Podoplanin`s expression in cancer-associated fibroblasts close to tumor budding of invasive edge

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INTRODUCTION

Tumor budding was declared as independent prognostic factor for early cancer in 2016. Tumor-associated fibroblasts (CAFs) are one of the main components of tumor microenvironment. Plenty of different markers are used for detection of CAFs, including podoplanin (POD).

OBJECTIVE

The aim of this study is to identify correlation between tumor budding, that indicates tumor invasive potential and is considered to be a negative prognostic factor, and CAFs near tumor buds using POD.

MATERIAL AND METHODS

43 cases of colon adenocarcinoma are included in the study. Double staining immunohistochemical technology with PCK and POD was used for detection of tumor budding and evaluation of POD expression near tumor buds.

RESULTS

Significant correlations are revealed between tumor budding and depth of tumor invasion (p=0.023)/regional lymph node metastasis (p=0.068), but between POD expression and depth of tumor invasion (p=0.088) only a tendency of correlation is identified. These facts demonstrate critical prognostic value of tumor budding instead POD expression near tumor buds. It was found that intensity of POD expression near tumor buds is statistically analogous to POD expression in the invasive margin (p=0.0016). That means it is not necessary to evaluate POD expression exactly near tumor buds. For the first time stronger POD expression near mucin complexes is reported. That, probably, will allow to use mucin complexes as alternative prognostic factor instead tumor budding, as tumor buds are absent in mucinous adenocarcinoma.

Keywords:

colorectal cancer

tumor budding

cancer-associated fibroblasts

podoplanin

Authors:

N.A. Oleynikova

Lomonosov Moscow State University

SPIN RSCI: 4076-2637
Scopus AuthorID: 57193513523
ResearcherID: H-7672-2014
ORCID: 0000-0001-8564-8874

I.A. Mikhailov

Center for Expertise and Quality Control of Medical Care;
Lomonosov Moscow State University

SPIN RSCI: 5798-0749
Scopus AuthorID: 57203900904
ResearcherID: I-9035-2017
ORCID: 0000-0001-8020-369X

T.V. Polushkina

Lomonosov Moscow State University

ORCID: 0000-0001-5074-3513

N.V. Danilova

Lomonosov Moscow State University

SPIN RSCI: 6878-2025
Scopus AuthorID: 36613033400
ResearcherID: H-6477-2014
ORCID: 0000-0001-7848-6707

O.A. Kharlova

Lomonosov Moscow State University

SPIN RSCI: 9795-899
Scopus AuthorID: 56606237500
ResearcherID: L-5119-2015
ORCID: 0000-0002-5909-1248

Received:

29.03.2021

Accepted:

28.05.2021

List of references:

WHO Classification of digestive system tumours. 5th ed. WHO Press; 2019.
Oleynikova NA, Malkov PG, Danilova NV. Changes in the classification of malignant colon epithelial neoplasms (WHO, 2019, 5TH EDITION). Archive of Pathology=Arkhiv patologii. 2020;82(3):38-46. (In Russ.). https://doi.org/10.17116/patol20208203138
Tommelein J, Verset L, Boterberg T, Demetter P, Bracke M, De Wever O. Cancer-associated fibroblasts connect metastasis-promoting communication in colorectal cancer. Front Oncol. 2015;5:63. https://doi.org/10.3389/fonc.2015.00063
Darby IA, Laverdet B, Bonte F, Desmouliere A. Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol. 2014;7:301-311. https://doi.org/10.2147/CCID.S50046
Lugli A, Kirsch R, Ajioka Y, Bosman F, Cathomas G, Dawson H, El Zimaity H, Fléjou JF, Hansen TP, Hartmann A, Kakar S, Langner C, Nagtegaal I, Puppa G, Riddell R, Ristimäki A, Sheahan K, Smyrk T, Sugihara K, Terris B, Ueno H, Vieth M, Zlobec I, Quirke P. Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016. Mod Pathol. 2017;30(9):1299-1311. https://doi.org/10.1038/modpathol.2017.46
Oe S, Hasegawa K, Nagase S, Kato R, Torii Y, Udagawa Y. Expression of podoplanin in epithelial ovarian carcinomas and its potential as a marker for clear cell adenocarcinoma. Int J Gynecol Pathol. 2010;29(5):405-410. https://doi.org/10.1097/PGP.0b013e3181d3261e
Beaton C, Twine CP, Williams GL, Radcliffe AG. Systematic review and meta-analysis of histopathological factors influencing the risk of lymph node metastasis in early colorectal cancer. Colorectal Dis. 2013;15(7):788-797. https://doi.org/10.1111/codi.12129
Sugai T, Uesugi N, Kitada Y, Yamada N, Osakabe M, Eizuka M, Sugimoto R, Fujita Y, Kawasaki K, Yamamoto E, Yamano H, Suzuki H, Matsumoto T. Analysis of the expression of cancer-associated fibroblast- and EMT-related proteins in submucosal invasive colorectal cancer. J Cancer. 2018;9(15):2702-2712. https://doi.org/10.7150/jca.25646
Choi SY, Sung R, Lee SJ, Lee TG, Kim N, Yoon SM, Lee EJ, Chae HB, Youn SJ, Park SM. Podoplanin, alpha-smooth muscle actin or S100A4 expressing cancer-associated fibroblasts are associated with different prognosis in colorectal cancers. J Korean Med Sci. 2013;28(9):1293-1301. https://doi.org/10.3346/jkms.2013.28.9.1293
Koriyama H, Ishii G, Yoh K, Neri S, Morise M, Umemura S, Matsumoto S, Niho S, Ohmatsu H, Tsuboi M, Goto K, Ochiai A. Presence of podoplanin-positive cancer-associated fibroblasts in surgically resected primary lung adenocarcinoma predicts a shorter progression-free survival period in patients with recurrences who received platinum-based chemotherapy. J Cancer Res Clin Oncol. 2015;141(7):1163-1170. https://doi.org/10.1007/s00432-014-1891-0
Park CK, Jung WH, Koo JS. Expression of cancer-associated fibroblast-related proteins differs between invasive lobular carcinoma and invasive ductal carcinoma. Breast Cancer Res Treat. 2016;159(1): 55-69. https://doi.org/10.1007/s10549-016-3929-2
Schoppmann SF, Jesch B, Riegler MF, Maroske F, Schwameis K, Jomrich G, Birner P. Podoplanin expressing cancer associated fibroblasts are associated with unfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis. 2013;30(4):441-446. https://doi.org/10.1007/s10585-012-9549-2

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Introduction

Tumor budding is determined by the presence of single tumor cells or small clusters of up to 4 cells in the invasive edge. This sign was first declared as an independent prognostic factor of early cancers in 2016. Moreover, tumor budding was included as mandatory characteristic of colon cancer into WHO guidelines (2019) [1]. Tumor budding in the invasive edge is associated with the loss of intercellular contacts between tumor cells and increase of their mobility. These features determine epithelial-mesenchymal transition as important and necessary aspect in tumor invasion and metastases [2]. Recently, various researchers are inclined towards the concept of significance of tumor microenvironment for growth and progression of the neoplasm. It is believed that tumor stroma remodeling results an acquisition of "immortality" by epithelial cells and changes in genotype and phenotype, and not vice versa [3]. One of the components of tumor microenvironment are cancer-associated fibroblasts (CAF). Various markers were described for detection of these cells including podoplanin (POD) [4].

The purpose of this study was to assess the relationships between tumor budding as an unfavorable prognostic factor reflecting invasive potential of tumor and adjacent CAF expressing POD.

Material and methods

The study included 43 paraffin blocks of colon adenocarcinoma. We had performed a synchronous immunohistochemical study with antibodies to podoplanin (POD, Abcam, clone EPR22182) to detect CAF and pan-cytokeratin (PCK, Dako, rabbit polyclonal) for reliable contrast enhancement of tumor budding. Duplex label detection was performed with the use of a Double Stain IHC Kit: M&R on human tissue (HRP/DAB&AP/Red, Abcam ab210059) according to the manufacturer's recommendations. Both quantitative (color separation and label area analysis in the LAS X program) and semi-quantitative (significant, moderate, weak or negative reaction) analysis of POD expression were carried out. POD expression was assessed around tumor budding (“hot spot” according to the guidelines [5]) and in invasive edge without tumor budding. These procedures were performed eather if tumor budding was not found, or if budding was in another field of view.

We paid attention to fusiform cells within invasive edge of tumor and around tumor budding which could potentially be CAF. We did not consider all fusiform cells which can comprise quiescent fibroblasts, activated and cancer-associated fibroblasts. On the contrary, fibroblasts expressing POD were analyzed only since POD was described as a marker of CAF. Considering CAF heterogeneity and a large number of markers, we cannot exclude potential coexistence of other POD-negative CAF subpopulations in invasive edge (and around tumor budding) in each case. Their assessment was not an objective of this study. POD expression normally observed in lymphatic endothelium was not considered in this study.

Statistical analysis was performed using Statistica 10 software package (StatSoft, Inc., USA). Shapiro — Wilk test was applied to test the null hypothesis about normal distribution of data. Contingency tables and Pearson chi-square test were used to analyze between-group differences of qualitative variables, Kruskal — Wallis test — for analysis of quantitative parameters in several groups with subsequent a posteriori pairwise comparison using Mann — Whitney test.

Results

During the study we have compared the following parameters: POD expression in CAF of the invasive edge and (separately) around tumor budding — with mucinous component in tumor; POD expression in the invasive edge with reaction around budding; the presence of tumor budding with clinical and morphological characteristics of tumors.

POD expression in CAF and presence of mucinous component

Among 43 cases of adenocarcinoma, mucinous component was 11 times observed. POD expression in the invasive edge considering mucinous component is shown in Fig. 1. In almost half of cases, POD reaction was negative in both groups (Fig. 2, b).

Fig. 1. Intensity of POD expression in the invasive margin depending on presence of mucinous component.

Fig. 2. POD expression in invasive margin. Immunohistochemical study with PCK (brown) and POD (red), ×20.

a — tumor budding near invasive margin, negative POD expression in the surrounding stroma; b — no tumor budding near invasive margin, negative POD expression in the surrounding stroma; c — tumor budding near invasive margin, weak POD expression in the surrounding stroma; d — tumor budding near invasive margin, strong POD expression in the surrounding stroma.

Tumor buddings were found in 53.5% of cases (Fig. 2, a). POD expression was also various around these structures (Fig. 3).

Fig. 3. Intensity of POD expression near tumor buds depending on presence of mucinous component.

We found no significant differences in POD expression depending on the presence of mucinous component using chi-square test (p = 0.678) either for invasive edge or individual tumor buddings. However, we observed prominent POD expression around mucinous complexes and "lakes of mucus" in most adenocarcinomas with a mucinous component (Fig. 4). We failed to confirm this pattern statistically due to small sample size (p = 0.181). However, these data indicate a possible statistical trend and this pattern may be confirmed in a larger sample.

Fig. 4. Intensity of POD expression in mucinous adenocarcinoma of the colon.

Immunohistochemical study with PCK (brown) and POD (red), ×20; a, b — strong POD expression near mucin complexes.

We found no studies devoted to POD in stroma of mucinous adenocarcinoma. However, S. Oe et al. [6] reported higher incidence of POD expression in ovarian clear cell adenocarcinomas compared to other histological types. These data confirm potential tropism of POD to tumors containing polysaccharides.

Comparison of POD expression in CAF of different areas of the invasive edge

There were significant between-group differences in POD expression around tumor buddings and in invasive edge without buddings (p <0.01). A posteriori pairwise comparison revealed the following facts (Fig. 5):

— reaction around buddings (active vs. moderate) significantly differed in tumors with active and moderate reaction in the invasive edge (p = 0.012);

— reaction around buddings (moderate vs. weak) significantly differed in tumors with moderate and weak reaction in the invasive edge (p = 0.017);

— reaction around buddings (moderate vs. negative) significantly differed in tumors with moderate and negative reaction in the invasive edge (p = 0.031).

Fig. 5. Comparison of intensity of POD expression near tumor buds and in the invasive margin.

These differences indicate no need to evaluate POD expression specifically around tumor buddings and POD tropism to these structures.

In addition to semi-quantitative assessment, we additionally applied quantitative analysis using color separation in the LAS X program to determine POD expression in the invasive edge. Kruskal — Wallis test (Fig. 6) showed a significant relationship (p = 0.0016) between qualitative and quantitative parameters. Thus, we can conclude that semi-quantitative method is adequate and correct and can be used instead of more laborious quantitative method.

Fig. 6. Distribution of the POD expression level depending on the expression level around the tumor buddies (the Kruskal—Wallis test was used to establish the significance of the differences).

Presence of tumor buddings and clinical and morphological characteristics of neoplasms

There are contradictory literature data on statistical relationship between tumor budding and metastases in regional lymph nodes in patients with early colorectal cancer [7, 8]. This fact can be due to non-standardized assessment of tumor budding since standardization protocol was adopted only in 2016 [5]. Chi-square test was used in this study to establish the relationships. We found a significant correlation (p = 0.023) between tumor budding and invasion depth (stage T3-T4) and a tendency towards significant relationship (p = 0.068) between tumor budding and metastases in regional lymph nodes, but not their number (Fig. 7).

Fig. 7. Clinical and morphological tumor characteristics depending on tumor budding.

POD reaction in CAF and clinical and morphological characteristics

There was also a tendency to relationship between POD expression around tumor buddings and invasion depth (p = 0.088), but not the presence of metastases (Fig. 8). It is noteworthy that no significant relationships were found between POD expression in the invasive edge and clinical and morphological characteristics.

Fig. 8. Revealed statistical correlations between clinical and morphological characteristics.

Blue arrows demonstrate correlations between tumor budding and depth of tumor invasion/regional lymph node metastasis. Red circle shows zone of POD expression near tumor buds. Red arrows demonstrate correlations between POD expression near tumor buds and depth of tumor invasion/POD expression in the invasive margin.

Discussion

Considering correlation between POD expression in the invasive edge and around tumor budding, one gets an impression not of prognostic significance of POD expression around tumor budding, but of prognostic significance of tumor budding per se. The last fact was also confirmed by statistical analysis and corresponds to WHO data [2]. Moreover, no correlation between POD expression around tumor budding and lymph node metastases indicates the lack of prognostic significance of POD.

There are literature data on adverse prognostic effect of the absence of POD expression in colorectal cancer due to higher incidence of lymph node metastases and worse relapse-free survival [9]. A later study demonstrated high incidence of regional lymph node metastases in submucous colorectal cancers with high expression of CAF and EMT markers (α-SMA, CD10, POD, FSP1, AEBP1, ZEB1, and TWIST1) [8]. Despite the absence of direct correlation between lymph node metastases and POD expression, significantly higher POD expression in the group with higher incidence of metastases indirectly indicates its unfavorable effect. It is also typical for many malignancies including adenocarcinoma of the lung, esophagus, invasive breast cancer [10–12]. Active expression of POD in the invasive edge may indicate high concentration of CAF in these tumors because POD is one of the markers of these cells. This conclusion is especially significant for mucinous tumors with higher POD expression near the "lakes of mucus", since tumor buddings are rarely formed in these neoplasms and their assessment according to WHO recommendations is difficult [2].

Conclusion

We first studied POD expression in CAF around tumor buddings in the invasive edge of colon adenocarcinoma using duplex labeling technology. It was shown that POD expression in CAF around tumor budding is similar to reaction in the invasive edge (p <0.01). Thus, there is no need to analyze POD expression around tumor buddings.

Correlations between tumor budding and invasion depth / regional lymph node metastases confirm their prognostic value. No statistical relationship between POD expression in CAF around tumor buddings and invasion depth / metastases indicates no predictive value of POD-positive CAFs. For the first time, active expression of POD in CAF near mucinous complexes was described. However, the cause and possible clinical significance of this fact are still unclear.

Author contribution:

Concept and design of the study — N.A. Oleynikova

Collection and analysis of data — N.A. Oleynikova, O.A. Kharlova

Statistical analysis — I.A. Mikhailov

Writing the text — N.A. Oleynikova

Editing — P.G. Malkov, N.V. Danilova

The research was financially supported by the Russian Foundation for Basic Research (RFBR, grant "Perspective" No. 19-315-60006). The authors used equipment purchased within the development program of the Lomonosov Moscow State University until 2020.