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.

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

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

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

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

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).
Lithium Carbonate Shield
Diagram of the thermal column of the RA-3
Irradiation position
Thermal column
Shutter External
Shield
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).

DOSIMETRIC CONSIDERATIONS
Restrictions in clinical radiotherapy with photons
Organ Condition Vol. Crit
(cc)
1 fraction
Threshold Gy Max. Gy
Heart
Spinal cord
Skin
Lung (R&L)
Lung (R&L)
Lung (R&L) pericarditis < 15
Myelitis ulceration
<0,35
<10 basic function 1500
Pneumonitis 1000
<10%
16
10
23
7
7,4
20
22
14
26
CBE
RBE tumor skin others
3,8
3,2
Blood
Metastasis
Lung
Spinal cord
Heart
Tissue
Metastasis / Lung
Metastasis / Blood
Trivillin et al., ARI 2014
BPA
(46.5 mg 10 B /Kg) ip
13.7
14.9
±
±
2.3 (n=5)
22.9 ± 7.2 (n=48)
12.2 ± 7.2 (n=9)
5.5 ± 2.4 (n=8)
3.7 (n=4)
BPA (31 mg 10 B/Kg) ip
+
GB-10 (34.5 mg 10 B/Kg) i.v
31.9 ± 5.9 (n=4)
32.8 ± 8.7 (n=32)
28.3 ± 5.6 (n=6)
4.7 ± 2.0 (n=5)
18.9
±
3.0 (n=3)
1.9
1.7
1.2
1.0
2,5
3,2
1,4
3,2

Prescribed absorbed dose for different protocols.
Absorbed dose (Gy) for BNCT mediated by BPA (46.5
mg 10 B/Kg), iv (low dose).
Heart
Minimum
4.4
Spinal Cord 1.0
Mean
5.8
1.9
Maximum
6.9
3.3
Lung
Tumor
3.0
4.0
4.6
6.3
6.2
9.0
Absorbed dose (Gy) for BNCT mediated by BPA (31 mg 10 B/Kg)
+ GB-10 (34.5 mg 10 B/Kg), iv (low dose).
Heart
Spinal Cord
Lung
Tumor
Minimum
4.0
0.8
3.7
4.0
Mean
5.3
1.6
5.9
6.4
Maximum
6.3
2.6
8.3
9.3
Absorbed dose (Gy) for BNCT mediated by BPA (46.5
mg 10 B/Kg), iv (high dose).
Minimum Mean Maximum
Heart 8.8
Spinal Cord 2.0
Lung
Tumor
6.1
8.0
11.6
3.9
9.1
12.7
13.8
6.6
12.4
17.9
Absorbed dose (Gy) for BNCT mediated by BPA (31 mg
10 B/Kg) + GB-10 (34.5 mg 10 B/Kg), iv (high dose).
Minimum Mean Maximum
Heart 8.0
Spinal Cord 1.6
Lung
Tumor
7.3
8.0
10.7
3.1
11.8
12.9
12.7
5.1
16.7
18.7

Parameters evaluated as an indicator of tumor response.
Experimental groups
Normal
TO
Sham
Beam Only LD (Low dose)
BPA-BNCT LD
Lung mass
(mean ± SD, N)
1.01 ± 0.20 N=12
1.58 ± 0.89 N=10
3.47 ± 1.65 N=13
2.62 ± 1.03 N=5
1.01 ± 0.25 N=5
1.35 ± 0.51 N=5 (BPA+GB-10)-BNCT LD
BNCT LD (pooled) 1.18 ± 0.42 N=10
Beam Only HD (High dose) 2.98 ± 1.47 N=6
BPA-BNCT HD
(BPA+GB-10)-BNCT HD
BNCT HD (pooled)
1.38 ± 0.22 N=5
1.42 ± 0.62 N=4
1.39 ± 0.41 N=9
N: Number of rats
Lung mass / body mass * 100
(mean ± SD, N)
0.40 ± 0.05 N=12
0.79 ± 0.38 N=10
1.87 ± 0.91 N=13
1.48 ± 0.64 N=5
0.56 ± 0.11 N=5
0.75 ± 0.30 N=5
0.65 ± 0.24 N=10
2.12 ± 1.19 N=6
0.80 ± 0.16 N=5
0.75 ± 0.25 N=4
0.78 ± 0.19 N=9

Percentage of lung mass / body weight for different protocols (mean ± SD).
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
**
**
**
* *** * * **
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

4,0
3,0
2,0
1,0
0,0
6,0
5,0
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

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

Representative examples of the microscopic appearance
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

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.

Prescribed absorbed dose for different protocols for the low dose level.
BPA (46.5 mg 10 B/Kg)
Heart
Spinal Cord
Lung
Tumor
BPA (31 mg
Dose (GyW)
Minimum Mean Maximum
6.3
1.1
4.2
10.5
8.5
2.6
6.7
18.6
10.4
4.7
9.5
27.7
10
Heart
Spinal Cord
Lung
Tumor
B/Kg) + GB-10 (34,5 mg 10 B/Kg)
Dose (GyW)
Minimum Mean Maximum
5.7
0.9
5.0
11.1
7.8
2.0
8.5
20.1
9.5
3.6
12.4
30.1
Absorbed dose
Absorbed dose
Heart
Spinal Cord
Lung
Tumor
Dose (Gy)
Minimum Mean Maximum
4.4
1.0
3.0
4.0
5.8
1.9
4.6
6.3
6.9
3.3
6.2
9.0
Heart
Spinal Cord
Lung
Tumor
Dose (Gy)
Minimum Mean Maximum
4.0
0.8
3.7
4.0
5.3
1.6
5.9
6.4
6.3
2.6
8.3
9.3

Prescribed absorbed dose for different protocols for the high dose level.
BPA (46.5 mg 10 B/Kg)
Heart
Spinal Cord
Lung
Tumor
Absorbed dose
Minimum
12.6
2.2
8.4
20.9
Heart
Spinal Cord
Lung
Tumor
Minimum
8.8
2.0
6.1
8.0
Dose (GyW)
Mean
17.1
5.1
13.5
37.2
Maximum
20.8
9.4
19.1
55.3
Dose (Gy)
Mean
11.6
3.9
9.1
12.7
Maximum
13.8
6.6
12.4
17.9
BPA (31 mg 10 B/Kg)
+
GB-10 (34,5 mg 10 B/Kg)
Heart
Spinal Cord
Lung
Tumor
Absorbed dose
Minimum
11.3
1.7
10.0
22.2
Dose (GyW)
Mean Maximum
15.6
4.0
17.0
40.2
19.1
7.3
24.8
60.3
Heart
Spinal Cord
Lung
Tumor
Minimum
8.0
1.6
7.3
8.0
Dose (Gy)
Mean
10.7
3.1
11.8
12.9
Maximum
12.7
5.1
16.7
18.7