The poor prognosis of patients with advanced bone and soft-tissue sarcoma has highlighted the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. The goal of this study is to screen for CTA expression, HLA expression, and tumor T-cell infiltration in human dedifferentiated liposarcoma (DDLPS) and osteosarcoma (OS) to establish their immune profile and to identify targetable immunogenic antigens for T-cell based immunotherapy.

Human tissue micro-arrays composed of 78 cores of OS and 50 cores of DDLPS were obtained, along with matched control tissues from the same patients. IHC for the cancer testis antigens NY-ESO-1, MAGE-A3, and SSX2, was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. IHC for CD8 and CD3 was also performed, and T-cell tumor infiltration was defined as either brisk, nonbrisk, or absent, as described in melanoma literature. Concurrently, evaluation of 38 human DDLPS specimens and 10 healthy human fat specimens by the Nanostring nCounter platform is underway for identification of novel antigen targets and to establish the immune profile of DDLPS.

Immunohistochemical analysis of CTA expression showed considerable inter- and intra-tumoral heterogeneity. DDLPS showed relatively low expression of all CTAs tested, with only 22% of samples exhibiting MAGE-A3 and one sample each (3.1%) showing expression of SSX2 and NY-ESO-1 in low percentages of tumor cells. By contrast, in osteosarcoma, 74% of samples expressed MAGE-A3 and 68% expressed SSX, both with >80% of positive cases showing moderate to high expression. NY-ESO-1 was expressed in 41% of OS samples, predominantly at low levels. Brisk infiltration of CD8+ T cells was observed in over 70% of both sarcoma types tested. Furthermore, all sarcoma samples tested were positive for HLA expression.

To date, these results show promising expression of CTAs MAGE-A3 and SSX in OS, which may be used as targets in the future development of an immunotherapy for sarcoma. DDLPS shows relatively low expression, highlighting the need for more exploratory study with NanoString. The data generated throughout this project will provide insight into the immune profile of DDLPS.

Periprosthetic joint infection (PJI) remains one of the most devastating complications that can occur following total joint arthroplasty. Failure rate of standard treatment for PJI is estimated to be around 40% at two years post revision surgery. A major clinical challenge contributing to treatment failure and antibiotics tolerance is the biofilm formation on implant surfaces. Lytic bacteriophages (phages) can target biofilm associated bacteria at localized sites of infection by penetrating and disrupting biofilm matrices; furthermore, phage replication within the biofilm leads to high local concentrations resulting in a powerful therapeutic effect. The aim of this study is to test if phage cocktail has better antimicrobial effect than vancomycin or a single agent phage against biofilm forming MRSA clinical strain Staphylococcus aureus (S. aureus).

S. aureus BP043 was utilized in this study. This strain is a PJI clinical isolate, methicillin resistant (MRSA) and biofilm-former. Three lytic phages, namely, 44AHJD, Team1 and P68, known to infect S. aureus, were tested for their efficiency against S. aureus BP043. The ability of the phages to eliminate S. aureus BP043 planktonic or biofilm cultures was tested either as singular phages or as a cocktail of the three phages. Planktonic cells were adjusted to ∼ 1×109 CFU/mL in tryptic soy broth (TSB) and each phage was added alone or as a cocktail at ∼ 1×109 PFU/mL with moi of 1 (a multiplicity of infection). Bacterial growth was assessed by measuring optical densities at 24hr and was compared to the control of S. aureus BP043 with no phage. BP043 biofilms was grown for 24hr on plasma sprayed titanium (Ti-6Al-4V) alloy disc surfaces. Mature biofilms were then treated with one of the three phages or a cocktail of the 3 phages for 24hr at ∼ 1×109 PFU/mL in TSB. Then, biofilms were dislodged, and bacterial survival was assessed by plating on tryptic soy agar plates. Survival in treated biofilms was compared to control biofilm that was exposed only to TSB.

Planktonic cells growth in the presence of phage 44AHJD was reduced significantly (p <0.0001) after 24hr compared to the control. The other two phages did not show a similar pattern when used alone. The reduction in growth was more pronounced when the three phages were combined together (p <0.0001, compared to the control, p=0.011 3, 44AHJD alone versus 3 phages). Exposing BP043 biofilm to the phage cocktail resulted in more than three logs (CFU/mL) reduction in bacterial load residing in the biofilm while no effect was detected when either vancomycin or each phage was used solely.

We have demonstrated that the usage of lytic phage cocktail contributes to better clearance of planktonic cultures of the S. aureus MRSA isolate. More importantly, viable bacteria in the biofilms that were grown on plasma sprayed titanium discs were reduced by more than 37% when a phage cocktail was used compared to using a single phage or vancomycin. This work is aimed at gathering preclinical evidence for using phage as a new therapeutic avenue to treat PJI.

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H₂O₂) and 50% zinc chloride (ZnCI₂) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions.

Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H₂O₂ and 50% ZnCI₂. The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures.

These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone.