Immunophenotype-Genotype Correlations in Clear Cell Sarcoma of Kidney-An Evaluation of Diagnostic Ancillary Studies.

INTRODUCTION: The purpose of this study was to establish a reliable panel of antibodies for immunohistochemical corroboration of a diagnosis of clear cell sarcoma of kidney (CCSK), taking into consideration the various genotypic subsets of CCSK. METHODS: We conducted full genotypic analysis for evidence of YWHAE-NUTM2, BCOR internal tandem duplication (ITD), and BCOR-CCNB3 in 68 archival cases of CCSK and then immunostained all cases for CCND1, TLE1, and BCOR along with 63 control samples representing tumor types that may enter into the differential diagnosis of CCSK, including 7 congenital mesoblastic nephromas, 2 desmoplastic small round cell tumors, 13 malignant rhabdoid tumors, 9 Ewing sarcomas/primitive neuroectodermal tumor, 5 synovial sarcomas, and 27 Wilms' tumors. RESULTS: Molecular assays showed that 54 CCSKs harbored a BCOR-ITD, 1 case expressed a YWHAE-NUTM2 fusion transcript while none expressed the BCOR-CCNB3 fusion. The remaining 13 CCSKs were designated "triple-negative" based on the molecular findings. CCND1 showed positive immunoreactivity across all subgroups. TLE1 was positive in 94% of cases, including 1 YWHAE-NUTM2 fusion-positive case. Three BCOR-ITD-positive tumors were TLE1-negative. BCOR immunostaining was most variable among subgroups, with triple-negative tumors showing the weakest staining. In all, 10/68 (15%) tumors did not stain for BCOR, of which 4 were triple-negative (4/13 = 31%) and 6 were BCOR-ITD-positive (6/54 = 11%). The single YWHAE-NUTM2-positive tumor showed strong staining for all 3 markers. No single case was negative for all 3 stains; however, 3 cases showed no reactivity for either BCOR or TLE1 of which 1 was triple-negative and 2 BCOR-ITD-positive. CONCLUSION: Having completed the first comprehensive evaluation of immunostaining of 68 fully genotyped CCSK tumors, we show herein that there is a rationale for the use of a small panel of antibodies to assist in the diagnosis of CCSK regardless of genotype, and we demonstrate that in combination CCND1, TLE1, and BCOR are compelling markers in aiding CCSK diagnosis.

Melan-A/MART-1 immunity in a EWS-ATF1 translocated clear cell sarcoma patient treated with sunitinib: a case report.

BACKGROUND: Clear cell sarcoma (CCS), initially named malignant melanoma of soft parts, is an aggressive soft tissue sarcoma (STS) that, due to MITF activation, shares with melanoma the expression of melanocyte differentiation antigens. CCS is poorly sensitive to chemotherapy. Multi-kinase inhibitors have been used as therapeutic agents. In the case we report here, treatment with sunitinib induced a long-lasting clinical response that was associated with an immune activation directed against Melan-A/MART-1 antigen. CASE PRESENTATION: A 28 years old female patient with an advanced molecularly confirmed CCS resistant to conventional chemotherapy was started in January 2012 on sunitinib, 37.5 mg/day, with evidence of radiologic and metabolic response at the primary and metastatic sites of disease. Pathologic response and loss of the Melan-A/MART-1 antigen were evidenced on residual tumor removed in April 2012. Immunological monitoring performed on patient's blood during pharmacological treatment revealed a systemic, Melan-A/MART-1 specific immunity and a low frequency of immunosuppressive cells. Sunitinib was restarted in May 2012, with a new response, and continued for 11 months although with repeatedly interruptions due to toxicity. Disease progression and new responses were documented at each treatment interruption and restart. Sunitinib was definitively interrupted in April 2013 for disease progression. CONCLUSION: The analysis of this case proves that antigens expressed by CCS, as for melanoma, can be immunogenic in vivo and that tumor-antigen specific T cells may exert anti-tumor activity in CCS patient. Thus, manipulation of the immune response may have therapeutic potential for this STS subtype and immunotherapy approaches, can be promising therapeutic options for these patients.

Co-expression of multiple cytokines and their receptors in primary clear cell sarcoma of the pubic bone with features of a small round cell tumor.

We present the case of an 81-year-old man with primary clear cell sarcoma (CCS) of the pubic bone with an associated aggressive clinical course. The patient's laboratory tests showed marked leukocytosis, elevated levels of C-reactive protein and multiple cytokines, including interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). Histological examination showed monomorphic small cells predominantly arranged as a diffuse sheet with morphological features of a small round cell tumor (SRCT). Immunohistochemical staining indicated that the tumor cells were positive for HMB45, S100, Melan A, IL-6, IL-6 receptor, G-CSF, and G-CSF receptor and negative for cytokeratin (AE1/AE3) and epithelial membrane antigen. To the best of our knowledge, this is the first case report of aggressive primary CCS of the pubic bone with features of SRCT showing the production and co-expression of multiple cytokines and their receptors. Thus, we suggest that proliferation through an IL-6- and G-CSF-associated autocrine mechanism may play an important role in the aggressive clinical course and poor prognosis of some CCSs showing features of SRCT.

Primary gastrointestinal clear cell sarcoma: report of 2 cases, one case associated with IgG4-related sclerosing disease, and review of literature.

Clear cell sarcoma (CCS) is a distinctive soft tissue sarcoma that shows melanocytic differentiation. Primary gastrointestinal (GI) CCSs have been rarely reported, but to our knowledge, no association between GI CCSs and immunoglobulin G4 (IgG4)-related sclerosing disease has been described in the literature. We experienced 2 cases of CCS that arose in the small intestine and metastasized to the liver. Histologic features and immunophenotype were typical of CCS. One of them showed a unique peritumoral sclerosing inflammatory reaction, which was highly reminiscent of IgG4-related sclerosing inflammatory disease. Dense lymphoplasmacytic infiltration with extensive sclerosis and obliterative phlebitis was observed in the immediate vicinity of the primary and metastatic tumors, but not in the distant areas from the tumor. The average number of IgG4-positive plasma cells was more than 50 per high-power field. We report 2 cases of primary GI CCS with one case showing a unique peritumoral IgG4-related lymphoplasmacytic sclerosing inflammation.

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Newly established MST-1 tumour cell line and tumour-infiltrating lymphocyte culture from a patient with soft tissue melanoma (clear cell sarcoma) and their potential applications to patient immunotherapy.

The establishment and characterisation of paired autologous tumour cell line (MST-1) and tumour-infiltrating lymphocyte (TIL) culture from a tumour mass of a 14-year-old Taiwanese girl with soft tissue melanoma are described. MST-1 cells grown in vitro were heterogeneous in morphology, ranging from floating round cells, loosely attached round/oval or elongated cells with prominent pseudopod-like processes, to well-attached spindle and elongated dendritic cells without obvious pseudopods. Immunostaining revealed that major melanoma-associated antigens, such as S100 protein, HMB-45, melanotransferrin, chondroitin sulphate proteoglycan, and the gangliosides GD2 and GD3, were consistently expressed by the tumour tissue, severe combined immunodeficiency (SCID) mouse xenograft and derived cell lines. Flow cytometric analysis of the tumour DNA content showed an index of 1.8 relative to normal peripheral blood lymphocyte DNA. Chromosome analysis revealed all cells at a hypotetraploid level with several clonal chromosome aberrations, including deletions at 10p and 12q, an addition at 12q, translocations t(1;14) and t(5;6). Electron microscopy showed melanosome structures. This observation and the expression of the major melanoma-associated antigens were all indicative of the melanocytic origin of MST-1 tumour. Interleukin-2 (IL-2) expanded TILs had the predominant CD8+ phenotype and the capacity to lyse cells of the cultured autologous tumour. The availability of the soft tissue melanoma cell line, the SCID mouse xenograft tumour system as well as autologous TILs described herein would provide useful materials for identifying T-cell-defined antigens as well as a model system for devising individualised cancer biotherapeutic strategies. This cell line can also be used for further studies aimed at uncovering the histogenesis of this rare cancer.