RSPO2 is a novel sensitive and specific biomarker in esophageal squamous intraepithelial neoplasia

doi:10.21203/rs.3.rs-1730879/v1

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RSPO2 is a novel sensitive and specific biomarker in esophageal squamous intraepithelial neoplasia

https://doi.org/10.21203/rs.3.rs-1730879/v1

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Background: Esophageal squamous intraepithelial neoplasia (ESIN) is considered a precursor lesion of esophageal squamous cell carcinoma (ESCC), High grade ESIN (HGESIN)has much higher risk of progression to ESCC comparing with low grade ESIN(LGESIN). the distinction between squamous cell dysplasia and reactive changes, and HGESIN and LGESIN can sometimes be subjective and challenging. RSPO2 is a member of the R-spondin family of proteins, and was identified as either oncogenes or tumor suppressors depending on the cancer type.

Methods: In the current study, we compared the immunohistochemistry(IHC) expression of newly identified marker RSPO2 with p53 and Ki-67 in different esophageal squamous lesions, and to evaluate correlations between their expression levels in these lesions.

Results: IHC stains of RSPO2, p53 and Ki-6 were performed on endoscopic submucosal dissection specimens from 71 patients including 96 HGESIN, 50 LGESIN, and 160 fragments of adjacent normal squamous esophageal mucosa. RSPO2 and Ki-67 showed high sensitivity and specificity for diagnosing ESIN from normal squamous epithelium. The sensitivity of high level expression of RSPO2 in distinguishing HGESIN from LGESIN was as high as 97.92%, but specificity was only 72%. The specificity of strong staining of RSPO2 in distinguishing HGESIN from LGESIN was as high as 100%, but the sensitivity was as low as 29.17%. The specificity of high level expression of Ki-67 and p53 in distinguishing HGESIN from LGESIN was 96% and 96% respectively, but sensitivity was reduced to 56.25% and 62.5% respectively.

Conclusions: This study demonstrates that both RSPO2 and Ki-67 can be useful in differential diagnosing ESIN.

Esophageal squamous intraepithelial neoplasia

Endoscopic submucosal dissection

RSPO2

p53

Ki-67

Esophageal cancer ranks as the 6th most common cause of cancer related death(544,076 deaths) worldwide in 2020^[1]. Esophageal squamous cell carcinoma (ESCC) accounts for nearly 90% of esophageal cancer cases worldwide^[2], which arises in the background of squamous epithelial dysplasia. The main purpose for ESCC screening is to detect early ESCC and esophageal squamous dysplasia and, to offer the best prognosis for ESCC. Epidemiologic studies found relative risks (95% confidence intervals) for incidence of ESCC, by initial histological diagnosis, were: normal 1.0 (reference), oesophagitis 0.8 (0.2–3.2), basal cell hyperplasia 1.9 (0.8–4.5), mild dysplasia 2.9 (1.6–5.2), moderate dysplasia 9.8 (5.3–18.3), severe dysplasia 28.3 (15.3–52.3), and carcinoma in situ 34.4 (16.6–71.4)^[3]. Two-tiered grading system (low-grade vs high-grade) of esophageal squamous intraepithelial neoplasia(ESIN) was recommended in the 2019 new WHO classification of digestive tumors (5th edition)^[4]. ESIN is considered a precursor lesion of esophageal ESCC^[5].In developing countries, the highest prevalence of ESIN has been observed in Iran (4%) and China (Taihang Mountain region, exceeding 20%.), similar to ESCC^[6].

Esophago-gastro endoscopy is the gold standard test for ESCC and the precursor lesions. Endoscopic manifestation is usually characteristic but not pathognomonic. Hence, a firm diagnosis depends on histopathologic findings and differentiation from other entities. However, the distinction between squamous cell dysplasia and reactive changes, and the low-grade and high-grade of squamous cell dysplasia can sometimes be subjective. Therefore, clinically useful adjunct markers for diagnosing precursor squamous lesions should be developed. Overexpression of p53 and Ki-67 have been reported in a few studies, indicating Ki-67 are sensitive and specific marker for dysplasia in mature squamous epithelium^[7], the sensitivity of p53 was much lower than that of Ki-67.

RSPO2 is a member of the R-spondin family of proteins, and was identified as an activator of the Wnt/β-catenin pathway^[8]. The R-Spondin family includes four members (R-Spondin1 through R-Spondin4), these four proteins have a common structural architecture and share 40–60% amino acid similarity^[9]. In humans, RSPO2 is located on chromosome 8q23.1. Evidence suggests that RSPO2 can function as either oncogenes or tumor suppressors depending on the cancer type^{[10, 11]}. High level Expression of RSPO2 was found to characterize a subset of colorectal cancers, and a subset of triple-negative breast cancers^[12]. While RSPO2 has also been attributed tumor suppressive activities to inhibit colorectal cancer cell migration, invasion, and metastasis^[13].Induction of RSPO2 secretion by keratinocyte– fibroblast interaction of the keloid cells might be contributing factors for the epidermal activation and thickening of keloids’ epidermis^[14]. RSPO2-LGR4 system contributes to the tumor growth, migration and invasion in tongue Squamous cell carcinoma via Wnt/β-catenin signaling^[15].

The purpose of this study was to investigate RSPO2, p53 and Ki-67 IHC expression in normal, hyperplastic, and dysplastic esophageal squamous epithelium, and to evaluate correlations between their expression levels in these lesions.

2.1 Case selection

In this study, the Ethics Committee of Nanfang Hospital of Southern Medical University approved the project. A total of 146 esophageal squamous lesions were retrieved from 71 patients underwent esophageal ESD (Endoscopic submucosal dissection), and normal adjacent mucosa were used as controls All cases were obtained from Nanfang Hospital of Southern Medical University, during the period from January 2020 to January 2022. Hematoxylin and eosin (H&E) stained slides were evaluated to confirm the diagnosis by three pathologists. The diagnoses were as follows: 96 high-grade squamous intraepithelial neoplasia (HGESIN), 50 low-grade squamous intraepithelial neoplasia (LGESIN), and 160 fragments of adjacent normal squamous esophageal mucosa in 71 patients.

2.2 Immunohistochemistry

Immunohistochemistry was performed by Ventana benchmark ultra (Ventana) with positive and negative controls. The primary antibodies used were as follows: RSPO2 (cat. no. ER61850; 1:500 dilution, HUABIO, Hangzhou, China), p53(clone DO-7; 1:2000 dilution; DAKO, Glostrup Denmark), Ki-67(clone MIB-1; 1:300; DAKO, Glostrup Denmark).

The intensity of IHC staining for all 3 markers were evaluated as negative, moderate, and strong. All IHC staining were reviewed independently by two pathologists. independently. For p53, and Ki-67,<25% staining or staining only in the lower quarter of the squamous epithelium was interpreted as negative ; >25% and <50% staining or staining involving less than half the thickness of the epithelium were considered as expression of low level; and >50% staining or occupying greater than half the thickness of the epithelium was interpreted as expression of high level^[7]. For RSPO2, no staining was regarded as negative; less than 50% of the thickness of squamous epithelium stained was interpreted as expression of low level; and staining present in more than half of squamous epithelium was scored expression of high level. Meanwhile, the staining intensity of RSPO2 was scored as follows: 0, no staining; 1, weak, light yellow; 2, moderate, yellow brown; and 3, strong, brown.

2.3 Statistical analysis

Comparisons of RSPO2, p53 and Ki-67 between 3 groups of foci were performed by the χ2 test or the Fisher exact test. The corresponding sensitivity and specificity were calculated between negative and intermediate/strong staining, as well as negative/intermediate and strong staining, to evaluate the differential diagnosis value of these three markers among normal/reactive hyperplasia and ESIN. Differences were considered significant if P < 0.05.

3.1 Immunohistochemical staining patterns of RSPO2, Ki-67 and p53 in normal squamous epithelium and ESIN

The expression level and staining pattern of these 3 markers were significantly different among normal squamous epithelium, LGESIN, and HGESIN (Table 1). For RSPO2, in normal squamous epithelium,146(91%) of the samples were negative(Fig. 1A,B),12(8%)showed low expression, and 2(4%) showed high expression. In LGESIN, 4(8%) of the samples were negative,32(64%) showed low expression(Fig. 1C,D), and 14(28%) showed high expression. In HGESIN, no case was negative,2(2%) showed low expression, and 94(98%) showed high expression(Fig. 1E-H). As for as the staining intensity of RSPO2, Fourteen were strong stained, either diffuse and moderate staining pattern with band-like strong staining (Fig. 1E,F), or diffuse and strong staining pattern (Fig. 1G,H). no foci were strongly stained In normal squamous epithelium and LGESIN (Table 2).

Table 1

Expression of RSPO2, p53 and Ki-67 in ESIN and normal squamous epithelium
	HGESIN (%),n = 96	LGESIN (%),n = 50	N (%),n = 160	P
RSPO2
Negative	0	4(8)	146(91)	< 0.001
Low	2(2)	32(64)	12(8)
High	94(98)	14(28)	2(1)
p53
Negative	28(29)	22(44)	154(96)	< 0.001
Low	8(8)	26(52)	6(4)
High	60(63)	2(4)	0
Ki-67
Negative	4(4)	16(32)	150(94)	< 0.001
Low	38(40)	32(64)	10(6)
High	54(56)	2(4)	0
Abbreviations: N, normal squamous epithelium.

Table 2

Sensitivity and specificity of different expression level of RSPO2, Ki-67 and p53 in ESIN
	Negative vs low/high expression		Negative/low vs high expression
	Sensitivity(%)	Specificity(%)	Sensitivity(%)	Specificity(%)
N vs ESIN
RSPO2	97.26	91.25	73.97	98.75
p53	65.75	96.25	42.47	100
Ki-67	86.3	93.75	38.36	100
N vs LGESIN
RSPO2	92	91.25	28	98.75
p53	56	96.25	4	100
Ki-67	68	93.75	4	100
LGESIN vs HGESIN
RSPO2	100	8	97.92	72
p53	70.83	44	62.5	96
Ki-67	95.83	32	56.25	96
Abbreviations: N, normal squamous epithelium.

For p53, In normal squamous epithelium, 154(96%)of the samples were negative(Fig. 1I,J), 6(4%)showed low expression, and no case showed high expression. In LGESIN, 22(44%) of the samples were negative,26(52%)showed low expression(Fig. 1K,L), and 2(4%) showed high expression. (Fig. 1M,N).

For Ki-67, In normal squamous epithelium, 150(94%)of the samples were negative(Fig. 1O,P),10(6%)showed low expression, and no case showed high expression. In LGESIN, 16(32%) of the samples were negative,32(64%)showed low expression(Fig. 1Q,R), and 2(4%) showed high expression. In HGESIN, 4(4%) of the samples were negative,38(40%)showed low expression, and 54(56%) showed high expression(Fig. 1S,T).

3.2 Low/high level expression of RSPO2 and Ki-67 are useful in distinguishing normal squamous epithelium from ESIN and LGESIN

We determined the diagnostic value (sensitivity, and specificity) of RSPO2, p53, and Ki-67 in distinguishing ESIN from normal squamous epithelium. RSPO2 and Ki-67 showed high sensitivity for diagnosing ESIN, 97.26% for RSPO2 and 86.3% for Ki-67, which were much more sensitive than p53 (65.75). RSPO2 and Ki-67 also showed high specificity in distinguishing normal squamous epithelium from dysplasia, 98.1% for Ki-67 and 96.2% for ProExC, whereas p53 showed a little higher specificity (96.25).

Next, we evaluated the diagnostic value of RSPO2, p53, and Ki-67 in distinguishing LGESIN from normal squamous epithelium. Low/high level expression of RSPO2 showed sensitivity of 92% in detecting LGESIN. But the sensitivity of Ki-67(68%) was obviously decreased. The specificities of RSPO2, p53, and Ki-67 remains the same contrasting with distinguishing normal squamous epithelium from ESIN (Table 2).

3.3 High level expression of RSPO2 was sensitive in distinguishing HGESIN from LGESIN

High level expression of RSPO2 was found in 94(98%) HGESIN, 14(28%) LGESIN and 2(1%) normal squamous epithelium. The sensitivity of high level expression of RSPO2 in distinguishing HGESIN from LGESIN was as high as 97.92%, but specificity was only 72%, much lower than those of p53 and Ki-67(96% for both marker) (Table 2).

3.4 Strong staining of RSPO2 was specific in Differentiating HGESIN from LGESIN

Strong staining of RSPO2 was found in 28(29%) HGESIN, but not in those of LGESIN and normal squamous epithelium. The specificity of Strong staining of RSPO2 in distinguishing HGESIN from LGESIN was as high as 100%, but sensitivity was as low as 29.17%(Table 3).

Table 3

Sensitivity and specificity of different staining pattern of RSPO2 in ESIN
RSPO2	HGESIN (%),n = 96	LGESIN (%),n = 50	N (%),n = 160	Sensitivity (%)	Specificity (%)
no staining	0	4(8)	146(100)	29.17	100
weak	14(15)	8(16)	0
moderate	54(56)	38(76)	0
strong	28(29)	0	0
Abbreviations: N, normal squamous epithelium.

3.5 High level expression of Ki-67 and p53 showed high specificity but lower sensitivity in distinguishing HGESIN from LGESIN

High level expression of Ki-67 and p53 was found in 54(56%) and 60(63%)HGESIN, respectively. Two (4%) LGESIN showed high level expression of both Ki-67 and p53, and no normal squamous epithelium was found with high level expression of these two markers. The specificity of high level expression of Ki-67 and p53 in distinguishing HGESIN from LGESIN was 96% and 96% respectively, but sensitivity was reduced to 56.25% and 62.5% respectively.

3.6 Correlation between expression level of different markers

Analysis for correlation between RSPO2 and Ki‑67 and p53 indicated that higher Ki-67(96.3%)and p53(96.67%) expression level in HGESIN associated with higher RSPO2 expression level (χ2 = 18.182, χ2 = 15.211, respectively; all P < 0.001) (Fig. 2).

Accurate diagnosis of HGESIN and its differentiation from LGESIN and non-ESIN is essential for the clinical management of patients who underwent biopsy.We studied the expression level of three biomarkers in ESIN. As is well known, Ki-67 and p53 are confirmed as useful markers for squamous lesions by many studies, but the diagnostic value of RSPO2 in squamous lesions was not confirmed. In this study, RSPO2 was found high level expression in 94(98%) HGESIN, 14(28%) LGESIN and 2(1%) normal squamous epithelium. Meanwhile, RSPO2 was strongly positive in in 28(29%) HGESIN, but not in those of LGESIN and normal squamous epithelium. To the best of our knowledge, the current study is the first to link RSPO2 to ESCC and ESIN.

ESIN is considered a precursor lesion of ESCC^[5]. Untreated patients in the high-risk Chinese population with ESIN (based on the 3-tiered system) was followed up, 5% of mild, 27% of moderate, and 65% of severe dysplasia were showed progression to ESCC after 3.5 years; and 24% of mild, 50% of moderate, and 74% of severe dysplasia after 13.5 years of follow-up, respectively^[16].Thus, severe and moderate dysplasia generally requires active treatment, such as endoscopic submucosal dissection or ablation ,while mild dysplasia may be followed up at longer intervals^[6].So, it is important to distinguish LGESIN from HGESIN.

Currently, ESIN is solely diagnosed based on morphological features, unavoidably, here was a certain level of subjectivity in some cases. RSPO2 is particularly useful for distinguishing LGESIN from HGESIN according to our study. Although the positive predictive value of high level expression for RSPO2 is 87%, much lower than that of Ki 67 (96.43%)and p53(96.77%). The positive predictive value of strong staining and negative predictive value no staining for RPSO2 are both 100%.

Multiple molecular markers have been identified in ESCC tumors, but ESIN have been studied much less than ESCC tumors^[6]. Several biomarkers, such as Ki67 and p16, were commonly characterized and found to be useful tools for the diagnosis of squamous dysplasia in the uterine cervix and the oral cavity; however, few such marker is developed for ESIN^[7].Overexpression of p53 protein in squamous dysplasia compared to normal esophagus mucosa.^[6] Other proteins shown to have increased expression in dysplasia include CD44; TGF beta I; PCNA; FADD, CDC25B, fascin, CK14, LAMC2, SPARC; p16, p15,p14, PTCH1^[6]. Decreased expression in dysplasia compared to normal has been observed for TGF beta receptor II; esophagin Fas, caspase 8, CK4, annexin 1^[6]. Overexpression of ProExC showed high sensitivity and specificity for distinguishing normal/reactive hyperplasia from ESIN, and normal/reactive hyperplasia from LGESIN^[7].

The nuclear nonhistone protein Ki-67 is a universally expressed protein in proliferating cells. Ki-67 IHC is helpful in distinguishing between LGESIN and HGESIN. In this study, the positive predictive value of high level expression for Ki-67 is 96.43%, and negative predictive value of negative expression is 80%, which is slightly lower than another study^[7].

The tumor suppressor protein p53 protein is crucial for regulating the G1/S cell cycle. P53-expressing cells are arrested in the G1 phase, thereby initiating apoptosis in response to DNA damage. p53 over expression has been reported in almost 50% LGESIN and 80% HGESIN^[7] comparing with normal esophageal squamous epithelium. In this study, p53 was found expression in 56% LGESIN and 71% HGESIN, similar to some previous studies^{[17, 18]}. p53 showed high specificity and low sensitivity for detecting ESIN comparing to RSPO2 and Ki-67.

Positive significant correlation between high expression level of RSPO2 and that of Ki‑67 and p53 indicates that there is increase in RSPO2 immunostaining with increase in Ki‑67 and p63 immunostaining in HGESIN. Some studies have tried to combine different markers to increase sensitivity or specificity, but these may not be a cost-effective strategy^[7]. In this study, RSPO2 and Ki-67 showed similar detecting power and significant advantage over p53, so we recommend to use either one of them as an adjunct tool for diagnosing difficult cases of HGSEIN.

Molecular characteristics of ESIN have been reported in a large number of studies, but, different designs and analytic approaches, generally small sample sizes, few repeated studies on the same subject, and insufficient objective and quantitative assessments limit the comparability and overall conclusions that can be drawn from the studies^[6]. Our study also has several limitations, due to its retrospective nature, potential biases may exist in the cases selection, the study was performed at a single center, and the study of expression of PRSO2 in ESCC was not performed.

In summary, we found both RSPO2 and Ki-67 show high sensitivity and specificity in distinguishing ESIN from normal squamous esophageal mucosa. High level expression and/or strong staining of RSPO2 is helpful in distinguishing LGESIN from HGESIN. Our study is the first series to investigate RSPO2 in esophageal squamous intraepithelial neoplasia and is also largest IHC series on these lesions.

IHC

Immunohistochemistry

ESIN

Esophageal squamous intraepithelial neoplasia

ESCC

Esophageal squamous cell carcinoma

HGESIN

High grade ESIN

LGESIN

low grade ESIN

RSPO2

R-spondin 2.

Acknowledgements

We would like to thank all patients who participated in the study. We would like to thank the Science and Technology Planning Project of Guangdong Province, the Scheme of Guangzhou Economic and Technological Development District for Leading Talents in Innovation and Entrepreneurship, the Scheme of Guangzhou for Leading Talents in Innovation and Entrepreneurship, and the Scheme of Guangzhou for Leading Team in Innovation.

Authors' contributions

QZP, TFC and JBF designed the study, collected the clinical, histopathological data, analyzed the data, and wrote the manuscript. QZP and JW performed immunohistochemistry study. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the Science and Technology Planning Project of Guangdong Province, China (Grant No. 2017B020226005), Scheme of Guangzhou Economic and Technological Development District for Leading Talents in Innovation and Entrepreneurship (Grant No. 2017-152), Scheme of Guangzhou for Leading Talents in Innovation and Entrepreneurship (Grant No. 2016007), and Scheme of Guangzhou for Leading Team in Innovation (Grant No. 201909010010).

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

This retrospective study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University (IRB No. NFEC-2019-015).

Consent for publication

All the patients involved in this study gave their written informed consent for publication of their case details.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

²Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China.

³Department of Pathology, Xi'an No.1 Hospital, Xi'an, China

⁴AnchorDx Medical Co., Ltd, Unit 502, No. 8, 3rd Luoxuan Road, International Bio-Island, Guangzhou, China.

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No competing interests reported.

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RSPO2 is a novel sensitive and specific biomarker in esophageal squamous intraepithelial neoplasia

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Case selection

2.2 Immunohistochemistry

2.3 Statistical analysis

3. Results

3.1 Immunohistochemical staining patterns of RSPO2, Ki-67 and p53 in normal squamous epithelium and ESIN

3.3 High level expression of RSPO2 was sensitive in distinguishing HGESIN from LGESIN

3.4 Strong staining of RSPO2 was specific in Differentiating HGESIN from LGESIN

3.6 Correlation between expression level of different markers

4. Discussion

Abbreviations

Declarations

References

Additional Declarations

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