Fibroblast Activation Protein Expression in Sarcomas

Objectives Fibroblast activation protein alpha (FAP) is highly expressed by cancer-associated fibroblasts in multiple epithelial cancers. The aim of this study was to characterize FAP expression in sarcomas to explore its potential utility as a diagnostic and therapeutic target and prognostic biomarker in sarcomas. Methods Available tissue samples from patients with bone or soft tissue tumors were identified at the University of California, Los Angeles. FAP expression was evaluated via immunohistochemistry (IHC) in tumor samples (n = 63), adjacent normal tissues (n = 30), and positive controls (n = 2) using semiquantitative systems for intensity (0 = negative; 1 = weak; 2 = moderate; and 3 = strong) and density (none, <25%, 25–75%; >75%) in stromal and tumor/nonstromal cells and using a qualitative overall score (not detected, low, medium, and high). Additionally, RNA sequencing data in publicly available databases were utilized to compare FAP expression in samples (n = 10,626) from various cancer types and evaluate the association between FAP expression and overall survival (OS) in sarcoma (n = 168). Results The majority of tumor samples had FAP IHC intensity scores ≥2 and density scores ≥25% for stromal cells (77.7%) and tumor cells (50.7%). All desmoid fibromatosis, myxofibrosarcoma, solitary fibrous tumor, and undifferentiated pleomorphic sarcoma samples had medium or high FAP overall scores. Sarcomas were among cancer types with the highest mean FAP expression by RNA sequencing. There was no significant difference in OS in patients with sarcoma with low versus high FAP expression. Conclusion The majority of the sarcoma samples showed FAP expression by both stromal and tumor/nonstromal cells. Further investigation of FAP as a potential diagnostic and therapeutic target in sarcomas is warranted.


Introduction
Sarcomas are a heterogenous family of malignancies arising from bone, cartilage, or connective tissues [1]. Tere are approximately 16,000 new cases of sarcoma and 6,500 deaths from sarcoma each year in the United States [2]. Despite multimodal treatment, which may include surgery, radiation, and/or chemotherapy, fve-year overall survival (OS) for patients with sarcomas is approximately 60 percent [3][4][5].
FAP is an important marker of CAFs. CAFs are heterogenous cells which are key components of tumor stroma or the tumor microenvironment [7,35]. Various phenotypic and functional subtypes have been described including myofbroblastic, infammatory, antigen-presenting, and vascular CAFs although there is not currently a consensus classifcation system or nomenclature for CAF subtypes [6,36,37]. As a group, CAFs shape the extracellular matrix (ECM) and contribute to metabolic and immune reprogramming [8,35]. CAFs have been shown to promote cancer cell survival, growth, metastasis, chemoresistance, and immune evasion [35,[38][39][40][41].
Given the diferential expression of FAP, with limited to absent expression in normal tissues and high expression in CAFs, approaches to diagnostically and therapeutically target FAP as a way to indirectly target cancer cells are of great interest. As sarcomas are derived from mesenchymal cells, FAP may also be used as a direct target for cancer cells in sarcomas. However, the literature on FAP expression in sarcomas is limited [25,27,42]. Tus, a more comprehensive understanding of FAP expression in sarcomas is critical to understand its potential utility as an imaging target for diagnosis, staging, treatment response evaluation, and as a potential therapeutic target. Tis includes understanding expression of FAP by CAFs and tumor cells in the sarcoma microenviroment.
Herein, our frst aim was to analyze the expression of FAP via immunohistochemistry (IHC) in tissue samples of patients with bone and soft tissue tumor subtypes, including sarcomas, available at our center. Our second aim was to analyze the expression of FAP by RNA sequencing and its association with OS using publicly available data.

Sample Selection.
We conducted an institutional review board approved (IRB #10-001857) retrospective chart review of pathology reports of patients with a diagnosis of a bone or soft tissue tumor made between January 1, 2007 to July 31, 2020 and pathology sample available at the University of California, Los Angeles. We identifed 73 available tumor samples from 59 patients with a pathologically confrmed bone or soft tissue tumor diagnosis. Samples were collected from various treatment time points including both pre-and postchemotherapy and/or radiation and were collected from both primary and metastatic sites. Additionally, we identifed 37 available adjacent normal tissue samples collected at the same time as the tumor biopsies or resections in 36 patients. All samples were re-reviewed by a pathologist (S.N.) with expertise in sarcoma to select optimal representative tissue blocks from tumor and adjacent normal tissues.

Immunohistochemistry. Archival
formalin-fxed parafn-embedded tissue specimens were obtained from tumor biopsies or resections. Staining for hematoxylin and eosin (H&E) and FAP were performed on the specimens at the UCLA Tissue Pathology Core Laboratory. Polyclonal rabbit anti-FAP alpha antibody from Abcam was utilized (catalog number ab207178). Immunostaining was performed on Leica Bond platform. A positive control (colon cancer tumor sample) was used. All slides were scanned at 20X magnifcation. Samples were reviewed using the Orbit Image Analysis software and Figure 1 was made using the QuPath software [43,44]. Seventeen of 110 samples (15%; n � 10 tumor; n � 7 normal) were excluded due to poor cellularity. Tus, 63 tumor samples from 53 patients and 30 adjacent normal tissue samples from 29 subjects were available for FAP IHC expression analysis. Assessment of IHC staining of each sample was performed by an oncologist (J.N.C.) and a pathologist (A.S.) and the consensus was reached. FAP staining intensity and density were graded semiquantitatively on a scale of 0-3 (0 � negative; 1 � weak; 2 � moderate; 3 � strong) and from 0--100% (none, <25%, 25-75%; >75%), respectively. Additionally, a qualitative overall FAP expression score was assigned to each sample as follows: not detected, low, medium, and high.
For tumor samples, the stromal cells and tumor cells were scored separately. For adjacent normal samples, the stromal and nonstromal cells were scored separately. Te average FAP intensity score was calculated for each of these cell types. Te percentage of tumor and normal samples with positive FAP staining was calculated. FAP positive staining was defned as FAP IHC intensity score of ≥2 and FAP IHC density score ≥25%.  form and the remainder of the data was processed through the toil pipeline in our laboratory. Toil is a computational pipeline through which raw RNA sequencing fles can be processed in a uniform manner in order to minimize batch efects in conducting meta-analyses of RNA sequencing data from diferent experimental runs or datasets [47]. Upper quartile normalized counts were generated from the toil processed data fles. All data were log transformed (log2(x + 1)) and FAP expression was compared across cancer subtypes. OS data for patients with sarcomas was downloaded from Xena Browser. Quality of the OS data from the TCGA has been previously reported [48]. Survival analysis was performed using the survival package in R. OS was compared between subjects with FAP expression in the upper and lower quartiles and p value was calculated based on chi-square testing.

Sample Selection.
A total of 63 tumor samples, 30 samples from normal tissue adjacent to the tumor samples, and 2 positive controls (colon cancer tumor samples) had adequate cellularity for review of H&E slides and quantifcation of FAP staining. A summary of tumor types and sample sources is shown in Table 1.

IHC Analysis.
Representative H&E and FAP stained images are shown in Figure 1, including tumor and normal samples with varying FAP intensity, density, and overall scores. Tis includes a case of ASPS with normal adjacent tissue (panels A and B, respectively), an additional case of ASPS (panel C), a case of DF with normal adjacent tissue (panels D and E, respectively), a case of MFS (panel F), a case of SFT (panel G), a case of SS (panel H), two cases of UPS (panels I and L), a case of osteosarcoma (panel K), and the positive control. Summaries of FAP scores are shown in Figure 2 and Table 2. Te details of the FAP IHC expression results for individual samples are listed in Supplemental File 1.
As shown in Table 2    OS and RNA sequencing data were available for 168 patients with sarcoma (9 SS, 45 leiomyosarcoma, 27 liposarcoma, 13 MFS, 29 UPS, 5 MPSNT, 2 DF, and 38 osteosarcoma). As shown in Figure 3(c), OS was not signifcantly diferent between the patients with sarcoma with low FAP expression (lower quartile Q1, n � 42) and the patients with sarcoma with high FAP expression (upper quartile Q4, n � 42) (p � 0.11). Given the diversity of clinical behavior of sarcoma subtypes, survival analysis was also performed for individual sarcoma subtypes with the largest numbers of patients (leiomyosarcoma, UPS, osteosarcoma), and again was not signifcantly diferent between the patients with FAP expression in the lower quartile compared to the upper quartile.

Discussion
Prior studies have shown that FAP is highly expressed by CAFs with limited to no expression in normal adult tissues [7, 8, 16-18, 27, 28]. Tis, coupled with the cancer supportive role of CAFs, makes FAP a promising diagnostic and therapeutic target. Te majority of the existing literature is focused on FAP expression in epithelial cancers and there is limited data on FAP expression in sarcomas. Te data in sarcomas include studies by Rettig  In the current study, the expression of FAP by IHC and RNA sequencing was shown to be variable across the diferent bone and soft tissue tumor subtypes with the highest expression occurring in DF, MFS, SFT, and UPS samples. DF, MFS, and SFT are classifed as fbroblastic and myofbroblastic tumors [49]. Tus, given the fbroblastic and myofbroblastic proliferations that characterize these tumors, it is not surprising that these tumor types show intense FAP expression. In epithelial cancers, FAP expression is largely limited to stromal cells although tumor cell expression of FAP has been reported in some epithelial cancer types including glioblastoma and uterine squamous cell carcinoma [8,14]. In the current study, the majority of sarcoma samples showed FAP expression by both stromal and tumor cells.
Prior studies, including a meta-analysis which evaluated multiple cancer types including osteosarcoma, have found that high expression of FAP is associated with poor outcomes [22]. However, in the current study we did not fnd a signifcant diference in overall survival when comparing subjects with RNA sequencing FAP expression in the upper and lower quartiles. It must be noted that this survival analysis was performed in a small number of subjects in a limited number of sarcoma subtypes. Tus, while FAP expression was not found to be a prognostic biomarker in our analysis, future analyses may further clarify if it is prognostic in certain sarcoma subtypes.
Advances in the management of sarcomas, including those subtypes with the highest FAP expression levels, is needed. DF does not have metastatic potential and is considered a locally aggressive soft tissue tumor rather than a true sarcoma. Still, it can impact vital structures and result in morbidity and death [50]. MFS is associated with high risk of local relapse [51]. SFT is considered an intermediate malignancy which generally behaves in a benign and indolent manner although a subset of SFTs has the propensity for local recurrence and metastasis [52]. UPS is high-grade aggressive soft tissue sarcoma that lacks a specifc line of diferentiation [53].
Fluorodeoxyglucose (FDG) positron emission tomography (PET) is used frequently in the staging of sarcomas but has variable utility among the diferent sarcoma subtypes [54]. Tus, there is a need for improved imaging approaches

Sarcoma 7
Previous studies have shown promising results of FAPI-PET tracers in sarcoma. [10,13,57]. Specifcally, a recent prospective observational trial including 47 patients with sarcoma imaged with 68-Gallium ( 68 Ga)-FAPI-PET showed a signifcant association between FAPI-PET uptake intensity and FAP histopathologic expression as well as high sensitivity and predictive positive value of FAPI-PET [10]. Additionally, in 45 patients with recurrent soft tissue sarcoma, Gu and colleagues showed that 68 Ga-DOTA-FAPI-04 PET detected more lesions and had improved sensitivity, specifcity, positive predictive value, and negative predictive value compared to 18F-FDG PET.
Further investigation is needed to understand if FAPI PET tracers may provide increased sensitivity for tumor detection compared to other imaging approaches in sarcoma. Currently, there are multiple active clinical trials recruiting patients for FAP-targeted imaging for which patients with sarcoma may be eligible as shown in Table 3.
Given the overall poor prognosis of sarcomas, novel therapeutics are greatly needed. Te cancer supportive functions of the tumor stroma and FAP expression by CAFs have stimulated the investigation of approaches to therapeutically target FAP in various cancer types [7,37,59,60]. Given the high expression of FAP in both stromal and tumor cells in various sarcoma subtypes, further studies to therapeutically target FAP in sarcomas are warranted. Te immunosuppressive efects of FAP and stromal cells further raise interest in immunotherapy approaches [39][40][41]. FAP is a potential target antigen for immunotherapeutic approaches including antibody-drug conjugates, bispecifc Tcell engagers (BiTE), and chimeric antigen receptor (CAR)-T cell therapies [9].
Clinical studies of FAP-targeting therapeutics are also underway. A phase 1 clinical trial investigating sibrotuzumab, a humanized monoclonal antibody directed against FAP, in various cancers known to have FAP positivity had no objective tumor responses but it was tolerable [69]. Additionally, in a phase I clinical trial of F19 FAP-CAR-Tcells in patients with malignant pleural mesothelioma (NCT01722149), no evidence of treatment toxicity was observed and there was persistence of CAR-T cells in the peripheral blood [66,70,71]. Additionally, Kratochwil et al. recently reported disease control in several patients with sarcomas treated with FAP-targeted radioligand therapies [72,73]. Table 4 shows ongoing therapeutic trials targeting FAP including for which patients with sarcoma may be eligible.
Further work to characterize FAP expression in normal tissue is needed to understand the potential for on-target oftumor efects with FAP-targeted interventions. In the current study, FAP staining was seen around blood vessels confrming prior reports [8,14]. FAP expression was also seen in a portion of the normal tumor-adjacent samples examined, though at lower levels than in the tumor samples,  Abbreviation key: FAP � fbroblast activation protein; CAR-T: chimeric antigen receptor (CAR)-T cell therapies.
8 Sarcoma which has been reported previously [14]. It is possible that the normal tumor-adjacent tissue is infuenced by the nearby tumor microenvionment. Terefore, we must be cautious in our interpretation of this data as the normal tissue samples obtained from specimen adjacent to the tumor may not be refective of normal healthy tissue [74].
Limitations of the current study include limited number of sarcoma subtypes and total samples included. Further work is needed including investigation of FAP expression in other sarcoma subtypes and further evaluation of expression in normal tissues.

Conclusion
In this study, the expression of FAP by IHC and RNA sequencing was shown to be variable across the diferent bone and soft tissue tumor subtypes with the highest expression occurring in DF, MFS, SFT, and UPS samples. FAP may serve as a potential diagnostic and therapeutic target in certain sarcomas subtypes.

Data Availability
Te results published here are in part based upon data generated by the Terapeutically Applicable Research to Generate Efective Treatments (TARGET) initiative (https://ocg.cancer. gov/programs/target) (data available at https://portal.gdc. cancer.gov/projects with dbGaP study accession number phs000218), Te Cancer Genome Atlas (TCGA) (https://www. cancer.gov/tcga) processed by the University of California Santa Cruz (data available at http://xena.ucsc.edu/under datahub TCGA Pan-Cancer), and the lab of Dr. Alejandro Sweet-Cordero (Sayles et al. paper; data available at https://egaarchive.org/under Dataset ID EGAS00001003201) [45,46]. Files of scanned IHC slides are available upon request from the corresponding author.

Disclosure
Jacquelyn N. Crane and Danielle S. Graham are co-frst authors. Tis work was presented as a poster at the virtual annual Connective Tissue Oncology Society Meeting in November 2021.

Conflicts of Interest
F.C.E. is on the Scientifc Advisory Board of Certis Oncology. T.G.G. has consulting and equity agreements with Auron Terapeutics, Boundless Bio, Coherus BioSciences, and Trethera Corporation. Te authors declare that they have no conficts of interest. erate Efective Treatments (TARGET) programs and the laboratory of Dr. Alejandro Sweet-Cordero for sharing RNA sequencing data. Te authors appreciate the time and efort made by the UCLA Tissue Pathology Core Laboratory staf in preparing our samples and performing the FAP IHC. Finally, the authors are deeply grateful to the patients, their families, and the opportunity to further our understanding of sarcomas. J.N.C. acknowledges that this work was supported by the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA Training Program and the UCLA Tumor Cell Biology Training Program Postdoctoral Fellowship (USHHS Ruth L. Kirschstein Institutional National Research Service Award #T32 CA009056). J.N.C, T.G.G, and N.C.F also acknowledge support from the Alan B. Slifka Foundation.

Supplementary Materials
Tis fle includes details about each sample including subject identifer, sample name, notes regarding relationship between tumor and adjacent normal samples, tumor diagnosis, FAP tumor/other cell intensity and density scores, FAP stromal cell intensity and density scores, FAP overall score, sample source (Primary tumor, metastasis, adjacent normal), and treatment status. (Supplementary Materials)