Cartilage oligomeric matrix protein overexpression is an independent poor prognostic indicator in patients with intrahepatic cholangiocarcinoma

Cartilage oligomeric matrix protein (COMP) interacts with various extracellular matrix proteins in tissues. Elevated COMP levels recently linked to worse overall survival in multiple cancer types. COMP's significance in intrahepatic cholangiocarcinoma (iCCA) remains uncertain. Here we report a retrospective study to explore COMP's impact on iCCA outcomes. We collected 182 patients' iCCA tumor tissues. COMP overexpression was associated with adverse factors like R1 resection (p = 0.008), advanced T stage (p < 0.001), large duct type (p = 0.004), and poorly differentiated histology (p = 0.002). COMP overexpression correlates with poorer DFS (HR, 3.651; p = 0.001), OS (HR, 1.827; p = 0.023), LRFS (HR, 4.077; p < 0.001), and MFS (HR, 3.718; p < 0.001). High COMP expression ties to worse overall survival (p = 0.0001), DSS (p < 0.0001), LRFS (p < 0.0001), and MFS (p < 0.0001). In conclusion, COMP overexpression links to poor prognosis and pathological features in iCCA, indicating its potential as a biomarker.

a variety of musculoskeletal disorders 1 .High COMP levels have also been associated to breast cancer, hepatocellular carcinoma, prostate cancer, and colon cancer.In these cancers, COMP overexpression was associated with increased tumor growth, cancer metastasis, cancer recurrence, and overall shorter survival [2][3][4][5] .Against this backdrop, the prognostic significance of COMP in intrahepatic cholangiocarcinoma (iCCA) remains unexplored.
After hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA) is the second most prevalent primary hepatic malignancy, accounting for around 15% of all primary liver tumors and 3% of all gastrointestinal cancers 6,7 .CCA is a diverse collection of malignant cancers that arise in various parts of the biliary tree.CCAs are divided into three types: intrahepatic (iCCA), perihilar (pCCA), and distal CCA (dCCA), each with its own etiologies, risk factors, prognosis, and clinical and therapies.Together, iCCA and pCCA account for more than 90% of all CCAs in the world 8 .CCA is a rare cancer, but its incidence (0.3-6 per 100,000 people per year) and mortality (1-6 per 100,000 people per year, globally, excluding specific regions with incidence > 6 per 100,000 people, such as South Korea, China, and Thailand) have been rising in recent decades, posing a global health problem [9][10][11] .Over the past few decades, the reported age-standardized incidence for iCCA has been steadily increasing in most locations worldwide, whereas the age-standardized incidence for dCCA has been decreasing 12 .Curative surgery remained the standard treatment for early CCA.Despite the improvement in CCA pathogenesis, diagnosis, and treatments over the last decade, patient prognosis has remained unchanged, with 5-year survival rates of 7-20 percent and tumor recurrence rates were as high as 48-56% after resection remaining unsatisfactory [13][14][15][16][17] .Several clinical markers, including as T stage, lymph node metastases, and histological grade, can be used to identify high-risk patients 16 .However, to establish further treatment plans after curative surgery, genomics-based prognostic biomarkers were warranted.
Given the rising incidence and mortality of iCCA globally, coupled with the challenges in improving patient prognosis, an investigation into COMP's role in iCCA assumes importance.Therefore, the primary aim of this study is to assess COMP expression levels in iCCA patients and determine its potential as a prognostic marker.In summary, this study investigates the expression of COMP in intrahepatic cholangiocarcinoma (iCCA) and its implications for prognostic evaluation.By shedding light on COMP's potential as a prognostic marker, our work seeks to contribute valuable insights into enhancing the management and treatment outcomes of iCCA patients.

Results
Upregulation of COMP gene links to extracellular matrix structure in the CCA transcriptome.For data mining, a published CCA transcriptome dataset (GSE26566) was used, which includes 104 patients who had radical surgery.We found 9 probes that covered 9 transcripts related to structural elements of the extracellular matrix (GO:0,005,201) (Table 1 and Fig. 1).When compared to neighboring liver tissue, COMP gene (ILMN 1,677,636) was elevated by up to 2.3107-fold log ratios (p < 0.0001) in CCA, as shown in Table 1.When compared to the normal intrahepatic bile duct, COMP was also enhanced in CCA, with 2.4308-fold log ratios (p < 0.0001).Thus, COMP was chosen for further analysis.

Correlations between COMP expression and pathological features in iCCA .
The association between COMP expression and various clinicopathological features was assessed using Pearson's chi-square test.As determined using immunohistochemistry, poorly differentiated and higher T stage cholangiocarcinoma had greater COMP immunoreactivity than well differentiated and lower T stage cholangiocarcinoma (Fig. 2).Table 2 summarized the relationships between COMP expression levels and clinicopathological parameters in iCCA cases.COMP overexpression was associated with R1 resection (p = 0.008), advanced T stage (p < 0.001), large duct type (p = 0.004) and poor differentiated histology (p = 0.002).

Prognostic significance of COMP expression. The predictive effects of COMP expression on cancer
metastasis and patient survival in iCCA were assessed using univariate and multivariate analyses.(Table 3).

COMP gene function prediction.
To recognize the potential functions of COMP in IHCC, we downloaded the top two hundred differentially expressed transcripts showing positive connections (Supplementary Table 1) or negative connections (Supplementary Table 2) with COMP from the cholangiocarcinoma dataset (TCGA, n = 51).Later, employing the Gene Ontology (GO) classification system, these transcripts were utilized to prognosticate the functions of COMP.The results revealed that, in respect to biological processes (Fig. 4A), the most significant term correlated with COMP upregulation was positive regulation of transcytosis (GO: 1,904,300, fold enrichment: > 100), and the platelet-activating factor receptor (PTAFR) gene was identified.

Discussion
In contrast to HCCs, mass-forming iCCAs are characterized by a significant desmoplastic and hypovascularized tumor stroma, which is frequently the predominant histological characteristic of the tumor 18 .Several research have studied not only the morphological differences, but also the molecular distinctions between iCCA, pCCA, and dCCA [19][20][21] .Nakamura et al. demonstrated a variation in tumor anatomical location, indicating IDH, EPHA2 and BAP1 mutations and FGFR2 fusions in iCCA, whereas extrahepatic tumors preferentially show PRKACA and PRKACB fusions as well as mutations in ELF3 and ARID1B.Based on these findings, cholangiocarcinoma in different anatomical locations should be treated and examined differently 20 .Furthermore, cholangiocarcinoma's highly desmoplastic nature, substantial support from a rich tumor microenvironment, and significant genetic heterogeneity all contribute to its treatment resistance 22 .COMP is a large pentameric cartilage protein and is found in articular cartilage, ligaments, and tendons.The protein can also be found in the skin, breast tissues, and liver tissues 3,23,24 .Although the function of COMP glycoprotein in a number of connective tissue illnesses has been well documented 25 , its function in carcinogenesis is still unknown.Evidence from recent studies has shown that COMP overexpression is associated with a poor prognosis and metastases in breast, prostate, thyroid, colon, and hepatocellular carcinoma 2,3,5,26-28 .In our previous study, we found COMP overexpression associated with poor survival and tumor invasiveness in urothelial carcinoma 29 .
As the extracellular matrix degrades with continued liver damage and fibrotic scar tissue builds up, the liver undergoes remodeling, which eventually results in fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC).Norman et el.demonstrated serum COMP could be an early marker of fibrosis, and that increased serum COMP levels could reflect the degree of cartilage breakdown during liver destruction and re-modeling, and also associated with HCC development 2 .Liver cirrhosis and viral hepatitis C and B have been recognized as risk factors for cholangiocarcinoma, especially intrahepatic disease [30][31][32][33] .The following associations were found in a meta-analysis 34  Based on this, we think that the COMP may also affect the prognosis of iCAA due to the shared etiology of HCC and iCCA.Moreover, cholangiocarcinoma is usually characterized by a prominent desmoplastic and hypovascularized stroma.There is now increasing evidence to suggest that the desmoplastic reaction, marked by a dramatic accumulation of α-smooth muscle actin positive cancer-associated fibroblasts (α-SMA + CAFs) with increased production of extracellular matrix proteins 18 .Based on these, we use the transcriptome dataset to assess the genes related to extracellular matrix structure constituent (GO:0005201).When compared to neighboring liver tissue, COMP gene was significantly elevated in CCA among these genes.Thus, COMP was chosen as a candidate for further survival analysis.To our knowledge, this is the first study to demonstrate COMP overexpression associated with poor survival in iCCA.
The Notch, WNT, and transforming growth factor (TGF) signaling pathways, among others, are highly active in iCCA.It is known that the Notch pathway has a role in biliary repair, growth, fibrosis, and stem cell niche maintenance.NOTCH3 overexpression was linked to the development and progression of iCCA, promoting cell survival through PI3K-AKT signaling 12,35,36 .Interestingly, Konstantinos et al. demonstrated that COMP regulates the cancer stem cell population through increasing the interaction between Notch3 and Jagged1, leading to increased activation of Notch3 signaling 37 .
In the other way, the epithelial to mesenchymal transition (EMT) is a critical biological step in the migration and invasion of malignant tumor cells 38 .It gives epithelial cancer cells the ability to develop mesenchymal characteristics with invasive capabilities, which promote colonization of metastatic sites 39 .The TGF-dependent pathway, whose signature has been discovered in iCCA stroma, is the prototypical inducer of EMT.In CCA, TGF either directly induces EMT or collaborates with other key EMT inducer pathways like EGFR [40][41][42] .Notably, transcription factors (EMT-TFs) that control the expression of epithelial and mesenchymal genes, including as the SNAIL, ZEB, and TWIST families, orchestrate EMT.Regardless of anatomical location, CCAs express EMT-TFs, which are linked to a poor prognosis 12,43,44 .Interestingly, COMP can stimulate tumor EMT, although the mechanism is unknown 27,45 .COMP glycoprotein has been demonstrated to be co-expressed with many EMT genes, and a clear association between high COMP expression and poor colon cancer survival has also been reported 45,46 .The evidences to support the link of NOTCH3 pathway, EMT pathway between COMP and iCCA are ambiguous initially but gradually apparent.However, the exact mechanism by which COMP acts as a poor prognostic factor in iCCA remains unclear and need further exploration.
Recently, microRNAs (miRs) and extracellular vesicles (EVs) have gained attention as promising noninvasive biomarkers for diagnosis and prognosis in cholangiocarcinoma 47 .MiRs can be secreted extracellularly or encapsulated in EVs, thereby taking part in intercellular communication 48 .Transcytosis, a type of transcellular mechanism for crossing of EVs through the interior of a cell, has also been implicated in various solid tumors 49 .Interestingly, the results of our bioinformatic analysis revealed that, in terms of biological processes, the most significant term correlated with COMP upregulation was positive regulation of transcytosis (Fig. 4A) that contains the PTAFR gene.Activation of the platelet-activating factor (PAF)/PTAFR pathways has also been suggested to cause hepatocellular carcinoma cell migration and invasion 50 .However, whether COMP may promote IHCC progression through transcytosis and PTAFR needs further investigation.Moreover, in terms of molecular functions, the most remarkable term correlated with COMP upregulation was platelet-derived growth factor binding (Fig. 4B) that includes the PDGFB, PDGFRB, COL1A1, COL1A2, COL3A1, and COL5A1 genes.The critical role of the PDGFB/PDGFRB axis in angiogenesis has been demonstrated in tumors 51 .PDGFD can also bind PDGFRB and activate cancer-associated fibroblasts (CAFs) that play crucial roles in modulating cholangiocarcinoma development 52 .The COL1A1, COL1A2, COL3A1, and COL5A1 genes are also identified as CAF signature in cholangiocarcinoma 53,54 .Accordingly, the associations among the expression levels of COMP, platelet-derived growth factors, and CAF-derived collagen in IHCC development are quite interesting and deserve further examination.
Our research has certain limitations.Firstly, it is a retrospective study conducted at a single institution and lack of experimental validation.Secondly, the exact molecular mechanism underlying disease progression and adverse outcomes in COMP-overexpressing IHCC remains unclear.Thirdly, there is currently no standardized immunostaining and scoring scheme for assessing COMP expression.Due to the lack of agreed staining standards, it is difficult to reach a consensus in this type of research.Fourthly, The GEO transcriptome dataset we use is from cholangiocarcinoma patients rather than iCCA patients, which may also cause some limitations in this study.Lastly, to validate our findings, prospective multicenter studies are required.
In conclusion, COMP overexpression was associated with worsening clinical-pathological characteristics.COMP is also related with poorer survival in iCCA, supporting its function as a biomarker for iCCA prognosis.This is the first study to our knowledge that clarifies the role of COMP in iCCA.To completely comprehend the mechanism and apply these findings to clinical practice, additional research is required.

Data mining of the gene expression omnibus (GEO) dataset. A transcriptome dataset (GSE26566)
containing 104 iCCA patients who received curative surgery was obtained from the NCBI Gene Expression Omnibus (GEO) database.Without pre-selection, all probe sets were used.The raw data was then transferred into the Nexus Expression 3 software, which was used to calculate gene expression levels.Comparative analyses were conducted to detect the significantly differently expressed genes connected to extracellular matrix structural constituents (GO:0,005,201) by comparing CCA part vs. normal surrounding liver and CCA part vs. normal intrahepatic duct.After analysis, differentially regulated genes in CCA part vs. normal surrounding liver and CCA part vs. normal intrahepatic duct were discovered (P < 0.0001 and log ratio > 2) (Fig. 1 and Table 1).

Study population.
Between 1990 and 2010, 182 patients with iCCA who received curative surgery were enrolled at Chi Mei Medical Center.The presence of lymph node involvement or distant metastasis was ruled out to guarantee curability.Only individuals with T1-3N0M0 disease were included.Two pathologists investigated tumor samples to rule out the possibility of other malignancies arising from the biliary system.In this study, we used anonymous patient sample information from biobank as approved by IRB.As a rule, inform consent has been sign by every patient before their sample/information collected into biobank.The study had been approved by the Institutional Review Board (IRB) of Chi Mei Medical Center with the approval number of 09,912,003.All research was performed in accordance with relevant guidelines/regulations.We gathered patients' retrospective

Figure 1 .
Figure 1.A published transcriptome dataset of intrahepatic cholangiocarcinoma (GSE26566) from GEO database showed gene expression associated with extracellular matrix structure constituent (GO:0005201).COMP is the most upregulated genes in cholangiocarcinoma compared to surrounding liver and normal biliary epithelium.

Figure 3 .
Figure 3. Kaplan-Meier analysis showed COMP overexpression associated with (A) worse overall survival (B) worse disease-specific survival (C) worse local recurrence free survival (D) worse metastasis free survival in iCCA patient.

Figure 4 .
Figure 4.The outstanding GO terms enriched in COMP upregulation.Employing the GO classification system based on (A) biological processes, (B) molecular functions, and (C) cellular components, the top 20 GO terms presenting positive correlations with COMP were displayed and ordered by fold enrichment.

Table 1 .
Summary of the alterations of gene associated with extracellular matrix structural constituent (GO:0005201) in cholangiocarcinoma (GSE26566).

Table 3 .
Univariate log-rank and multivariate analyses for overall and disease-specific survivals in primary localized iCCA.*Statistically significant.

Table 4 .
Univariate log-rank and multivariate analyses for local recurrence-free and metastasis-free survivals in primary localized iCCA.*Statistically significant.