The efficacies and biomarker investigations of anti-programmed death-1 (anti-PD-1)-based therapies for metastatic bone and soft tissue sarcoma

Objective: Sarcomas are a group of rare malignancies with various subtypes. Patients with metastatic sarcoma who have failed traditional treatments can possibly achieve better prognoses from using novel therapies, including anti-programmed death-1 (PD-1)-based therapies. Methods: We retrospectively analyzed clinical data of 24 metastatic sarcoma patients from June 15, 2016 to December 30, 2019. These patients mainly received angiogenesis inhibitors combined with anti-PD-1 therapy after they became resistant to traditional treatments. Furthermore, 8 patients underwent panel DNA and whole transcript sequencing. Results: Six patients received 2 cycles of anti-PD-1 therapy and were included in the safety evaluation only group. The median follow-up time was 5.77 months. The median progression-free survival was 7.59 months, the overall response rate was 16.7% and the disease control rate was 55.6%. Based on whole exome and transcript sequencing data, there was no association between TMB, TNB, MSI, HLA-LOH, and PD-L1 expressions and sarcoma types with clinical responses. Immunotherapy efficacy and bioinformatics analyses indicated higher intratumoral heterogeneity (ITH) in progressive disease (PD) patients and lower ITH in partial response (PR) and stable disease patients. A higher percentage of immune cell infiltration, especially monocytes, was observed in PR patients. Active stromal gene expression was increased in PD patients but decreased in PR patients. Enrichment analysis revealed that an increased TGF-β signaling pathway was reversely correlated with anti-PD-1 efficacy, while a decreased inflammatory response signaling pathway was positively correlated with anti-PD-1 efficacy. Conclusions: Our study showed PD-1 inhibitors combined with anti-angiogenesis agents were effective and well-tolerated. ITH, monocyte ratio, stroma subtypes, and the status of immune-associated signaling pathways may be related with anti-PD-1 based therapy.


Introduction
Sarcomas are rare tumors originating from mesenchymal tissue and account for approximately 1% of all adult malignancies, and 15% of all pediatric malignancies 1,2 . Based on their histological characteristics, the World Health Organization (WHO) classifies sarcomas into more than 100 subtypes, of which soft tissue sarcoma is divided into more than 50 subtypes 3 . Over 16,730 patients were diagnosed with bone and soft tissue sarcomas in USA in 2020, and approximately 7,070 patients have died of this disease 2,4 . The National Central Cancer Registry of China has estimated that there were 28,000 newly diagnosed bone sarcoma in China in 2015 and 20,700 deaths from this disease 5 . Approximately 39,900 new soft tissue sarcoma cases occurred nationwide in China in 2014, accounting for 1.05% of the overall cancer incidence. However, the mortality data still remains unknown. Although surgery, chemotherapy, radiotherapy, targeted therapy, and neoadjuvant therapy comprise the traditional treatments for sarcomas, they do not significantly improve the overall survival (OS), especially in patients with advanced stages. Thus, patients with stage IV sarcomas usually have a poor prognosis and cannot benefit from systemic therapy, with a median OS (mOS) time of approximately 12 months, and a 5-year survival of <10% [6][7][8] .
Recently, the use of immune-suppressive checkpoint inhibitors has facilitated better outcomes for patients with advanced sarcomas. Programmed death-1 (PD-1), a transmembrane protein on the surface of T cells, is an important inhibitory receptor and an important immunosuppressive molecule for the maintenance of autoimmune tolerance. As the major ligand of PD-1, programmed death-ligand 1 (PD-L1) is overexpressed in malignant tumor cells and expressed in antigen-presenting cells, lymphocytes, hematopoietic cells, and epithelial cells 9,10 . The interaction of PD-1 with PD-L1 suppresses T cells and blocks T cell attack on tumors. Tumor immunotherapy strategies that block PD-1 and/or PD-L1 have been shown to be effective in various malignancies, such as melanomas, lung cancers, and lymphoma [11][12][13] .
Although anti-PD-1 therapy is effective in other tumor types, clinical trials of anti-PD-1 therapies for sarcomas are still ongoing and are not currently being used in clinics. Groisberg et al. 14 analyzed the medical records of patients with advanced sarcomas referred to MD Anderson Phase I clinic, who received immunotherapeutic [checkpoint inhibitors (anti-PD1, anti-PD-L1, anti CTLA4, etc.) vaccines and cytokine-based therapies]. The results showed a median overall survival (OS) of 13.4 months (a target of 11.2 months was not achieved) and a median progression-free survival (mPFS) of 2.4 months [95% confidence interval (CI: 1.9-3.2 months)]. The best response in 2 patients with alveolar soft partial sarcoma (ASPS) was a partial response (PR) 14 . In SARC028 (NCT02301039), 40 patients (n = 84) with soft tissue sarcoma could be evaluated for responses; the overall response rate (ORR) was 18%, the mPFS was 18 weeks (95% CI: 8-68 weeks), and the mOS was 49 weeks (95% CI: 34-73 weeks). Of these cases, patients with undifferentiated pleomorphic sarcomas (UPSs) [4 of 10 (40%)] and dedifferentiated liposarcoma (LPS) [2 of 10 (20%)] had higher responses to anti-PD-1 therapy 15 . To further confirm the clinical efficacies of pembrolizumab in UPS and LPS patients, the study created 2 expansion cohorts with advanced UPSs and LPSs. The UPS cohort achieved its primary endpoint (ORR: 23%; mPFS: 3 months; and mOS: 12 months) 16 . The efficacy of pembrolizumab in UPS patients therefore deserves further evaluation in a randomized study. However, the efficacy of pembrolizumab was not confirmed in the LPS cohort (ORR: 10%; mPFS: 2 months; and mOS: 13 months) 16 . The Alliance A091401 study was a multicenter, open, non-comparative, randomized phase II clinical study of nivolumab combined with ipilimumab for unselected patients with advanced sarcoma after multiline treatment. The patients in the trial achieved an objective remission of 16%, which was comparable to the efficacy of standard chemotherapy 17 . In the same clinical trial, the objective remission with nivolumab was 5% 17 .
The overall efficacy of anti-PD-1 therapy alone is approximately 20% for most solid tumors. Many studies have been conducted using clinical trials to identify more effective combination therapies based on PD-1 inhibitors, such as combining anti-PD-1 therapies with chemotherapy, radiotherapy, or targeted therapies. Given the role of VEGF in cancer treatment, Wilky et al. 18 evaluated the efficiency of the VEGF receptor tyrosine kinase inhibitor, axitinib, plus the PD-1 inhibitor, pembrolizumab, in patients with sarcomas. The results showed that the ORR was 21.9%, the mPFS was 4.7 months, and the 3-month PFS was 65.6% (95% CI: 46.6-79.3) (n = 33). In addition, the majority of responses occurred in patients with ASPSs. The percentage of patients who achieved a clinical benefit was 72.7% (n = 8; 95% CI: 32.3-92.7) 18 . In a single-arm, open-label phase 2 clinical trial of apatinib in combination with carrilizumab (anti-PD1 therapy, SHR-1210) for osteosarcomas that progressed after advanced chemotherapy, the results showed a 6-month PFS of 50.9% (95% CI: 34.6%, 65.0%) at a median follow-up of 48.3 weeks. The final objective response rate was 20.9% (9/43) 19 . Patients with PD-L1 expressions ≥5% and with lung metastases tended to have a longer PFS than other patients (P = 0.004 and 0.017, respectively).
As studies on immunotherapy have become more extensive and complicated, corresponding clinical trials of immunotherapy-based combination therapies are attracting more attention from researchers. However, due to the low incidences of bone and soft tissue sarcomas in China, as well as the complex subtype classification, clinical research results on the immunotherapies for these disorders have been insufficient. Moreover, although PD-L1 expression, microsatellite instability (MSI), and tumor mutational burden (TMB)/tumor neoantigen burden (TNB) are recognized predictors of solid tumors, they vary greatly in sarcomas, even among different subtypes. Biomarkers for sarcoma immunotherapies have therefore still not been identified.
In this study, we described a clinical study that retrospectively analyzed several patients with distant metastases from bone and soft tissue sarcomas treated with anti-PD-1-based therapy after evaluations of efficacy and safety using conventional therapies. The results of the study showed an ORR of 22.2% (4/18), a disease control rate (DCR) of 72.2% (13/18) , and a progression-free rate (PFR) of 49.8% at 12 weeks. The mPFS was 7.59 months. Our study further reported that PD-1 inhibitor-based therapy was effective in patients with advanced sarcomas. More importantly, we found that intratumoral heterogeneity (ITH), monocyte ratio, stromal subtype, and the status of immune-related signaling pathways may be associated with the efficacy of anti-PD-1-based therapies in sarcoma patients. These results supported our establishment of the NCT04126993 clinical trial in advanced bone and soft tissue sarcomas, which will hopefully provide evidence of using anti-PD-1 based comprehensive therapy for patients with advanced sarcomas.

Patient information and treatment
We retrospectively summarized the demographic and clinical information of 24 metastatic bone and soft tissue sarcoma patients who received anti-PD-1 monotherapy or an angiogenesis inhibitor combined anti-PD-1 therapy at the Tianjin Medical University Cancer Institute & Hospital. The patients were diagnosed from June 2016 to December 2019. Before anti-PD-1 treatment, the 24 patients had received up to 3 previous lines of systemic anticancer therapy, such as radiotherapy and chemotherapy. Chemotherapy mainly included the AI regimen, MAID regimen, and variations based on the ADM regimen. The inclusion and exclusion criteria of patients are in Supplementary materials. This retrospective investigation was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Tianjin Medical University Cancer Institute & Hospital (Approval No. E2019144). All followed patients provided signed informed consents. The trial registration was NCT04126993.
Detailed information of anti-PD-1 based therapies are shown in Table 1. The anti-PD1 inhibitor, pembrolizumab, was given by intravenous infusion over 30 min at a dose of 3 mg/kg every 3 weeks. Another anti-PD-1 inhibitor, camrelizumab, was also given by intravenous infusion at the dose of 3 mg/kg every 3 weeks. A total of 20 patients were given anti-PD-1 therapy combined with apatinib, an angiogenesis inhibitor. Apatinib was administered at a starting dose of 500 mg daily. If intolerance appeared in patients, the dose was reduced to a maximum of 50%, to 375 mg and then to 250 mg, if necessary. Patients who could not tolerate the dose of 250 mg stopped receiving treatment.

Efficacy and safety evaluations
We collected information from 24 patients receiving pretreatments, including the results of physical examinations, clinical blood counts, blood chemistry panels and computed tomography scans of the measurable lesions at baseline. Toxicity was assessed monthly by medical records or by telephone call follow-ups. Measurable lesions were first assessed by computed tomography at 8 weeks, then confirmed at 12 weeks, and further evaluated every 2 months. The patients were followed-up until death, loss to follow-up, or the end of the observation period.
Clinical responses to the treatments were evaluated according to Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) 20 . Evidence of efficacy was agreed upon by two independent radiologists, who were blinded to the treatments. Each patient had at least 1 measurable extracranial lesion, and the responses were evaluated according to RECIST 1.1 21,22 . Some sarcoma patients with non-measurable lesions were also evaluated according to RECIST 1.1 21,22 . Nonmeasurable lesions included small lesions (longest diameter <10 mm or pathological lymph nodes with 10-15 mm short diameters) as well as truly non-measurable lesions. Lesions considered truly non-measurable included leptomeningeal disease, ascites, pleural or pericardial effusion, lymphangitis involvement of the skin or lung, or abdominal mass/ abdominal organomegaly that were identified by physical examination but were not measurable by reproducible imaging techniques [20][21][22] . Patients with these non-measurable lesions were evaluated as having a complete response (CR), progressive disease (PD), or non-CR/non-PD according to RECIST 1.1 21,22 . Non-CR/non-PD was preferred over SD (stable disease) for the patients with no target diseases 21,22 . To simplify and unify the evaluations, we replaced non-CR/ non-PD with SD in some patients with non-measurable lesions.
To accurately assess the biological activities and side effects of treatments, we defined PFS as the primary endpoint, and PFR, ORR, and DCR at 12 weeks as the secondary endpoints. PFS was defined as the time from initiating anti-PD-1 treatment until disease progression according to RECIST 1.1. Disease control was defined as CR, PR, or SD. The ORR was calculated as follows: (CR+PR)/the total number of cases × 100%. The DCR was calculated as (CR+PR+SD)/the total number of cases × 100%.
All the patients were included in the safety and toxicity analyses, using medical records or telephone interviews. Treatment-related adverse events (AEs) were assessed and graded based on the (National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0) 23 .

Immunohistochemistry (IHC) of PD-L1 expression
PD-L1 expression was assessed in selected patients who had available tissues for testing. Some patients (n = 13) received biomarker testing by the Yuce Bio Company (Guangdong, China) ( Figure 1A). This company performed the VENATA PD-L1 SP263 Assay using the Ventana BenchMark Ultra OptiView DAB IHC detection kit (Thermo Fisher Scientific, Waltham, MA, USA) with pathological tissue sections analyzed from formalin-fixed, paraffin-embedded tissue sections. The evaluation results used the index of %TC (the percentage of PD-L1 expressing tumor cells of any intensity) and %IC (the percentage of tumor area occupied by PD-L1 expression tumor-infiltrating immune cells of any intensity).

Bioinformatics analysis of DNA and RNA sequencing data
The data of gene sequencing were obtained with tissue samples from 8 patients. Samples from all 8 patients were  analyzed by whole exome sequencing (WES) and transcriptome data were obtained from 7 patients (Figure 1A, B). The raw reads of WES-seq were processed by SOAPnuke (version 1.5.6, parameters: -l 20 -q 0.1 -n 0.1) to remove ambiguous reads and/or low quality reads. These qualified sequence reads were then aligned to the human reference genome (UCSC hg38) using BWA-mem (BWA, version 0.7.12). Removal of duplicates by SAMBLASTER (Version 0.1.22) was used to reduce biases in downstream analyses. The single nucleotide variants were detected using VarScan, version 2.4. Single-nucleotide variants (SNVs) with an allele read count of less than 20 or with corresponding normal coverage of less than 20 reads were removed. The mutations were then filtered using a customized Perl script to eliminate false positives and annotated by SnpEff, version 4.3. TMB was calculated using non-silent somatic mutations, including coding base substitutions and indels. To analyze human lymphocyte antigen-I typing across patients, we followed the method developed by Yi 24 . All non-silent mutations were translated to 9-11 mer peptides, which were used to predict potential neoantigens, as previously described 25 . Tumor neoantigen burden (TNB) was measured as the number of mutations that were predicted to generate neoantigens per megabase.

Specific information of patients in biomarker exploring
The cancer cell fraction (CCF) of mutations was estimated using PyClone, version 0.13.0 with the tumor purity estimated by All-FIT. The ratios of subclone mutations to all mutations were interpreted as ITH. Microsatellite instable (MSI) scores were analyzed by interrogating 344 available genomic microsatellites using MSIsensor, Version 0.2. Tumor samples with MSI scores >20 were defined as MSI-H.
For raw reads of RNA-seq, preprocessing was conducted as described above. Clean data were then aligned on the hg19 genome using the STAR aligner with default parameters. Aligned reads were then counted using RESM. All differential gene analysis was conducted using the DESeq2 package. Differentially expressed genes (DEGs) were considered for further analysis with a P-value <0.05. The estimation of immune populations was done using the QuanTIseq algorithm.

Statistical analysis
All statistical analyses were conducted using Statistical Package for the Social Sciences (SPSS) software, Version 21.0 as previously described 26 . We observed and summarized the objective response based on the best response while on treatment. We used the Life Table method to estimate overall and progression-free survivals. Using the Kaplan-Meier method, we estimated the single factor effect. The ORR and DCR analyses were based on frequencies.

Maximum change in target lesion size
First, we evaluated the optimal response to anti-PD-1-based therapies. The maximum change in target lesion size was evaluated according to RECIST 1.1. Of these 24 patients, 6 patients received only 2 cycles of anti-PD-1 therapy, so data from only 18 patients were included in this evaluation. As a result, no patient achieved CR, 4 patients (22.2%, 4/18) achieved PR, 10 patients (55.6%, 10/18) achieved SD, and 4 patients (22.2%, 4/18) were PD at the time of best efficacy evaluation. Thus, up to 77.8% (14/18) of the patients responded to anti-PD-1-based therapies during the treatment period (Figure 2A). There was no significant difference in maximum change of tumor size between bone sarcomas (osteosarcomas and chondrosarcomas) and soft tissue sarcomas (Supplementary Figure S1A-B).

Overall response
After reviewing all the clinical data of the 18 sarcoma patients who had enough information for the final efficacy evaluations, 3 patients achieved PR, 7 patients achieved SD, 8 patients had PD, and 4 patients died of PD (Table 3 and Figure 2C). Finally, the ORR was 16.7% (3/18) and the DCR was 55.6% (10/18) ( Table 2). The median PFS was 7.59 months, and the median OS was not reached (Figure 2D, 2E). However, there was no significant difference between bone sarcomas and soft tissue sarcomas (Supplementary Figure S1C-D).
AEs were more complex in patients receiving combination therapy, when compared with those receiving single agent anti-PD-1 therapy. However, the grade was limited to grade 1 or 2. Two patients (OS and chondrosarcomas) discontinued apatinib therapy or had their dose reduced due to side effects, mainly consisting of uncontrolled wound healing. No more significant side effects were observed in patients treated in combination.

The genomic landscape of sarcoma patients
The mutational landscape was described by pretreatment WES data from 8 patients, combining TMB, TNB, MSI, HLA-LOH,  and PD-L1 expressions, sarcoma types, and anti-PD-1 clinical responses (Figures 1B and 3A). The top 3 genes with the highest mutation frequencies included ZNF729 (38%), TP53 (25%), and RB1 (25%), which are known drivers of soft tissue sarcomas (Figure 3A) 27 . As mentioned above and shown in Figure 3, it was difficult to find a significant association of TMB, TNB, MSI, HLA-LOH (loss of heterozygosity in human leukocyte antigens), PD-L1 expression, and sarcoma types with the clinical response of anti-PD-1-based therapy in our cohort.
By analyzing the mutated genes and clinical responses of each patient, 1 patient (KZP) carried a MDM2 amplification, which is a negative indicator associated with immunotherapy, indicating the risk of hyper progression. Overall, these results explained the patients' non-response to anti-PD-1 therapy.
HLA-LOH refers to deletion of HLA heterozygosity. A positive HLA-LOH represents heterozygous deletion or complete loss of function of the HLA gene. Loss of function results in loss of antigen presentation and inability of the immune system to recognize the tumor. HLA-LOH is an indicator of immune escape in monotherapy, but not in immunochemotherapy. In our results, the HLA-LOH phenomenon occurred in patients GJJ and MXW ( Table 5), but they both responded to anti-PD-1 treatment (PR and SD).

PD-L1 expression, TMB/TNB, MSI, and ITH
PD-L1 expression, and TMB and MSI play important roles as predictive biomarkers for immunotherapy 28,29 . We examined PD-L1 expressions in the tissues of 13 patients with subtypes including LMS, SS, MPNST, LPS, UPS, chondrosarcoma, and OS ( Figure 1A; Table 3). The results showed that only 3 patients (LPS, UPS, LMS; 23.07%; 3/13) had positive PD-L1 expressions without a tendency for a sarcoma type (Figure 1B;  Supplementary Figure S3J; Table 3). Even the 3 patients with positive PD-L1 expressions achieved PR or SD efficacy, and the osteosarcoma patients with negative PD-L1 expressions achieved PR. Fisher's exact test showed no significant correlations between PD-L1 expressions and responses to anti-PD-1 therapy in sarcoma patients (Fisher's exact test = 4.708; P = 0.185; Table 3).
Previous studies showed that TMB values of sarcomas were usually low 30 . Consistent with these studies, the TMB was also low in all patients in our cohort, ranging from 1.12-3.45 mutations/MBs. A similar situation was detected regarding TNB (Figures 3B-C). Similarly, MSI was detected by next-generation sequencing (NGS) in patients with microsatellite stable (MSS) tumors. Although PD-L1 expression, TMB, and MSI are recognized predictors of prognosis and immunotherapy efficacy in solid tumors, these biomarkers did not show a better prognostic significance in our cohort. Therefore, whether PD-L1 expression, TMB, or MSI can serve as biomarkers for anti-PD-1 therapy in sarcoma remains to be determined.
ITH refers to the presence of distinct tumor cell populations, which describes the differences in morphologies and expressions of histopathological markers in different subtypes of cancer. It has been reported to be associated with poor clinical prognoses of various tumors, such as non-small cell lung cancer (NSCLC) and melanoma [31][32][33] . It has been reported that patients with low levels of ITH always had improved responses to immunotherapy 34 . In the present, a higher ITH was observed in PD patients. It was also observed that the PR and SD of patients had a relatively lower level of ITH based on the relationship between ITH and the efficacy of immunotherapy ( Figure 3D).

Immune microenvironment
The tumor microenvironment is a complex ecosystem, composed of cancerous and non-cancerous cells, including stromal cells and immune cells 35 . The immune cells constitute the tumor immune microenvironment, and it has been reported that these tumor-associated immune cells may possess tumor-antagonizing or tumor-promoting functions 36 .
Using the immune-related gene set from ImmPort data, we analyzed the expressions of immune-related genes in the samples. Even though 2 PR patients had decreased expressions of immune-related genes, clustering analysis revealed no significant differences between patients with different clinical responses ( Figure 4A). Dividing the patients into a response group (PR patients, n = 2) and a non-response group (SD/ PD patients, n = 5), we quantitated the scores of 10 immune cell types from the pretreatment RNA-seq data ( Figure 4B). The heat map also showed that monocytes were significantly elevated in the response group, while the others showed no clear pattern (Figure 4B-D). The immune microenvironment of PD patients also showed a deficiency of immune cells, especially T cells (Figure 4B-C). The differences in cell abundance between the reactive and non-reactive groups were compared, and monocytes were found to be significantly higher in the reactive group (Figure 4E). Monocytes are known to have Table 5 The testing results of common biomarkers HLA mutation -   Figure 4 Continued phagocytic, antigen-presenting, and cytokine producing functions 37 , so the higher number monocytes might indicate better immunotherapy responses. However, this positive result needs to be validated using more samples. Besides immunocytes, tumor tissues also have a certain percentage of infiltrating stroma cells, which have been reported to affect the immune microenvironment. It had been reported that the "activated" stroma subtype may describe the activated inflammatory stromal microenvironment, which has a shorter overall survival than normal stroma subtypes 38,39 . The 7 patients were clustered on the heat map according to the expression levels of stromal signature genes ( Figure 4F). It was found that PR patients had decreased stromal gene expressions, while PD patients had active stromal gene expressions, thus PR patients had a normal stromal subtype while PD patients had an active stromal subtype. PR patients may therefore exhibit normal stromal subtypes while PD patients exhibit active stromal subtypes, suggesting that in sarcoma patients, an activated stromal subtype may indicate a poor response to immunotherapy.

The gene expressions of important tumor and immune associated signaling pathways
Seven patients were analyzed for differences in gene expressions between efficacy groups at the RNA level before treatment (selected P < 0.05, |Fold change| >2) (Figure 1)  372 genes with increased prominent expressions and 318 with decreased prominent expressions ( Figure 5A). We also performed enrichment analysis of differentially-expressed genes to identify the affected pathways. The genes with increased expressions mainly belonged to cytokine-cytokine receptor interactions, cell adhesion molecules (CAMs), calcium signaling pathways, and proteoglycans in cancer (Figure 5B), while genes with decreased expressions mainly belonged to neuroactive ligand-receptor interactions and calcium signaling pathways ( Figure 5C). We then investigated the relationship between immune-related pathways such as TGF-β, inflammatory responses, TNFA signaling via NFKB, IL6-JAK-STAT3,   (Figure 5D). We found that an increase in the TGF-β signaling pathway was inversely correlated with anti-PD1 efficacy (Figure 5D), while a decrease in the inflammatory response signaling pathway was positively correlated with anti-PD1 efficacy ( Figure 5D). However, most of the key tumor-related signaling pathways such as PI3K-AKT, PTEN, and MAPK did not show differences among patients with different responses (Figure 5E).
Some patients were treated with anti-PD-1 plus apatinib, and some studies reported that the antitumor effects of apatinib might be associated with VEGF, VEGFR2, angiogenesis, apoptosis, autophagy, EMT/MET, and multiple drug resistance (MDR) signaling pathways 40,41 ; therefore, we also measured levels of these keys signaling pathways before treatment ( Table 6; Supplementary Figure S2A-M). However, we did not find a significant pattern of these pathways. In the signal pathways of VEGF, AXL and ITPR3 showed low expressions  in the response groups, while PAK3 and PRKCA showed high expressions in the response groups (Supplementary Figure  S2A). In the signal pathway of VEGFR2, ITPR3 showed low expression in the response groups, and PRKCA showed high expression in the response groups (Supplementary Figure  S2B). We also found that PRKCA showed increasing expressions in the VEGF, VEGFR2, and BioCarta-PKC groups, which belonged to the MDR signaling pathways (Supplementary Figure S2A, S2B, S2H). PRKCA is 1 of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoints, and cell volume control.

Discussion
With the effectiveness of anti-PD-1 therapy in malignant melanomas and lung cancers, more attention has been directed to immunotherapy. Sarcomas are not included in the approved indications of anti-PD-1 agents, but its immunotherapy still needs further study. Our results showed that ORR −12w was 22.2%, the DCR −12w was 72.2%, the PFR −12w was 49.8%, and the median PFS was 7.59 months. These results are consistent with previous reports on the efficacy of PD-1 inhibitors in sarcomas 15,18,42 . Our data suggested PD-1 inhibitors combined with anti-angiogenesis agents were novel and effective treatments for metastatic sarcomas after failure of conventional therapies. More importantly, we found that the ITH, monocyte ratio, stroma subtype, and the status of immune-associated signaling pathways may have correlations with anti-PD-1-based therapies. Although PD-L1 expression (>50%) can serve as an indicator of pembrolizumab in NSCLC 43 , PD-L1 expression level alone cannot be used as an independent criterion for predicting the effectiveness of anti-PD-1 therapies in all tumors. In sarcomas, studies have evaluated the expression levels of PD-L1 in osteosarcomas and soft tissue sarcomas, but patients with high PD-L1 expressions did not achieve better outcomes after receiving immunotherapy [44][45][46] . The degree of PD-1 positivity in tumor-infiltrating lymphocytes, and PD-L1 expressions in tumor specimens from 105 cases of soft tissue --sarcomas correlated with a poor prognosis and aggressive disease 47 . Furthermore, PD-L1 expression of the same sarcoma subtypes was not exactly the same in different trials 48,49 . This may be due to the standardization process of testing, different antibodies used for PD-L1 detection, the number of proprietary companion diagnostics, different samples used for specific sarcoma subtypes, and the lack of clear definitions of a "positive" tumor in terms of PD-L1 staining by immunohistochemistry (IHC) 50 . In prospective clinical trials, anti-PD-1 therapy for bone and soft tissue sarcomas resulted in minimal responses 14,15,51,52 . Furthermore, PD-L1 is a useful predictor of poor prognoses in patients with bone and soft tissue sarcomas, but is insufficient as a predictor of anti-PD-1 therapy efficacy 44,52 . Our results showed that only 3 out of 13 patients (23.07%) had positive PD-L1 expressions without sarcoma type tendencies. PD-L1 expression also had no significant correlation with the response to anti-PD-1 treatments in patients with sarcomas. As a result, more investigations will be necessary to determine whether PD-L1 expression can serve as a reliable biomarker for anti-PD-1 treatment in bone and soft tissue sarcomas. Because the subtypes of sarcomas are numerous and complicated, it is important to determine which subtypes are sensitive to immunotherapy. Although extended cohort studies have shown that UPSs and ASPSs are sensitive subtypes to anti-PD-1 therapies 15 , our results suggested that leiomyosarcoma, osteosarcoma, and chondrosarcoma may also respond to anti-PD-1 therapies. Recent genomic studies have confirmed the different mutation burdens of different tissue types 47 . Many types of sarcomas in adults, as well as osteosarcomas in pediatric patients, have high mutation burdens but lack consistent targetable underlying events. The therapeutic effects of immunosuppressive checkpoint inhibitors on different subtypes of sarcomas differ [53][54][55] , with the differences between subtypes of sarcomas, including pathological alterations such as translocated genes, fusion genes, and mutation burden, suggesting that anti-PD-1 therapy is an option 53 . Studies have shown that most subtypes with a low mutation burden tended to be translocation-associated sarcomas, including Ewing sarcoma, rhabdomyosarcomas, and synovial sarcomas. However, the high mutation burden subtypes include osteosarcomas, leiomyosarcoma, and undifferentiated pleomorphic sarcomas 8 . TMB differences also include the genetic instability of some specific subtypes acquired during metastasis or recurrence 53 . The differences of subtypes in etiology and mutation burden result in differences of effectiveness.
The therapy based on or combined with PD-1 inhibitors, such as radiotherapy, chemotherapy, and anti-angiogenesis, needs additional study. In our data, a PD-L1 positive LMS patient received PR at the same time of radiotherapy. The efficacy was the result of monotherapy against PD-1, or the synergistic effect of radiotherapy. The "abscopal effect" of radiotherapy may increase the burden of mutation and stimulate immunity. The expression of PD-L1 in human sequencetagged sites (STS) and tumor-associated macrophages (TAMs) can increase after preoperative radiotherapy 53 . Radiation therapy can induce increased antigenic expression, release proinflammatory cytokines that recruit immune cells, promote antigen cross-presentation, and induce tumor expression of death receptors 54,55 . Considering the synergistic effects of anti-PD-1 treatment and radiotherapy, the reasons mentioned above should not be excluded. We should therefore consider PD-1 inhibitor-based therapy, especially anti-PD-1 therapy combined with radiotherapy, as a promising and effective strategy. This combination may produce synergistic effects. At the same time, imRecist should be used to evaluate the effect of immunotherapy. Treatments of PD-L1 inhibitors combined with angiogenesis inhibitors have resulted in increased interest in this process.
In a 68-year-old female patient with osteosarcoma, the results of gene sequencing from biopsy tissue could not explain the treatment response at the molecular level. Gene sequencing results showed that the mutation load was 2.3 mutations per megabase (muts/mb), and IHC results showed that PD-L1 was negative. These results suggested that the patient may not have responded to anti-PD-1 therapy. However, the efficacy of anti-PD-1 treatment has been confirmed clinically. We also screened the role of PTEN InDel in the PI3K signal pathway by whole genome sequencing of the patient. The change of PTEN in the PI3K signal transduction pathway can change the tumor microenvironment. It has been reported that the relationship between PTEN deletion and immunotherapy resistance suggested that PTEN gene deletion may be one of the driving mechanisms of tumor resistance to PD-1/PD-L1 inhibition. Therefore, even if the expression of PD-L1 is negative, TMB is low, MSI is stable, and the PTEN InDel is positive in sarcomas, it is difficult to explain why this osteosarcoma patient obtained the PR effect.
We found that the expressions of TMB/TNB, MSI, HLA-LOH, and PD-L1 could not be used as independent predictors of anti-PD-1 therapy. Immunotherapy efficiency and bioinformatics analysis showed that the levels of ITH in PD patients was higher than that in PR and SD patients. Furthermore, PR patients may have a higher percentage of immune cells, especially monocytes. The expressions of active matrix gene decreased in PR patients and increased in PD patients. Based on the enrichment analysis of RNA sequencing data, it was found that TGF-β, an increase of the signal pathway was negatively correlated with the efficacy of anti-PD-1, while a decrease of the inflammatory signal pathway was positively correlated with the efficacy of anti-PD-1. The status of ITH, monocyte ratio, stromal subtypes, and immune related signaling pathways may therefore be correlated with the efficacy of anti-PD-1 treatments for sarcoma, but more samples and analyses are needed to confirm these results. Our ongoing clinical trial of NTC04126993 in the treatment of advanced bone and soft tissue sarcomas in China is therefore of great importance.
Although our study confirmed that PD-1 inhibitors were effective in the treatment of advanced sarcomas, this observational study had shortcomings and deficiencies. First, this was a retrospective analysis of anti-PD-1 treatments. There were not enough cases to be divided into groups according to the use of monotherapy or the combination of PD-1 inhibitors. However, these results provided us with some ideas, such as combination therapy strategies for sarcomas, but more prospective clinical trials are needed to confirm these findings. Second, due to uncontrollable factors, some patients did not review on time, so the evaluations of efficacy and adverse events were insufficient. In addition, due to economic problems, we were unable to obtain gene sequencing results for all patients, and we could not compare and predict treatment differences at the molecular level. Even in drug-resistant patients, we could not compare point mutations or gene sequence changes. In addition, only 8 patients had gene sequencing data, and the others had no pathological tissue because surgery was performed in other hospitals. The results of PD-L1 expression should only be considered when using different PD-L1 antibodies. In addition, the cut-off point for positive outcomes must be considered.

Conclusions
In this retrospective study of the largest cohort of sarcomas in China, we reported that PD-1 inhibitor-based therapies for advanced sarcomas were effective, suggesting that PD-1 inhibitor combined with anti-angiogenic drugs is a new and effective treatment for metastatic sarcomas after the failure of chemotherapy. Most importantly, we showed that although PD-L1 expression, MSI, TMB/ NB, and HLA-LOH were difficult to use as independent biomarkers for anti-PD-1 therapy in metastatic sarcomas, ITH, monocyte ratio, and interstitial subtypes, and the status of immune related signaling pathways may be related to the efficacy of anti-PD-1 therapy in sarcomas. Although there are many questions still to be answered, we look forward to the results of the NCT04126993 clinical trial for the treatment of advanced sarcoma, in order to identify more effective treatment options for patients with sarcoma.

Conflict of interest statement
No potential conflicts of interest are disclosed.