
==== 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

38938104
10.5146/tjpath.2024.13521
Original Article
The Importance of Stroma and Stromal Sma Expression in Pancreatic Ductal Adenocarcinoma*
Akbas Gamze
Bagcı Pelin *
Department of Pathology, Marmara University, School of Medicine, Istanbul, Turkey
* E-mail: pelinbagci@gmail.com
Concept: GA, PB, Design: PB, Supervision: PB, Materials: GA,PB, Data collection and/or processing: GA,PB, Analysis and/or interpretation: GA,PB, Literature search: GA, Writing: GA,PB, Approval: PB.

2024
02 9 2024
40 3 181189
16 5 2023
04 6 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: Pancreatic stellate cells (PSC) have been defined to be the key players in pancreatic fibrogenesis and carcinogenesis. They undergo myofibroblast-like differentiation, express α-smooth muscle actin (α-SMA), and play a crucial role in injury and inflammation sites. This study aims to evaluate the relationship between α-SMA expression and histopathological parameters of pancreatic ductal adenocarcinoma (PDAC), and investigate their association with prognosis.

Material and Methods: Eighty-one consecutive pancreatectomies diagnosed as usual pancreatic ductal adenocarcinoma were included. The stromal density was scored as loose, moderate, or dense, and α-SMA expression was evaluated immunohistochemically.

Results and Conclusion: Mean survival was 19.6 months. Male gender, larger tumor diameter (>3.7 cm), and older age (>64 years) were identified as independent poor prognostic factors. Perineural invasion significantly effected survival. A statistically significant correlation was found between high α-SMA expression and the presence of angioinvasion (p=0.01). Stromal α-SMA expression in PDAC may help determine the risk of angioinvasion.

Pancreas
Cancer
Stellate cell
Tumor stroma
α-SMA
Angioinvasion
==== Body
pmcINTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) ranks as the fourth leading cause of cancer-related deaths in males and the third in females (1,2). The primary reasons for its poor survival rates include low operability at the time of diagnosis and the resistance to conventional chemotherapy protocols. Hence, there is an urgent need for new targeted therapeutic approaches (1).

The stroma of cancers is a recent hot topic of research. Colorectal tumors are the first to be proven to have significant prognostic outcomes related to their stroma (3). Ueno et al. classified the desmoplastic reaction as mature, intermediate, or immature based on the presence of hyalinized keloid-like collagen and myxoid stroma at the extramural desmoplastic front. The prognostic power of this categorization in stratifying relapse-free survival was greater than any other conventional prognostic factors, including TNM stage, venous invasion, and tumor grade (4,5). Subsequently, Wang et al. applied an undefined scoring system using H&E and Masson’s trichrome stains for PDACs (6).

The stroma of PDAC constitutes more than 50% of the tumor mass and includes various components such as extracellular matrix (ECM), pancreatic stellate cells (PSC), fibroblasts, macrophages, blood and lymphatic vessels, pericytes, stem cells, and inflammatory cells. Recent research has focused on developing treatment strategies targeting stromal elements, particularly PSCs, which play a central role in pancreatic fibrogenesis (7–12). PSCs are star-shaped, vitamin A-storing cells, which comprise approximately 4% of all pancreatic cells and show a periacinar distribution in a healthy pancreas. In response to several injuries (such as cancer or inflammation), PSCs undergo an activation process via exhibiting a myofibroblastic-like phenotype, expressing α-smooth muscle actin (α-SMA). Later they become the key cells in pancreatic fibrogenesis (13,14).

In many studies, PSCs have been shown to be present since the early stages of preneoplastic transformation. PSCs are activated and exhibit myofibroblastic morphology during epithelial carcinogenesis. This observation can be explained by three hypotheses: 1) PSCs may interact with tumor cells from the early stages of carcinogenesis, 2) they may attempt to circumvent and confine tumor cells from early preneoplastic stages, or 3) PSCs may activate around genetically defective cells in an initially limiting manner, then synthesize elements of the extracellular matrix (ECM) to make the stroma more fibrotic. This fibrosis can lead to hypoxic conditions in subsequent stages, resulting in the release of reactive oxygen species and genetic instability in epithelial cells (15).

Although there are clinical (oncology, surgery) and preclinical (biochemistry, pharmacology) studies related to PDAC stroma in the literature (15–18), publications based on histopathological examinations are few and serve only to complement the gaps between these studies (6,19–21). This study aims to evaluate the relationship between α-SMA expression and the histopathological parameters of pancreatic ductal adenocarcinoma (PDAC), as well as to investigate the effects of these parameters on the prognosis.

MATERIALS and METHODS

All pancreatic resections examined in the Department of Pathology between 2011 and 2016 were re-evaluated retrospectively. All consecutive cases with a diagnosis of usual (classical or conventional) PDAC were included in the study (n=81). PDACs with nonconventional subtypes and those associated with intraductal papillary mucinous neoplasm (IPMN) or mucinous cystic neoplasm (MCN) were excluded. A Whipple resection had been performed in 62 cases, a total pancreatectomy in 6 cases, a distal pancreatectomy in 10 cases, and partial excisions in 3 cases. Information on histological tumor subtypes, grade, location, diameter, and lymphatic, vascular, and perineural invasion, as well as surgical margin status, was retrieved from pathology reports. After revision of all tumor slides, the pattern of stromal density was classified based on all hematoxylin and eosin (H&E) stained tumor slides as loose (myxoid), moderate (keloid-like), or dense (mature) according to the relevant literature (4–6). The definition of the final stromal score in these studies was not clear enough. Therefore, the scoring of the stromal pattern was based on the dominant density (≥ 50% of the total tumor stroma) for each case. Loose stroma was defined as a loose fibroblastic and myxoid stroma containing short, faint collagen fibers (Figure 1). Moderate density stroma was composed of haphazard bands of keloid-like collagen (Figure 1). Dense stroma was characterized by the dominance of mature, thick collagen fibers that resembled scar tissue (Figure 1). For statistical purposes, the stroma was grouped as mixed (loose + moderate) and dense. The best representative formalin-fixed paraffin-embedded (FFPE) tumor block was selected for immunohistochemistry. This block showed the dominant type of stromal density and did not contain necrosis.

Figure 1 A) Myxoid or loose stroma of pancreatic ductal carcinoma. The periglandular area is pale, has less myofibroblasts, and looks edematous (Hematoxylin & Eosin, x10). B) Moderate or keloid-like stroma of pancreatic ductal carcinoma. Interrupted bands of dense eosinophilic keloid-like collagen are seen in the picture. This collagen is the product of activated myofibroblasts (Hematoxylin & Eosin, x40). C) Strong or mature stroma of pancreatic ductal carcinoma. Collagen replaced everything between and around the tumor cells. There are a few tumor cells at the top center of the figure (Hematoxylin & Eosin, x40).

Immunohistochemical Analysis

The tumor blocks were sectioned at 4 µm thickness as positively charged slides. For the processes, the Leica Bond-Max automatic immunohistochemistry device was used. To block the endogenous peroxidase activity, the sections were incubated for 10 min with three steps of at room temperature. Antigen retrieval was achieved with tris-EDTA buffer (pH 8) for α-SMA. The slides incubated with α-SMA (1:200, Clone 1A4, Cell Marque) for 45 minutes at room temperature. After primary antibody, secondary antibody and poly-HRP solutions (Bond Polymer Detection Kit; DS9800, Leica) were applied in the device. Diaminobenzidine (DAB) was used as the chromogen for colored visualization of the antigens. The sections were then counterstained with hematoxylin and dehydrated with ethanol.

Two observers blinded to the clinical outcome interpreted the slides. α-SMA was expressed in the stroma only. Fine granular cytoplasmic staining was regarded as positive and the percentage (extensity) was scored as follows: 0 = 0-10%; 1 = 11-50%; 2 = 51-75%; 3 = 76-100%. The intensity of staining was graded as: 0 = none; 1 = weak; 2 = moderate; 3 = strong. An overall score for α-SMA was calculated as (extensity + intensity): negative = 0–1; low = 2-3; moderate = 4-5; high = 6 (Table 1).

Table 1 Scoring method of immunohistochemical staining for stromal α-SMA*

Stromal score

	Extensity (%)

	Intensity

	
0

	0-10

	None

	
1

	11-50

	Weak

	
2

	51-75

	Moderate

	
3

	76-100

	Strong

	
Overall Stromal score =

Extensity score + Intensity score

(negative = 0–1; low = 2-3; moderate = 4-5; high = 6)

	
* α-SMA: alpha smooth muscle actin

Statistical Analysis

Histopathologic and clinical variables were compared in terms of the SMA score. Categorical variables were compared using the Pearson Chi-Square test. Continuous variables were compared using the Kruskal-Wallis test. Cox-regression and Kaplan-Meier estimates were used to calculate the factors effecting survival. A multivariate Cox proportional hazards model was employed to identify independent prognostic factors. Statistical significance was set at a p-value of <0.05. All analyses were performed using jamovi (version 2.3) (22).

RESULTS

The Relationship Between Immunohistochemical and Clinicopathological Findings and Overall Survival (OS)

The mean age of the patients was 64, with a female/male ratio of 30/51. Most of the tumors were grade 2 (n=55, 68%) and were located in the head of the pancreas (n=67, 83%). The mean tumor diameter was 3.7 cm (range: 0.5- 10.2 cm). The distribution of pathological T stages according to the recent classification by the American Joint Committee on Cancer (AJCC) 2017 was T1/T2/T3/T4: 7/41/30/3, respectively. The N stages were N0/N1/N2: 20/23/37. Lymphatic invasion was found in 73 cases (90%). Vascular invasion was present in 55 cases (68%). Perineural invasion was found in 78 cases (96.3%). Forty-two cases (52%) had positive margins (R1) (Table 2).

Table 2 The baseline characteristics of the patients and histopathological findings (n = 81)

		Mean (range) or n (%)

	
Age

		64 (34-85)

	
Gender

	Female

	30 (37)

	
Male

	51 (63)

	
Grade

	Grade 1

	8 (10)

	
Grade 2

	55 (68)

	
Grade 3

	18 (22)

	
Location

	Head

	67 (83)

	
Corpus&tail

	14 (17)

	
Tumor diameter (cm)

		3.7 (0.5-10.2)

	
T (AJCC* 2017)

	T1

	7 (9)

	
T2

	41 (51)

	
T3

	30 (37)

	
T4

	3 (4)

	
N (AJCC 2017)

	N0

	20 (25)

	
N1

	23 (28)

	
N2

	37 (46)

	
Lymphatic invasion (present)

		73 (90)

	
Angioinvasion (present)

		55 (68)

	
Perineural invasion (present)

		78 (96)

	
Surgical margin

	R0

	39 (48)

	
R1

	42 (52)

	
*AJCC: American Joint Committee on Cancer (T: Tumor stage, N: Lymph node stage)

The total score of α-SMA immunohistochemistry was “low” in 2 patients (2%), “moderate” in 25 patients (31%), and “high” in 54 patients (67%) (Figure 2) (Table 3).

Table 3 Distribution of immunohistochemical staining for stromal α-SMA in the cohort

Extensity Scores (n, %)

	1

	1 (1)

	
2

	20 (25)

	
3

	60 (74)

	
Intensity Scores (n, %)

	1

	1 (1)

	
2

	13 (16)

	
3

	67 (83)

	
Total Score (n, %)

	Low

	2 (2)

	
Moderate

	25 (31)

	
High

	54 (67)

	

Figure 2 A) Low expression of α-SMA in the stroma of pancreatic ductal carcinoma (α-SMA antibody, x10). B) Moderate expression of α-SMA in the stroma of pancreatic ductal carcinoma (α-SMA antibody, x10). C) High expression of α-SMA in the stroma of pancreatic ductal carcinoma (α-SMA antibody, x10).

The stromal pattern exhibited mixed stromal density in 38 cases (47%), while 43 cases (53%) had a pure mature (dense) type stroma. Among the cases with pure mature (dense) stroma, 12 had low or moderate α-SMA scores, whereas 31 had high α-SMA scores. Of the cases with mixed stromal density, 15 had low or moderate α-SMA scores, and 23 had high α-SMA scores (Table 4).

Table 4 Distribution of stromal pattern and α-SMA scores

	Stromal Pattern

	
α-SMA Scores

	Mixed stroma

(n=38, 47%)

	Pure mature (dense) stroma (n=43, 53%)

	
Low + Moderate α-SMA score

	15

	12

	
High α-SMA score

	23

	31

	

α-SMA was found to be strongly positive around the glandular tumoral component in 56 cases (Figure 3), as well as around the normal perilobular area in 24 cases (Figure 3). Staining was also stronger in peritumoral chronic pancreatitis in 27 cases. Additionally, the stromal components of metastatic foci in lymph nodes were positive for α-SMA in 12 cases (Figure 3).

Figure 3 A) α-SMA staining was stronger at the immediate borders of the glandular tumoral component, and the interglandular area was less dense (α-SMA antibody, x10). B) Activated pancreatic stellate cells stained with α-SMA highlighting the normal perilobular area (α-SMA antibody, x40). C) Lymph node metastasis of a pancreatic ductal carcinoma. The same strong staining pattern was also seen in metastatic areas (α-SMA antibody, x10).

The total scores of α-SMA did not reveal any significant correlation when compared with other clinicopathological parameters. However, α-SMA levels were found to be higher in patients with angioinvasion than in those without angioinvasion (p = 0.01) (Table 5).

Table 5 Relation of α-SMA and other pathological parameters

Variables

	n

	High α-SMA Score

n=54

	Low+Moderate α-SMA Score

n=27

	p-values

	
Gender: Male

	81

	31

	20

	p=0.141

	
Age (mean)

	81

	62.5

	68.0

	p=0.202

	
Grade

	81

			p=0.871

	
1

		6

	2

		
2

		36

	19

		
3

		12

	6

		
Location

	81

			p=0.061

	
Body

		2

	5

		
Distal

		4

	3

		
Head

		48

	19

		
Tumor Diameter (cm, mean)

	81

	3.5

	3.5

	p=0.832

	
pT

	81

			p=0.451

	
T1

		4

	3

		
T2

		28

	13

		
T3

		21

	9

		
T4

		1

	2

		
pN

	80

			p=0.251

	
N0

		12

	8

		
N1

		13

	0

		
N2

		28

	9

		
Lymphatic invasion (Present)

	81

	49

	24

	p=0.791

	
Angioinvasion (Present)

	81

	42

	13

	p=0.011

	
Perineural invasion (Present)

	81

	53

	25

	p=0.211

	
Positive surgical margin (R1)

	81

	28

	14

	p=1.001

	
Higher grade (Grade 3)

	81

	12

	6

	p=1.001

	
Higher T stage (T3+T4)

	81

	22

	11

	p=1.001

	
1Pearson. 2Wilcoxon. (p<0,05)

The mean overall survival (OS) was 19.6 months (range: 2-107 months), with a median survival time of 12 months. Male gender was associated with worse OS [HR: 1.51, 95% CI: 0.94-2.41, p=0.089]. Patients older than 64 years of age were associated with a worse prognosis [HR:1.02, 95% CI: 1.00-1.05, p=0.059)]. Patients with positive surgical margins had significantly worse OS [HR: 1.76, 95% CI: 1.11-2.78, p=0.016]. The presence of perineural invasion had a statistically significant negative effect on OS [HR: 4.30, 95% CI: 1.04-17.78, p=0.044]. The presence of angioinvasion was associated with worse outcomes [HR: 1.45, 95% CI: 0.89-2.35, p=0.136], and lymphatic invasion also worsened OS [HR: 1.23, 95% CI: 0.59-2.58, p=0.576]. Tumors located in the head of the pancreas showed worse survival compared to those in the distal and body locations [HR: 0.91, 95% CI: 0.41-1.99, p=0.809]. Tumors larger than the mean tumor diameter (3.7 cm) showed worse prognosis [HR: 1.33, 95% CI: 1.11-1.60, p=0.002], although this did not affect higher tumor stages. Tumors with higher stages (T3 and T4) had a worse prognosis [HR: 1.42, 95% CI: 0.90-2.25, p=0.132]. N2 cases had a worse prognosis compared to N1 cases [HR: 1.39, 95% CI: 0.79-2.45, p=0.259]. Tumors with low+moderate SMA scores had a worse prognosis compared to high SMA scores [HR: 1.20, 95% CI: 0.75-1.92, p=0.457)] (Table 6).

Table 6 Multivariate survival analysis

Dependent: Survival

	Levels

	All

	HR (univariate)

	HR (multivariate)

	
Age

	Mean (SD)

	63.8 (9.8)

	1.02 (1.00-1.05, p=0.059)

	1.03 (1.00-1.05, p=0.038)

	
Gender

	Female

	30

			
	Male

	50

	1.51 (0.94-2.41, p=0.089)

	1.88 (1.07-3.31, p=0.029)

	
Surgical Margin

	R0

	39

			
	R1

	41

	1.76 (1.11-2.78, p=0.016)

	1.63 (0.97-2.76, p=0.066)

	
Perineural Invasion

	Absent

	3

		-

	
	Present

	77

	4.30 (1.04-17.78, p=0.044)

	3.76 (0.65-21.69, p=0.139)

	
Angioinvasion

	Absent

	26

			
	Present

	54

	1.45 (0.89-2.35, p=0.136)

	1.08 (0.57-2.06, p=0.806)

	
Lymphatic Invasion

	Absent

	8

			
	Present

	72

	1.23 (0.59-2.58, p=0.576)

	0.58 (0.20-1.70, p=0.320)

	
Location

	Body

	7

			
	Distal

	7

	0.97 (0.34-2.80, p=0.962)

	0.71 (0.23-2.25, p=0.567)

	
	Head

	66

	0.91 (0.41-1.99, p=0.809)

	1.62 (0.67-3.91, p=0.282)

	
Tumor Diameter

	Mean (SD)

	3.7 (1.4)

	1.33 (1.11-1.60, p=0.002)

	1.75 (1.22-2.53, p=0.003)

	
T Stage

	T1+T2

	47

			
	T3+T4

	33

	1.42 (0.90-2.25, p=0.132)

	0.49 (0.18-1.35, p=0.168)

	
N Stage

	N0

	20

			
	N1

	23

	1.24 (0.67-2.29, p=0.492)

	1.40 (0.69-2.84, p=0.355)

	
	N2

	37

	1.39 (0.79-2.45, p=0.259)

	1.15 (0.57-2.29, p=0.697)

	
Sma Score

	High

	53

		-

	
	Low + Moderate

	27

	1.20 (0.75-1.92, p=0.457)

	1.28 (0.71-2.30, p=0.414)

	

Older age (>64 years), male gender, and larger tumor diameter (>3.7 cm) were found to be independent factors in multivariate analysis (with p-values of 0.038, 0.029, and 0.003, respectively). However, tumor diameter did not have a significant effect on the T stage of the tumor. Surgical margin status approached significance but was not an independent factor (p=0.066) (Table 6).

Oncological Follow-Up

Forty-five cases were followed up at our institution. Two of them received neoadjuvant therapy: one received FOLFIRINOX, and the other received gemcitabine in combination with radiotherapy. Adjuvant therapy was administered to 45 patients, with two receiving FOLFIRINOX, 41 receiving gemcitabine, and two receiving a combination of capecitabine and gemcitabine.

Two cases that received neoadjuvant therapy had high α-SMA scores, with lifetimes of 37 and 42 months, respectively. Among the cases treated with adjuvant FOLFIRINOX, two had high α-SMA scores, with lifetimes of 16 and 37 months. Forty-one cases treated with adjuvant gemcitabine had moderate or high stromal α-SMA scores, with a mean lifetime of 24.7 months (range: 3-107 months) (Table 7). Two cases treated with the adjuvant gemcitabine and capecitabine combination had moderate-high SMA scores and the lifetimes were 32 and 10 months respectively. Due to the small number of cases treated with FOLFIRINOX (only two), and combination therapies (only two), the α-SMA scores could not be compared for different therapy regimens.

Table 7 Therapy types and SMA score distribution

n=45

	Chemotherapy Type

	SMA Score

	Lifetime (months)

	
Neoadjuvant CT

n=2

	1- Folfirinox

1- Gemcitabine

	High

High

	37

42

	
Adjuvant CT

n=45

	2- Folfirinox

	High

	16 and 37

	
41, Gemcitabine

2, Capecitabine + Gemcitabine

	Moderate/High

	Mean=24,7(3-107)

32 and 10

	

Sixteen cases experienced distant metastasis, with 11 of them spreading to the liver or lungs. Additionally, 24 cases had local recurrence. All of these cases with metastasis or local recurrence had moderate or high α-SMA scores in their primary tumor.

DISCUSSION

The stroma of colorectal tumors has been the first to be proven to significantly influence the prognostic outcome. Ueno et al. classified the desmoplastic reaction in colon tumors as mature, intermediate, or immature based on the presence of hyalinized keloid-like collagen and myxoid stroma at the extramural desmoplastic front. The prognostic power of this reaction categorization in stratifying survival was superior to that of any other conventional prognostic factors such as TNM stage, venous invasion, and tumor grade (4,5).

Bever et al. utilized computer-based image analysis of collagen-stained slides to calculate a specific stromal density score for 66 PDAC cases who underwent adjuvant chemotherapy. However, they did not observe any correlation with prognosis (23). Although it appeared to be an accurate method for calculation, this was reported as an expensive and non-reproducible method for scoring the stroma of PDAC.

Afterward, Wang et al. applied a modified method using hematoxylin-eosin with Masson’s trichrome, and α-SMA for PDACs (6). They found that high stromal density was associated with a significantly better clinical outcome compared to patients with intermediate or low stromal density in multivariate analysis. The stromal pattern was defined as an independent poor prognostic factor (6).

Methods for examining stromal density vary widely in the literature, ranging from eyeballing to computer-based image analysis. We believe that a specific, reproducible, easy, and cost-effective method for assessing stromal density needs to be established through larger studies.

Pancreatic stellate cells (PSCs) become activated in situations such as inflammation and carcinogenesis, leading them to express α-SMA (13,14,24). We also observed stronger α-SMA staining in peritumoral chronic pancreatitis, which supports the thesis that pancreatic stellate cells (PSCs) are triggered by chronic inflammation (not shown here).

Fujita et al. studied 109 resection specimens of PDACs and discovered that high levels of α-SMA mRNA were associated with a worse prognosis. However, it is worth noting that the adjuvant chemotherapy regimens in this study were heterogeneous (19). Similar results were obtained in the CONKO-001 study (25). However, a “tissue microarray” assay was used for immunohistochemical staining in this study, which we believe may not adequately demonstrate the heterogeneous distribution of stromal α-SMA. The therapy regimens were classified as treated with or without gemcitabine, and the antibody clone used was different from ours (Clone: M0874). In contrast to these studies, Özdemir et al. utilized 53 genetically altered mice with decreased α-SMA-expressing myofibroblasts. The response to injury was a decrease in the amount of extracellular matrix (ECM), leading to increased tumor progression. The final result was a worse tumor prognosis (26). Erkan et al. proposed an “activated stromal index” combining high levels of α-SMA and low levels of collagen, and found a relationship with a worse prognosis. However, they could not reveal a significant effect of α-SMA levels alone on survival (20). Wang et al. found that the effect of α-SMA expression on the survival of 145 resected PDACs treated with adjuvant gemcitabine was significant in univariate analysis, but they did not observe any significant results in multivariate analysis (6).

In our study, there was no significant association between the immunohistochemical expression of α-SMA and overall survival (univariable/multivariable: p=0.457/p=0.414). Only 45 cases were followed up in our institution, with 43 receiving gemcitabine-based adjuvant chemotherapy and 2 receiving FOLFIRINOX. Among them, 24 experienced recurrence, and the drug types chosen for recurrence varied (gemcitabine, XELOX, FOLFOX, paclitaxel, etc.). Since most of the cases were treated with the same first-line gemcitabine-based regimen, statistical analyses to assess the relationship between the dominant stromal pattern and/or α-SMA density with response to therapy could not be performed.

Lymph nodes were not within the scope of our study; however, in some cases (n=12), they were present on the slides selected for immunohistochemistry. We observed that the stromal components of metastatic foci in lymph nodes were also strongly positive for α-SMA, indicating that the tumor carries these stromal features wherever it spreads. We believe that examining the stroma in metastatic areas such as the liver, lung, and lymph nodes should be the next step.

The major outcome of our study was the significant correlation between higher α-SMA scores and the presence of angioinvasion (p=0.01). Previous similar studies did not report this kind of correlation (6,19). Pancreatic stellate cells are also recognized to have angiogenic or antiangiogenic effects in both the early and late stages of the disease (27). The presence of such an association may suggest that tumors with higher α-SMA scores are at a higher risk for vascular invasion and local recurrence and/or distant metastasis. This result could be valuable in predicting the metastatic capacity of the tumor. However, due to the small size of our cohort, this preliminary finding did not reach statistical significance in the multivariate analysis (p=0.136).

We believe that the disparate results regarding the effects of α-SMA on survival in the literature are primarily attributable to the retrospective nature of the studies, as well as variations in tissue types, tissue sizes, antibody clones, and the quality of the cohorts. To mitigate these discrepancies, prospective studies and clinical trials should be designed based on analyses and scoring of stromal α-SMA. Additionally, our study primarily focuses on α-SMA expression at the protein level. Integrating molecular analyses, such as gene expression profiling or mutation status, could provide deeper insights into the underlying biological mechanisms driving the observed associations.

Conflict of Interest

The authors declare that they have no conflict of interest for this article.

Funding

The financial support was received from the Marmara University Committee of Scientific Research Project (BAPKO) (Project No: SAG-C-TUP-110915-04).

Ethics Approval

Ethical approval was obtained from the local human ethics committee at Marmara University Institute of Health Sciences (Protocol number: 88-15).

The authors would like to thank Serdar Balci, MD for the statistical analyses; https://github.com/sbalci/ClinicoPathJamoviModule/

* Oral poster presentation at the 30th European Congress of Pathology, 8-12 September, 2018, Bilbao, Spain.
==== Refs
Haqq Jonathan , Howells Lynne M. , Garcea Giuseppe , Metcalfe Matthew S. , Steward Will P. , Dennison Ashley R. Eur J Cancer Pancreatic stellate cells and pancreas cancer: current perspectives and future strategies 10 2014
50 2570 2582 10.1016/j.ejca.2014.06.021
American Cancer Society 2024

Loughrey Maurice B. , Webster Fleur , Arends Mark J. , Brown Ian , Burgart Lawrence J. , Cunningham Chris , Flejou Jean-Francois , Kakar Sanjay , Kirsch Richard , Kojima Motohiro , Lugli Alessandro , Rosty Christophe , Sheahan Kieran , West Nicholas P. , Wilson Richard H. , Nagtegaal Iris D. Ann Surg Dataset for Pathology Reporting of Colorectal Cancer: Recommendations From the International Collaboration on Cancer Reporting (ICCR) 03 2022
275 e549 e561 10.1097/SLA.0000000000005051 34238814
Ueno Hideki , Shinto Eiji , Shimazaki Hideyuki , Kajiwara Yoshiki , Sueyama Takahiro , Yamamoto Junji , Hase Kazuo Ann Surg Oncol Histologic categorization of desmoplastic reaction: its relevance to the colorectal cancer microenvironment and prognosis 05 2015
22 1504 1512 10.1245/s10434-014-4149-9 25395146
Ueno Hideki , Kanemitsu Yukihide , Sekine Shigeki , Ishiguro Megumi , Ito Eisaku , Hashiguchi Yojiro , Kondo Fukuo , Shimazaki Hideyuki , Mochizuki Satsuki , Kajiwara Yoshiki , Shinto Eiji , Yamamoto Junji Am J Surg Pathol Desmoplastic Pattern at the Tumor Front Defines Poor-prognosis Subtypes of Colorectal Cancer 11 2017
41 1506 1512 10.1097/PAS.0000000000000946
Wang Lai Mun , Silva Michael A. , D'Costa Zenobia , Bockelmann Robin , Soonawalla Zahir , Liu Stanley , O'Neill Eric , Mukherjee Somnath , McKenna W. Gillies , Muschel Ruth , Fokas Emmanouil Oncotarget The prognostic role of desmoplastic stroma in pancreatic ductal adenocarcinoma 01 2016
7 4183 4194 10.18632/oncotarget.6770
Lunardi Serena , Muschel Ruth J. , Brunner Thomas B. Cancer Lett The stromal compartments in pancreatic cancer: are there any therapeutic targets? 02 2014
343 147 155 10.1016/j.canlet.2013.09.039
Luo Guopei , Long Jiang , Zhang Bo , Liu Chen , Xu Jin , Ni Quanxing , Yu Xianjun Biochim Biophys Acta Stroma and pancreatic ductal adenocarcinoma: an interaction loop 08 2012
1826 170 178 10.1016/j.bbcan.2012.04.002 22521638
Yoshida Go J. J Exp Clin Cancer Res Regulation of heterogeneous cancer-associated fibroblasts: the molecular pathology of activated signaling pathways 06 2020
39 112 112 10.1186/s13046-020-01611-0 32546182
Wu Yang , Zhang Chun , Jiang Kuirong , Werner Jens , Bazhin Alexandr V. , D'Haese Jan G. Front Oncol The Role of Stellate Cells in Pancreatic Ductal Adenocarcinoma: Targeting Perspectives 2020
10 621937 621937 10.3389/fonc.2020.621937 33520728
Chakkera Mohana , Foote Jeremy B. , Farran Batoul , Nagaraju Ganji Purnachandra Biochim Biophys Acta Rev Cancer Breaking the stromal barrier in pancreatic cancer: Advances and challenges 01 2024
1879 189065 189065 10.1016/j.bbcan.2023.189065
Wang Yuehui , Zhang Arun , Li Quanwang , Liu Chuanbo J Ethnopharmacol Modulating pancreatic cancer microenvironment: The efficacy of Huachansu in mouse models via TGF-β/Smad pathway 05 2024
326 117872 117872 10.1016/j.jep.2024.117872 38325667
Omary M. Bishr , Lugea Aurelia , Lowe Anson W. , Pandol Stephen J. J Clin Invest The pancreatic stellate cell: a star on the rise in pancreatic diseases 01 2007
117 50 59 10.1172/JCI30082
Erkan M. , Reiser-Erkan C. , Michalski C. W. , Kleeff J. Exp Oncol Tumor microenvironment and progression of pancreatic cancer 09 2010
32 128 131 21403605
Gillies Robert J. , Verduzco Daniel , Gatenby Robert A. Nat Rev Cancer Evolutionary dynamics of carcinogenesis and why targeted therapy does not work 06 2012
12 487 493 10.1038/nrc3298 22695393
Shen Jianjun , Person Maria D. , Zhu Jijiang , Abbruzzese James L. , Li Donghui Cancer Res Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry 12 2004
64 9018 9026 10.1158/0008-5472.CAN-04-3262
Olive Kenneth P. , Jacobetz Michael A. , Davidson Christian J. , Gopinathan Aarthi , McIntyre Dominick , Honess Davina , Madhu Basetti , Goldgraben Mae A. , Caldwell Meredith E. , Allard David , Frese Kristopher K. , Denicola Gina , Feig Christine , Combs Chelsea , Winter Stephen P. , Ireland-Zecchini Heather , Reichelt Stefanie , Howat William J. , Chang Alex , Dhara Mousumi , Wang Lifu , Rückert Felix , Grützmann Robert , Pilarsky Christian , Izeradjene Kamel , Hingorani Sunil R. , Huang Pearl , Davies Susan E. , Plunkett William , Egorin Merrill , Hruban Ralph H. , Whitebread Nigel , McGovern Karen , Adams Julian , Iacobuzio-Donahue Christine , Griffiths John , Tuveson David A. Science Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer 06 2009
324 1457 1461 10.1126/science.1171362 19460966
Conroy Thierry , Gavoille Céline , Adenis Antoine Curr Opin Oncol Metastatic pancreatic cancer: old drugs, new paradigms 07 2011
23 390 395 10.1097/CCO.0b013e3283473610 21505335
Fujita Hayato , Ohuchida Kenoki , Mizumoto Kazuhiro , Nakata Kohei , Yu Jun , Kayashima Tadashi , Cui Lin , Manabe Tatsuya , Ohtsuka Takao , Tanaka Masao Pancreas alpha-Smooth Muscle Actin Expressing Stroma Promotes an Aggressive Tumor Biology in Pancreatic Ductal Adenocarcinoma 11 2010
39 1254 1262 10.1097/MPA.0b013e3181dbf647
Erkan Mert , Michalski Christoph W. , Rieder Simon , Reiser-Erkan Carolin , Abiatari Ivane , Kolb Armin , Giese Nathalia A. , Esposito Irene , Friess Helmut , Kleeff Jörg Clin Gastroenterol Hepatol The activated stroma index is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma 10 2008
6 1155 1161 10.1016/j.cgh.2008.05.006
Askan Gokce , Sahin Ibrahim Halil , Chou Joanne F. , Yavas Aslihan , Capanu Marinela , Iacobuzio-Donahue Christine A. , Basturk Olca , O'Reilly Eileen M. BMC Cancer Pancreatic cancer stem cells may define tumor stroma characteristics and recurrence patterns in pancreatic ductal adenocarcinoma 04 2021
21 385 385 10.1186/s12885-021-08123-w 33836674
Balcı Serdar sbalci/ClinicoPathJamoviModule: justice for all 08 2020
10.5281/ZENODO.3997188
Bever Katherine M. , Sugar Elizabeth A. , Bigelow Elaine , Sharma Rajni , Laheru Daniel , Wolfgang Christopher L. , Jaffee Elizabeth M. , Anders Robert A. , De Jesus-Acosta Ana , Zheng Lei HPB (Oxford) The prognostic value of stroma in pancreatic cancer in patients receiving adjuvant therapy 04 2015
17 292 298 10.1111/hpb.12334 25250696
Bachem Max G. , Schünemann Marion , Ramadani Marco , Siech Marco , Beger Hans , Buck Andreas , Zhou Shaoxia , Schmid-Kotsas Alexandra , Adler Guido Gastroenterology Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells 04 2005
128 907 921 10.1053/j.gastro.2004.12.036 15825074
Sinn M. , Denkert C. , Striefler J. K. , Pelzer U. , Stieler J. M. , Bahra M. , Lohneis P. , Dörken B. , Oettle H. , Riess H. , Sinn B. V. Br J Cancer α-Smooth muscle actin expression and desmoplastic stromal reaction in pancreatic cancer: results from the CONKO-001 study 11 2014
111 1917 1923 10.1038/bjc.2014.495
Özdemir Berna C. , Pentcheva-Hoang Tsvetelina , Carstens Julienne L. , Zheng Xiaofeng , Wu Chia-Chin , Simpson Tyler R. , Laklai Hanane , Sugimoto Hikaru , Kahlert Christoph , Novitskiy Sergey V. , De Jesus-Acosta Ana , Sharma Padmanee , Heidari Pedram , Mahmood Umar , Chin Lynda , Moses Harold L. , Weaver Valerie M. , Maitra Anirban , Allison James P. , LeBleu Valerie S. , Kalluri Raghu Cancer Cell Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival 06 2014
25 719 734 10.1016/j.ccr.2014.04.005 24856586
Erkan Mert , Adler Guido , Apte Minoti V. , Bachem Max G. , Buchholz Malte , Detlefsen Sönke , Esposito Irene , Friess Helmut , Gress Thomas M. , Habisch Hans-Joerg , Hwang Rosa F. , Jaster Robert , Kleeff Jörg , Klöppel Günter , Kordes Claus , Logsdon Craig D. , Masamune Atsushi , Michalski Christoph W. , Oh Junseo , Phillips Phoebe A. , Pinzani Massimo , Reiser-Erkan Carolin , Tsukamoto Hidekazu , Wilson Jeremy Gut StellaTUM: current consensus and discussion on pancreatic stellate cell research 02 2012
61 172 178 10.1136/gutjnl-2011-301220
