOXP3 regarding most parameters probably reflect the reported role of TAMs in the recruitment of Tregs.[43]

FOXP3 expression in tumor cells was investigated in a narrower scope, data in literature were controversial and inconsistent likely reflecting different study protocols involving population selection, technique of assessment (gene versus protein expression) as well as protocol of assessment (nuclear versus cytoplasmic staining). Takenaka et al., Ladoire et al. and Lopes et al. reported the absence of a statistically significant association between FOX3p+ cancer cells and tumor prognostic indices.[20],[44],[45] On the other hand, many others reported the contrary.[46],[47],[48] Our results agreed with the latter group and showed a statistically significant association of FOXP3 expression in tumor cells with tumor grade (absent expression in grade I, versus positive 2+/3+ scores in grade III), likewise, Ladoire et al. highlighted that among their investigated prognostic parameters, only tumor grade showed an isolated statistically significant association of FOX3p+ cancer cells.[44] Association of FOXP3 cancer cells staining with histologic and biologic tumor type also proved statistically significant (medullary subtype with FOX3p: 3+ score, metaplastic and mucinous subtypes with FOX3p: 2+ score, while pleomorphic lobular showed absent expression); moreover, TNBC phenotype showed FOX3p score: 2+/3+, Lopes et al., and others suggested that FOXP3 expression in cancer cells exerts a repressor or silencing effect on Her2 neu gene.[45],[49],[50] their suggestion echoes in our results since both the Her2-neu expressing groups (Her2+-EBC and TPBC) exhibited negative or low FOXP3 expression in cancer cells. FOXP3 expression also showed statistically significant association with tumor stage T (all of T3 tumors were FOXP3 +), additionally, FOXP3 expression in tumor cells was the only indicator that showed statistically significant association with pTN (nodal metastasis) albeit only in pN3 stage for which all cases showed positive FOXP3 cancer cells expression. Furthermore, it also showed a highly statistical association with lymphovascular invasion synchronizing with the high FOXP3 expression profiles in Tregs. Takenaka et al., emphasized that when taken together, FOXP3 cytoplasmic staining in cancer cells and in Tregs represent a strong prognostic determinant.[20]

Expression of both CD163 and FOXP3 in the periductal stroma of DCIS showed a high statistically significant correlation. A few studies addressed this scope, they emphasized that periductal FOXP3 expressing TILs are predictive of increased risk of recurrence[25] Hoskoppal and Reisenbichler reported non-significant association of periductal stromal TAM with DCIS nuclear grade despite denser infiltrate was observed in higher nuclear grade DCIS.[51]

   Conclusion Top

Our assessment methodology was guided by the international immuno-oncology biomarkers working group recommendations to consider TS immune infiltrate rather than TN infiltrate, They based such recommendations on evidence based prognostic analytic studies. We hereby add another rationale to support such selection based on practical application and observational reproducibility. In our study, we noticed that assessing TN infiltrates could only be credible in tumors with histology showing substantial sheets or clusters of tumor cells; in contrast, it was considerably arbitrary in tumors showing histology of Indian files or thin strands of cells with or without tubular patterns. This is attributed to the restricted identification and accurate quantification of their intermingled immune cells.

This study also highlighted differential infiltrates of TAM and Tregs. in BC types and emphasized their significant associations with some prognostic indices as tumor grade, stage, focality and LVI thus supporting prior publications signifying the pro-tumor role of TAMs and Tregs. in BC particularly in the TNBC group, their consistent concurrence in most cases also emphasizes the close pathogenic interactions among these two populations. Additionally, we guardedly raise the suggestion that TAM and Tregs presentations might also show variation among BC histopathological subtypes; an under-investigated area for which the limited representative numbers of our cases couldn't be fulfilled; thus we encourage larger scope studies to address this region. Moreover, both TAM and Tregs populations were invariably seen in periductal specialized stroma of DCIS, such encasement of the pre-invasive precursor might reflect a permissive role of these cells in promoting cancer invasion, all our DCIS cases included an associated invasive component, studying this hypothesis in a proper unbiased DCIS focused cohort might provide valuable predictive insights thus inspire novel preventative strategies.

Our findings regarding the additive value of FOXP3 staining in cancer cells as well as the disappointing inconsistency and deficiency in studies investigating this point advocate for shedding more light on this aberrant expression and its possible predictions.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics. CA Cancer J Clin 2014;64:9-29.  Back to cited text no. 1
Bates JP, Derakhshandeh R, Jones L, Webb TJ. Mechanisms of immune evasion in breast cancer. BMC Cancer 2018;18:556.  Back to cited text no. 2
Coffer PJ, Burgering BM Forkhead-box transcription factors and their role in the immune system. Nat Rev Immunol 2004;4:889-99.  Back to cited text no. 3
Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor FOXP3. Science 2003;299:1057-61.  Back to cited text no. 4
Obeid E, Rita Nanda R, Fu YX, Olopade OI. The role of tumor-associated macrophages in breast cancer progression (Review). Int J Oncol 2013;43:5-12.  Back to cited text no. 5
Ruffella B, Au A, Rugo HS, Esserman LJ, Hwang ES, Coussensa LM. Leukocyte composition of human breast cancer. PNAS 2012;109:2796-801.  Back to cited text no. 6
Mittal D, Gubin MM, Schreiber RD, Smyth MJ. New insights into cancer immunoediting and its three component phases—elimination, equilibrium and escape. Curr Opin Immunol 2014;27:16-25?.  Back to cited text no. 7
Coussens LM, Pollard JW. Leukocytes in mammary development and cancer. Cold Spring Harb Perspect Biol 2011;3:a003285.  Back to cited text no. 8
Medrek C, Pontén F, Jirström K, Leandersson K. The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients. BMC Cancer 2012;12:306.  Back to cited text no. 9
Cho EY, Chang MH, Choi YL, Lee JE, Nam SJ, Yang JH, et al. Potential candidate biomarkers for heterogeneity in triple-negative breast cancer (TNBC). Cancer Chemother Pharmacol 2011;68:753-61.  Back to cited text no. 10
Adams S, Gray RJ, Demaria S, Goldstein L, Perez EA, Shulman LN, et al. Prognostic value of tumor-infiltrating lymphocytes (TILs) in Triple Negative Breast Cancers (TNBC) from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. J Clin Oncol 2014;32:2959-66.  Back to cited text no. 11
Engels CC, Fontein DB, Kuppen PJ, de Kruijf EM, Smit VT, Nortie JW, et al. Immunological subtypes in breast cancer are prognostic for invasive ductal but not for invasive lobular breast carcinoma. Br J Cancer 2014;111:532-8.  Back to cited text no. 12
Mougiakakos D, Johansson CC, Trocme E, All-Ericsson C, Economou MA, Larsson O, et al. Intratumoral forkhead box P3-positive regulatory T cells predict poor survival in cyclooxygenase-2-positive uveal melanoma. Cancer 2010;116:2224-33.  Back to cited text no. 13
Menetrier-Caux C, Curiel T, Faget J, Manuel M, Caux C, Zou W. Targeting regulatory T cells. Targert Oncol 2012;7:15-28.  Back to cited text no. 14
Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, et al. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 2004;16:1643-56.  Back to cited text no. 15
Vasilescu F, Arsene D, Cionca F, Comanescu M, Enache V, Iosif C, et al. FOXP3 and IL17 expression in tumor infiltrating lymphocytes (TIL) and tumor cells – correlated or independent factors? Rom J Morphol Embryol 2013;54:43-9.  Back to cited text no. 16
Laoui D, Movahedi K, Van Overmeire E, Van den Bossche J, Schouppe E, Mommer C, et al. Tumor-associated macrophages in breast cancer: Distinct subsets, distinct functions. Int. J. Dev. Biol 2011;55:861-67.  Back to cited text no. 17
Krneta T, Gillgrass A, Poznanski S, Chew M, Lee AJ, Kolb M, et al. M2-polarized and tumor-associated macrophages alter NK cell phenotype and function in a contact-dependent manner. J Leukoc Biol 2017;101:285-95.  Back to cited text no. 18
DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, et al. CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell 2009;16:91-102.  Back to cited text no. 19
Takenaka M, Seki N, Toh U, Hattori S, Kawahara A, Yamaguchi T, et al. FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes is associated with breast cancer prognosis. Mol Clin Oncol 2013;1:625-32.  Back to cited text no. 20
Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 2014;515:558-62.  Back to cited text no. 21
Chen DS, Mellman I. Oncology meets immunology: The Cancer-Immunity Cycle. Immunity 2013;39:1-10.  Back to cited text no. 22
Nakasone ES, Hurvitz SA, McCann KE. Harnessing the immune system in the battle against breast cancer. Drugs Context 2018;7:212520.  Back to cited text no. 23
Hendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. Assessing tumor infiltrating lymphocytes in solid tumors: A practical review for pathologists and proposal for a standardized method from the International Immuno-Oncology Biomarkers Working Group:Part 1: Assessing the host immune response, TILs in invasive breast carcinoma and ductal carcinoma in situ, metastatic tumor deposits and areas for further research. Adv Anat Pathol 2017;24:235–51.  Back to cited text no. 24
Dieci MV, Radosevic-Robin N, Fineberg S, van den Eynden G, Ternes N, Penault-Llorca F, et al. Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer. Semin Cancer Biol 2018;52:16-25.  Back to cited text no. 25
Postow MA, Callahan MK, Wolchok JD. Immune checkpoint blockade in cancer therapy. J Clin Oncol 2015;33:1974-82.  Back to cited text no. 26
Wang K, Xu J, Zhang T, Xue D. Tumor-infiltrating lymphocytes in breast cancer predict the response to chemotherapy and survival outcome: A meta-analysis. Oncotarget 2016;7:44288-98.  Back to cited text no. 27
Cheng Z, Zhang D, Gong B, Wang P, Liu F. CD163 as a novel target gene of STAT3 is a potential therapeutic target for gastric cancer. Oncotarget 2017;14:87244-62.  Back to cited text no. 28
Lu Y-C, Robbins PF. Cancer immunotherapy targeting neoantigens. Semin Immunol 2016;28:22-7.  Back to cited text no. 29
Campbell MJ, Tonlaar NY, Garwood ER, Huo D, Moore DH, Khramtsov AI, et al. Proliferating macrophages associated with high grade, hormone receptor negative breast cancer and poor clinical outcome. Breast Cancer Res Treat 2011;128:703-11.  Back to cited text no. 30
Mahmoud SM, Lee AH, Paish EC, Macmillan RD, Ellis IO, Green AR. Tumour-infiltrating macrophages and clinical outcome in breast cancer. J Clin Pathol 2012;65:159-63.  Back to cited text no. 31
Stanton SE, Adams S, Disis ML. Variation in the incidence and magnitude of tumor-infiltrating lymphocytes in breast cancer subtypes: A systematic review. JAMA Oncol 2016;2:1354-60.  Back to cited text no. 32
Liu F, Lang R, Zhao J, Zhang X, Pringle GA, Fan Y, et al. CD8+ cytotoxic T cell and FOXP3+ regulatory T cell infiltration in relation to breast cancer survival and molecular subtypes. Breast Cancer Res Treat 2011;130:645-55.  Back to cited text no. 33
Bohling SD, Allison KH. Immunosuppressive regulatory T cells are associated with aggressive breast cancer phenotypes: A potential therapeutic target. Mod Pathol 2008;21:1527-32.  Back to cited text no. 34
Tian W, Wang L, Yuan L, Duan W, Zhao W, Wang SH, et al. A prognostic risk model for patients with triple negative breast cancer based on stromal natural killer cells, tumor-associated macrophages and growth-arrest specific protein 6. Cancer Sci 2016;107:882-9.  Back to cited text no. 35
Miyan M, Schmidt-Mende J, Kiessling R, Poschke I, de Boniface J. Differential tumor infiltration by T-cells characterizes intrinsic molecular subtypes in breast cancer. J Transl Med 2016;14:227.  Back to cited text no. 36
Jeong H, Hwang I, Kang SH, Shin HC, Kwon SY. Tumor-associated macrophages as potential prognostic biomarkers of invasive breast cancer. J Breast Cancer 2019;22:38-51.  Back to cited text no. 37
Segovia-Mendoza M, Morales-Montor J. Immune tumor microenvironment in breast cancer and the participation of estrogen and its receptors in cancer physiopathology. Front Immunol 2019;10:348.  Back to cited text no. 38
Fortis SP, Sofopoulos M, Sotiriadou NN, Haritos C, Vaxevanis CK, Anastasopoulou EA, et al. Differential intratumoral distributions of CD8 and CD163 immune cells as prognostic biomarkers in breast cancer. J immunother Cancer 2017;5:39.  Back to cited text no. 39
Jagtap SV. Evaluation of CD4+T-cells and CD8+T-cells in triple-negative invasive breast cancer. Indian J Pathol Microbiol 2018;61:477.  Back to cited text no. 40
Miyoshi Y, Shien T, Ogiya A, Ishida N, Yamazaki K, Horii R, et al. Associations in tumor infiltrating lymphocytes between clinicopathological factors and clinical outcomes in estrogen receptor-positive/human epidermal growth factor receptor type 2 negative breast cancer. Oncol Lett 2019;17:2177-86.  Back to cited text no. 41
Du T, Zhu L, Levine K, Tasdemir N, Lee A, Vignali D, et al. Invasive lobular and ductal breast carcinoma differ in immune response, protein translation efficiency and metabolism. Sci Rep 2018;8:7205.  Back to cited text no. 42
Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell 2010;141:39-51.  Back to cited text no. 43
Ladoire S, Arnould L, Mignot G, Coudert B, Rébé 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;1:65-72.  Back to cited text no. 44
Lopes LF, Guembarovski RL, Guembarovski AL, Kishima MO, Campos CZ, Oda JM, et al. FOXP3 Transcription factor: A candidate marker for susceptibility and prognosis in triple negative breast cancer. Biomed Res Int 2014;2014:341654. doi: 10.1155/2014/341654.  Back to cited text no. 45
Merlo A, Casalini P, Carcangiu ML, Malventano C, Triulzi T, Mènard S, et al. FOXP3 expression and overall survival in breast cancer. J Clin Oncol 2009;27:1746-52.  Back to cited text no. 46
Triulzi T, Tagliabue E, Balsari A, Casalini P. FOXP3 expression in tumor cells and implications for cancer progression. J Cell Physiol 2013;228:30-5.  Back to cited text no. 47
Kim M H, Koo J S, Lee S. FOXP3 expression is related to high Ki-67 index and poor prognosis in lymph node-positive breast cancer patients. Oncology 2013;85:128-36.  Back to cited text no. 48
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;129:1275-86.  Back to cited text no. 49
Zuo T, Liu R, Zhang H, Chang X, Liu Y, Wang L, et al. FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2. J Clin Invest 2007;117:3765-73.  Back to cited text no. 50
Hoskoppal D, Reisenbichler ES. Can tumor-associated macrophages in ductal carcinoma in situ on biopsy predict invasive carcinoma on excision? Hum Pathol 2018;82:158-62.  Back to cited text no. 51

Correspondence Address:
Lobna S Shash
58 Abdullah Ibn Taher St., Nasr City, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_210_20

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