PMC5498771_01
(A): The tumor lesion at presentation. A large ulcerated neoplastic lesion affected the hoof of the horse hind leg. (B): Surgical debulking of the hoof neoplasm. (C, D): Ultrasound guided isolated limb perfusion chemotherapy. (E, F, G): Delivery of permeabilizing electric pulses by means of plate and different needle array electrodes (H): The patient 1 year after the last ECT session: there is no gross evidence of cancer disease in the hoof.
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F1
The horse exhibited a large ulcerative and proliferative lesion of the right hind hoof (Fig. 1A) and had a grade 4 lameness according to the American Association of Equine Practitioners (AAEP) lameness grading system (Table 1), evaluated through a dedicated veterinary software (EquineTec Pro Advanced Edition, EquineTec, Monroe, USA). Surgical debulking was performed, reaching the underlying digital cushion and coffin bone (Fig. 1B). Thirty milligrams of Bleomycin (Bleomicina Solfato, Sanofi-Aventis, Milan, Italy) were diluted in 60 ml of NaCl 0.9% solution, and administered over 40 minutes, under ultrasonographic guidance (Fig. 1C, 1D). ECT was performed using a clinical electroporator (Onkodisruptor  Biopulse S.r.l., Naples, Italy) using needle array (double couple array for the center of the lesion, paired needles for the periphery), followed by booster with plate electrodes (Patent application EP2221086A1) (Fig. 1E, 1F, 1G). The patient was still in remission after one year from the completion of the ECT protocol (Fig. 1H) and her gait greatly improved moving from a grade 4 to a grade 2 as measured by dedicated software (Huguet and Duberstein, 2012) (Table 1).
(A): Histological appearance of the neoplasia at presentation: cellular invasion of the laminar corium, a high tumor cell mitotic index, and the formation of several keratin pearls are visible (Hematoxylin and Eosin staining; original magnification X20). (B): Histopathology exam after the third ECT session showing partial tumor regression (arrow) and local inflammation and fibrosis (asterisk). (C): Histopathology exam after the fifth ECT session showing tumor regression and osteomyelitis. (D): Histopathology exam three months after the fifth ECT session showing complete tumor regression and its replacement by fibrovascular tissue.
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F2
The hoof wall mass revealed cellular invasion of the laminar corium, a high tumor cell mitotic index, and the formation of several keratin pearls; all features characteristics of well-differentiated squamous cell carcinoma (Fig. 2A). A biopsy taken at the time of the third surgery showed histopathologic evidence of partial substitution of tumor tissue with fibrotic tissue and inflammatory reaction (Fig. 2B). After the fifth session of ECT there was necrosis of the connective tissues with secondary osteomyelitis (Fig. 2C), but no detectable tumor tissue. A histopathology exam of bioptic tissue at three months showed complete remission of the tumor and its replacement by fibrovascular tissue (Fig. 2D).