Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma

Pratistha Koirala, Michael E Roth, Jonathan Gill, Sajida Piperdi, Jordan M Chinai, David S Geller, Bang H Hoang, Amy Park, Michael A Fremed, Xingxing Zang, Richard Gorlick, Pratistha Koirala, Michael E Roth, Jonathan Gill, Sajida Piperdi, Jordan M Chinai, David S Geller, Bang H Hoang, Amy Park, Michael A Fremed, Xingxing Zang, Richard Gorlick

Abstract

Osteosarcoma patient survival has remained stagnant for 30 years. Novel therapeutic approaches are needed to improve outcomes. We examined the expression of Programmed Death Ligand 1 (PD-L1) and defined the tumor immune microenvironment to assess the prognostic utility in osteosarcoma. PD-L1 expression in osteosarcoma was examined in two patient cohorts using immunohistochemistry (IHC) (n = 48, n = 59) and expression was validated using quantitative real time PCR (n = 21) and western blotting (n = 9). IHC was used to determine the presence of tumor infiltrating lymphocytes and antigen-presenting cells (APCs) in the tumor. Expression of PD-L1 was correlated with immune cell infiltration and event-free-survival (EFS). The 25% of primary osteosarcoma tumors that express PD-L1 were more likely to contain cells that express PD-1 than PD-L1 negative tumors (91.7% vs 47.2%, p = 0.002). Expression of PD-L1 was significantly associated with the presence of T cells, dendritic cells, and natural killer cells. Although all immune cell types examined were present in osteosarcoma samples, only infiltration by dendritic cells (28.3% vs. 83.9%, p = 0.001) and macrophages (45.5% vs. 84.4%, p = 0.031) were associated with worse five-year-EFS. PD-L1 expression was significantly associated with poorer five-year-EFS (25.0%. vs. 69.4%, p = 0.014). Further studies in osteosarcoma are needed to determine if targeting the PD-L1:PD-1 axis improves survival.

Figures

Figure 1. PD-L1 is expressed in some…
Figure 1. PD-L1 is expressed in some primary osteosarcoma tumors.
(A) PD-L1 mRNA expression in cell lines derived from primary osteosarcoma tumors. The PD-L1 expressing breast cancer cell line MDA-MB-231 was used as the positive control. (B) PD-L1 mRNA expression in osteosarcoma tumor specimens. Dashed lines represent the level of PD-L1 expression in the positive control. (C) Western blot validation confirms that PD-L1 is expressed in primary osteosarcoma tumors (30%) (D) IHC on selected osteosarcoma tumor specimens show areas of heterogeneous PD-L1 expression. Scale bar represents 100 μm. Neg = negative, pos = positive.
Figure 2. Expression of the immune checkpoint,…
Figure 2. Expression of the immune checkpoint, PD-L1, is associated with the presence of tumor infiltrating lymphocytes and antigen presenting cells.
Each individual column represents a unique patient; of the 28 patients included in the study, 37 patients had known clinical outcomes, and 29 patients had both clinical outcome data and as well as demographic data available. Rows represent either PD-L1 expression or tumor infiltration by an immune cell type, as determined by immunohistochemistry. PD-L1 expression was significantly associated with infiltration by PD-1+ immune cells, CD3+ T cells, CD56+ natural killer cells, CD68+ cells, and CD1a+ dendritic cells. Presence of immune cells or positive PD-L1 expression was defined as IHC staining of >1% the tumor volume. * p 

Figure 3. PD-L1 expression is consistent throughout…

Figure 3. PD-L1 expression is consistent throughout the tumor mass.

( A , C )…

Figure 3. PD-L1 expression is consistent throughout the tumor mass.
(A,C) All four slides from the tumor mass of patient A did not demonstrate PD-L1 staining. Few immune cells are present in this tumor map. (B,D) All of the slides from the tumor mass of patient B stained PD-L1 positive with the exception of one section outside the tumor mass (not shown). Numerous immune cells are present throughout all sections of this tumor map. ARM = anterior resection margin, DCS = distal cross section.

Figure 4. PD-L1 expression is prognostic in…

Figure 4. PD-L1 expression is prognostic in osteosarcoma.

( A ) PD-L1 expression in the…

Figure 4. PD-L1 expression is prognostic in osteosarcoma.
(A) PD-L1 expression in the TMA trends with poorer EFS. (B) PD-L1 expression in the whole slide specimens is significantly associated with poorer EFS.

Figure 5. APC infiltration is prognostic in…

Figure 5. APC infiltration is prognostic in osteosarcoma.

Presence of APCs, specifically ( A )…

Figure 5. APC infiltration is prognostic in osteosarcoma.
Presence of APCs, specifically (A) CD68 positive macrophages or (B) CD1a positive dendritic cells are significantly associated with poorer EFS in osteosarcoma. Over 50% of osteosarcoma samples have APCs present.
Figure 3. PD-L1 expression is consistent throughout…
Figure 3. PD-L1 expression is consistent throughout the tumor mass.
(A,C) All four slides from the tumor mass of patient A did not demonstrate PD-L1 staining. Few immune cells are present in this tumor map. (B,D) All of the slides from the tumor mass of patient B stained PD-L1 positive with the exception of one section outside the tumor mass (not shown). Numerous immune cells are present throughout all sections of this tumor map. ARM = anterior resection margin, DCS = distal cross section.
Figure 4. PD-L1 expression is prognostic in…
Figure 4. PD-L1 expression is prognostic in osteosarcoma.
(A) PD-L1 expression in the TMA trends with poorer EFS. (B) PD-L1 expression in the whole slide specimens is significantly associated with poorer EFS.
Figure 5. APC infiltration is prognostic in…
Figure 5. APC infiltration is prognostic in osteosarcoma.
Presence of APCs, specifically (A) CD68 positive macrophages or (B) CD1a positive dendritic cells are significantly associated with poorer EFS in osteosarcoma. Over 50% of osteosarcoma samples have APCs present.

References

    1. Damron T. A., Ward W. G. & Stewart A. Osteosarcoma, chondrosarcoma, and Ewing’s sarcoma: National Cancer Data Base Report. Clin Orthop Relat Res. 459, 40–47, 10.1097/BLO.0b013e318059b8c9 (2007).
    1. Sweetnam R. Osteosarcoma. Br J Hosp Med. 28(112), 116–121 (1982).
    1. Ottaviani G. & Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res. 152, 3–13, 10.1007/978-1-4419-0284-9_1 (2009).
    1. Carrle D. & Bielack S. Osteosarcoma lung metastases detection and principles of multimodal therapy. Cancer Treat Res. 152, 165–184, 10.1007/978-1-4419-0284-9_8 (2009).
    1. Harting M. T. & Blakely M. L. Management of osteosarcoma pulmonary metastases. Semin Pediatr Surg. 15, 25–29, 10.1053/j.sempedsurg.2005.11.005 (2006).
    1. Link M. P. et al.. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity. N Engl J Med 314, 1600–1606, 10.1056/NEJM198606193142502 (1986).
    1. Meyers P. A. et al.. Addition of pamidronate to chemotherapy for the treatment of osteosarcoma. Cancer 117, 1736–1744, 10.1002/cncr.25744 (2011).
    1. Mirabello L., Troisi R. J. & Savage S. A. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer 115, 1531–1543, 10.1002/cncr.24121 (2009).
    1. Bielack S. S. et al.. Second and subsequent recurrences of osteosarcoma: presentation, treatment, and outcomes of 249 consecutive cooperative osteosarcoma study group patients. J Clin Oncol. 27, 557–565, 10.1200/JCO.2008.16.2305 (2009).
    1. Hawkins D. S. & Arndt C. A. Pattern of disease recurrence and prognostic factors in patients with osteosarcoma treated with contemporary chemotherapy. Cancer 98, 2447–2456, 10.1002/cncr.11799 (2003).
    1. Gajewski T. F., Schreiber H. & Fu Y. X. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 14, 1014–1022, 10.1038/ni.2703 (2013).
    1. Finn O. J. Immuno-oncology: understanding the function and dysfunction of the immune system in cancer. Ann Oncol. 23 Suppl 8, viii6-9, 10.1093/annonc/mds256 (2012).
    1. Grivennikov S. I., Greten F. R. & Karin M. Immunity, inflammation, and cancer. Cell 140, 883–899, 10.1016/j.cell.2010.01.025 (2010).
    1. Schreiber R. D., Old L. J. & Smyth M. J. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331, 1565–1570, 10.1126/science.1203486 (2011).
    1. Champiat S., Ferte C., Lebel-Binay S., Eggermont A. & Soria J. C. Exomics and immunogenics: Bridging mutational load and immune checkpoints efficacy. Oncoimmunology 3, e27817, 10.4161/onci.27817 (2014).
    1. Broadhead M. L., Clark J. C., Myers D. E., Dass C. R. & Choong P. F. The molecular pathogenesis of osteosarcoma: a review. Sarcoma 2011, 959248, 10.1155/2011/959248 (2011).
    1. Gorlick R. Current concepts on the molecular biology of osteosarcoma. Cancer Treat Res. 152, 467–478, 10.1007/978-1-4419-0284-9_27 (2009).
    1. Kansara M., Teng M. W., Smyth M. J. & Thomas D. M. Translational biology of osteosarcoma. Nat Rev Cancer 14, 722–735, 10.1038/nrc3838 (2014).
    1. Jones M. J. & Jallepalli P. V. Chromothripsis: chromosomes in crisis. Dev Cell 23, 908–917, 10.1016/j.devcel.2012.10.010 (2012).
    1. Stephens P. J. et al.. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 144, 27–40, 10.1016/j.cell.2010.11.055 (2011).
    1. Chen X. et al.. Recurrent somatic structural variations contribute to tumorigenesis in pediatric osteosarcoma. Cell Rep. 7, 104–112, 10.1016/j.celrep.2014.03.003 (2014).
    1. Lussier D. M. et al.. Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions. J Immunother 38, 96–106, 10.1097/CJI.0000000000000065 (2015).
    1. Shen J. K. et al.. Programmed cell death ligand 1 expression in osteosarcoma. Cancer Immunol Res. 2, 690–698, 10.1158/2326-6066.CIR-13-0224 (2014).
    1. Carreno B. M. & Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol. 20, 29–53, 10.1146/annurev.immunol.20.091101.091806 (2002).
    1. Dong H. & Chen L. B7-H1 pathway and its role in the evasion of tumor immunity. J Mol Med (Berl) 81, 281–287, 10.1007/s00109-003-0430-2 (2003).
    1. Keir M. E., Butte M. J., Freeman G. J. & Sharpe A. H. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 26, 677–704, 10.1146/annurev.immunol.26.021607.090331 (2008).
    1. Ansell S. M. et al.. PD-1 blockade with nivolumab in relapsed or refractory Hodgkin’s lymphoma. N Engl J Med. 372, 311–319, 10.1056/NEJMoa1411087 (2015).
    1. Garon E. B. et al.. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 372, 2018–2028, 10.1056/NEJMoa1501824 (2015).
    1. McDermott D. F. & Atkins M. B. PD-1 as a potential target in cancer therapy. Cancer Med. 2, 662–673, 10.1002/cam4.106 (2013).
    1. Mullard A. New checkpoint inhibitors ride the immunotherapy tsunami. Nat Rev Drug Discov. 12, 489–492, 10.1038/nrd4066 (2013).
    1. Swaika A., Hammond W. A. & Joseph R. W. Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy. Mol Immunol. 67, 4–17, 10.1016/j.molimm.2015.02.009 (2015).
    1. Afanasiev O. K. et al.. Merkel polyomavirus-specific T cells fluctuate with merkel cell carcinoma burden and express therapeutically targetable PD-1 and Tim-3 exhaustion markers. Clin Cancer Res. 19, 5351–5360, 10.1158/1078-0432.CCR-13-0035 (2013).
    1. Chen B. J. et al.. PD-L1 expression is characteristic of a subset of aggressive B-cell lymphomas and virus-associated malignancies. Clin Cancer Res. 19, 3462–3473, 10.1158/1078-0432.CCR-13-0855 (2013).
    1. Herbst R. S. et al.. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515, 563–567, 10.1038/nature14011 (2014).
    1. Wimberly H. et al.. PD-L1 Expression Correlates with Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy in Breast Cancer. Cancer Immunol Res. 3, 326–332, 10.1158/2326-6066.CIR-14-0133 (2015).
    1. Lussier D. M., Johnson J. L., Hingorani P. & Blattman J. N. Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J Immunother Cancer 3, 21, 10.1186/s40425-015-0067-z (2015).
    1. Chen D. S., Irving B. A. & Hodi F. S. Molecular pathways: next-generation immunotherapy–inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res. 18, 6580–6587, 10.1158/1078-0432.CCR-12-1362 (2012).
    1. Zhang Q. W. et al.. Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature. PLoS One 7, e50946, 10.1371/journal.pone.0050946 (2012).
    1. Gwak J. M., Jang M. H., Kim D. I., Seo A. N. & Park S. Y. Prognostic value of tumor-associated macrophages according to histologic locations and hormone receptor status in breast cancer. PLoS One 10, e0125728, 10.1371/journal.pone.0125728 (2015).
    1. Laoui D. et al.. Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions. Int J Dev Biol. 55, 861–867, 10.1387/ijdb.113371dl (2011).
    1. Leek R. D. et al.. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res. 56, 4625–4629 (1996).
    1. Quatromoni J. G. & Eruslanov E. Tumor-associated macrophages: function, phenotype, and link to prognosis in human lung cancer. Am J Transl Res. 4, 376–389 (2012).
    1. Jersmann H. P. Time to abandon dogma: CD14 is expressed by non-myeloid lineage cells. Immunol Cell Biol. 83, 462–467, 10.1111/j.1440-1711.2005.01370.x (2005).
    1. Buddingh E. P. et al.. Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clin Cancer Res. 17, 2110–2119, 10.1158/1078-0432.CCR-10-2047 (2011).
    1. Kleinerman E. S. et al.. Phase II study of liposomal muramyl tripeptide in osteosarcoma: the cytokine cascade and monocyte activation following administration. J Clin Oncol. 10, 1310–1316 (1992).
    1. Pahl J. H. et al.. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-gamma. J Exp Clin Cancer Res. 33, 27, 10.1186/1756-9966-33-27 (2014).
    1. Endo-Munoz L., Evdokiou A. & Saunders N. A. The role of osteoclasts and tumour-associated macrophages in osteosarcoma metastasis. Biochim Biophys Acta 1826, 434–442, 10.1016/j.bbcan.2012.07.003 (2012).
    1. Endo-Munoz L. et al.. Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases. Cancer Res. 70, 7063–7072, 10.1158/0008-5472.CAN-09-4291 (2010).
    1. Avnet S. et al.. Increased osteoclast activity is associated with aggressiveness of osteosarcoma. Int J Oncol. 33, 1231–1238 (2008).
    1. Akiyama T. et al.. Systemic RANK-Fc protein therapy is efficacious against primary osteosarcoma growth in a murine model via activity against osteoclasts. J Pharm Pharmacol. 62, 470–476, 10.1211/jpp/62.04.0009 (2010).
    1. Fang X., Jiang C. & Xia Q. Effectiveness evaluation of dendritic cell immunotherapy for osteosarcoma on survival rate and in vitro immune response. Genet Mol Res. 14, 11763–11770, 10.4238/2015.October.2.10 (2015).
    1. Himoudi N. et al.. Lack of T-cell responses following autologous tumour lysate pulsed dendritic cell vaccination, in patients with relapsed osteosarcoma. Clin Transl Oncol. 14, 271–279, 10.1007/s12094-012-0795-1 (2012).
    1. Kawano M., Nishida H., Nakamoto Y., Tsumura H. & Tsuchiya H. Cryoimmunologic antitumor effects enhanced by dendritic cells in osteosarcoma. Clin Orthop Relat Res. 468, 1373–1383, 10.1007/s11999-010-1302-z (2010).
    1. Kawano M. et al.. Dendritic cells combined with anti-GITR antibody produce antitumor effects in osteosarcoma. Oncol Rep. 34, 1995–2001, 10.3892/or.2015.4161 (2015).
    1. Muraro M. et al.. Interactions between osteosarcoma cell lines and dendritic cells immune function: An in vitro study. Cell Immunol. 253, 71–80, 10.1016/j.cellimm.2008.05.002 (2008).
    1. Edwards J. R. et al.. Lymphatics and bone. Hum Pathol. 39, 49–55, 10.1016/j.humpath.2007.04.022 (2008).
    1. Bacci G. et al.. Neoadjuvant chemotherapy for osteosarcoma of the extremities with metastases at presentation: recent experience at the Rizzoli Institute in 57 patients treated with cisplatin, doxorubicin, and a high dose of methotrexate and ifosfamide. Ann Oncol. 14, 1126–1134 (2003).
    1. Hattori H. & Yamamoto K. Lymph node metastasis of osteosarcoma. J Clin Oncol 30, e345–e349, 10.1200/JCO.2012.42.3384 (2012).
    1. Sowers R. et al.. Impairment of Methotrexate Transport Is Common in Osteosarcoma Tumor Samples. Sarcoma 2011, 834170, 10.1155/2011/834170 (2011).
    1. Abdeen A. et al.. Correlation between clinical outcome and growth factor pathway expression in osteogenic sarcoma. Cancer 115, 5243–5250, 10.1002/cncr.24562 (2009).
    1. Osborne T. S. et al.. Evaluation of eIF4E Expression in an Osteosarcoma Specific Tissue Microarray. Journal of pediatric hematology/oncology 33, 524–528, 10.1097/MPH.0b013e318223d0c1 (2011).
    1. Roth M. et al.. Targeting Glycoprotein NMB With Antibody-Drug Conjugate, Glembatumumab Vedotin, for the Treatment of Osteosarcoma. Pediatr Blood Cancer 63, 32–38, 10.1002/pbc.25688 (2016).
    1. Kubo T. et al.. Platelet-derived growth factor receptor as a prognostic marker and a therapeutic target for imatinib mesylate therapy in osteosarcoma. Cancer 112, 2119–2129, 10.1002/cncr.23437 (2008).

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner