PD-1 and PD-L1 expression in bone and soft tissue sarcomas.

PD-1 and its ligands have been shown to play a significant role in evasion of malignant tumour cells from the immune system. Last year, the Unites States Food and Drug Administration (FDA) approved anti-PD-1 inhibitors for treatment of non-small cell lung carcinoma and recently expanded the use of immunotherapy for metastatic urothelial cell carcinoma and Hodgkin lymphoma. However, studies on expression of PD-1 and its ligand in malignant bone and soft tissue sarcoma are sparse. In this study, we evaluated PD-1 and PD-L1 expression on variants of liposarcomas and rhabdomyosarcomas, osteosarcomas and chondrosarcomas. Tissue microarrays (TMAs) for liposarcomas (well differentiated, myxoid/round cell, and pleomorphic), rhabdomyosarcomas (alveolar, embryonal, pleomorphic, and spindle cell), conventional osteosarcomas and chondrosarcomas were stained for PD-1 and PD-L1 antibodies. Adipose tissue, skeletal muscle, bone, osteochondroma and lipoma were used as control and benign counterparts. Western blot was performed to evaluate expression of PD-1 and PD-L1 in four sarcoma cell lines. Osteosarcomas, chondrosarcomas, and all variants of liposarcomas and rhabdomyosarcomas over-expressed PD-1 relative to normal tissue. Expression of PD-1 in rhabdomyosarcomas was associated with higher tumour stage. Only one case of pleomorphic liposarcoma, one case of pleomorphic rhabdomyosarcoma and two cases of alveolar rhabdomyosarcomas were positive for PD-L1. Normal adipose tissue, skeletal muscle, and bone were negative for both PD-1 and PD-L1 and lipomas and osteochondroma weakly expressed PD-1 but not PD-L1. Western blot confirmed the presence of PD-1 protein in all four sarcoma cell lines. Overall, our results showed cytoplasmic expression of PD-1 in the bone and soft tissue sarcomas, while PD-L1 was negative. Whether these data are an indication for effectiveness of immunotherapy in the management of malignant bone and soft tissue sarcomas remains to be elucidated.

Sclerostin expression in skeletal sarcomas.

Sclerostin (SOST) is an extracellular Wnt signaling antagonist which negatively regulates bone mass. Despite this, the expression and function of SOST in skeletal tumors remain poorly described. Here, we first describe the immunohistochemical staining pattern of SOST across benign and malignant skeletal tumors with bone or cartilage matrix (n=68 primary tumors). Next, relative SOST expression was compared to markers of Wnt signaling activity and osteogenic differentiation across human osteosarcoma (OS) cell lines (n=7 cell lines examined). Results showed immunohistochemical detection of SOST in most bone-forming tumors (90.2%; 46/51) and all cartilage-forming tumors (100%; 17/17). Among OSs, variable intensity and distribution of SOST expression were observed, which highly correlated with the presence and degree of neoplastic bone. Patchy SOST expression was observed in cartilage-forming tumors, which did not distinguish between benign and malignant tumors or correlate with regional morphologic characteristics. Finally, SOST expression varied widely between OS cell lines, with more than 97-fold variation. Among OS cell lines, SOST expression positively correlated with the marker of osteogenic differentiation alkaline phosphatase and did not correlate well with markers of Wnt/ß-catenin signaling activity. In summary, SOST is frequently expressed in skeletal bone- and cartilage-forming tumors. The strong spatial correlation with bone formation and the in vitro expression patterns are in line with the known functions of SOST in nonneoplastic bone, as a feedback inhibitor on osteogenic differentiation. With anti-SOST as a potential therapy for osteoporosis in the near future, its basic biologic and phenotypic consequences in skeletal tumors should not be overlooked.

Lactoferrin immuno-expression in human normal and neoplastic bone tissue.

Lactoferrin (Lf) expression was investigated by using a Lf monoclonal antibody in 50 formalin-fixed and paraffin-embedded human bone tumours [10 giant cell tumours (GCTs), 7 osteoid osteomas, 6 ossifying fibromas, 19 enchondromas, 2 chondroblastomas, 2 chondrosarcomas, 2 chondroblastic osteosarcomas, 1 myeloma and 1 adamantinoma] as well as in 8 samples of adult and foetal human normal bone specimens. In addition, the immunohistochemical expression of the estrogen receptor (ER), progesterone receptor (PR) and Ki-67 antigen was analysed on parallel sections from the same specimens. Quantification of Lf immunoreactivity was performed by using an Intensity Distribution (ID) score. Lf immuno-expression with a variable ID score was encountered in 19/50 tumours and specifically in 10/10 GCTs, in 5/7 osteoid osteomas, in 2/2 chondroblastomas as well as in the adamantinoma and in the myeloma. With reference to normal bone samples, Lf was expressed by the osteoblasts only in the foetal bone. No immunoreactivity for ER and PR was encountered in all neoplastic samples, and no correlation was found between Lf and sex steroid hormone receptor (ER and PR) immuno-expression. Even more, no association was evidenced between Lf immuno-reactivity and the growth fraction of the tumours, reflected by the Ki-67 labelling index. Lf expression in the osteoblastic lineage of bone-forming tumours, together with its presence in the osteoblasts of foetal bone, requires further investigations, although it cannot be ruled out that Lf might be involved in the bone formation in humans, similarly to what has been demonstrated in other species.