In total, 111 mastectomy specimens were collected from HER2-positive breast-cancer patients who had received no pre-operative therapy. Patients with invasive lobular carcinoma, mixed invasive ductal and lobular carcinoma, invasive micropapillary carcinoma, mucinous carcinoma, or inadequate tissue blocks (<5 % tumor/stroma) were excluded12.

Data were abstracted from medical records and included age, tumor size, histology, histologic grade, vascular invasion, lymph-node metastasis, pathological stage, estrogen-receptor (ER) expression, progesterone-receptor (PR) expression, HER2 expression, tumor-cell proliferation index (Ki-67 expression), and treatment regimen.

Unfavorable outcomes comprised ipsilateral locoregional invasive breast-tumor recurrence, contralateral invasive breast cancer, distant disease recurrence, or death from any cause. Overall-survival (OS) and disease-free-survival (DFS) data were obtained from medical records or by patient contact.

All data were collected at the National Cancer Hospital (Hanoi, Vietnam) between 1 January 2017 and 31 December 2021, with follow-up completed by 31 December 2024.

The study was approved by the Hanoi Medical University Ethics Committee (code: IRB-VN01.001/IRB00003121/FWA 00004148) and was conducted in accordance with the Declaration of Helsinki.

The hematoxylin-and-eosin (H&E) staining protocol is provided in Supplementary S1.

Whole sections of H&E-stained slides were used to evaluate stromal TILs in strict accordance with the criteria proposed by the International TIL Working Group13. The percentage of all mononuclear cells (including lymphocytes and plasma cells) in the stromal compartment within the border of the invasive tumor was assessed visually. TILs were scored in 5 % increments and classified as <50 % or ≥50 %, following Swisher14.

The immunohistochemical (IHC) protocol is provided in Supplementary S2.

Whole-section slides were scanned with a Leica Aperio AT2 (40×). CD8 expression on the membrane and in the cytoplasm was analyzed across the entire tumor area using QuPath software15. High CD8 expression was defined as ≥10 % CD8-positive lymphocytes among nucleated stromal cells, as described by L. Zong12.

Transgelin expression in CAFs: nuclear and/or cytoplasmic staining was graded as 0 (no staining), 1 (light brown), 2 (brown), or 3 (dark brown). Grades 0–1 were considered weakly positive, whereas grades 2–3 were considered strongly positive11.

Transgelin expression in TCs: a result was deemed positive when ≥10 % of tumor cells showed dark-brown cytoplasmic staining7. All assessments were performed by two pathologists; discrepancies were resolved by a third pathologist.

Data were entered in EpiData 4.6.0.4 (EpiData Association, Denmark) and analyzed in R 4.1.2 (https://www.r-project.org). Associations between transgelin expression and clinicopathological characteristics were examined with the χ² test or Fisher’s exact test. Survival was analyzed with the Kaplan–Meier method and log-rank test. Variables with p < 0.05 in univariate Cox regression and post-hoc power ≥80 % were included in the multivariate model. Model fit was evaluated with −2 log-likelihood and the AIC. Risk groups were compared with the Kaplan–Meier method. A p-value <0.05 was considered statistically significant.

Table 1

Parameters	N	Percent
Age (years)
< 50	51	45.9
≥ 50	60	54.1
Tumor size (cm)
< 2	32	28.8
2-5	79	71.2
Histology
Ductal carcinoma	107	96.4
Others	4	3.6
Histologic grade
1 - 2	56	50.5
3	55	49.5
Vascular invasion
No	73	65.8
Yes	38	34.2
Lymph node metastases
< 2	92	82.9
≥ 2	19	17.1
Pathological stage
Early stage (IA, IIA, T2N1)	87	78.4
Locally advanced stage (IIB, T3N0, IIIA-C)	24	21.6
ER expression
Negative	56	50.5
Positive	55	49.5
PR expression
Negative	64	57.7
Positive	47	42.3
HER2 expression
IHC 2+ ISH +	31	27.9
IHC 3+	80	72.1
Ki67 expression
Low (< 20%)	7	6.3
High (≥ 20%)	104	93.7
Transgelin expression in TCs
Negative (< 10%)	90	81.1
Positive (≥ 10%)	21	18.9
Transgelin expression in CAFs
Weak (Grade 0,1)	74	66.7
Strong (Grade 2,3)	37	33.3
CD8 expression
Low (< 10%)	80	72.1
High (≥ 10%)	31	27.9
TILs
Low (< 50%)	98	88.3
High (≥ 50%)	13	11.7
Trastuzumab
No	59	53.2
Yes	52	46.8

Abbreviations: CAFs: Cancer-associated fibroblasts, ER: Estrogen receptor, HER2: Human epidermal growth factor receptor 2, IHC: immunohistochemistry, ISH: In situ hybridization, Ki67: Tumor cell proliferation index, PR: Progesterone receptor, TCs: Tumor cells, TILs: Tumor-infiltrating lymphocytes.

The distribution of clinicopathological characteristics and transgelin expression is shown in Table 1. The median patient age was 50 years (range, 29–74 years). Most tumors were invasive ductal carcinomas; three were tubular carcinomas and one was metaplastic carcinoma, not otherwise specified. All patients were treated in accordance with Vietnam’s breast-cancer guidelines and underwent surgery. Hormone receptor-positive cases received tamoxifen plus chemotherapy, whereas hormone receptor-negative cases received chemotherapy alone. Regimens consisted of four cycles of AC (doxorubicin/cyclophosphamide) or six cycles of FAC (5-FU/doxorubicin/cyclophosphamide). Fifty-two patients received trastuzumab. In the entire cohort, the median percentages of TILs and CD8⁺ cells were 17.4% (interquartile range [IQR], 0.0%–80.0%) and 8.9% (IQR, 0.0%–66.7%), respectively (Figure 1 and Supplementary Fig.).

Figure 1

Table 2

Parameters	Transgelin in TCs positive N (%)	P-value	Strong transgelin expression in CAFs N (%)	P-value
<50	9 (17.6)	0.752	17 (33.3)	1.000
≥50	12 (20.0)		20 (33.3)
Tumor size (cm)
<2	5 (15.6)	0.573	8 (25.0)	0.236
≥2	16 (20.3)		29 (36.7)
Histologic grade
1 - 2	7 (12.5)	0.081	20 (35.7)	0.591
3	14 (25.5)		17 (30.9)
Vascular invasion
No	11 (15.1)	0.151	31 (33.7)	0.859
Yes	10 (26.3)		6 (31.6)
Lymph node metastases
<2	17 (18.5)	0.755a	14 (15.2)	0.507a
≥2	4 (21.1)		4 (21.1)
Pathological stage
Early stage (IA, IIA, T2N1)	13 (14.9)	0.073a	28 (32.3)	0.625
Locally advanced stage (IIB, T3N0, IIIA-C)	8 (33.3)		9 (37.5)
ER expression
Negative	16 (28.6)	0.009	19 (33.9)	0.893
Positive	5 (9.1)		18 (32.7)
PR expression
Negative	17 (26.6)	0.016	21 (32.8)	0.892
Positive	4 (8.5)		16 (34.0)
Ki67 expression
Low (<20 %)	0 (0.0)	0.343a	3 (42.9)	0.864a
High (≥20 %)	21 (20.2)		34 (32.7)
Transgelin expression in TCs
Negative (<10 %)			29 (32.2)	0.607
Positive (≥10 %)			8 (38.1)
Transgelin expression in CAFs
Weak (Grade 0,1)	13 (17.6)	0.607
Strong (Grade 2,3)	8 (21.6)
CD8 expression
Low (<10%)	13 (16.3)	0.249	36 (45.0)	<0.001a
High (≥10%)	8 (25.8)		1 (3.2)
TILs
Low (<50%)	18 (18.4)	0.709a	37 (37.8)	0.004a
High (≥50%)	3 (23.1)		0 (0.0)

Abbreviations: CAFs: Cancer-associated fibroblasts, ER: Estrogen receptor, Ki67: Tumor cell proliferation index, PR: Progesterone receptor, TCs: Tumor cells, TILs: Tumor-infiltrating lymphocytes. a Fisher's exact test.

Figure 2

Figure 3

As shown in Table 2, strong transgelin expression in CAFs was negatively associated with CD8 expression (p < 0.001) and TILs (p = 0.004). Transgelin expression in TCs was positively associated with ER status (p = 0.009) and PR status (p = 0.016) (Figure 2 and Figure 3).

Table 3

Parameters		Log rank	Univariable Cox regression		Multivariate Cox regression
			HR (95%CI)	p-value	HR (95%CI)	p-value
Age (years)	< 50	0.352	1
	≥50		1.882 (0.486 – 7.283)	0.360
Histologic grade	1 - 2	0.708	1
	3		1.247 (0.359 – 4.518)	0.708
Vascular invasion	No	0.740	1
	Yes		1.239 (0.349 - 4.396)	0.740
Pathological stage	Early stage (IA, IIA, T2N1)	0.032	1		1
	Locally advanced stage (IIB, T3N0, IIIA-C)		3.561 (1.030 – 12.312)	0.045	4.001 (1.154 – 13.873)	0.029
ER expression	Negative	0.656	1
	Positive		1.326 (0.382 – 4.606)	0.657
PR expression	Negative	0.263	1
	Positive		0.423 (0.090 – 1.997)	0.277
Ki67 expression	Low (<20 %)	0.506	1
	High (≥20 %)		0.502 (0.063 – 3.978)	0.514
Transgelin expression in TCs	Negative (<10 %)	0.543	1
	Positive (≥10 %)		0.619 (0.131 – 2.935)	0.546
Transgelin expression in CAFs	Weak (Grade 0,1)	0.014	1		1
	Strong (Grade 2,3)		5.559 (1.180 – 26.191)	0.030	6.082 (1.286 – 28.758)	0.023
CD8 expression	Low (<10%)	0.165	1
	High (≥10%)		0.258 (0.033 – 2.033)	0.198
TILs	Low (<50%)	0.288	1
	High (≥50%)		0.042 (0.000 – 349.497)	0.492
Trastuzumab	No	0.955	1
	Yes		0.965 (0.278 – 3.348)	0.955

Abbreviations: CAFs: Cancer associated fibroblasts, CI: Confidence interval, ER: Estrogen receptor, HER2: Human epidermal growth factor receptor 2, HR: Hazard ratio, Ki67: Tumor cell proliferation index, PR: Progesterone receptor, TCs: Tumor cells, TILs: Tumor-infiltrating lymphocytes.

The analysis included 81 women with a median follow-up of 65 months (range, 29–96 months). Ten patients (12.3%) experienced recurrence and three (3.7%) died. The 5-year OS and DFS rates were 93% and 84%, respectively. Owing to the low number of deaths, we focused on associations between transgelin expression, clinicopathological characteristics, and DFS. Univariate analysis (Table 3) identified two variables associated with shorter DFS: strong transgelin expression in CAFs (p = 0.030) and a locally advanced pathological stage (p = 0.045). Post-hoc power analysis yielded 98.9% for transgelin expression in CAFs and 88.4% for pathological stage (α = 0.05). In multivariate analysis (Table 3), transgelin expression in CAFs (hazard ratio [HR] = 6.082; 95% confidence interval [CI], 1.286–28.758; p = 0.023) and pathological stage (HR = 4.001; 95% CI, 1.154–13.873; p = 0.029) were independent prognostic factors for DFS. Adding transgelin expression in CAFs to the pathological-stage model improved model performance, lowering the −2 log-likelihood from 80.815 to 74.062 (p = 0.004) and the AIC from 82.81 to 78.06 (Table 4). Patients classified as high-risk (strong transgelin expression in CAFs and a locally advanced pathological stage) had a significantly shorter DFS than the low-risk group (p < 0.0001; Figure 4).

Figure 4

Table 4

Model variables	-2 Log Likelihood	Likelihood ratio P value	AIC
Pathological stage	80.815	0.032	82.81
Pathological stage + transgelin expression in CAFs	74.062	0.004	78.06

Abbreviations: AIC: Akaike information criterion, CAFs: Cancer-associated fibroblasts.

This study found that strong transgelin expression in CAFs was negatively associated with CD8⁺ cells and TILs. Transgelin expression in TCs was positively associated with ER and PR status. We confirmed that transgelin expression in CAFs and pathological stage were independent prognostic factors for DFS. Including transgelin expression in CAFs in the predictive model provided additional prognostic information beyond pathological stage alone. All patients received no neoadjuvant therapy; stained sections were obtained from resected tumors, accurately reflecting the TME.

In our study, CD8 expression was positively associated with TILs. However, like Liu (2011)16, we did not identify a significant association between CD8 expression and improved DFS. This discrepancy may be attributed to variations in CD8 evaluation methods across studies. Whereas our analysis focused on CD8 in the stromal compartment, other studies have assessed intratumoral compartments or both together17, 18, potentially leading to differing prognostic implications. Furthermore, chronic antigen stimulation within the TME can lead to exhaustion of CD8 effector T cells, reducing their antitumor activity19.

Several studies have investigated the relationship between CAFs and transgelin, a cytoskeletal protein that plays a role in fibroblast activation and tumor progression. Transgelin expression varies between TCs and the surrounding stroma, with significant up-regulation in CAFs compared with TCs. These findings suggest that transgelin is a key marker of fibroblast activation and may contribute to the pro-tumor behavior of CAFs9, 10. Our study showed that transgelin expression was higher in CAFs than in TCs and was significantly associated with short DFS, similar to findings reported previously6, 7, 8

In our study, strong transgelin expression in CAFs was negatively associated with CD8 and TILs, consistent with previous research20, 21. CAFs shape the TME by modulating CD8 responses through antigen presentation and up-regulating immune checkpoints, leading to T-cell impairment and tumor immune evasion22.

Transgelin expression in TCs was positively associated with ER and PR status, in agreement with earlier studies23, 24. However, unlike its prognostic value in CAFs, transgelin expression in TCs was not linked to DFS, suggesting different roles in the stromal and tumor compartments.

Although the number of DFS events was limited, we performed a post-hoc power analysis and model comparisons to enhance the reliability of the findings. Both transgelin expression in CAFs and pathological stage demonstrated sufficient power to detect meaningful associations and were confirmed as independent prognostic factors in the multivariate analysis. Their combination improved model performance, as shown by a lower −2 log-likelihood and the lowest AIC among the tested models. Patients in the high-risk group had a shorter DFS than those in the low-risk group.

This study has several limitations. The small number of DFS events may reduce statistical power and affect the stability of the multivariate analysis. The retrospective, single-center design may also limit generalizability. Finally, the wide confidence intervals (particularly for transgelin) suggest uncertainty in the effect estimates.

Transgelin expression in CAFs may help predict DFS in patients with HER2-positive breast cancer. Further multicenter studies with larger cohorts are needed to validate these results.

AIC- Akaike information criterion, CAFs- Cancer-associated fibroblasts, CI- Confidence interval, DFS- Disease-free survival, ER- Estrogen receptor, H&E- Hematoxylin and eosin, HER2- Human epidermal growth factor receptor 2, HR- Hazard ratio, IHC- Immunohistochemistry, ISH- In situ hybridization, IQR- Interquartile range, Ki67- Tumor cell proliferation index, OS- Overall survival, PR- Progesterone receptor, TCs- Tumor cells, TILs- Tumor-infiltrating lymphocytes, TME- Tumor microenvironment.

The authors thank Nguyen Canh Hiep, Nguyen Thi Khuyen, Nguyen Van Tuan, Nguyen Ngoc Duong, and Mai Thi Nhung for their help during the study.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

The study was approved by the Ethics Committee of Hanoi Medical University (IRB-VN01.001/IRB00003121/FWA 00004148) and adhered to the Helsinki Declaration.

Written informed consent was obtained from the patient’s mother for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

The authors declare that they have not used generative AI (a type of artificial intelligence technology that can produce various types of content including text, imagery, audio and synthetic data. Examples include ChatGPT, NovelAI, Jasper AI, Rytr AI, DALL-E, etc.) and AI-assisted technologies in the writing process before submission.

The authors declare that they have no competing interests.