Carl V. Hamby

Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.

Melanoma is a lethal melanocytic neoplasm. Unfortunately, the histological diagnosis can be difficult at times. Distinguishing ambiguous melanocytic neoplasms that are benign nevi from those that represent true melanoma is important both for treatment and prognosis. Diagnostic biomarkers currently used to assist in the diagnosis of melanoma are usually specific only for melanocytic neoplasms and not necessarily for their ability to metastasize. Traditional prognostic biomarkers include depth of invasion and mitotic count. Newer diagnostic and prognostic biomarkers utilize immunohistochemical staining as well as ribonucleic acid, micro-ribonucleic acid, and deoxyribonucleic acid assays and fluorescence in situ hybridization. Improved diagnostic and prognostic biomarkers are of increasing importance in the treatment of melanoma with the development of newer and more targeted therapies. Herein, the authors review many of the common as well as newer diagnostic and prognostic biomarkers used in melanoma.

Endothelial progenitor cells (EPCs) have been shown to participate in tissue repair under diverse physiological and pathological conditions. It is unknown whether EPCs are mobilized in response to acute renal injury. The aim of this study was to characterize EPC mobilization and homing in the course of acute renal ischemia. Mice were subjected to unilateral renal artery clamping (UC) for 25 min. At 10 min, 3, 6, 24 h, and 7 days after UC, the pool of circulating and splenic CD34+/Flk-1+ cells within the monocytic population was detected by flow cytometry. For ischemic preconditioning (IPC), the first UC was performed 7 days before the repeated ischemic episode. For EPC detection in the kidney, cryosections were stained for c-Kit+/Tie-2+ cells. The number of circulating EPCs was not significantly affected at any time after UC compared with sham-operated or control mice. IPC did not significantly change the circulating pool of EPCs. Splenectomy performed before UC resulted in a surge of circulating EPCs. Accordingly, splenic EPCs were significantly increased after UC at 3 and 6 h, but not at later times. EPC homing to the spleen was absent in IPC animals. Immunohistochemical analysis of the kidneys showed a sixfold increase in the number of c-Kit+/Tie-2+ cells localized in the medullopapillary region in mice by day 7 after ischemia. Enriched population of c-Kit+/Tie-2+ cells from the medullopapillary parenchyma of Tie-2green fluorescent protein chimeric mice subjected to IPC was isolated and transplanted to wild-type mice with acute renal ischemia. This procedure resulted in the improvement of renal function in recipients. In conclusion, 1) renal ischemia rapidly (within 3-6 h) mobilizes EPCs, which transiently home to the spleen, acting as a temporary reservoir of mobilized EPCs; 2) the late phase of IPC is associated with the mobilization of the splenic pool and accumulation of EPCs in the renal medullopapillary region; and 3) transplantation of EPC-enriched cells from the medullopapillary parenchyma afforded partial renoprotection after renal ischemia, suggesting the role of the recruited EPCs in the functional rescue.

BACKGROUND: beta-catenin plays a crucial role in the function of cell adhesion molecules and also participates in growth regulatory signalling pathways that may be involved in malignant transformation. OBJECTIVES: To examine beta-catenin expression in lesions of melanocytic origin for associations with clinicopathological markers of disease progression and for its significance as a predictor of disease recurrence and prognosis. METHODS: beta-catenin expression was examined by immunoperoxidase staining in 50 melanocytic naevi and 91 primary and 50 metastatic melanomas. RESULTS: beta-catenin was expressed in 96% of melanocytic naevi, in 94% and 65%, respectively, of radial and vertical growth phase primary melanomas, and in 38% of metastatic melanomas. Benign and malignant melanocytic lesions had distinct patterns of beta-catenin localization. Most lesions expressing beta-catenin exhibited cytoplasmic staining; however, over 40% of benign lesions also displayed nuclear staining, which was present only in 10% of primary and 15% of metastatic melanomas. Absent or weak expression of beta-catenin in primary melanomas was associated with several markers of disease progression, including tumour thickness and presence of lymph node metastases. A similar but not statistically significant trend was observed for the association of beta-catenin expression with disease recurrence and prognosis. CONCLUSIONS: These results suggest that loss or downregulation of beta-catenin expression in melanoma cells plays a significant role in progression of the disease.

Patients with metastatic or recurrent and refractory sarcomas have a dismal prognosis. Therefore, new targeted therapies are urgently needed. This study was designed to evaluate chimeric antigen receptor (CAR) T cells targeting the type I insulin-like growth factor receptor (IGF1R) or tyrosine kinase-like orphan receptor 1 (ROR1) molecules for their therapeutic potential against sarcomas. Here, we report that IGF1R (15/15) and ROR1 (11/15) were highly expressed in sarcoma cell lines including Ewing sarcoma, osteosarcoma, alveolar or embryonal rhabdomyosarcoma, and fibrosarcoma. IGF1R and ROR1 CAR T cells derived from eight healthy donors using the Sleeping Beauty (SB) transposon system were cytotoxic against sarcoma cells and produced high levels of IFN-γ, TNF-α and IL-13 in an antigen-specific manner. IGF1R and ROR1 CAR T cells generated from three sarcoma patients released significant amounts of IFN-γ in response to sarcoma stimulation. The adoptive transfer of IGF1R and ROR1 CAR T cells derived from a sarcoma patient significantly reduced tumor growth in pre-established, systemically disseminated and localized osteosarcoma xenograft models in NSG mice. Infusion of IGF1R and ROR1 CAR T cells also prolonged animal survival in a localized sarcoma model using NOD/scid mice. Our data indicate that both IGF1R and ROR1 can be effectively targeted by SB modified CAR T cells and that such CAR T cells may be useful in the treatment of high risk sarcoma patients.