
==== Front
Turk Patoloji Derg
Turk Patoloji Derg
Turk Patoloji Derg
Turkish Journal of Pathology
1018-5615
1309-5730
1018-5615
Federation of Turkish Pathology Societies Ankara, Turkey

38801127
10.5146/tjpath.2024.12805
Original Article
Investigation of the Relationship Between Tumor Microenvironment and Prognostic Parameters in Invasive Breast Carcinomas of No Special Type: A Retrospective Analysis
Ozsen Mine *
Tolunay Sahsine
Senol Kazım
Deligonul Adem
Gokgoz Sehsuvar
Evrensel Turkkan
Department of Pathology, Bursa Uludag University, Faculty of Medicine, Bursa, Türkiye
Department of General Surgery, Bursa Uludag University, Faculty of Medicine, Bursa, Türkiye
Department of Medical Oncology, Bursa Uludag University, Faculty of Medicine, Bursa, Türkiye
* E-mail: m.isikoglu@hotmail.com
Concept: MO, ST, Design: MO, ST, Supervision: ST, AD, SG, TE, Materials: MO, ST, KS, SG, Analysis and/or interpretation: MO, ST, KS, SG, Literature search: MO, ST, Writing: MO, ST, Approval: MO, ST, KS, AD, SG, TE.

2024
02 9 2024
40 3 170180
03 5 2023
02 5 2024
Copyright © 2024 The Author(s).
2024
The Author(s)
https://creativecommons.org/licenses/by/4.0/ This is an open-access article published by Federation of Turkish Pathology Societies under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.
Objective: The tumor microenvironment is a heterogeneous and constantly changing territory that plays an active role in tumor formation and progression. It constantly interacts with tumor cells, plays an active role in tumor development, and even appears as a parameter of prognostic importance, and the importance of the tumor microenvironment in breast cancer has been emphasized by recent studies. In this study, we aimed to retrospectively evaluate the relationship between the tumor microenvironment and prognostic parameters in invasive breast carcinomas of no special type.

Material and Methods: A total of 271 cases diagnosed as invasive breast carcinoma of no special type from resection materials in our center between 2007 and 2015 were included in the study. Hematoxylin-eosin stained slides with a thickness of 4-5 micrometers were evaluated in terms of tumor infiltrating lymphocytes, peritumoral and intratumoral desmoplastic reaction, intratumoral and peritumoral tumor budding, stromal features, and tumor growth pattern.

Results: When parameters related to the tumor microenvironment were compared with other prognostic parameters, there was a significant relationship between TILs and tumor grade, size, stage, immunohistochemical subgroup and Ki-67 proliferation index. A significant relationship was detected between intratumoral stromal reaction and tumor grade, size, molecular subgroup and the Ki-67 proliferation index (p<0.05). When stroma and other prognostic parameters were compared, tumors with desmoplastic stroma had higher grades and higher Ki-67 proliferation indexes, and they were observed more frequently in the triple negative molecular subgroup.

Conclusion: We believe that including parameters related to tumor microenvironment in breast cancer reports, which hold a prognostic and predictive importance, will contribute to patient management. Considering the fact that these can be easily evaluated from routinely used hematoxylin-eosin stained slides, this does not cause additional costs or excessive time loss.

Breast cancer
Tumor budding
Tumor infiltrating lymphocytes
Tumor microenvironment
Prognosis
==== Body
pmcIntroduction

Over the years, research has been conducted to reveal the relationship between the prognosis and various histopathological (invasive tumor size, histopathological type, grade, lymphovascular invasion, and axillary lymph node status), immunohistochemical (estrogen receptor, progesterone receptor, c-erbB2 status, and ki-67 proliferative index) and molecular (HER2 status) parameters in breast cancer (1,2).

Another parameter whose impact on the prognosis has been investigated is the tumor microenvironment. The tumor microenvironment can be defined as a heterogeneous and constantly changing territory that plays an active role in tumor formation and progression. Studies have shown that the tumor microenvironment, consisting of tumor cells and various non-neoplastic cells (fibroblasts, immune cells, endothelial cells, inflammatory cells, adipocytes, signaling molecules, extracellular matrix components) plays a role in the emergence, development and treatment response of breast cancer (3). Therefore, we aimed to investigate the relationship between the tumor microenvironment and prognostic parameters in invasive breast carcinoma of no special type (IBC-NST) cases that had not undergone any neoadjuvant treatment.

Materials and Method

All cases diagnosed with IBC-NST from resection materials (lumpectomy, segmental mastectomy, modified radical mastectomy, breast-conserving surgical material) in our center, between 2007 and 2015, were included in the study. Cases where the slides were not available or not suitable for re-evaluation, cases who received neoadjuvant treatment, cases diagnosed from non-resection materials, and cases histologically diagnosed as other than IBC-NST were excluded from the study.

Demographic (age) and clinicopathological (tumor site, size, grade, presence of lymphovascular invasion, perineural invasion, immunohistochemical subgroups (according to immunohistochemical staining results; ER positive and low ki-67 proliferation index, ER positive and high ki-67 proliferation index, HER2 positive and triple negative), presence of metastasis, site of metastases if present, stage, presence of recurrence, survival status, recurrence-free, total survival and follow-up periods) of the cases were obtained from patient files and pathology reports.

Hematoxylin-eosin (H&E) stained slides with a thickness of 4-5 micrometers included in the study were evaluated in terms of parameters related to the tumor microenvironment (tumor infiltrating lymphocytes (TILs), peritumoral and intratumoral desmoplastic reaction, intratumoral and peritumoral tumor budding, stromal features and tumor growth pattern).

Two different methods were used when evaluating the TIL. The first was based on the evaluation technique recommended by the World Health Organization (WHO) Breast Tumors Classification in 2019. Accordingly, the ratio of mononuclear inflammatory cells to the stromal area in the intratumoral compartment was taken as the basis. The entire tumor area was evaluated and a net percentage value was obtained for each tumor from a single slide. The percentage values were grouped as <10%, between 10% and 50%, and >50%, based on the recommended threshold values (1,4,5). The second evaluation method included evaluating the distribution of mononuclear inflammatory cells in the tumor stroma. Tumors were divided into two groups as diffuse and non-diffuse TIL.

When evaluating the peritumoral desmoplastic reaction, the stromal area around the tumor was considered. Desmoplasia was categorized as absent, mild, moderate and prominent. Similar grading was used for the evaluation of the intratumoral desmoplastic reaction, except for including the stromal area within the tumor and not the tumor periphery.

Tumor budding was defined as isolated tumor cells or groups of less than 5 tumor cells. Tumor budding was noted as present or absent. It was evaluated from hotspots of two separate sites, using 400x magnification. One site was the border between invasive tumor and the surrounding stroma (peritumoral), and the other was within the invasive tumor (intratumoral) (6).

Stromal features were evaluated and recorded, and the stroma was divided into three categories as myxoid, desmoplastic and hyalinized.

Growth patterns were assessed based on publications that report growth patterns to be associated with the prognosis in metastatic liver tumors. Accordingly, tumor growth patterns were defined as “desmoplastic” if there was a desmoplastic rim between the tumor cells and the surrounding stroma, “replacement” if tumor cells were observed between normal structures without destroying the main architecture, and “pushing” if tumor cells were growing by pushing the normal structures (7).

Approval for the research, dated 25 November 2020 and numbered 2020-21/10, was obtained from the local Clinical Research Ethics Committee.

Statistical Analysis

The SPSS 25.0 package program was used for statistical analyses. Categorical measurements were given as numbers and percentages. Continuous measurements were calculated as mean and standard deviation (median and minimum-maximum where appropriate). The chi-square test or Fisher’s exact test was used to compare categorical variables. The distribution of the groups was checked for comparison of continuous measurements. Student’s T test was used for parameters with a normal distribution. Anova was used for the comparison of more than two variables. The Mann-Whitney U test was used for parameters that did not show a normal distribution and the Kruskal-Wallis test was used for the comparison of more than two variables. The Kaplan-Meier method was used to evaluate the survival curve and the Long-rank test was used to calculate the difference of survival between groups. A statistical significance level of 0.05 was determined in all tests.

Results

A total of 271 cases diagnosed with IBC-NST from resection materials in our center, between 2007 and 2015, and which met the study criteria were identified. The general characteristics of the cases and the routine parameters evaluated in resection materials are summarized in Table 1 and Table 2 (Table 3, Table 4 and Table 5).

Table 1 General characteristics of the cases (n=271)

	Mean + SD

	Min-Max

	
Age (years)

	54.8±12.1

	27-83

	
Size (cm)

	2.6±1.5

	0.3-10

	
Follow-up period

	69.6±33.9

	1-156

	
Recurrence-free survival (months)

	88.3±30.3

	3-156

	
Total survival (months)

	91.7±28.1

	3-156

	
	n (%)

	
Stage

1

2

3

4

	78 (28.8)

144 (53.1)

38 (14)

11 (4.1)

	
Recurrence

Absent

Present

	251 (92.6)

20 (7.4)

	
Status

Dead of disease

Alive

	59 (21.8)

212 (78.2)

	

Table 2 Histopathological features of the cases (n=271)

	n (%)

	
TILs

<10%

10-50%

>50%

	183 (67.5)

44 (16.3)

44 (16.2)

	
TILs

Diffuse

Non-diffuse

	193 (71.2)

78 (28.8)

	
Peritumoral stromal reaction

Absent

Mild

Moderate

Prominent

	196 (72.3)

65 (24)

3 (1.1)

7 (2.6)

	
Intratumoral stromal reaction

Absent

Mild

Moderate

Prominent

	3 (1.1)

50 (18.5)

56 (20.7)

162 (59.8)

	
Peritumoral tumor budding

Absent

Present

	98 (36.2)

173 (63.8)

	
Intratumoral tumor budding

Absent

Present

	78 (28.8)

193 (71.2)

	
Stroma

Myxoid

Desmoplastic

Hyalinized

	67 (24.7)

91 (33.6)

113 (41.7)

	
Growth pattern

Replacement

Pushing

Desmoplastic

	244 (90)

19 (7)

8 (3)

	
Grade

1

2

3

	9 (3.3)

115 (42.4)

147 (54.2)

	
Lymphovascular invasion

Absent

Present

	197 (72.7)

74 (27.3)

	
Perineural invasion

Absent

Present

	198 (73.1)

73 (26.9)

	
Immunohistochemical subgroup

ER positive

HER-2 positive

Triple negative

	187 (69)

19 (7)

65 (24)

	
Ki-67 percentage

≤14%

>14%

	77 (28.4)

194 (71.6)

	
Axillary metastasis

Absent

Present

	147 (54.2)

124 (45.8)

	

Table 3 Relation of Tumor Microenvironment to Prognostic Parameters (n=271)

TIL

	
	≤10 n (%)

	10-50 n (%)

	≥50   n (%)

	p

	
Grade

	1

	8

	4.4

	1

	2.3

	0

	0.0

	0.0001

	
2

	93

	50.8

	12

	27.3

	10

	22.7

	
3

	82

	44.8

	31

	70.5

	34

	77.3

	
Lvi

	Absent

	134

	73.2

	28

	63.6

	35

	79.5

	0.236

	
Present

	49

	26.8

	16

	36.4

	9

	20.5

	
Perineural Invasion

	Absent

	129

	70.5

	33

		36

	81.8

	0.299

	
Present

	54

	29.5

	11

	25.0

	8

	18.2

	
Immunohistochemical Groups

	ER positive

	150

	82.0

	26

	59.1

	11

	25.0

	0.0001

	
HER2 positive

	8

	4.4

	4

	9.1

	7

	15.9

	
Triple Negative

	25

	13.7

	14

	31.8

	26

	59.1

	
Ki-67 (%)

	≤14

	65

	35.5

	7

	15.9

	5

	11.4

	0.001

	
>14

	118

	64.5

	37

	84.1

	39

	88.6

	
Axillary Metastasis

	Absent

	94

	51.4

	24

	54.5

	29

	65.9

	0.220

	
Present

	89

	48.6

	20

	45.5

	15

	34.1

	
Stage

	1

	59

	32.2

	8

	18.2

	11

	25.0

	0.030

	
2

	101

	55.2

	21

	47.7

	22

	50.0

	
3

	18

	9.8

	12

	27.3

	8

	18.2

	
4

	5

	2.7

	3

	6.8

	3

	6.8

	
Presence of Recurrence

	Absent

	166

	90.7

	43

	97.7

	42

	95.5

	0.205

	
Present

	17

	9.3

	1

	2.3

	2

	4.5

	
Survival Status

	Exitus

	141

	77.0

	35

	79.5

	36

	81.8

	0.768

	
Alive

	42

	23.0

	9

	20.5

	8

	18.2

	
Intratumoral stromal reaction

	
	Absent n (%)

	Mild n (%)

	Moderate n (%)

	p

	
Grade

	1

	0

	0.0

	1

	1.8

	8

	4.9

	0.001

	
2

	11

	20.8

	26

	46.4

	78

	48.1

	
3

	42

	79.2

	29

	51.8

	76

	46.9

	
Lvi

	Absent

	42

	79.2

	41

	73.2

	114

	70.4

	0.451

	
Present

	11

	20.8

	15

	26.8

	48

	29.6

	
Perineural Invasion

	Absent

	44

	83.0

	38

	67.9

	116

	71.6

	0.164

	
Present

	9

	17.0

	18

	32.1

	46

	28.4

		
Immunohistochemical Groups

	ER positive

	20

	37.7

	39

	69.6

	128

	79.0

	0.0001

	
HER2 positive

	5

	9.4

	6

	10.7

	8

	4.9

	0.451

	
Triple Negative

	28

	52.8

	11

	19.6

	26

	16.0

		
Ki-67 (%)

	≤14

	4

	7.5

	16

	28.6

	57

	35.2

	0.001

	
>14

	49

	92.5

	40

	71.4

	105

	64.8

		
Axillary Metastasis

	Absent

	33

	62.3

	30

	53.6

	84

	51.9

	0.415

	
Present

	20

	37.7

	26

	46.4

	78

	48.1

	0.451

	
Stage

	1

	14

	26.4

	18

	32.1

	46

	28.4

	0.419

	
2

	26

	49.1

	26

	46.4

	92

	56.8

		
3

	10

	18.9

	11

	19.6

	17

	10.5

		
4

	3

	5.7

	1

	1.8

	7

	4.3

	0.415

	
Presence of Recurrence

	Absent

	49

	92.5

	51

	91.1

	151

	93.2

	0.869

	
Present

	4

	7.5

	5

	8.9

	11

	6.8

	0.451

	
Survival Status

	Exitus

	41

	77.4

	40

	71.4

	131

	80.9

	0.768

	
Alive

	12

	22.6

	16

	28.6

	31

	19.1

		
Intratumoral tumor budding

	
	Absent n (%)

	Present n (%)

	p

	
Immunohistochemical Groups

	ER positive

	42

	53.8

	145

	75.1

	0.0001

	
HER2 positive

	12

	15.4

	7

	3.6

	
Triple Negative

	24

	30.8

	41

	21.2

	
Axillary Metastasis

	Absent

	51

	65.4

	96

	49.7

	0.022

	
Present

	27

	34.6

	97

	50.3

	
Peritumoral tumor budding

	
	Absent n (%)

	Present n (%)

	p

	
Lvi

	Absent

	79

	80.6

	118

	68.2

	0.033

	
Present

	19

	19.4

	55

	31.8

	
Stage

	1

	42

	42.9

	102

	59.0

	0.023

	
2

	21

	21.4

	17

	9.8

	
3

	4

	4.1

	7

	4.0

	
4

	31

	31.6

	47

	27.2

	
Stroma

	
	Myxoid n (%)

	Desmoplastic n (%)

	Hyalinized n (%)

	p

	
Lateralisation

	Left

	31

	46.3

	34

	37.4

	65

	57.5

	0.016

	
Right

	36

	53.7

	57

	62.6

	48

	42.5

	
Grade

	1

	3

	4.5

	1

	1.1

	5

	4.4

	0.003

	
2

	40

	59.7

	29

	31.9

	46

	40.7

	
3

	24

	35.8

	61

	67.0

	62

	54.9

	
Immunohistochemical Groups

	ER positive

	61

	91.0

	53

	58.2

	73

	64.6

	0.0001

	
HER2 positive

	3

	4.5

	8

	8.8

	8

	7.1

	
Triple Negative

	3

	4.5

	30

	33.0

	32

	28.3

	
Ki-67 (%)

		38

	56.7

	13

	14.3

	26

	23.0

	0.0001

	
	29

	43.3

	78

	85.7

	87

	77.0

	0.0001

	
Stage

	1

	5

	7.5

	33

	36.3

	40

	35.4

	0.001

	
2

	48

	71.6

	43

	47.3

	53

	46.9

	
3

	13

	19.4

	10

	11.0

	15

	13.3

	
4

	1

	1.5

	5

	5.5

	5

	4.4

	
Growth Pattern

	
	Desmoplastic n (%)

	Replacement n (%)

	Pushing n (%)

	p

	
Grade

	1

	0

	0.0

	9

	3.7

	0

	0.0

	0.028

	
2

	3

	37.5

	110

	45.1

	2

	10.5

	
3

	5

	62.5

	125

	51.2

	17

	89.5

	
Immunohistochemical Groups

	ER positive

	6

	75.0

	177

	72.5

	4

	21.1

	0.0001

	
HER2 positive

	1

	12.5

	18

	7.4

	0

	0.0

	
Triple Negative

	1

	12.5

	49

	20.1

	15

	78.9

	
Ki-67 (%)

	≤14

	2

	25.0

	75

	30.7

	0

	0.0

	0.016

	
>14

	6

	75.0

	169

	69.3

	19

	100.0

	
Axillary Metastasis

	Absent

	4

	50.0

	127

		16

	84.2

	0.025

	
Present

	4

	50.0

	117

	48.0

	3

	15.8

	

Table 4 Relationship of tumor microenvironment-related parameters with recurrence-free survival rates

	Estimated Meana

	Std. Error

	95% Confidence Interval

	5-year recurrence-free survival %

	p

	
Lower Bound

	Upper Bound

	
Recurrence-free Survival

	146.8

	2.0

	142.9

	150.7

	93.8

	-

	
TIL (%)

	
≤10

	144.2

	2.7

	138.9

	149.6

	91.9

	0.202

	
10-50

	117.9

	2.1

	113.9

	121.9

	97.6

	
≥50

	129.2

	2.6

	124.1

	134.4

	97.6

	
TIL

							
Diffuse

	145.7

	2.4

	140.8

	150.6

	92.9

	0.384

	
Non-diffuse

	141.9

	2.9

	136.1

	147.8

	94.4

	
Intratumoral Stromal Reaction

	
Absent - Mild

	126.1

	3.3

	119.6

	132.7

	93.7

	0.881

	
Moderate

	122.8

	4.0

	114.9

	130.7

	90.7

	
Severe

	147.4

	2.5

	142.5

	152.4

	93.4

	
Intratumoral Tumor Budding

	
Absent

	137.8

	3.3

	131.3

	144.2

	93.0

	0.903

	
Present

	146.7

	2.4

	142.0

	151.4

	93.5

	
Peritumoral Tumor Budding

	
Absent

	136.8

	3.1

	130.7

	143.0

	93.3

	0.661

	
Present

	147.2

	2.4

	142.4

	152.1

	94.0

	
Stroma

	
Myxoid

	110.7

	2.4

	105.9

	115.5

	95.3

	0.132

	
Desmoplastic

	150.7

	2.6

	145.7

	155.8

	96.2

	
Hyalinized

	133.4

	3.3

	126.8

	139.9

	90.1

	
Growth Pattern

	
Desmoplastic

	-

	-

	-

	-

	100

	0.278

	
Replacement

	147.3

	2.1

	143.2

	151.3

	94.4

	
Pushing

	110.5

	7.2

	96.4

	124.6

	83.3

	

Table 5 Relationship of tumor microenvironment-related parameters with total survival rate

	Estimated Meana

	Std. Error

	95% Confidence Interval

	1-year survival %

	3-year survival %

	5-year survival %

	p

	
Lower Bound

	Upper Bound

	
Total Survival

	126.2

	3.9

	118.4

	133.9

	97.8

	93.4

	88.2

	-

	
TIL (%)

	
≤10

	126.1

	4.7

	116.7

	135.3

	97.8

	91.3

	86.9

	0.780

	
10-50

	105.3

	4.7

	95.9

	114.6

	95.5

	90.9

	84.0

	
≥50

	117.5

	5.1

	107.5

	127.5

	95.5

	93.2

	88.5

	
TIL

	
Diffuse

	125.3

	4.9

	115.6

	134.9

	98.4

	92.2

	87.6

	0.457

	
Non-diffuse

	121.8

	6.3

	109.5

	134.1

	94.9

	88.5

	87.2

	
Peritumoral Stromal Reaction

	
Absent

	126.134

	5.086

	116.165

	136.103

	98.0

	92.9

	88.3

	0.977

	
Mild

	114.453

	4.568

	105.500

	123.405

	96.9

	90.8

	87.7

	
Moderate

	120.000

	0.000

	120.000

	120.000

	100

	100

	100

	
Severe

	99.286

	20.270

	59.556

	139.016

	85.7

	71.4

	71.4

	
Intratumoral Stromal Reaction

	
Absent - Mild

	112.9

	5.3

	102.6

	123.3

	94.3

	90.6

	88.6

	0.331

	
Moderate

	109.9

	5.1

	99.9

	119.9

	98.2

	92.9

	93.9

	
Severe

	132.1

	4.3

	123.7

	140.5

	98.1

	92.6

	87.7

	
Intratumoral Tumor Budding

	
Absent

	127.1

	4.8

	117.6

	136.4

	96.2

	92.3

	89.7

	0.235

	
Present

	120.9

	5.3

	110.4

	131.3

	97.9

	92.2

	86.5

	
Periumoral Tumor Budding

	
Absent

	120.8

	4.7

	111.6

	129.9

	95.9

	91.8

	86.7

	0.875

	
Present

	124.7

	5.8

	113.3

	136.1

	98.8

	91.9

	88.4

	
Stroma

	
Myxoid

	99.5

	3.8

	91.8

	107.1

	95.5

	91.0

	82.1

	0.288

	
Desmoplastic

	122.0

	6.2

	109.8

	134.3

	95.6

	87.9

	84.6

	
Hyalinized

	126.7

	3.9

	118.9

	134.5

	99.1

	96.5

	92.0

	
Growth Pattern

	
Desmoplastic

	110.3

	15.4

	80.1

	140.5

	87.5

	87.5

	72.9

	0.987

	
Replacement

	124.9

	4.7

	115.6

	134.2

	98.0

	92.2

	87.3

	
Pushing

	116.0

	6.9

	102.3

	129.7

	94.7

	89.2

	89.2

	

The rate of TILs was less than 10% in 183 (67.5%), between 10% and 50% in 44 (16.2%) and more than 50% in 44 (16.2%) cases. 193 (71.2%) tumors showed diffuse TILs and 78 (28.8%) showed non-diffuse TILs (Figure 1).

Figure 1 Assessment of tumor-infiltrating lymphocytes in the breast cancer microenvironment. High magnification view of one stromal area included in the scoring, estimated as <10% sTILs (A, H&Ex400). High magnification view of one stromal area included in the scoring, estimated as 10-50% sTILs (B, H&Ex200). High magnification view of one stromal area included in the scoring, estimated as >50% sTILs (H&Ex400).

Peritumoral stromal reaction was absent in 196 (72.3%), mild in 65 (24%), moderate in 3 (1.1%), and prominent in 7 (2.6%) tumors. Intratumoral stromal reaction was absent in 3 (1.1%), mild in 50 (18.5%), moderate in 56 (20.7%), and prominent in 162 (59.8%) tumors (Figure 2).

Figure 2 Assessment of intratumoral stromal reaction in the breast cancer microenvironment. Areas with evaluation of intense intratumoral stromal reaction on high magnification view (A, H&Ex400). Areas with evaluation of low intratumoral stromal reaction on high magnification view (B, H&Ex400).

Peritumoral tumor budding was not detected in 98 (36.2%), and was present in 173 (63.8%) cases. Intratumoral tumor budding was absent in 78 (28.8%) and present in 193 (71.2%) tumors (Figure 3).

Figure 3 Assessment of tumor budding in the breast cancer microenvironment (A,B, H&Ex400).

Stroma was myxoid in 67 (24.7%), desmoplastic in 91 (33.6%), and hyalinized in 113 (41.7%) tumors.

A replacement growth pattern was observed in 90% of the tumors, while a pushing pattern and desmoplastic pattern was present in 7% and 3%, respectively.

Comparison of TILs and Other Prognostic Parameters

When features related to the tumor microenvironment were compared with other prognostic parameters, a statistically significant relationship was found between TILs and grade, tumor size, molecular subgroup, ki-67 percentage, and tumor stage. Accordingly, the rate of TILs was higher in tumors with a larger size, higher grade, and a higher ki-67 proliferation index. TILs and size, grade, and ki-67 percentage were directly proportional (p=0.014, p=0.0001, p=0.001). In addition, an inverse relationship was found between TILs and tumor stage. As the rate of TILs decreased, the tumor stage increased (p=0.030). The rate of TILs was significantly higher in the Her2 and triple negative molecular subgroups compared to the luminal subgroup (p=0.0001). When TILs were categorized as diffuse and non-diffuse, significant statistical results could not be obtained.

Comparison of Intratumoral Stromal Reaction and Other Prognostic Parameters

A statistically significant relationship was present between intratumoral stromal reaction intensity and tumor grade, size, molecular subgroup, and ki-67 proliferation index. Grade, tumor size and ki-67 proliferation index increased as the intensity of intratumoral stromal reaction decreased (p=0.007, p=0.001, p=0.001). The stromal reaction intensity in Her2 and triple negative molecular subgroups was lower than in the luminal subgroup (p=0.0001).

Comparison of Tumor Budding and Other Prognostic Parameters

Incidence of axillary metastasis was higher in cases showing intratumoral tumor budding. The lymphovascular invasion incidence was higher in cases showing peritumoral tumor budding (p=0.022, p=0.033).

Comparison of Stromal Features and Other Prognostic Parameters

Tumors with a desmoplastic stroma were higher grade tumors, the ki-67 proliferation indices were higher, and were most frequently in the triple negative subgroup (p=0.003, p=0.0001 and p=0.0001). Tumors with a myxoid stroma were predominantly in the luminal subgroup, whereas tumors with a hyalinized stroma had a higher ki-67 proliferation index compared to tumors with a myxoid stroma (p=0.003, p=0.0001).

Comparison of Growth Pattern and Other Prognostic Parameters

In cases with axillary metastases, a desmoplastic growth pattern was more frequent and the pushing growth pattern was seen at a lower rate (p=0.025).

Comparison of Peritumoral Stromal Reaction and Other Prognostic Parameters

A statistically significant correlation was not detected between peritumoral stromal reaction intensity and other prognostic parameters.

Relationship Between Tumor Microenvironment and Survival

The mean follow-up period was 69.6±33.9 months. Recurrence-free survival and total survival periods ranged from 3 to 156 months. Mean recurrence-free survival period was 88.3±30.3 months. Mean total survival period was 91.7±28.1 months. A total of 59 patients died during the follow-up period. The 1, 3, and 5-year recurrence-free survival rates were 98.9%, 95.8%, and 93.8%, while overall survival rates were 97.8%, 93.4%, and 88.2%, respectively. Recurrence-free and total survival periods were not statistically related to the parameters of the tumor microenvironment (p>0.05).

Discussion

The tumor microenvironment is constantly interacting with tumor cells, plays an active role in tumor development and even provides prognostic information in some tumors. It is formed as a result of genetic changes in the tumor cells. The tumor microenvironment is observed differently in every tumor type and is tumor-specific. It has been investigated thoroughly in malignant melanoma and lung and colorectal carcinomas where immune cell infiltration is more frequent (8–10). Although immune cell infiltration is less common in breast cancer, it has been reported that the tumor microenvironment is effective in tumor development, progression, and treatment response (11,12). After its first report in 1922, the assessment of TILs was included in the 5th edition of the WHO classification of breast tumors, published in 2019. Accordingly, the evaluation is based on the ratio of mononuclear inflammatory cells to the stromal area, in the intratumoral stromal compartment. The evaluation should include the entire tumor area and should be done on a 20x or 40x objective on a single slide (1,13). Various studies on non-mammary tumors show that tumors with high TILs rates have a better prognosis compared to tumors with low TILs rates (10). The same results were obtained from similar studies on breast carcinomas. Accordingly, higher TILs rates are associated with better prognosis and better treatment responses, especially in HER2 and triple-negative tumors, known to have poorer prognoses (1,3,12,14). It has been reported that every 10% increase in TILs rate reduces the risk of recurrence or death by 13%, the risk of distant organ metastasis or death by 17%, and the risk of death by 16% (15). Although a clear cutoff value for TILs related to the prognosis is not reported in the WHO Classification of Breast Tumors, a threshold of 50-60% is generally accepted (1). In our study, no relationship was found between TILs and survival rates, but the rate of TILs was significantly higher in HER2 and triple negative subgroups. In addition, the tumor stage was higher in tumors with lower TILs, which can be interpreted as a poorer clinical course for tumors showing low TILs.

Another parameter that has not been widely investigated in breast cancer, but has been found to be an independent prognostic and predictive marker in various malignancies such as colorectal and pancreoticobiliary system cancers, is the feature of the stroma (16,17). It has been reported that stromal reaction in breast tumors is associated with the rate of lymph node and distant organ metastasis and has an effect on prognosis (18). In their study of triple-negative breast cancers, Zakhartseva and Yanovytska determined the tumor stroma ratio to be an independent prognostic parameter associated with both disease-free survival and total survival periods (19). Roeke et al. have shown that a high tumor/stroma ratio is an independent prognostic parameter for total survival rate, distant metastasis, and recurrence-free survival rates. The clinical course is worse in cases with a high tumor stroma ratio. At the same time, this ratio is associated with advanced age and larger tumor size (20). In our study, the stroma was evaluated independently in the intratumoral and peritumoral areas. Contrary to other studies, the ratio of intratumoral stroma and tumor grade, size and ki-67 proliferation index were found to be inversely proportional. This is thought to be due to the difference of evaluation. In addition to the intensity of the stromal reaction, different histomorphological features of the stroma such as desmoplastic, hyalinized, or myxoid, are also known to have an effect on the prognosis. Yanai et al. found fibrotic stroma to be associated with a higher venous invasion rate and tumor grade. In addition, a fibrotic stroma is associated with a worse recurrence-free and total survival rate in triple-negative tumors (21). Similarly in our study, we found that tumors with a desmoplastic stroma were of higher grade, had higher Ki-67 proliferation indices and were more frequent in triple negative cases. Tumor budding is another important feature of the tumor microenvironment. It has been evaluated since it was first described in 1954 and found to be strongly related to the prognosis. In contrast to pancreatic, lung, and gastrointestinal system tumors, research on tumor budding is limited in breast cancer (22–25). However, studies show that tumor budding is an important parameter associated with lymphovascular invasion, lymph node metastasis and survival in this cancer (25,26). The study of Kumarguru et al. has shown a significant correlation between tumor budding and lymphovascular invasion, lymph node metastasis, necrosis, and tumor stage (27). Liang et al. have determined that the tumor size was larger, lymph node metastasis was more common, and the overall survival was shorter in cases with higher numbers of budding (28). Evaluation of tumor budding with different methods leads to different results. The evaluation in our study was done in a way similar to Renuka et al.’s study, in two separate areas as intratumoral and peritumoral. Intratumoral tumor budding was associated with lymph node metastasis, while peritumoral tumor budding was associated with lymphovascular invasion (26).

The relationship between tumor microenvironment and various prognostic parameters was studied in our study, but a relationship with recurrence-free and total survival rate was not detected. Despite the high number of cases included, this may suggest the need for a larger studies on breast cancer. The relatively low number of HER2 positive and triple negative cases is one of the limitations of this study. The prolongation of the total survival periods and the decrease in recurrence rates in breast cancer with the help of various treatment methods, also brings up the need of conducting surveillance studies with longer follow-up periods. In our study, the cases were evaluated over a 5-year follow-up period. This is one of the limitations of this study and longer follow-up periods may change the results. The tumor microenvironment is an important prognostic and predictive parameter. The fact that the evaluation can be made from H&E stained slides and that it does not cause additional costs and time loss makes it possible to easily evaluate these parameters worldwide. The relatively limited number of studies in this area in breast cancer prevents routine evaluation and the inclusion of these parameters in reporting formats. However, we believe that it will be possible to set a worldwide standard as the number of studies in this area increases.

Funding

There were no external sources of funding for the present study.

Ethics Committee Approval

Approval for the research, dated 25 November 2020 and numbered 2020-21/10, was obtained from the local Clinical Research Ethics Committee.

Conflict of Interest

The authors declare that they have no conflict of interest.
==== Refs
Lokuhetty D , White VA , Watanabe R , Cree IA WHO Classification of Tumours Invasive breast carcinoma: General Overview: Breast Tumours IARC Press Lyon, France 2019
82 101
Schnitt SJ , Collins LC Biopsy Interpretation of the Breast Invasive Breast Cancer Philadelphia 2018
311 400
Mittal Suruchi , Brown Nicola J. , Holen Ingunn Expert Rev Mol Diagn The breast tumor microenvironment: role in cancer development, progression and response to therapy 03 2018
18 227 243 10.1080/14737159.2018.1439382 29424261
International Immuno-Oncology Biomarker Working Group on Breast Cancer 2018

Salgado R. , Denkert C. , Demaria S. , Sirtaine N. , Klauschen F. , Pruneri G. , Wienert S. , Eynden G. Van den , Baehner F. L. , Penault-Llorca F. , Perez E. A. , Thompson E. A. , Symmans W. F. , Richardson A. L. , Brock J. , Criscitiello C. , Bailey H. , Ignatiadis M. , Floris G. , Sparano J. , Kos Z. , Nielsen T. , Rimm D. L. , Allison K. H. , Reis-Filho J. S. , Loibl S. , Sotiriou C. , Viale G. , Badve S. , Adams S. , Willard-Gallo K. , Loi S. , International TILs Working Group 2014 Ann Oncol The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014 02 2015
26 259 271 10.1093/annonc/mdu450 25214542
Li Xiaoxian , Wei Bo , Sonmez Ceyda , Li Zaibo , Peng Limin Hum Pathol High tumor budding count is associated with adverse clinicopathologic features and poor prognosis in breast carcinoma 08 2017
66 222 229 10.1016/j.humpath.2017.06.008 28655638
Dam Pieter-Jan van , Stok Eric P. van der , Teuwen Laure-Anne , Eynden Gert G. Van den , Illemann Martin , Frentzas Sophia , Majeed Ali W. , Eefsen Rikke L. , Braak Robert R. J. Coebergh van den , Lazaris Anthoula , Fernandez Maria Celia , Galjart Boris , Laerum Ole Didrik , Rayes Roni , Grünhagen Dirk J. , Paer Michelle Van de , Sucaet Yves , Mudhar Hardeep Singh , Schvimer Michael , Nyström Hanna , Kockx Mark , Bird Nigel C. , Vidal-Vanaclocha Fernando , Metrakos Peter , Simoneau Eve , Verhoef Cornelis , Dirix Luc Y. , Van Laere Steven , Gao Zu-Hua , Brodt Pnina , Reynolds Andrew R. , Vermeulen Peter B. Br J Cancer International consensus guidelines for scoring the histopathological growth patterns of liver metastasis 11 2017
117 1427 1441 10.1038/bjc.2017.334
Junttila Melissa R. , Sauvage Frederic J. de Nature Influence of tumour micro-environment heterogeneity on therapeutic response 09 2013
501 346 354 10.1038/nature12626 24048067
Badalamenti Giuseppe , Fanale Daniele , Incorvaia Lorena , Barraco Nadia , Listì Angela , Maragliano Rossella , Vincenzi Bruno , Calò Valentina , Iovanna Juan Lucio , Bazan Viviana , Russo Antonio Cell Immunol Role of tumor-infiltrating lymphocytes in patients with solid tumors: Can a drop dig a stone? 09 2019
343 103753 103753 10.1016/j.cellimm.2018.01.013 29395859
Yasuda Koji , Nirei Takako , Sunami Eiji , Nagawa Hirokazu , Kitayama Joji Radiat Oncol Density of CD4(+) and CD8(+) T lymphocytes in biopsy samples can be a predictor of pathological response to chemoradiotherapy (CRT) for rectal cancer 05 2011
6 49 49 10.1186/1748-717X-6-49 21575175
Deepak K. G. K. , Vempati Rahul , Nagaraju Ganji Purnachandra , Dasari Venkata Ramesh , S Nagini , Rao D. N. , Malla Rama Rao Pharmacol Res Tumor microenvironment: Challenges and opportunities in targeting metastasis of triple negative breast cancer 03 2020
153 104683 104683 10.1016/j.phrs.2020.104683 32050092
Savas Peter , Salgado Roberto , Denkert Carsten , Sotiriou Christos , Darcy Phillip K. , Smyth Mark J. , Loi Sherene Nat Rev Clin Oncol Clinical relevance of host immunity in breast cancer: from TILs to the clinic 04 2016
13 228 241 10.1038/nrclinonc.2015.215 26667975
Laenkholm Anne-Vibeke , Callagy Grace , Balancin Marcelo , Bartlett John M. S. , Sotiriou Christos , Marchio Caterina , Kok Marleen , Dos Anjos Carlos Henrique , Salgado Roberto Virchows Arch Incorporation of TILs in daily breast cancer care: how much evidence can we bear? 01 2022
480 147 162 10.1007/s00428-022-03276-w
Bareche Yacine , Buisseret Laurence , Gruosso Tina , Girard Edwina , Venet David , Dupont Floriane , Desmedt Christine , Larsimont Denis , Park Morag , Rothé Françoise , Stagg John , Sotiriou Christos J Natl Cancer Inst Unraveling Triple-Negative Breast Cancer Tumor Microenvironment Heterogeneity: Towards an Optimized Treatment Approach 07 2020
112 708 719 10.1093/jnci/djz208 31665482
Loi S. , Michiels S. , Adams S. , Loibl S. , Budczies J. , Denkert C. , Salgado R. Ann Oncol The journey of tumor-infiltrating lymphocytes as a biomarker in breast cancer: clinical utility in an era of checkpoint inhibition 10 2021
32 1236 1244 10.1016/j.annonc.2021.07.007
Wang Kai , Ma Wei , Wang Jianbo , Yu Liang , Zhang Xiaomei , Wang Zhenbo , Tan Bingxu , Wang Nana , Bai Bing , Yang Shengsi , Liu Houqiang , Zhu Shengjie , Cheng Yufeng J Thorac Oncol Tumor-stroma ratio is an independent predictor for survival in esophageal squamous cell carcinoma 09 2012
7 1457 1461 10.1097/JTO.0b013e318260dfe8 22843085
Kramer C. J. H. , Vangangelt K. M. H. , Pelt G. W. van , Dekker T. J. A. , Tollenaar R. a. E. M. , Mesker W. E. Breast Cancer Res Treat The prognostic value of tumour-stroma ratio in primary breast cancer with special attention to triple-negative tumours: a review 01 2019
173 55 64 10.1007/s10549-018-4987-4
Catteau Xavier , Simon Philippe , Noël Jean-Christophe BMC Cancer Myofibroblastic stromal reaction and lymph node status in invasive breast carcinoma: possible role of the TGF-β1/TGF-βR1 pathway 07 2014
14 499 499 10.1186/1471-2407-14-499 25011545
Zakhartseva Liubov M. , Yanovytska Mariia A. Wiad Lek PROGNOSTIC VALUE OF TUMOR STROMA RATIO IN TRIPLE NEGATIVE BREAST CANCER 2021
74 565 571 33843614
Roeke Toni , Sobral-Leite Marcelo , Dekker Tim J. A. , Wesseling Jelle , Smit Vincent T. H. B. M. , Tollenaar Rob A. E. M. , Schmidt Marjanka K. , Mesker Wilma E. Breast Cancer Res Treat The prognostic value of the tumour-stroma ratio in primary operable invasive cancer of the breast: a validation study 11 2017
166 435 445 10.1007/s10549-017-4445-8
Yanai Hirotsugu , Yoshikawa Katsuhiro , Ishida Mitsuaki , Tsuta Koji , Sekimoto Mitsugu , Sugie Tomoharu PLoS One Presence of myxoid stromal change and fibrotic focus in pathological examination are prognostic factors of triple-negative breast cancer: Results from a retrospective single-center study 2021
16 e0245725 e0245725 10.1371/journal.pone.0245725 33571189
Puppa Giacomo , Senore Carlo , Sheahan Kieran , Vieth Michael , Lugli Alessandro , Zlobec Inti , Pecori Sara , Wang Lai Mun , Langner Cord , Mitomi Hiroyuki , Nakamura Takatoshi , Watanabe Masahiko , Ueno Hideki , Chasle Jacques , Conley Stephen A. , Herlin Paulette , Lauwers Gregory Y. , Risio Mauro Histopathology Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using virtual microscopy 10 2012
61 562 575 10.1111/j.1365-2559.2012.04270.x
Masugi Yohei , Yamazaki Ken , Hibi Taizo , Aiura Koichi , Kitagawa Yuko , Sakamoto Michiie Hum Pathol Solitary cell infiltration is a novel indicator of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer 08 2010
41 1061 1068 10.1016/j.humpath.2010.01.016 20413143
Taira Tetsuhiko , Ishii Genichiro , Nagai Kanji , Yoh Kiyotaka , Takahashi Yusuke , Matsumura Yuki , Kojima Motohiro , Ohmatsu Hironobu , Goto Koichi , Niho Seiji , Takashima Hiroshi , Inoue Hiromasa , Ohe Yuichiro , Ochiai Atsushi Lung Cancer Characterization of the immunophenotype of the tumor budding and its prognostic implications in squamous cell carcinoma of the lung 06 2012
76 423 430 10.1016/j.lungcan.2011.11.010 22153829
Gujam F. J. A. , McMillan D. C. , Mohammed Z. M. A. , Edwards J. , Going J. J. Br J Cancer The relationship between tumour budding, the tumour microenvironment and survival in patients with invasive ductal breast cancer 09 2015
113 1066 1074 10.1038/bjc.2015.287 26263482
Venkata Renuka Inuganti , Madhavi K , Premalatha P , KrishnamacharyuluYulu Pav , Vaishnavi R JDPO Tumor budding in invasive carcinoma of breast of No Special Type (NST): Value as a prognostic factor 06 2019
4 125 129 10.18231/j.jdpo.2019.024
Kumarguru B. N. , Ramaswamy Anikode S. , Shaik Shahanuma , Karri Aruna , Srinivas Venugopal Sandeep , Prashant B. M. Indian J Pathol Microbiol Tumor budding in invasive breast cancer - An indispensable budding touchstone 02 2020
63 S117 S122 10.4103/IJPM.IJPM_731_18
Liang Fenli , Cao Wei , Wang Yili , Li Linrui , Zhang Guanjun , Wang Zhuo Pathol Res Pract The prognostic value of tumor budding in invasive breast cancer 05 2013
209 269 275 10.1016/j.prp.2013.01.009 23561623
