Intralobular Distribution of Ovarian-like Stroma in Pancreatic Mucinous Cystic Neoplasms: Hypothesis on Its Tumorigenesis

doi:10.21203/rs.3.rs-627775/v1

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Intralobular Distribution of Ovarian-like Stroma in Pancreatic Mucinous Cystic Neoplasms: Hypothesis on Its Tumorigenesis

https://doi.org/10.21203/rs.3.rs-627775/v1

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Pancreatic mucinous cystic neoplasm (MCN) harbors two histological components, tumor epithelia and ovarian-like stroma (OLS). To examine the tumorigenesis of pancreatic MCNs, this study analyzed the distribution, amount, immunohistochemical phenotype, presence of theca cells of the OLS, and the alteration of tumor epithelium of 29 surgically resected MCN cases and compared them with tumor sizes. Non-mucinous type epithelium was present in all low-grade MCNs but its ratio decreased with tumor size (p < 0.05), suggesting that epithelial mucinous changes are a progression phenomenon. The intralobular distribution of OLS was observed in 27.6 % of MCN cases and its existence related to a smaller size (p< 0.05), suggesting intralobular generation of MCNs. Nuclear expression of β-catenin was observed for OLS of everywhere, suggesting consistent activation of the Wnt pathway for OLS. Three MCN cases (10.3%) contained a-smooth muscle actin (SMA)-negative OLS, where OLS surrounding dilated pancreatic ducts or MCN cysts were a-SMA-positive and otherwise negative, suggesting that a-SMA-positivity is an acquired phenomenon of OLS. With this study, we could hypothesize that pancreatic MCNs may generate intralobularly. Epithelial mucinous change and a-SMA-positivity of OLS may be progression phenomena. This is the first study to show the intralobular distribution of OLS.

Health Economics & Outcomes Research

Pathology

mucinous cystic neoplasm (MCN)

OLS

intralobular distribution

tumor

Mucinous cystic neoplasm (MCN) is a cyst-forming mucinous tumor that arises mainly in the ovary, pancreas, liver, and rarely in the mesentery, retroperitoneum and spleen.^1–3 In the pancreas, MCN primarily occurs in the pancreatic body or tail of middle-aged women, does not communicate with the pancreatic duct, and, unlike branch duct intraductal papillary mucinous neoplasms (IPMNs), is always a single lesion.^4,5 MCN harbors two histological components, tumor epithelia and ovarian-like stroma (OLS). The OLS expresses ER and PgR, sometimes containing inhibin a-positive theca cells and is the most specific histological feature of MCN.^6–8 However, despite such specific pathological features of MCN, the tumorigenesis of this tumor is not well known to date. Currently, there are two hypotheses regarding the origin of MCN: (i) pancreatic remnant/attachment of primordial germ cells during their migration to the gonad (ectopic ovarian tissue),^9–13 and (ii) endodermal-derived epithelium and primitive mesenchyme in the pancreas proliferation under the stimuli of female sex steroids.^12,13 Among these two theories, recent molecular studies have favored the former.^9–11

In this study, to examine the progression and alteration of histology and immunophenotype and to examine the tumorigenesis of pancreatic MCNs, we analyzed the distribution, amount, immunohistochemical phenotype, presence of theca cells of the OLS, and pathological features of the tumor epithelium of MCN tumors.

Materials

Twenty-nine pancreatic MCN cases (28 female cases and one male case) were analysed. All patients underwent surgical resection, and 15, 11 and three patients underwent surgery at the Department of Hepatobiliary Pancreatic Surgery of Juntendo University Hospital, Tokyo Medical and Dental University Hospital, and Department of Gastroenterological Surgery of Kagawa University Hospital, respectively, between 2005 and May, 2021. Among them, MCNs were located at the pancreatic body/tail in 28 cases and at the pancreatic head in one case. OLS, confirmed by immunohistochemistry for ER and PgR, was observed in all cases. In each case, the entire pathological specimen was cut to 5-μm thickness and microscopic examination was performed with haematoxylin and eosin（HE）staining. Representative one to three sections per case were utilized for immunohistochemical evaluation.

Methods

Clinicopathological data, including patients’ age, sex, and tumor site, were collected, and tumor size, segment, and multi- or mono-locularity of the cyst were determined by macroscopic inspection of the tumor, where the maximal diameter of the MCN cyst was recorded for tumor size; when the tumor was small enough and localized into one half of the cut section, we recorded in which half the tumor belonged (anterior, posterior, superior, or inferior). The histological grade of the MCN epithelium was classified into low, high, or high with associated invasive carcinoma, according to the World Health Organization classification.⁶ The epithelium of MCN was categorized into mucinous or non-mucinous type according to the previous study.⁹ Briefly, the pancreatic intraepithelial neoplasia-like columnar cells with pale pink mucin were classified as mucinous type; whereas, flat to cuboidal to short columnar without obvious mucin or goblet cells were classified as non-mucinous type. The ratio of non-mucinous epithelia among the total cyst-lining epithelia was recorded in 10% increments by analyzing the entire tumorous lesion for each case, where the part in which no lining epithelia were seen was not counted. Since high-grade epithelial components of MCNs showed almost exclusively mucinous-type epithelium, the ratio of non-mucinous type epithelium was analyzed only for low-grade MCN cases.

Assessment of OLS

Regarding OLS, their distribution and amount were determined by HE staining and immunohistochemistry for ER and PgR. Herein, the OLS was defined as the stroma cells similar to ovarian stroma and showing positive for ER/PgR, immunohistochemically. The distribution of OLS was categorized into the cyst wall (C), septum of the cyst (S), inside the pancreatic lobule (IL), perilobular area (PL), and extrapancreatic site (EX). When OLS was distributed in the acinar cell-containing areas, clearly separated from MCN cyst, it was determined as IL, and when OLS was seen between pancreatic lobules or between pancreatic duct and pancreatic lobule, it was determined as PL. The distinction between pancreatic duct and a part of the MCN cyst was as follows: when the epithelia-lined structure was far enough from the main MCN cyst, we considered it a duct, and when the structure was continuous or close enough to the main MCN cyst, we considered a part of the cyst. The amount of OLS was classified as scarce when the maximum thickness of the OLS was <100 mm; moderate, when it was ≥100 mm, but <1000 mm; abundant, when it was ≥1000 mm. The presence of theca cells was determined by HE staining and immunohistochemistry for inhibin a, and when present, their distribution was classified as C, S, IL, PL, and EX in a similar way as OLS. Immunohistochemistry for a-smooth muscle actin (SMA) and b-catenin (nuclear expression) was performed to determine the difference in immunophenotype of OLS at different sites. This study was approved by the Ethics Committee of Juntendo University, Tokyo, Japan, in November 2018 (#J-2018076) and was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants.

Statistical analysis

To determine the relationship between tumor size and each clinicopathological feature of MCN, single regression and single logistic analyses were performed for continuous data and ordinal/nominal data, respectively, utilizing JMP 14.2.0 statistical software (SAS Institute, Incorporation, Cary, NC).

Clinicopathological data of MCN patients, cyst, tumor epithelium, and OLS are shown in Supplementary Table 1. Briefly, the mean age of the MCN patients was 46.5 years (range 22–78 years); all MCN patients, except one, were female; mean tumor size was 60.7 mm (range 12–150 mm). All MCNs, except one, were located at the pancreatic body/tail, and 24 (82.8 %) and five (17.2 %) MCNs were multilocular and monolocular, respectively. Seven MCN cases (24.1 %) were localized in one half (or smaller) of the cut sections, which belonged to the inferior half (n = 4), including the anterior-inferior (n = 1) and the posterior-inferior (n = 1), and the superior half (n = 3), including the anterior-superior. Eight of the 27 MCN cases (27.6 %) contained high-grade or associated invasive carcinoma components. Non-mucinous type epithelia were seen in all 21 low-grade MCN cases (100 %), and the mean ratio of non-mucinous type was 48.0 % (rage 10–80 %) in low-grade MCN cases.

All MCN cases contained OLS showing positivity for both ER and PgR immunohistochemically, and 51.7 % (15/29), 31.0 % (9/29), and 17.2 % (5/29) cases of MCN contained abundant, moderate, and scarce amounts of OLS, respectively. Regarding the OLS distribution, 96.6 % (28/29), 89.7 % (26/29), 27.6 % (8/29), and 3.4% (1/29) of MCN cases contained OLS at C, S, IL, and PL, respectively. However, no MCN cases contained OLS at EX (Fig. 1). Theca cells immunohistochemically confirmed with inhibin a positivity were present in 37.9 % (11/29) of MCN cases and among the 11 MCN cases with theca cells, 81.8 % (9/11), 9.1 % (1/11), and 18.2 % (2/11) MCN cases contained theca cells at C, S, and IL, respectively (Fig. 1).

Immunohistochemically, most OLS was positive for a-SMA irrespective of OLS distribution sites or MCN size, whereas three MCN (10.3%) cases contained some areas composed of a-SMA-negative OLS. In these three cases, a-SMA-negative OLS was observed in the IL (n = 2), C (n = 1), and S (n = 1) (Supplementary Table 1). At IL, C and S, OLS surrounding dilated pancreatic ducts or MCN cysts was a-SMA-positive, but otherwise negative (Fig. 2). Nuclear b-catenin accumulation was observed in OLS and theca cells, but not in tumor epithelia at all locations (C, S, IL, or PL) of all MCN cases (Fig. 3).

Logistic analyses showed that the histological higher grade (high or high grade with invasion) was related to larger tumor size (p < 0.05), higher ratio of non-mucinous type epithelium was related to smaller tumor size (p < 0.01), and the intralobular distribution of OLS was related to smaller tumor size (p < 0.0005) (Table 1).

Table 1

The relationship between clinicopathological data and tumor size of pancreatic MCN
		p-value (vs tumor size)*
Age		NS (p = 0.113)
mean	46.5
range	22–78
Cyst		NS (p = 0.490)
Monolocular	5
Multilocular	24
Grade		p = 0.0249
Low	21	(R² = 0.147, b= -0.0228)
high+	8
Ratio of non-mucinous type		P = 0.0088
mean	48% (n = 21**)	(R² = 0.0826, b = 0.0308)
range	10–80
OLS amount		NS (p = 0.410)
abundant	15
moderate	9
scarce	5
OLS at IL		p = 0.0003
present	8	(R² = 0.382, b = 0.0843)
absent	21
Theca cell		NS (p = 0.357)
present	10
Absent	19
*p-value, R² score (contribution rate), and b-value (coefficient) by regression/logistic analysis were shown.
**Ratio of non-mucinous type epithelium was analyzed for low grade-MCN cases (n = 21).

The exact mechanism of tumorigenesis of MCN has not yet been elucidated. To examine its tumorigenesis, we analyzed size-dependent histological and immunohistochemical changes of MCN in this study, hypothesizing that smaller MCN group harbors histological features of the early phase of MCN development. The relationship of histological features and tumor size of the MCN was analyzed.

Regarding MCN epithelia, our study showed larger MCNs more often contained high-grade or high-grade with invasive carcinoma components with statistical significance. Non-mucinous-type epithelium was seen in all low-grade MCN cases, and the ratio of non-mucinous epithelium among the entire epithelium was significantly higher in smaller MCNs. Our results showed that the non-mucinous epithelium was a common finding in pancreatic MCN, and that smaller MCN contained more non-mucinous type epithelium, which is concordant with a previous study,¹⁴ indicating that the mucinous change of the epithelium is a “progression” phenomenon in pancreatic MCNs.

Regarding OLS, our study showed a tendency that smaller MCNs more often contained abundant amount of OLS and showed smaller MCNs more often contained OLS at intralobular sites with statistical significance. The presence of OLS is the most specific histological feature of MCN, and OLS is located immediately beneath or associating with the neoplastic epithelium.¹⁵ To the best of our knowledge, the intralobular distribution of the OLS in pancreatic MCN has never been reported. However, this study revealed that 27.6 % of our MCN cohort, or 75.0 % of MCN cases sized ≤ 30mm contained OLS at intralobular sites, which are clearly distant sites, or apparently with no association with the neoplastic epithelium. Although it is difficult to determine the origin of MCN epithelia or OLS in this study, the frequent intralobular distribution of OLS in small-size MCN suggests that OLS or initial MCN may be generated in pancreatic lobules.

Since pancreatic MCN is usually detected or surgically resected when it is large in size, it is generally difficult to see where or in which part of the pancreatic parenchyma MCN has generated. Although it is well known that pancreatic MCNs do not communicate with pancreatic ducts, it is possible that MCNs may have communicated with small ducts, such as intralobular or intercalated ducts or acinus in the early phase of MCN development. One experimental study by Sano et al., reported the intralobular generation of pancreatic MCN;¹⁶ the authors showed the development of MCN with ER- and PgR-positive OLS by introducing Wnt1 into pancreatic acinar cells and acinar cell progenitors in LSL-Kras^G12D, Ptf1a-cre mice. The authors reported the importance of the interaction between acinar cells and OLS in MCN development.

In this study, 7 MCN cases were small enough to localize in either half of the pancreatic cut section, and the locations of these 7 cases were relatively diverse; four cases (57.1 %) belonged to the inferior half portion of the pancreatic cut section and 3 cases (42.9 %) to the superior; where one case was localized in the anterior/inferior, one in the posterior/inferior, and another in the anterior/superior quarter. In contrast to the diversity of MCN location, no OLS was detected at the extrapancreatic site in this study, suggesting that early MCN may start at the site of pancreatic parenchyma, mostly at pancreatic body-tail, but not at the extrapancreatic site.

One of the supported mechanisms for MCN development is the pancreatic attachment of primordial germ cells during their migration to the gonad (ectopic ovary),^9–11 and if this theory is true, our result of diverse localization of MCNs indicates the complicated process of organogenesis. Since there is a time overlap between the migration of primordial germ cells through the dorsal mesentery to the gonad and the rotation of pancreatic anlage, both of which are around the fifth week after conception, we can consider that such complicated organogenesis may contributed to the diverse location of pancreatic MCNs. Our finding that no OLS was seen at the extrapancreatic site may seem inconsistent with the diverse localization of MCNs, and one possible reason for this may be that some microenvironment of pancreatic parenchyma is necessary for MCN to survive or extrapancreatic sites may be unsuitable for MCN to survive.

Using immunohistochemistry, nuclear expression of β-catenin for OLS was observed for all MCN cases and for all OLS sites. Several studies have suggested the activation of Wnt signaling pathway in OLS and its contribution in MCN development.^16–18 Sano et al. has shown the activation of Wnt signaling pathway in OLS by introducing Wnt1 and KRAS^G12D to the epithelial component in mice.¹⁶ Fukushima et al. have shown the overexpression of Wnt2B and secreted Frizzled-related protein, a moderator of Wnt pathway in OLS¹⁷ and Treek et al. have shown the upregulation of Wnt and the Hedgehog pathway at OLS.¹⁸ Our result that all OLS showed nuclear accumulation of b-catenin irrespective of cyst size or OLS site, suggests consistent activation of Wnt pathway for OLS, that is, the contribution of Wnt pathway activation in OLS is not only for the early developmental phase, but also for the late phase.

The present study detected three (10.3 %) MCN cases containing areas composed of a-SMA-negative OLS, although the most OLS were a-SMA-positive. The immunoreactivity of OLS for a-SMA has been reported in several studies.^1,7,15 Shiono et al., have suggested that OLS obtains myofibroblastic differentiation in response to MCN development by analyzing OLS with electron microscopy and comparing the stroma of ovarian MCN and normal ovary.¹ Our observation that pericystic or periductal OLS was a-SMA-positive and otherwise a-SMA-negative suggest that OLS might be a-SMA-negative at the time of MCN generation and later acquires myofibroblastic features, possibly because of the ductal pressure. Further studies with more MCN cases containing abundant OLS are needed to confirm our hypothesis.

In conclusion, our study showed an increase in histological grade and a decrease in the ratio of non-mucinous type epithelium with an increase of tumor size. We clarified that OLS can be observed at intralobular sites mainly in small-size pancreatic MCNs, suggesting that initial MCN may be generated inside the pancreatic lobules. The fact that some OLS are immunonegative for a-SMA, especially in areas distant from the duct/cyst epithelium, suggests that a-SMA-positivity of OLS may be an acquired phenomenon during MCN progression. Our hypothesis on the development and tumorigenesis of pancreatic MCN is shown in Fig. 4. The limitation of this study is the nature of the study; this is an observational study of histology and immunohistochemical data. Analyses of the size-dependent alterations in the genetic profile of OLS/tumor epithelia are required for future studies on pancreatic MCNs.

Acknowledgements

We thank Editage co. Ltd, for proofreading the manuscript. We also thank Ms. Shuko Nojiri, Associate Professor, Medical Technology Innovative Center, Clinical Research and Trial Center, Juntendo University, for statistical advice. One case of MCN (male case) in this study cohort was reported as a case report by Tomishima et al.¹⁹

Author contributions

Y.F., Y.K., Y.M., M.Tak. and M.To. substantially contributed to the conception, data analysis and design of the manuscript. Y.F., Y.K., Y.M., M.M., A.S., M.Tan., K.O., and Y.S. contributed to the acquisition and interpretation of data. Y.F., M.To, and K.K. drafted the article including figures, and Y.K., Y.M., M.Tak. and T.Y. have revised it critically and substantially. All authors have approved the final version and accepted accountability for all aspects of the study.

Shiono S, Suda K, Nobukawa B, et al. Pancreatic, hepatic, splenic, and mesenteric mucinous cystic neoplasms (MCN) are lumped together as extra ovarian MCN. Pathol Int 2006; 56: 71-77.
Izumo A, Yamaguchi K, Eguchi T, et al. Mucinous cystic tumor of the pancreas: immunohistochemical assessment of “ovarian-type stroma”. Oncol Rep 2003; 10: 515-525.
Zamboni G, Scarpa A, Bogina G et al. Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and relationship to other mucinous cystic tumors. Am J Surg Pathol 1999; 23: 410-422.
Yamao K, Yanagisawa A, Takahashi K, et al. Clinicopathological features and prognosis of mucinous cystic neoplasm with ovarian-type stroma: a multi-institutional study of the Japan pancreas society. Pancreas 2011; 40: 67-71.
Crippa S, Salvia R, Warshaw AL, et al. Mucinous cystic neoplasm of the pancreas is not an aggressive entity: lessons from 163resected patients. Ann Surg 2008; 247: 571-579.
Basturk O, Esposito I, Fukushima N er al. Pancreatic mucinous cystic neoplasm. In the WHO Classification of Tumours Editorial Board ed., Who classification of Tumours Digestive system tumours. 5^th edition 2019: 319-321, IARC.
Farrell JJ. Prevalence, diagnosis, and management of pancreatic cystic neoplasms: current status and future directions. Gut Liver 2015; 9: 571-589.
Lam MM, Swanson PE, Upton MP, et al. Ovarian-type stroma in hepatobiliary cystadenomas and pancreatic mucinous cystic neoplasms. An immunohistochemical study. Am J Clin Pathol 2008; 129: 211-218.
Erdogan D, Kloek J, Lamers WH, et al. Mucinous cystadenomas in liver: management and origin. Dig Surg 2010 27: 19-23.
Elias KM, Tsantoulis P, Tille JC, et al. Primordial germ cells as a potential shared cell of origin for mucinous cystic neoplasms of the pancreas and mucinous ovarian tumors. J Pathol 2018 246: 459-169.
Kumata H, Murakami K, Ishida K, et al. Steroidogenesis in ovarian-like mesenchymal stroma of hepatic and pancreatic mucinous cystic neoplasms. Hepatol Res 2018; 48: 989-999.　
Zamboni G, Scarpa A, Bogina G, et al. Mucinous cystic tumors of the pancreas: Clinicopathological features, prognosis, and relationship to other mucinous cystic tumors. Am J Surg Pathol 1999 23: 410-422.　
Fukushima N, Zamboni G. Mucinous cystic neoplasms of the pancreas: Update on the surgical pathology and molecular genetics. Sem Diagn Pathol 2014 31: 467-474.
Zhelnin K, Xue Y, Quigley B, et al. Nonmucinous biliary epithelium is a frequent finding and is often the predominant epithelial type in mucinous cystic neoplasms of the pancreas and liver. Am J Surg Pathol 2017 41: 116-120.
Thompson L, Rpbert B, Ronald P, et al. Mucinous cystic neoplasm (mucinous cystadenocarcinoma of low-grade malignant potential of the pancreas. A clinicopathologic study of 130 cases. Am J Surg Pathol 1999 23: 1-16.
Sano M, Driscoll DR, Jesus-Monge WED, et al. Activated wnt signaling in stroma contributes to development of pancreatic mucinous cystic neoplasms. Gastroenterology 2014 146: 257-267.
Fukushima N, Sato N, PrasadN, et al. Characterization of gene expression in mucinous cystic neoplasms of the pancreas using oligobucleotide microarrays. Oncogene 2004; 23: 9042-9051.
Van Treeck BJ, Lotfalla M, Czeczok TW, et al. Molecular and immunohistochemical analysis of mucinous cystic neoplasm of the liver. Am J Clin Pathol 2020; 154: 837-847.
Tomishima K, Fujisawa T, Fukumura Y, et al. Mucinous cystadenocarcinoma of the pancreas with cyst infection in a male patient. Intern Med 2020 59: 2383-2389.

No competing interests reported.

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Intralobular Distribution of Ovarian-like Stroma in Pancreatic Mucinous Cystic Neoplasms: Hypothesis on Its Tumorigenesis

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