V.A. Trivillin 1,2, M.A. Garabalino 1, L.L. Colombo 2,3,4, E.C.C. Pozzi 1, A. Monti Hughes 1, P Curotto 1, S Thorp 1, R.O. Farías 1,2, S.J. González 1,2, S. Bortolussi 5, S. Altieri 5, M.E. Itoiz 1,6, R.F. Aromando 6, D.W. Nigg 7, A.E. Schwint 1,2 1 Comisión Nacional de Energía Atómica (CNEA); 2 CONICET; 3 Instituto de Oncología Angel H Roffo; 4 Universidad Abierta Interamericana (UAI), Argentina; 5 Dipartimento di Fisica Nucleare e Teorica dell’ Università, Pavia and Istituto Nazionale di Fisica Nucleare (INFN), Pavia, Italia; 6 Facultad de Odontología, Universidad de Buenos Aires (UBA), Argentina; 7 Idaho National Laboratory, EE.UU.

 BNCT has been proposed for the treatment of non resectable, diffuse tumors in lung.  The lung is the most frequent (and sometimes unique) site of metastases for several tumor types.  Surgical resection is not an option when metastases are multiple, and chemotherapy is often ineffective. In these cases the short-term mortality rate is 100%.

 The aim of the present study was to perform BNCT studies in an experimental model of lung metastases to assess therapeutic efficacy and potential toxicity.

 Based on the work of Bortolussi et al. (ARI 2011), we adapted an experimental model of disseminated lung metastases in rats to perform experimental BNCT studies devoted to optimizing the therapeutic advantage of ex-situ or in-situ BNCT for lung tumors.  This work is part of the multi-institutional project (CNEA, Maimonides Univ., Inst. A. Roffo and Favaloro Foundation) whose primary objective is to study the feasibility of applying BNCT ex-situ to patients with multiple metastases in both lungs.

At different times post-injection of the tumor cells, the animals were sacrificed, the lungs were removed and fixed in Bouin's solution. Weeks 2 Weeks Between 1 to 6 x 10 6 DHD/K12/TRb colon carcinoma cells in 0.5 ml of F10-DMEM were injected in the jugular vein. The selected tumor cell load was 3 x 10 6 cells. The colon carcinoma cells DHD/K12/TRb were maintained in vitro in culture medium F10-DMEM supplemented with 10% fetal bovine serum. Weeks 3 Weeks Weeks 4 Weeks Weeks 5 Weeks Experimental Model: different experimental conditions were tested to optimize the model for biodistribution studies and for in vivo BNCT studies.

Syngeneic inoculation of colon cancer cells (DH/DK12/TRb) in BDIX rats in the jugular vein Development of lung metastases Administration of boron compounds Irradiation in the RA-3 Nuclear Reactor Sacrifice of the group T0 Sacrifice of the experimental groups Parameters evaluated: *Body weight *Lung mass *Histology *Surface with metastatic nodules (ongoing work) 3 weeks 3 h. 2 weeks The general state of the animals was evaluated in terms of body weight, neurological symptoms and clinical signs throughout the process.

Without boron compound administration. Irradiation at RA-3 Nuclear Reactor. Sacrificed 5 weeks post-inoculation and 2 post therapy. Experimental Groups: 1. Control groups In all groups (except NORMAL), BDIX rats were inoculated with 3 * 10 6 cells (DH/DK12/TRb). Sacrificed 3 weeks post inoculation (pre-treatment). Without boron compound administration. Same manipulation without irradiation. Sacrificed 5 weeks post inoculation. Beam only T0 Sham Without inoculation of cancer cells. Without boron compound administration. Euthanized to assess normal lung mass. NORMAL

Administration of BPA (46.5 mg 10 B/Kg) iv. Irradiation at RA-3 Nuclear Reactor. Sacrificed 5 weeks after inoculation and 2 post therapy. Administration of BPA (31 mg 10 B/Kg) + GB-10 (34.5 mg 10 B/Kg) iv. Irradiation at RA-3 Nuclear Reactor. Sacrificed 5 weeks after inoculation and 2 post therapy. BPA-BNCT (BPA+GB-10) -BNCT Low dose: minimum absorbed dose to tumor 4 Gy. High dose: minimum absorbed dose to tumor 8 Gy. Low dose: minimum absorbed dose to tumor 4 Gy. High dose: minimum absorbed dose to tumor 8 Gy. Experimental Groups: 2. BNCT groups In all groups, BDIX rats were inoculated with 3 * 10 6 cells (DH/DK12/TRb).

Thermal column ShutterExternal Shield Irradiation position Feasibility of BNCT for experimental lung metastases in the RA-3 Nuclear Reactor Based on potentially useful administration protocols assayed in previous biodistribution studies (Trivillin et al., 2013), dosimetric calculations were performed to carry out irradiations at RA-3. It was necessary to design and build a shield to protect the animal body while the lung is exposed through an opening. Razetti et al. designed, constructed and characterized an adequate thermal neutron shield of lithium carbonate (enriched in lithium-6). Design, construction and application of a neutron shield for the treatment of diffuse lung cancer in rats using BNCT. A. Razetti, R.O. Farías, S.I. Thorp, V.A. Trivillin, E.C.C. Pozzi, P. Curotto, A.E. Schwint, S.J. González. Applied Radiation and Isotopes (in press). Diagram of the thermal column of the RA-3 Lithium Carbonate Shield

Restrictions in clinical radiotherapy with photons Organ Condition Vol. Crit (cc) 1 fraction Threshold GyMax. Gy Heartpericarditis< Spinal cordMyelitis<0, Skinulceration< Lung (R&L)basic function15007 Lung (R&L)Pneumonitis10007,4 Lung (R&L)<10%20 tumorskinothers CBE 3,82,51,4 RBE 3,2 Tissue BPA (46.5 mg 10 B /Kg) ip BPA (31 mg 10 B/Kg) ip + GB-10 (34.5 mg 10 B/Kg) i.v Blood13.7 ± 2.3 (n=5)31.9 ± 5.9 (n=4) Metastasis22.9 ± 7.2 (n=48)32.8 ± 8.7 (n=32) Lung12.2 ± 7.2 (n=9)28.3 ± 5.6 (n=6) Spinal cord5.5 ± 2.4 (n=8)4.7 ± 2.0 (n=5) Heart14.9 ± 3.7 (n=4)18.9 ± 3.0 (n=3) Metastasis / Lung Metastasis / Blood DOSIMETRIC CONSIDERATIONS Trivillin et al., ARI 2014

Prescribed absorbed dose for different protocols. Absorbed dose (Gy) for BNCT mediated by BPA (46.5 mg 10 B/Kg), iv (low dose). MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose (Gy) for BNCT mediated by BPA (31 mg 10 B/Kg) + GB-10 (34.5 mg 10 B/Kg), iv (low dose). MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose (Gy) for BNCT mediated by BPA (46.5 mg 10 B/Kg), iv (high dose). MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose (Gy) for BNCT mediated by BPA (31 mg 10 B/Kg) + GB-10 (34.5 mg 10 B/Kg), iv (high dose). MinimumMeanMaximum Heart Spinal Cord Lung Tumor

Parameters evaluated as an indicator of tumor response. Experimental groups Lung mass (mean ± SD, N) Lung mass / body mass * 100 (mean ± SD, N) Normal 1.01 ± 0.20 N= ± 0.05 N=12 TO 1.58 ± 0.89 N= ± 0.38 N=10 Sham 3.47 ± 1.65 N= ± 0.91 N=13 Beam Only LD (Low dose) 2.62 ± 1.03 N=51.48 ± 0.64 N=5 BPA-BNCT LD 1.01 ± 0.25 N=50.56 ± 0.11 N=5 (BPA+GB-10)-BNCT LD 1.35 ± 0.51 N=50.75 ± 0.30 N=5 BNCT LD (pooled) 1.18 ± 0.42 N= ± 0.24 N=10 Beam Only HD (High dose) 2.98 ± 1.47 N=62.12 ± 1.19 N=6 BPA-BNCT HD 1.38 ± 0.22 N=50.80 ± 0.16 N=5 (BPA+GB-10)-BNCT HD 1.42 ± 0.62 N=40.75 ± 0.25 N=4 BNCT HD (pooled)1.39 ± 0.41 N=90.78 ± 0.19 N=9 N: Number of rats

Percentage of lung mass / body weight for different protocols (mean ± SD). BNCT corresponds to data pooled for BPA-BNCT and (BPA+GB-10)-BNCT Statistical significance vs. Sham, *p<0.05 **p<0.01 ***p<0.001; Normal vs. T0 **p<0.01 ** ******

Lung mass for different protocols (mean ± SD). BNCT corresponds to data pooled for BPA-BNCT and (BPA+GB-10)-BNCT Statistical significance vs. Sham, *p<0.05 **p<0.01 ***p<0.001; Normal vs. T0 **p<0.01 * * * ** ***

Sham (no treatment, 5 weeks post-inoculation) BNCT (5 weeks post-inoculation, 2 weeks post-treatment) T0 (pre-treatment, 3 weeks post-inoculation) Beam only (5 weeks post- inoculation, 2 weeks post-treatment) Representative examples of the macroscopic appearance of left lung lobes

Representative example of metastatic dissemination in lung (low magnification) Sham: viable tumor nodule with glandular differentiation BNCT: tumor nodule exhibiting areas of viable cells, areas of necrosis and/or fibrosis and pleomorphic cells with radioinduced damage Representative examples of the microscopic appearance

Discussion and Conclusions  BNCT induced a partial, consistent and significant control of lung metastases, 2 weeks post-irradiation, with no associated toxicity.  The BNCT groups did not exhibit significant differences with T0, revealing that BNCT halted tumor growth.  No clinical, macroscopic or histological changes were observed in normal lung in any of the groups.  One problem so far is that we do not have a system for noninvasive monitoring of lung metastases. That is why we worked with the T0 group, to have a representative value of pretreatment lung mass.  BNCT allows the treatment of all nodules and scattered cells, without the need to know the exact number, distribution or shape (Bortolussi, 2011). Furthermore, the application of BNCT in lung tumors has the advantage that it is not necessary to adjust for the movement of breathing associated with the organ.

Acknowledgements  This study was partially supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. GB-10 was kindly provided by Dr. David W. Nigg of Idaho National Laboratory, USA.

Thanks !!!

Prescribed absorbed dose for different protocols for the low dose level. Dose (GyW) MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose Dose (Gy) MinimumMeanMaximum Heart Spinal Cord Lung Tumor BPA (46.5 mg 10 B/Kg) Dose (GyW) MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose Dose (Gy) MinimumMeanMaximum Heart Spinal Cord Lung Tumor BPA (31 mg 10 B/Kg) + GB-10 (34,5 mg 10 B/Kg)

Dose (GyW) MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose Dose (Gy) MinimumMeanMaximum Heart Spinal Cord Lung Tumor Dose (GyW) MinimumMeanMaximum Heart Spinal Cord Lung Tumor Absorbed dose Dose (Gy) MinimumMeanMaximum Heart Spinal Cord Lung Tumor BPA (46.5 mg 10 B/Kg) BPA (31 mg 10 B/Kg) + GB-10 (34,5 mg 10 B/Kg) Prescribed absorbed dose for different protocols for the high dose level.