Various Expression and Clinical Signi�cance of Checkpoint Molecules in Advanced Osteosarcoma: Possibilities for Novel Immunotherapy

10 Background: The fact that studies on anti-programmed cell death 1 (PD-1) or its 11 relevant ligand 1 (PD-L1) have yielded such few responses greatly decreases the 12 confidence in immunotherapy with checkpoint inhibitors for advanced osteosarcoma. 13 We examined 35 advanced osteosarcoma specimens, characterized the expression of 14 various checkpoint molecules with immunohistochemistry and analyzed the 15 relationship of the expression of these checkpoint molecules with patients’ clinical 16 courses. 17 Methods: Immunohistochemistry for B7-H3, CD47, PD-L1, TIM3, TGF- β, CXCR 4, 18 CD27, IDO1, KIRs and SDF-1 was performed on 35 resected advanced osteosarcoma 19 specimens. Associations between the marker levels and clinicopathological variables 20 and survival were evaluated. 21 Results: The positive rates of B7-H3, CD47, PD-L1, TIM3, and TGF- β expression in 22 this sample of 35 heavily treated osteosarcomas were 29% (10/35), 15% (5/35), 9% 23 (3/35), 6% (2/35) and 6% (2/35), respectively, and diverse staining intensities were

data analysis was performed using GraphPad Prism V.5.03 (GraphPad Software La 148 Jolla, California, USA) statistical package and R language (V.3.6.1). Interdependence 149 between staining and clinical data was calculated using the χ 2 displayed by 150 cross-tables. Student's t-test was used for the comparison of data between two groups. 151 The Cox proportional hazards regression model and Kaplan-Meier plots were 152 generated by the survival package, and the log-rank test was used to compare survival 153 curves between different groups. Statistical significance was defined as a P < 0.05. 154

Patient clinicopathological characteristics and outcome 156
The clinicopathological characteristics of the patients included in the study are 157 summarized in Table 1. All these patients were in a metastatic or refractory state, and 158 their disease had progressed upon first-line chemotherapy [24,25]. The median age at 159 diagnosis was 14 (interquartile range (IQR), 6, 22) years. The male-to-female ratio 160 was approximately 4:3. Among this population, 30/35 (85.7%) had conventional 161 osteosarcoma, while 29/35 (82.9%) of the primary tumors were located at the 162 extremities. Twenty-five of the 35 specimens (71.4%) were resected from 163 musculoskeletal lesions, 7/35 (20.0%) were resected from pulmonary metastatic 164 lesions, and 3/35 (8.6%) were resected from osteosarcoma lymph node metastases. 165 We usually conducted our first-line chemotherapy following the Peking University 166 People's Hospital-Osteosarcoma (PKUPH-OS 02) regimen (Additional file 3: Fig. S1) 167 [24,25], which included high-dose methotrexate, doxorubicin, cisplatin, and 168 ifosfamide. Our second-line chemotherapy was usually ifosfamide (1.8 g/m 2 /d d1-5) 169 and etoposide (100 mg/m 2 /d d1-5 Q3W), while our third-line systemic therapy was and regorafenib [24,25]. For this group of patients, 14/35 (40%) had been confirmed 172 to be refractory to first-and second-line chemotherapy, while 4/35 (11.4%) had been 173 progressive upon TKI therapy. We tried anti-PD-1 antibodies in combination therapy 174 in some of our patients, but in clinical evaluations, we found confirmed efficacy of 175 this treatment in only 4/35 (11.4%) patients, with progression-free survival (PFS) of 176 more than 1 year, while this treatment was firmly believed to be invalid in 9/35 177 (25.7%) of patients. A total of 35 patients (83.3%) were followed up for an average of 178 34.7 months, which ranged from 14.7 to 110.1 months. At the time of the last 179 follow-up, 4 deaths had occurred among the 35 patients. 180

Checkpoint ligands expression on advanced osteosarcoma 181
First, we examined the expression of immune checkpoint ligands on tumor cells. As 182 shown in Fig. 1, the patterns of PD-L1 staining were predominantly membranous. 183 B7-H3 was also detected to be the most commonly expressed checkpoint and was 184 mainly expressed on the cell membrane of tumor cells. In some studies, PD-L1 was 185 also observed in vascular endothelial cells and tumor-infiltrating immune cells. 186 However, in this study, according to the specifications of the B7-H3 antibody, 187 pathologists agreed to judge the expression as negative if tumor cells and background 188 were all stained positive. As shown in Tables 1 and 2, in this sample  We conducted a statistical analysis of the correlation between staining and baseline 199 clinicopathological characteristics, but we found no significant correlation. However, 200 for this small sample, a tendency for benefiting from anti-PD-1 therapy was observed 201 for patients with positive B7-H3 expression (P = 0.057) ( Table 1). 202 <Fig. 1 near here> 203 <Table 2 near here> 204

Expression of B7 family proteins in tumor-associated immune cells 205
Next, we evaluated checkpoint molecule expression on tumor-associated immune 206 cells. Only TIM3 was found to be 6% (2/35) expressed on the TILs with low staining 207 intensity. No positive expression was found for the checkpoints CD27, IDO1 and 208 SDF-1. Intriguingly, we noticed that a large portion of our samples (more than half) 209 had background staining, which had then been identified as negative by our 210 pathologists. TIM3, a receptor within the subfamily of TIM (T 211 cell-immunoglobulin-mucin domain) proteins, is another T cell-expressed IgSF 212 protein of significant interest. Multiple TIM3 ligands have been described, including 213 phosphatidylserine, galectin 9 (GAL9), high mobility group protein B1 (HMGB1) and 214 carcinoembryonic antigen-related cell adhesion molecule (CEACAM1). Because the 215 blocking characteristics of therapeutic TIM3-specific antibodies have not been 216 completely described, their mechanism of action is poorly understood, and we did not 217 further explore ligand expression. Interestingly, both of these patients who had TIM3 218 expression also showed evidence of the coexpression of PD-L1 at low intensity levels, 219 and one even had moderate-intensity expression of B7-H3. 220 Clinical significance of B7 family protein coexpression in selected osteosarcoma 221 patients 222 We further analyzed the relationship between these high-risk osteosarcoma patients' 223 clinical prognoses and their B7 family ligand/receptor expression. No obvious 224 expression of these molecules and PFS (for chemotherapy resistance) and overall 225 survival (OS) had an inverse association (P all > 0.05) (shown in Table 3). However, 226 in this small subset of the study population, we noticed that females and telangiectatic 227 subtypes of osteosarcoma had a tendency for poorer PFS or OS, respectively, which 228 was not in accordance with the long-term follow-up results of European and American 229 Osteosarcoma Study-1 (EURAMOS-1). One of the 5 patients who showed evidence 230 of the coexpression of more than one checkpoint molecule had high levels of B7-H3, 231 CD47 and TGFβ but was negative for PD-L1 ( Fig. 1a- On the basis of our experience, only sporadic patients could truly benefit from anti-PD-1 therapy, and these patients usually showed evidence of the coexpression of 296 multiple checkpoint molecules. In clinical practice, with so few patients who are 297 responsive to immunotherapy, we usually need to observe patients who are on 298 combination therapy with checkpoint inhibitors, such as chemotherapy or targeted 299 therapy, for longer than a year. In addition, PFS was a more appropriate indicator than 300 the objective response rate for advanced osteosarcoma clinical evaluation because 301 most of our target lesions had calcification and ossification [5]. Although tumor cells 302 sometimes vanished pathologically after treatment, the remains of ossification were 303 still there for clinical evaluation. One of the 5 patients who had evidence of the 304 coexpression of more than one checkpoint molecule had high-intensity staining for 305 B7-H3, CD47 and TGFβ but was negative for PD-L1 (shown in Fig. 1), however, two 306 years ago, he had high expression of PD-L1 on his tumor cells. The case of this 307 patient demonstrates the evolutionary course of secondary drug resistance to 308 anti-PD-1 antibodies. Later, anti-TGFβ, anti-B7-H3 or anti-CD47 therapy might be 309 practicable for this patient. 310 The limitations of our analysis are that our study was performed at a single 311 institution, utilized a retrospective cohort, and had a relatively small sample size, 312 which precluded us from clarifying whether subtypes with a poor prognosis express 313 checkpoint molecules more frequently than others. Second, we did not collect patient 314 paired samples from biopsy samples before treatment or progressive samples after 315 multiple treatments to observe the evolution of the expression of these checkpoint 316 molecules. Third, we did not examine the expression of tumor-associated checkpoint 317 molecules together with their TILs expressing corresponding receptors for 318 confirmation, which could make the results be more convincing. Fourth, TILs were 319 subjectively identified microscopically by two pathologists individually. The pathologists did not use CD3 or CD8 staining to identify those TILs, which could 321 make the interpretation difficult and the results unreliable. Finally, the follow-up time 322 for all these patients was not long enough to observe their OS (4/35 (11.4%) had died 323 from their disease at the time of the last follow-up), which made univariate Cox 324 proportional hazards analysis of OS impossible. 325

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In summary, the present study highlights that only a small subset of progressive 327 osteosarcomas, which had been heavily treated, expressed tumor immune-associated 328 checkpoint molecules. Those osteosarcomas that had ever been responsive to 329 anti-PD-1 therapy usually had evidence of the coexpression of multiple checkpoint 330 molecules, which might also be the reason for secondary drug resistance.

Availability of data and materials 368
We promised to ensure patients' data confidentially. However, patient data will be 369 made available in a disguised manner, including data dictionaries, for approved data 370 sharing requests. Individual data will be shared that underlie the results reported in 371 this article after the deidentification and normalization of information (text, tables, 372 figures, and supplementaries). The statistical analysis plan will also be available upon 373 request. Anonymized data will be available beginning 3 months later and ending 2 374 years after the publication of this article to researchers after methodological review of 375    10-<50% = moderate, and >50% = high) was applied for the evaluation of immune checkpoint ligand expression; If these checkpoint molecules 523 were expressed on the tumor-infiltrating lymphocytes, a semiquantitative scoring system (0-<1% = negative/rare, 1-<10% = low, 10-<50% = 524 moderate, and >50% = high) was applied for the evaluation of immune checkpoint receptor expression on the surface of the CD3-positive 525 tumor-infiltrating lymphocytes (TILs). 526 527  Negative expression of B7-H3 but with background staining in one sample(200×)