1. A. Monti Hughes 1, E.C.C. Pozzi 1, S. Thorp 1, P. Curotto 1, V.A. Medina 2,3, D.J. Martinel Lamas 2, E.S. Rivera 2, M.A. Garabalino 1, E.M. Heber 1, M.E. Itoiz 1,4, R.F. Aromando 1,4, D.W. Nigg 5, V.A. Trivillin 1,3, A.E. Schwint 1,3. 1 National Atomic Energy Commission (CNEA), Argentina; 2 School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), Argentina; 3 National Research Council (CONICET), Argentina; 4 Faculty of Dentistry, UBA, Argentina, 5 Idaho National Laboratory (INL) USA.

2. Squamous cell carcinoma of the head and neck (HNSCC) remains a major cause of morbidity and mortality worldwide. The relatively poor overall 5-year survival rate for malignancies of the oral cavity poses the need for more effective and selective therapies. To date, BNCT clinical results have shown a potential therapeutic advantage, with room for improvement. Studies in appropriate experimental models are pivotal to progress in this field. HEAD AND NECK CANCER SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL CANCER MODEL CANCER MODEL SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL CANCER MODEL CANCER MODEL Mesocricetus auratus

3. Non-cancerized hamster cheek pouch (everted). Mesocricetus auratus SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL CANCER MODEL Widely known animal system that closely mimics events involved in the development of premalignant and malignant human oral lesions. Similar to human oral mucosa (in histological, histochemical and ultrastructural terms). Resembles a pocket of oral mucosa in the thickness of the hamster´s cheek. It does not develop spontaneous tumors. It can be easily everted for local irradiation and follow-up.

4. Applied without anesthesia. Cancerized hamster cheek pouch with a tumor (arrow) surrounded by precancerous tissue. Unlike models of implanted tumor cells in normal tissue, it mimics the spontaneous process of malignant transformation. surrounded by precancerous tissue Provides a tumor model surrounded by precancerous tissue which gives rise to the formation of additional tumors, as occurs in field cancerized human oral mucosa in head and neck cancer. SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL CANCER MODEL CARCINOGENESIS PROTOCOL: Repeated topical application of the carcinogen dimethyl-1,2-benzanthracene (DMBA) Topical application of DMBA 0.5% in mineral oil.

5. The study of precancerous tissue in our hamster model is useful because: The development of tumors from precancerous tissue would model the development of second primary tumors and recurrences in field cancerized tissue in human oral mucosa in head and neck cancer, a frequent cause of therapeutic failure. Toxicity in precancerous tissue mimics the constraints imposed on therapeutic protocols by the dose limiting nature of field cancerized tissue in humans.

6. Short-term (1 month) therapeutic efficacy of BPA-BNCT, GB-10-BNCT and (GB-10+BPA)-BNCT to treat hamster cheek pouch oral cancer with no normal tissue radiotoxicity, and slight/moderate mucositis in dose-limiting precancerous tissue around tumors (Kreimann et al, 2001a; Trivillin et al, 2006; Pozzi et al., 2009). Previous studies Classical carcinogenesis protocol (12 weeks, twice a week) short-term tumor control studies Classical carcinogenesis protocol (12 weeks, twice a week) was useful for short-term tumor control studies (1 month follow up) but: Does not allow Does not allow long-term studies of precancerous tissue, which are essential to model (with constraints) a clinical scenario in which the risk of developing second primary tumors jeopardizes therapeutic efficacy; Does not mimic Does not mimic the kinetics of human oral carcinogenesis. Oral precancer model (6 weeks, twice a week) for long-term follow up (8 months) Oral precancer model (6 weeks, twice a week) for long-term follow up (8 months) (Heber et al., 2010) Long-term therapeutic efficacy of Double (GB-10+BPA)-BNCT 10 Gy total absorbed dose to precancerous tissue (in two 5 Gy doses, 4 weeks apart). ( Monti Hughes et al., 2013)

7. In a clinical scenario, oral mucositis limits the dose delivered to head and neck tumors and affects patients’ quality of life. Despite therapeutic success, BNCT-induced mucositis in precancerous tissue was dose limiting and favored, in some cases, tumor development (Monti Hughes et al., 2013). Despite many years of research there are few radiation protectors in use, whose clinical value is limited due to their toxicity. Nowadays mucositis continues to represent an important unmet medical need in oncology practice.

8. The aim of the present study was to evaluate the effect of the administration of radioprotective agents, seeking to reduce the incidence of severe BNCT-induced mucositis in precancerous tissue. RADIOPROTECTIVE AGENTS: HISTAMINE J 77771 20 J NJ77771 20 J NJ101915 84 RADIOPROTECTIVE AGENTS: HISTAMINE J 77771 20 J NJ77771 20 J NJ101915 84 Protect small intestine, bone marrow and salivary gland from gamma radiation in mice and rats, with no local or systemic side effects. (Medina et al., 2007, 2011; Martinel Lamas et al., 2013)

9. DMBA-cancerized animals (Oral precancer carcinogenesis protocol: 6 weeks, twice a week) treated with BPA-BNCT 5 Gy total absorbed dose AND: RADIOBIOLOGICAL STUDIES Group 1) HISTAMINE LOW CONCENTRATION (1mg/kg, sc, n=6 animals) Group 2) HISTAMINE HIGH CONCENTRATION (5mg/kg, sc, n=6 animals) CONTROL GROUP: vehicle (saline solution) (n=11 animals) Group 3) J NJ77771 20 (10mg/kg, sc, n=7 animals) Group 4) J NJ101915 84 (10mg/kg, sc, n=3 animals) Cancerized, sham irradiated animals (no BNCT or radioprotective treatment) (n=88 animals) All radioprotectors were applied 1 day before BNCT, on the day of BNCT (concomitantly with BPA injection) and daily for 14 days after BNCT. BPA-BNCT protocol: the incidence of severe mucositis in field cancerized tissue was highest with this protocol in our previous studies (Monti Hughes et al., 2013).

10. Thermal column Shutter External shield water core Irradiation position Sample insertion RA-3 (Buenos Aires, Argentina) Thermal column Sample insertion Irradiation position Sample insertion The animals were irradiated at the RA-3 thermal facility. The animals are inserted into a near-isotropic neutron field while the reactor is in normal operation. RADIOBIOLOGICAL STUDIES – RA-3 thermal facility

11. The animals were placed inside a Lithium-6 carbonate shielding to protect their bodies while the cheek pouch is everted out of the enclosure onto a protruding shelf for exposure. RADIOBIOLOGICAL STUDIES – Animal shielding

12. RADIOBIOLOGICAL STUDIES Absorbed Doses (Gy) for each of the Dose Components: Fast neutrons Gamma photons Boron (precancerous tissue) (per ppm) Induced protons BPA-BNCT (15.5 mg 10 B/kg b.w.) Cancerized cheek pouch -0.36±0.090.30±0.070.9±0.1 Boron concentration in precancerous tissue Irradiation time Thermal neutron flux at the irradiation position Mean absorbed dose 12.5±2.6 ppm7.9 min(7.7±0.6)*10 9 n.cm -2.seg -1 5.0±1.7 Gy Dose calculations were performed using the “Hamster Tool” software developed by Farías R and Gonzalez S.

13. Must be minimally toxic itself. Must be minimally toxic itself. IDEAL RADIOPROTECTOR Must reduce the incidence of severe mucositis. Must reduce the incidence of severe mucositis. Must not compromise BNCT therapeutic effect. Must not compromise BNCT therapeutic effect.

14. FOLLOW UP 8 months after BNCT Grade 0: healthy appearance, no erosion or vasodilation. Grade 1: erythema and/or edema and/or vasodilation, no evidence of mucosal erosion. Grade 2: severe erythema and/or edema, vasodilation and/or superficial erosion. Grade 3: severe erythema and/or edema, vasodilation and formation of ulcers <2 mm in diameter. Grade 4: severe erythema and/or edema, vasodilation and formation of ulcers ≥2 mm and <4 in diameter, and/or necrosis areas <4mm in diameter. Grade 5: severe erythema and/or edema, vasodilation and formation of ulcers ≥4mm or multiple ulcers ≥2mm, and/or necrosis areas ≥4mm in diameter. Grading was based on the most severe feature. Radiotoxicity in terms of mucositis in precancerous tissue: six grade oral mucositis scale based on macroscopic features adapted from other studies in humans and non-cancerized hamsters (Lopez-Castaño et al., 2005; Sonis et al., 2000): Inhibitory effect of BNCT on the development of new tumors in precancerous tissue. Local and systemic side effects. SEVERE MUCOSITIS: G4/G5

15. % of animals affected by severe mucositis (G4/G5) and % of animals with new tumors after irradiation (8 months follow up). RESULTSRESULTS For all radioprotective agents: only local signs of irritation. His LOW was able to reduce the incidence of severe BNCT induced mucositis vs Control group His LOW was able to reduce the incidence of severe BNCT induced mucositis vs Control group. Both JNJ treatments and His HIGH were not able to reduce it. None of the protocols compromised the therapeutic effect of BNCT None of the protocols compromised the therapeutic effect of BNCT in terms of inhibition of tumor development from precancerous tissue at 8 months after treatment.

16. Hamster cheek pouch treated with BPA-BNCT without radioprotective treatment (saline sol.) vs. BPA-BNCT + His LOW at 14 days post treatment (time at which maximum mucositis was observed). BPA-BNCT 5Gy + His LOW GRADE 2 BPA-BNCT 5Gy + saline solution GRADE 5 Necrosis RESULTSRESULTS Focal erosion

17. “n” is number of samples; * 2 animals; **4 animals. BIODISTRIBUTION STUDIES Treatment with His low did not significantly alter gross boron concentration from BPA in tumor, precancerous tissue, normal pouch tissue or blood (p≥0.11).

18. The use of histamine LOW concentration reduced the incidence of BNCT-induced severe mucositis associated with BPA-BNCT at 5 Gy total dose without compromising the inhibitory effect of BNCT on tumor development. This could be due to: > The role of Histamine as a radical oxygen species (ROS) scavenger. > The capacity of histamine to act as a growth factor, favoring proliferation and repair and inhibiting apoptosis. DISCUSSIONDISCUSSION Biodistribution studies showed that His low did not affect gross boron biodistribution in tumor or field cancerized tissue. This is an asset in terms of preserving therapeutic efficacy. Long periods of high histamine concentration (5 mg/kg) and JNJ compounds (10mg/kg) failed to exert a radioprotective effect. This could be due to: > A dose dependent effect on tumor development. An increase in proliferation rate could be enhancing radiosensitivity and consequently an increase in severe mucositis. > Extensive removal of ROS would affect the healing process of mucositis.

19. Two assets to consider histamine as a radioprotector in BNCT of head and neck tumors: Medina et al. (2011b) have shown that histamine also prevents functional and morphological radioinduced alterations of submandibular glands. A synthetic derivative of histamine, histamine dihydrochloride, is currently safely used in clinical trials (Romero et al., 2009). DISCUSSIONDISCUSSION

20. This study suggests the potential use of treatment with histamine LOW concentration (1mg/kg) to minimize the incidence of severe mucositis in field cancerized tissue associated with BPA-BNCT at a therapeutic absorbed dose of 5 Gy, in an oral precancer model. These results may be of clinical value in reducing radiation radiotoxicity in head and neck cancer patients undergoing BNCT treatment. CONCLUSIONCONCLUSION

21. Los Alerces National Park; Patagonia ARGENTINA

22. ACKNOWLEDGMENTS

23. Medina et al. (2011b) have shown that histamine also prevents functional and morphological alterations of submandibular glands induced by ionizing radiation and completely reversed the radiation-induced reduced salivation in rats. Overall, histamine would not only reduce oral mucositis in BNCT of head and neck cancer but could also be used to preserve submandibular salivary glands and prevent xerostomia in patients with salivary gland carcinoma and head and neck cancers. A synthetic derivative of histamine, histamine dihydrochloride, is currently safely used in clinical trials as an adjuvant for the potential treatment of different cancers, exhibiting no unexpected or irreversible side effects (Romero et al. 2009). This would be an asset in considering the use of histamine as a radioprotector in BNCT of head and neck tumors. DISCUSSIONDISCUSSION

24. JNJ7777120 JNJ10191584

25. Human oral mucosa Hamster oral mucosa Thin non-keratinized or parakeratinized layer. Epithelium witdh: 200 mm. Below: Connective tissue, muscule and the skin of the cheek. Approximately: 2000  m of thickness. Orthokeratinized layer. Epithelium witdh: 30 - 50 mm. Below: Connective tissue, interrupted muscule and a loose adventitia. Approximately: 300 - 600  m of thickness.