Immunotherapy for Osteosarcoma

 

PBF status*
 
Number

Negative

Low

High

Bone tumors

Osteosarcoma
    
 Primary lesion

83

7

27

49

 Lung metastatic lesion

8

1

2

5

Chondrosarcoma

5

2

0

3

Malignant fibrous histiocytoma

2

1

0

1

Soft tissue tumors

Malignant fibrous histiocytoma

15

2

7

6

Liposarcoma

5

1

0

4

Rhabdomyosarcoma

3

0

0

3

Clear-cell sarcoma

3

0

1

2

Ewing’s sarcoma

2

0

0

2

Synovial sarcoma

2

0

1

1

Leiomyosarcoma

2

0

1

1

Alveolar soft-part sarcoma

2

0

0

2

MPNST

1

0

0

1

Total

133

14

39

80


MPNST malignant peripheral nerve sheath tumor

*Expression of PBF was graded according to the number of tumor cells stained positively with the anti-PBF antibody; negative (<5 % positive cells), low (5–50 % positive cells), or high (>50 % positive cells)



PBF is a transcription factor containing a Zn finger domain. It was first reported to regulate transcriptional activity in the human papillomavirus type 8 genome DNA [6]. However, osteosarcoma is not related to papillomavirus infection. Therefore, we hypothesized that PBF might regulate cell survival or apoptosis in osteosarcoma cells. To characterize the function of PBF, we performed yeast two-hybrid screening using a cDNA library of OS2000 and PBF as the bait to isolate the binding partners of PBF. As a result, we obtained cDNA clone 93 encoding Scythe/BAT3 (currently designated BAG6) as the associated molecule of PBF [7]. Scythe/BAT3 was reported to be an essential factor for cell proliferation [8] and inhibitor of apoptosis [9]. Surprisingly, overexpression of PBF could induce apoptosis in 293EBNA cells and OS2000 cells. However, coexpression of Scythe/BAT3 could inhibit cell death induced by PBF. Moreover, PBF and Scythe/BAT3 colocalized in the nuclei of osteosarcoma cells but in the cytoplasm of normal tissues. These results suggested that colocalization of PBF and BAG6 in nuclei might be important for the survival of osteosarcoma cells. Recently, PBF was reported to be associated with innate immunity [10] and adipogenesis [11]. The function of PBF is variable and other characteristics might be revealed in the future.



3.2.4 Expression Status of HLA Class I in Osteosarcoma


For peptide-based immunotherapy for osteosarcoma, expression of target molecules in tumor cells is required. However, the expression of HLA class I molecules is also important to present antigenic peptides toward T-cell receptors of CTLs recognizing tumor cells. To assess this issue, we analyzed the expression status of HLA class I molecules on formalin-fixed paraffin-embedded sections of primary osteosarcoma tissues by immunohistochemistry using the pan-HLA class I monoclonal antibody EMR8-5 [12]. HLA class I molecules were detected as high grade (positive cells >50 %) in 48 %, low grade (5 ≤ positive cells ≤50 %) in 32 %, and negative (positive cells <5 %) in 20 % of primary lesions of osteosarcoma. Surprisingly, the overall survival and event-free survival of the patients with HLA class I-positive osteosarcoma were significantly better than those with HLA class I-negative osteosarcoma [13]. These findings suggested that osteosarcoma might be an immunogenic tumor surveyed by the human cellular immune system. In addition, peptide-based immunotherapy might be able to elicit the response of CTLs naturally reacting with tumor cells.


3.2.5 Identification of CTL Epitopes Presented by HLA-A24 and HLA-A2


As described above, we identified the antigenic peptide encoded by PBF in the context of HLA-B*55:02. However, this epitope is not adequate to clinically immunize patients with osteosarcoma because the frequency of the allele is too low (less than 2 %) in Japanese. Therefore, we identified the CTL epitopes of PBF in the context of HLA-A*24:02 and HLA-A*02:01. At first, ten candidate peptides each for HLA-A*24:02 and HLA-A*02:01 were selected using the peptide motif prediction system BIMAS. Using in vitro peptide binding assay, we selected peptides PBF A24.2 (AYRPVSRNI) and PBF A2.2 (ALPSFQIPV) as candidates to assess their immunogenicity. Next, we synthesized MHC/peptide tetramers (HLA-A*24:02/PBF A24.2 and HLA-A*02:01/PBF A2.2) and assessed the frequency of tetramer-positive cells in peripheral mononuclear cells of patients with osteosarcoma. We found that the frequencies of tetramer-positive cells ranged from 5 × 10−7 to 7 × 10−6 and from 2 × 10−7 to 5 × 10−6 in HLA-A*24:02-positive and HLA-A*02:01-positive patients, respectively [14]. These frequencies were compatible with that of anti-MAGE-3 peptide CTL in preimmunized HLA-A1 patients with melanoma (<1.3 × 10−6) [15]. Moreover, tetramer-positive cells could recognize allogeneic osteosarcoma cell lines in the context of HLA-A*24:02 or HLA-A*02:01. Therefore, peptides PBF A24.2 and PBF A2.2 were used for studies of clinical vaccination.


3.2.6 Clinical PBF-Derived Peptide Vaccination Study in HLA-A24-Positive and HLA-A2-Positive Patients with Osteosarcoma


Under approval by the IRB, we started a clinical phase I peptide vaccination trial in 2008. To date, five HLA-A24-positive patients and five HLA-A2-positive patients with osteosarcoma have been enrolled in the study. Three had shown stable disease (SD) and the other seven had progressive disease (PD). One HLA-A2-positive patient with metastatic lesions in the bilateral lungs and subcutaneous region of the right lower back vaccinated with peptide PBF A2.2 survived for 31 months without any other therapeutic intervention. The patient received the peptide PBF A2.2 vaccination under three protocols: Protocol 1 (six subcutaneous vaccinations with 1 mg of peptide PBF A2.2 mixed with incomplete Freund’s adjuvant [IFA] at 14-day intervals), Protocol 2 (vaccination with 10 mg of the peptide mixed with IFA), and Protocol 3 (vaccination with 1 mg of the peptide mixed with IFA and subcutaneous injection of interferon-α on the same day and 3 days after the vaccination). Although Protocol 1 and Protocol 2 were successfully completed, Protocol 3 was discontinued after the 1st vaccination because of leukopenia that might have been a side effect of interferon-α. Vaccine peptide-specific immunological responses were observed by ELISpot assay [16] in Protocols 1, 2, and 3 (Fig. 3.1a). Protocol 2 showed the best immunological response. High-dose vaccination of the peptide seemed to be more important than combination with interferon-α. Although clinical responses were evaluated as PD due to the appearance of new metastatic lesions in the lung at the end of Protocol 1, a subcutaneous metastatic lesion showed marginal calcification (Fig. 3.1b, c). Such marginal calcification was occasionally observed after chemotherapy and considered to be a partial response. For Protocol 2 and Protocol 3, clinical responses were evaluated as SD. Considering the good immunological responses and the clinical observation of marginal calcification of the subcutaneous lesion, we investigated a resected specimen of the subcutaneous metastatic lesion after Protocol 1 (Fig. 3.1d). Microscopically viable tumor cells were observed. However, immunohistochemistry reveled CD8+ T-cell infiltration into the metastatic tumor, which was not observed in the primary biopsy specimen before the vaccination therapy. Obviously, a natural nonspecific response could not be denied. Nevertheless, we believe that the tumor-infiltrating lymphocytes were elicited by the peptide vaccination and contributed to killing the tumor for a long time. The clinical phase I trial is still continuing in our institute. After the trial, we are planning a vaccination trial for HLA-A24 patients with sarcoma with high risk for metastasis, including osteosarcoma, synovial sarcoma, and Ewing’s sarcoma in the adjuvant setting using a peptide cocktail. In addition to peptide PBF, an inhibitor of apoptosis protein-derived peptide survivin 2B [16] is also used.

A323285_1_En_3_Fig1_HTML.gif


Fig. 3.1
Immunological and clinical responses against PBF peptide vaccination. (a) ELISpot assay. PBMC obtained from peripheral blood samples of the patient was in vitro stimulated with the peptide PBF A2.2 and used as responders for ELISpot assay. T2 cells pulsed with indicated peptides were used as stimulators. (b, c) Computed tomography of the bilateral lungs (b) and right lower back (c) before and after vaccination in Protocol 1. Metastatic lesions were indicated in red arrows. Marginal calcification appeared after 6th vaccination was indicated in a white arrow. (d) Immunohistochemistry of the resected subcutaneous lesion. Tumor-infiltrating CD8-positive lymphocytes were demonstrated. Original magnification was ×100



3.3 Future Perspectives of Immunotherapy for Osteosarcoma


Recently, monoclonal antibodies against immune checkpoint molecules (ipilimumab for CTLA-4 and nivolumab for PD-1) showed dramatic clinical responses in patients with melanoma [17, 18]. However, the response might be limited to some cancers having the characteristics of natural immunogenicity. In melanoma, many mutated antigens are recognized by tumor-infiltrating lymphocytes [19, 20]. In addition, melanoma antigens could be presented to and efficiently prime specific CTLs by Langerhans cells existing abundantly in the skin around the tumor. Indeed, ipilimumab did not show any clinical benefit for patients with synovial sarcoma [21]. However, we found that chromosomal translocation SYT-SSX-derived peptide vaccination could induce an immune response and provide clinical benefits including long SD (>20 months) [22]. On the other hand, adoptive cell transfer using engineered T lymphocytes expressing TCR directed to cancer-testis antigen NY-ESO-1 showed a clinical response in synovial sarcoma [23]. These findings suggest that synovial sarcoma cells are sensitive to CTL and also support the idea that specific antigenic stimulation is still required for the effectiveness of immune checkpoint antibodies, especially for sarcomas.

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Jun 4, 2017 | Posted by in ORTHOPEDIC | Comments Off on Immunotherapy for Osteosarcoma

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