Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation

Figures & data

Table 1. Number of sarcomas analyzed by assay method

	LPS	LMS	MFS	UPS	OSA^a	Total
Total	n = 25	n = 24	n = 57	n = 33	n = 87	n = 226
Non-metastatic	24	20	50	29	70	193
Metastatic at diagnosis	1	4	7	4	17	33
IHC	25	24	25	25	9	108
Non-metastatic	24	20	22	22	9	97
Metastatic at diagnosis	1	4	3	3	0	11
RT-PCR	NA	NA	57	33	79	169
Non-metastatic			50	29	65	144
Metastatic at diagnosis			7	4	14	25
IHC and RT-PCR	NA	NA	20	22	9	51
Non-metastatic			17	19	9	45
Metastatic at diagnosis			3	3	0	6

^a10 additional recent OSA cases tested for IHC and RT-PCR only.

Table 2. Characteristics of UPS, MFS and OSA patient cohorts presenting with non-metastatic disease

		UPS cohort (n = 29)		MFS cohort (n = 50)		OSA cohort (n = 55)
Clinical parameter		Number	%	Number	%	Number	%
Gender
	Male	14	48.3	26	52.0	37	67.3
	Female	15	51.7	24	48.0	18	32.7
Tumor size
	≤9 cm	10	34.5	22	44.0	23	41.8
	>9 cm	19	65.5	26	52.0	28	50.9
	Missing	0	0.0	2	4.0	4	7.3
Grade
	Grade 1	0	0.0	0	0.0	0	0.0
	Grade 2	1	3.4	9	18.0	9	16.4
	Grade 3	28	96.6	41	82.0	46	83.6
Depth
	Deep	25	86.2	34	68.0	NA	NA
	Superficial	4	13.8	16	32.0	NA	NA
Follow-up status
	ANED	17	58.6	26	52.0	31	56.4
	AWED	1	3.5	0	0.0	2	3.7
	DECEASED	4	13.8	8	16.0	1	1.8
	DOD	7	24.1	16	32.0	21	38.1
Necrosis after preoperative chemotherapy
	≤90%	NA	NA	NA	NA	37	67.3
	>90%	NA	NA	NA	NA	8	14.6
	Missing	NA	NA	NA	NA	10	18.1
Age at diagnosis (years)
	Mean	63.6		68.1		28.3
	SD	12.0		15.9		17.9
	Minimum	31		32		7
	Maximum	83		95		89
Follow-up (months)
	Median	66		58		79
	Range	(12, 179)		(1, 201)		(12, 270)

ANED: alive no evidence of disease; AWED: alive with evidence of disease, DOD: died of disease; NA: not applicable.

Figure 1. IHC of PD-L1; (a) UPS case with high PD-L1, (b) UPS case with low PD-L1, (c) MFS case with high PD-L1, (d) MFS case with low PD-L1, (e) OSA case with high PD-L1 and (f) OSA case with low PD-L1. All immunohistochemical-stained images were taken at the equivalent of x20 magnification

Figure 2. Distribution of IHC scoring in soft-tissue sarcomas by dot plots: (a) PD-L1 tumor, PD-L1 immune, PD-1 immune in 25 UPS, 25 MFS, 25 LPS, 24 LMS; (b) CD3, CD8, CD4, CD20 in 25 UPS, 25 MFS, 25 LPS, 24 LMS; (c) CD68, CD163 in 25 UPS, 25 MFS; (d) PD-L1 tumor, PD-L1 immune in 9 osteosarcomas

Table 3. Spearman correlations for immunohistochemical staining across STS subtypes

	CD3	CD8	CD4	CD20	CD68	CD163	PD-L1 tumor	PD-L1 immune	PD-1 immune
CD3	1	0.81**	0.51**	0.29	0.45*	0.53**	0.43**	0.50**	0.47**
CD8		1	0.51**	0.24	0.36	0.38	0.52**	0.46**	0.55**
CD4			1	0.13	0.23	0.32	0.35*	0.34*	0.37*
CD20				1	0.16	0.23	0.05	0.14	0.32
CD68					1	0.90**	0.23	0.20	0.38
CD163						1	0.27	0.28	0.42
PD-L1 tumor							1	0.66**	0.31
PD-L1 immune								1	0.39**
PD-1 immune									1

**p < .0001; *p < .001.

Figure 3. Expression of PD-L1 relative to STAM 2 in: (a) 33 UPS, (b) 57 MFS, (c) 79 osteosarcomas. RT-qPCR was performed in duplicate and the average of two independent experiments (N = 2) was utilized to calculate standard deviation and relative PD-L1 values

* indicates cases that were metastatic at diagnosis.

Figure 4. Scatterplot of log2 RNAseq versus log2RT-qPCR generated PD-L1 values for (a) osteosarcoma (N = 11, rho = 0.821, p-value = 0.002) and (b) soft-tissue sarcoma (N = 14, rho = 0.937, p-value = 7.7e-07)

Table 4. Association between PD-L1 RNA level and clinical parameters in 29 UPS patients

	PD-L1
	High (≥median) (n = 13)		Low (<median) (n = 16)
Clinical parameter	n	%	n	%	P-value
Tumor size
≤9 cm	3	23.1	7	43.8	0.4335*
>9 cm	10	76.9	9	56.2
Grade
Grade 2	1	7.7	0	0.0	0.4483*
Grade 3	12	92.3	16	100.0
AJCC stage
Stage 2	2	15.4	5	31.2	0.4100*
Stage 3	11	84.6	11	68.8
Gender
Female	9	69.2	6	37.5	0.0890**
Male	4	30.8	10	62.5
Age at diagnosis
Mean (years)	65.5		62.0		0.4562***
SD	9.1		14.2
Minimum	51		31
Maximum	82		83

*Fisher’s exact test; **Chi-square test; *** t-test.

Table 5. Association between PD-L1 RNA level and clinical parameters in 55 OSA patients

PD-L1
	High (≥median) (n = 28)		Low (<median) (n = 27)
Clinical parameter	n	%	n	%	P-value
Tumor size
≤9 cm	13	50.0	10	40.0	0.4731*
>9 cm	13	50.0	15	60.0
Grade
Grade 2	6	21.4	3	11.1	0.4688**
Grade 3	22	78.6	24	88.9
Necrosis after preoperative chemotherapy
≤90%	15	71.4	22	91.7	0.1205**
>90%	6	28.6	2	8.3
Gender
Female	10	35.7	8	29.6	0.6307*
Male	18	64.3	19	70.4
p53 status
Mutation	4	28.6	7	46.7	0.3156*
Wild type	10	71.4	8	53.3
Age at diagnosis
Mean (years)	26.1		30.6		0.3578***
SD	14.5		20.9
Minimum	7		8
Maximum	58		89

*Chi-square test; **Fisher’s exact test; *** t-test.

Table 6. Overall survival (OS) analysis by Cox proportional hazards model^# for UPS patients (n = 29)

	Univariate			Multivariate
	HR	95% CI		HR	95% CI
PD-L1*
Low vs. high	4.47^#	0.93	43.1	6.27^#	1.05	79.1
Gender
Male vs. female	1.68	0.41	7.57	0.63	0.06	5.86
Age at diagnosis	1.02	0.95	1.10	1.03	0.96	1.14
Size
>9 cm vs. ≤9 cm	1.23	0.30	6.83	1.45	0.30	8.82

^#Inference by Firth-type bias correction: Likelihood Ratio Test for PD-L1 (univariate p = 0.062; multivariate p = 0.035).

*Log2PDL1-dichotomized at the median.

PD-L1: programmed death-ligand 1; UPS: undifferentiated pleomorphic sarcoma.

Table 7. Overall survival (OS) analysis by Cox proportional Hazards Model^# for MFS patients (n = 50)

	Univariate			Multivariate
	HR	95% CI		HR	95% CI
PD-L1*
Low vs. high	1.16^#	0.44	3.13	1.01^#	0.36	2.91
Gender
Male vs. female	1.22	0.46	3.22	1.30	0.48	3.56
Age at diagnosis	1.00	0.97	1.04	1.00	0.97	1.04
Size
>9 cm vs. ≤9 cm	1.36	0.52	3.84	1.50	0.51	4.66

^#Inference by Firth-type bias correction: likelihood ratio test for PD-L1 (univariate p = 0.76; multivariate p = 0.99).

*Log2PDL1-dichotomized at the median.

PD-L1: programmed death-ligand 1; UPS: undifferentiated pleomorphic sarcoma.

Figure 5. Kaplan–Meier overall survival (OS) curves for (a) 29 UPS cases; (b) 50 MFS cases based on PD-L1 expression high (green) versus low (red) determined by RT-qPCR

Table 8. Overall survival analysis by Cox proportional Hazards model^# for 45 osteosarcoma patients

	Univariate			Multivariate
	HR	95% CI		HR	95% CI
PD-L1*
Low vs. high	3.10	1.21	9.19	2.71	1.03	8.19
Grade
3 vs. 2	1.90	0.48	17.3	1.60	0.37	14.9
Gender
Male vs. Female	0.93	0.38	2.53	0.94	0.38	2.60
Age at diagnosis	1.01	0.99	1.03	1.00	0.97	1.02
Size
>9 cm vs. ≤9 cm	2.94	1.11	9.59	2.17	0.81	7.09
Necrosis following preoperative chemotherapy ≤90% vs >90%	9.70	1.33	1234.8	6.79	0.87	875.5

^#Inference under Firth-type bias correction.

Likelihood ratio tests for PD-L1 (univariate p = 0.0173; multivariate p = 0.0192).

*Log2PDL1-dichotomized at the median.

Figure 6. Kaplan–Meier overall survival (OS) curves for 55 osteosarcoma cases based on PD-L1 expression high (green) versus low (red) determined by RT-qPCR

Figure 7. Distribution of the number of non-silent SSNVs (orange – missense variants; blue – nonsense variants; purple – splice site variants) identified in the coding regions of the UPS and MFS tumor genomes analyzed by DNA next-generation sequencing. The UPS and MFS tumors with high PD-L1 expression are indicated

Figure 8. Venn diagrams indicating differentially expressed genes comparing PD-L1 high versus PD-L1 low expression in UPS, MFS and OSA