Correlation between TACS and FAP with Prognosis of Clear Cell Renal Cell Carcinoma Patient

doi:10.21203/rs.3.rs-1634379/v1

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Correlation between TACS and FAP with Prognosis of Clear Cell Renal Cell Carcinoma Patient

https://doi.org/10.21203/rs.3.rs-1634379/v1

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Background: Despite recent promising findings from immunotherapy and other targeted medicines, individuals with metastatic Clear Cell Renal Cell Carcinoma (mCCRCC) still have a poor prognosis. Biomarkers associated with metastatic status in CCRCC are important for early detection and for the identification of new therapeutic targets. Fibroblast activation protein (FAP) expression is associated with development of early metastases and worse cancer-specific survival. Tumor-associated collagen signature (TACS) is type of collagen that develop during tumor growth and associated with tumor invasion.

Methods: 26 metastatic clear cell renal cell carcinoma patients that underwent nephrectomy were admitted to this study. Data regarding age, sex, Fuhrman’s grade, tumor diameter, staging, FAP expression, and TACS grading were collected. Spearman rho test was used to correlate FAP expression and TACS grading in both primary tumors and metastases, and with patients age and sex.

Results: FAP expression correlated positively with TACS degree (Spearman rho test r = 0.51; p = 0.0001). FAP was positive in 25 (96%) of all intratumor samples and positive in 22(84%) of all stromal sample.

Conclusion: FAP can be used as prognostic factor in metastatic CCRCC, its presence can predict aggressiveness of mCRCC and poorer outcome in patient. Furthermore TACS can be also used for prediction of aggressiveness and metastasis due to the changes are necessary for tumor to invade other organs.

clear cell renal cell carcinoma

fibroblast activation protein

metastasis

tumor-associated collagen signature

Based on histopathological and molecular features, the renal cell tumors represent a group of heterogeneous tumors, with different sets of genetic and epigenetic abnormalities [1]. The renal cell carcinoma (RCC) is one of the most common form of kidney cancer, comprising 65–70% of all adult RCC, also claiming more than 14,000 lives annually in the United States itself [1, 2].

Roughly, 25% of patients have been reported to have locally progressed or metastatic stages at the time of diagnosis, and approximately 33% of organ-confined tumors may acquire metastatic disease [3]. Despite recent promising findings from immunotherapy and other targeted medicines, individuals with metastatic clear cell renal cell carcinoma (mCCRCC) could still have poor prognosis [4]. Therefore, finding a significant method for early detection via biomarkers associated with metastatic stages within the CCRCC may contribute in new approach in identification of new therapeutic targets [4, 5].

Fibroblast activation protein (FAP, seprase) is a serine protease with enzymatic activities due to the presence of post-proline dipeptidyl peptidase and endopeptidase. The FAP is upregulated differently in several types of tumors, while its genetic expression in healthy adult tissues remains scarce [6]. A correlation between RCC recurrence and/or progression with a wide series of tissue biomarkers has been also reported. In a recent analysis, Solano-Itturi and co-workers have investigated the association between the fibroblast activation protein-α (FAP- α) and the development of early metastases, in which the FAP- α expression were found on fibroblast surface of ccRCC, pRCC, and chRCC, however; it was not found in renal oncocytoma tissue. Furthermore, their findings also have implied association between high expressions of FAP and the development of early metastase [7]. In clear CRCC, stromal FAP expressions (found in 23% of patients) were linked to aggressiveness markers and shorter survival [8]. Meanwhile, in metastatic CRCC, stromal FAP expressions were detected in 36% of primary lesion and 44% of metastatic lesions, and these expressions were also associated with several parameters of tumor aggressiveness and worsened survival features [9].

Other changes that occurred during tumor growth is collagen that has distinct characteristics, termed as tumor-associated collagen signature (TACS). TACS can be a marker for tumor in detection of in-situ method. In the beginning, there is an increase amount of collagen in the surrounding tissue (TACS-1), later on this collagen fibers will align parallelly to tumor (TACS-2). Ultimately, in invasive tumor, due to parallel features on the tumor border (TACS-3) [10, 11], the realignment could create collagen void in extracellular matrix during migration of tumor cells. Thus, this collagen realignment has been shown to precede invasion and has caused active traction forces and led to cellular tension. Normally, in absence of this tension tumor cells, invasion is locally suppressed by the local extracellular matrix.

In this study we aim to study the correlation between FAP and TACS with metastasis clear cell renal carcinoma prognosis

Total of 26 patients with mCCRCC is included in this study. The data was obtained from medical records and histopathological data, issued by the pathological anatomy department of tertiary referral hospital. All of the samples were collected from patients that underwent nephrectomy. Follow-up was obtained from clinical histories one year post surgery. All cases were reviewed by pathologists that interpreted the histopathological data using Fuhrman’s nuclear grade for CCRCC. Immunohistochemistry method was performed by using carbonic anhydrase, CK7, and CD 117 to confirm CCRCC in cases with unclear histopathology result.

The FAP expression evaluation was carried out by using tissue microarrays, and tumor tissue at the front of invasion into renal parenchyma was selected in each case. The FAP antibody was evaluated in the stromal fibroblast adjacent to neoplastic nest. Tris-EDTA was used for antigen retrieval, and negative controls were microscopic sample which was not exposed to primary antibody. The analysis for the Tris-EDTA group was conducted with any antigen retrieval in all cases. Negative controls were slides, which were not exposed to the primary antibody, and these were incubated in the PBS and then processed under the same conditions as the test slides. The analysis was performed using Olympus CX 22 instrument.

TACS analysis was performed by using second harmonic generation (SHG) imaging using Olympus FV1000. SHG images were then overlaid and shared among reviewer who were in term of blind-reviewed to the patient outcome. Every reviewer then rated the sample for the presence of TACS rating. The rating was taken from established definition of collagen. The confirmation of collagen fiber visualization of the image and the corresponding image of H&E-stained microarray samples were simultaneously observed.

The evaluated variables were age, sex, Fuhrman’s grade, tumor diameter, staging, FAP expression, and TACS rating.

SPSS® 21.0 software was used for the statistical analysis. Spearman rho test was used to correlate FAP expression and TACS grading in both primary tumors and metastases, and with patients age and sex.

Table 1

Clinical Characteristic the Subject
	TACS 1	TACS 2	TACS 3	P value
Age (mean,SD)	53 ± 8,4	52 ± 15,1	45 ± 17,8	0,563
Gender (male n,%)	4 (50%)	6 (75%)	10 (71%)	0,000

Table 2

FAP and TACS relationship
TACS degree	FAP		P value
TACS degree	Intratumor	Stroma	P value
TACS 1	4	3	0,000
TACS 2	8	7	0,000
TACS 3	13	12	0,000

FAP expression correlated positively with TACS degree (Spearman rho test r = 0.51; p = 0.0001). FAP was positive in 25 (96%) of all intratumor samples and positive in 22(84%) of all stromal samples.

Metastatic of CRCC is observed in a third from total patients with clear status of renal cell carcinoma, in which the possibility of CCRCC relapse even after nephtrectomy. Metastatic disease has conventional prognostic factor that predict its prognosis factor such as tumor size, staging and grading, but its utility is currently decreasing. It is of importance to be able to find other means of early detection for metastatic condition in patient with renal cell carcinoma. Cancer development and progression is associated with its tumour microenvironment and its effect on normal cells [12].

FAP is a marker of activated fibroblasts that is found to be more abundant in tumor with invasive and aggressive phenotype than those in non-invasive and aggressive types. Studies have suggested that with FAP expression is associated with poorer prognosis. Whilst, to detect the effect of malignancy in MCCRC can be seen using TACS grading in which can be assessed by the changes in collage structure; thus, these changes could be used as prediction for invasiveness of MCCRC [13].

As a multifunctional protein, FAP regulates tumor growth and invasiveness in independent status due to its catalytic activity. From one of its effects, homologous enzyme such as the dipeptidyl-peptidase (DPPIV) may be formed, which facilitates ECM degradation and promotes cancer cell invasion. In addition, both FAP and DPPIV can also act as adhesive molecules, which could interact to integrins and influence the intracellular signal. As a result, an increase in DPPIV level would lead to advanced stage of CCRCC, which indicates association of poor survival rates. From these mechanisms, it is suggested that FAP may have complementary roles in renal carcinogenesis [14, 15].

In this study, we try to describe FAP expression prevalence rate both in stroma and intratumor of mCCRC patient. Other studies have shown that FAP presence in mCCRC patient is correlated with high stage, higher grade and necrotic tumors with 10-years survival. Furthermore, it is found that 96% of all intratumor samples and 84% of all stromal sample in metastatic renal cell carcinoma is found to have positive FAP expression, implying to poorer prognosis among majority of the patients in our center. Our data showed majority of cancer mCCRC patients are positive prevalence of expression for FAP. This finding is in line with other study that found immunostaining FAP is 100% found in patient with sarcomatoid mCCR [15].

Collagen characteristics in RCC are not well described due to its pattern that has been linked to tumor behavior and clinical factors in other malignancies. In the invasive phase of malignancy, it is found that collagen fibers are aligned perpendicularly to the tumor boundary. These changes have significant differences in both collagen density and fiber alignment between those in grade 1 and grade 4 RCC. This demonstrates that the ability in reorganizing collagen fibers within random collagen matrix is shown by the tumor in order to facilitate the invasion. This ability in organizing a collagen as “a scaffold” may suggest an important step in tumorigenesis. Although this active process has not yet been investigated, it is significant to determine the greatest collagen alignment within the most aggressive of RCCs. However, the current two collagen features cannot be directly used as diagnostic tools since both features lack the applicable discriminant ability [16, 17].

In our investigation, it is found that intratumoral TACS grading was 15% in TACS 1, 32% in TACS 2, and 50% in TACS 50%. Meanwhile, the stromal TACS grading was 11% in TACS 1, 26% in TACS 2, and 46% in TACS 3.

It is found that, FAP status is correlated with TACS grading (r = 0,51 p = 0.001). Via the FAP, finding both in intratumour and stromal lesion can be used as marker for possible metastasis to the other organs. A high TACS grading indicated collagen perpendicular formation, which will inform the occurrence of metastasis.

FAP can be used as prognostic factor in metastatic CCRCC, and its presence can be used to predict aggressiveness of mCRCC. Furthermore, TACS can be also used for prediction of aggressiveness and metastasis due to the changes, which are also necessary to determine tumor invasion to other organs.

CCRCC: clear cell renal cell carcinoma; DPP IV: dipeptidyl-peptidase IV; FAP: fibroblast activation protein; H&E: hematoxylin and eosin; RCC: renal cell carcinoma; SHG: second harmonic generation; TACS: tumor-associated collagen signature

Ethics approval

This study is based on outpatient clinic registry, and ethical statement has been received from Ethical Committee Faculty of Medicine, Universitas Sumatera Utara with ethical clearance reference number 330/KEPK/USU/2022.

Competing interests

The authors have declared that no competing interests exist.

Funding

This research did not receive any financial support.

Authors' contributions

Warli SM – the concept of article, data collection, text editing. Putrantyo II - data collection and analysis, writing the text of manuscript. Laksmi LI – conducting histology study, text editing. All authors read and approved the final manuscript.

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No competing interests reported.

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Correlation between TACS and FAP with Prognosis of Clear Cell Renal Cell Carcinoma Patient

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