1. “Sequential” BNCT in an oral precancer model: a novel BNCT approach to treat tumors and inhibit the development of second primary tumors from surrounding precancerous tissue” A. Monti Hughes 1, E.C.C. Pozzi 1, S. Thorp 1, P. Curotto 1, M.A. Garabalino 1, E.M. Heber 1, M.E. Itoiz 1,3, R.F. Aromando 1,3, D.W. Nigg 4, V.A. Trivillin 1,2, A.E. Schwint 1,2 1 National Atomic Energy Commission (CNEA), Argentina 2 National Research Council (CONICET), Argentina 3 Faculty of Dentistry, University of Buenos Aires (UBA), Argentina 4 Idaho National Laboratory, 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 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. 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) 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). Although dose escalation would conceivably serve to optimize therapeutic outcome, it is limited by mucositis in the dose- limiting precancerous tissue. Previous studies Sequential-24h-BNCT (BPA-BNCT followed by GB-10-BNCT 24 h later) Novel approach to BNCT (Molinari et al., 2011) Allows for higher doses to be delivered to tumor, enhancing tumor response at no extra cost in terms of toxicity in the dose limiting precancerous tissue, at short term follow up (1 MONTH).

7. Why SEQUENTIAL–24h-BNCT? 1)The use of 2 boron agents with different properties and complementary mechanisms of action is a strategy that contributes to boron targeting homogeneity. 2)High interstitial fluid pressure impairs drug delivery and can promote tumor progression. A decrease in interstitial fluid pressure after the first part of the treatment favors the subsequent distribution of blood-borne therapeutic agents such as GB-10 and would favor therapeutic efficacy. 3)The interval between BPA-BNCT and GB-10-BNCT is short enough to preclude tumor cell repopulation and could favor re-targeting of the tumor cells that were refractory to the first application. 4)The delivery of high doses in two applications rather than all in one was demonstrated to induce a lower incidence of animals with severe mucositis. BNCT-induced mucositis in precancerous tissue was dose limiting and favored, in some cases, tumor development (Monti Hughes et al., 2013).

8. Previous studies Classical carcinogenesis protocol (12 weeks, twice a week) was useful for short-term tumor control studies (1 month) but: Does not allow long-term studies of tumor development in 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 the kinetics of human oral carcinogenesis. Oral precancer model (6 weeks, twice a week) for long term follow up (8 months) long term follow up (8 months) (Heber et al., 2010) Oral precancer model (6 weeks, twice a week) for long term follow up (8 months) long term follow up (8 months) (Heber et al., 2010) We studied long term therapeutic effect of different BNCT protocols in this model. The results were encouraging but mucositis in precancerous tissue was dose-limiting in some cases. THUS, THERE WAS ROOM FOR IMPROVEMENT

9. Study mucositis and therapeutic efficacy of Sequential-24h-BNCToral precancer Sequential-24h-BNCT in our model of oral precancer, at longer follow up times (3 months instead of 1 month).AIM CLINICAL RELEVANCE In a clinical scenario, controlling tumor growth while inhibiting tumor development in surrounding precancerous tissue, without significant radiotoxic effects, would be a very useful approach.

10. SEQ-24H-BNCT 9Gy total absorbed dose was compared to previous BNCT studies performed in the oral hamster cheek pouch precancer model, exposed to similar total absorbed doses (Monti Hughes et al., 2013) : 8 Gy total absorbed dose Single (GB-10+BPA)-BNCT 8 Gy total absorbed dose. 10 Gy total absorbed dose Double (GB-10+BPA)-BNCT 10 Gy total absorbed dose (5 Gy each irradiation, 4 week interval). SEQ-24H-BNCT 9Gy total absorbed dose was compared to previous BNCT studies performed in the oral hamster cheek pouch precancer model, exposed to similar total absorbed doses (Monti Hughes et al., 2013) : 8 Gy total absorbed dose Single (GB-10+BPA)-BNCT 8 Gy total absorbed dose. 10 Gy total absorbed dose Double (GB-10+BPA)-BNCT 10 Gy total absorbed dose (5 Gy each irradiation, 4 week interval). RADIOBIOLOGICAL STUDIES BPA-BNCT 2.6 Gy 2.6 GyGB-10-BNCT 6.8 Gy 24h 3 months follow up 1.SEQUENTIAL–24h-BNCT 9 Gy total absorbed dose 2.Beam only–24h-Beam only 3.Control group: Cancerized, sham irradiated animals. DMBA-cancerized animals with the oral precancer carcinogenesis protocol (6 weeks, twice a week):

11. Thermal column Shutter External shield water core Irradiation position Sample insertion RADIOBIOLOGICAL STUDIES – RA-3 thermal facility 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.

12. RADIOBIOLOGICAL STUDIES – Animal shielding 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.

13. RADIOBIOLOGICAL STUDIES Boron concentration in precancerous tissue Irradiation time Thermal neutron flux at the irradiation position Mean absorbed dose BPA: 12.5±2.6 ppm4.0 min(7.4±0.6)*10 9 n.cm -2.seg -1 2.6±0.3 Gy GB-10: 24.4±9.7 ppm6.6 min(7.3±0.6)*10 9 n.cm -2.seg -1 6.8±1.7 Gy Absorbed Doses (Gy) for each of the Dose Components Absorbed Doses (Gy) for each of the Dose Components Fast neutrons Gamma photons Boron (precancerous tissue) Induced protons BPA-BNCT (15.5 mg 10 B/kg) Precancerous tissue --0.18±0.01 Gy1.9±0.3 Gy0.45±0.03 Gy GB-10-BNCT (50 mg 10 B/kg) Precancerous tissue --0.27±0.02 Gy5.8±1.7 Gy0.69±0.05 Gy Seq-24h-BO Precancerous tissue (BPA-BNCT) --0.18±0.01 Gy--0.45±0.03 Gy Precancerous tissue (GB-10-BNCT) --0.27±0.02 Gy--0.69±0.05 Gy Dose calculations were performed using the “Hamster Tool” software developed by Farías R and Gonzalez S.

14. FOLLOW UP 3 months after BNCT 2. Radiotoxicity in terms of mucositis in precancerous tissue: six grade oral mucositis scale based on macroscopic features adapted for other studies made on humans and hamsters (Lopez-Castaño et al., 2005; Sonis et al., 2000) : Grading was based on the most severe feature. 3. Inhibitory effect of BNCT on the development of new tumors in precancerous tissue. 1. Clinical status of the animals. SEVERE MUCOSITIS: G4/G5 Three end-points assessed weekly:

15. Percentage of animals affected by severe mucositis (G4/G5), Necrosis size (mm 2 ) and percentage of animals with new tumors after irradiation (3 months follow up). RESULTSRESULTS Protocols (“n” number of animals) Severe mucositis (Grade 4/Grade 5) Necrosis Mean±SD (mm 2 ) % animals with “new” tumors 3 months after BNCT Cancerized, sham irradiated animals (n=88) 0%--79% SBeam only (n=6) 0%--100% DBeam only (n=6) 0%--83% Seq-24h-BO (n=5) 0%--80% S(GB-10+BPA)-BNCT 8 Gy (n=6) (Monti Hughes et al., 2013) 100%74±22 40% (remaining pouch tissue) Seq-24h-BNCT 9 Gy (n=7) THIS STUDY 57%36±2029% D(GB-10+BPA)-BNCT 10 Gy (n=6) (Monti Hughes et al., 2013) 33%16±533% Seq-24h-BNCT and D(GB-10+BPA)-BNCT protocols showed significant lower percentages of animals with « new » tumors versus cancerized, sham irradiated animals (p=0.0093; p=0.0275, respectively). Significant reduction in size of necrosis between Seq-24hs-BNCT 9Gy vs S(GB- 10+BPA)-BNCT 8Gy (p=0.0244) and D(GB-10+BPA)-BNCT 10Gy vs S(GB-10+BPA)- BNCT 8Gy(p=0.0125). Seq-24h-BNCT 9Gy vs D(GB-10+BPA)-BNCT 10Gy exhibited similar sizes of necrosis (p=0.2574). S(GB-10+BPA)-BNCT 8 Gy exhibited severe mucositis in all animals with important tissue loss. Instead, Seq-24h-BNCT 9Gy and D(GB-10+BPA)- BNCT 10 Gy exhibited a reduction in the incidence of severe mucositis.

16. Hamster cheek pouch treated with Single (GB-10+BPA)-BNCT 8 Gy total absorbed dose treatment vs. Sequential-24h-BNCT 9 Gy total absorbed dose at 14 days post treatment (time at which maximum mucositis was observed). RESULTSRESULTS Single (GB-10+BPA)-BNCT 8 Gy: Grade 5Sequential-24h-BNCT 9 Gy: Grade 0 Necrosis

17. RESULTSRESULTS Single (GB-10+BPA)-BNCT 8 Gy Necrosis: 139mm 2 Sequential-24h-BNCT 9 Gy Necrosis: 21mm 2 Significant reduction in size of necrosis in Seq-24hs-BNCT 9 Gy vs S(GB-10+BPA)-BNCT 8 Gy Hamster cheek pouch treated with Single (GB-10+BPA)-BNCT 8 Gy total absorbed dose treatment vs. Sequential-24h-BNCT 9 Gy total absorbed dose at 14 days post treatment (time at which maximum mucositis was observed).

18. Sequential-24h-BNCT minimizes the incidence of severe mucositis and significantly inhibits tumor development at 3 months after BNCT in precancerous tissue, in our oral precancer model. In a clinical scenario, this is an asset in terms of allowing higher doses to be delivered to tumor, enhancing tumor response, while inhibiting tumor development in surrounding precancerous tissue. DISCUSSIONDISCUSSION

19. DISCUSSIONDISCUSSION Both protocols, D(GB-10+BPA)-BNCT 10 Gy total absorbed dose (5 Gy each application, 4 weeks apart) and Seq-24hs-BNCT 9 Gy total absorbed dose were shown to be useful to treat tumors (Molinari et al., 2011) and precancerous tissue, with a lower incidence of severe mucositis. Each protocol could be used for different clinical purposes: Double BNCT Double BNCT could conceivably reduce large tumors to improve dose distribution for the second application. However, some patients are too fragile to be included in a double application protocol with a 4 week interval. Sequential-24h-BNCT Sequential-24h-BNCT could deliver a high, therapeutically effective dose with only a one day interval. This protocol would be more appropriate for fragile patients and would be more effective for smaller tumors.

20. In a separate study we demonstrated that Histamine significantly reduced the incidence of severe BNCT-induced mucositis in our oral precancer model without impairing therapeutic efficacy (Monti Hughes et al., ICNCT 16th congress). Our next goal would be to improve therapeutic efficacy and reduce, even more, the incidence of severe mucositis induced by Seq-BNCT and Double BNCT protocols, by combining them with treatment with Histamine. In a clinical scenario, this strategy could allow for an increase in the dose delivered to head and neck tumors without enhancing mucositis. FUTURE STUDIES

21. Buenos Aires city, Argentina

22. ACKNOWLEDGMENTS

23. Absorbed doses for each dose component (Gy) for SBNCT and DBNCT protocols (Monti Hughes et al., 2013). For DBNCT experiment, data is quoted for each of two similar irradiations. Fast neutrons Gamma photons Boron (per ppm) Induced protons S(GB-10+BPA)-BNCT (GB-10: 34.5 mg 10 B/kg, BPA: 31 mg 10 B/kg) Precancerous tissue -- 0.69±0.07 Gy0.20±0.04 Gy0.56±0.12 Gy SBeam only Precancerous tissue -- 1.25±0.13 Gy--1.0±0.2 Gy D(GB-10+BPA)-BNCT (GB-10: 34.5 mg 10 B/kg, BPA:31 mg 10 B/kg) Precancerous tissue -- 0.16±0.03 Gy0.12±0.06 Gy0.39±0.03 Gy DBeam only Precancerous tissue -- 0.35±0.07 Gy--0.84±0.06 Gy

24. “Hamster Tool” (Farías R and Gonzalez S)

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 mm of thickness. Orthokeratinized layer. Epithelium witdh: 30 - 50 mm. Below: Connective tissue, interrupted muscule and a loose adventitia. Approximately: 300 - 600 mm of thickness.