Dr#Giorgio#Russo,#Ricercatore#IBFM4CNR#

New$Projects$for$in$vivo$irradia2ons$

IBFM – METHODS

Proteogenomic#Analysis##

Hadron#Therapy#

Data#CorrelaBon#

CEFALU’

CATANIA

Breast#Cancer#4#Radiotherapy#OpBmizaBon#of#Breast#Cancer#(BCa)#radiotherapy#can#be#categorized#in#three#aspects:##

1.  improvement#of#dose#homogeneity#within#the#target#volume#

2.  maximum#reducBon#of#cardiopulmonary#dose##

3.  minimizing#risks#of#second#malignancy#inducBon##

Local#radiotherapy#limited#to#the#breast#aSer#breast4conserving#surgery#(BCS),#or#to#the#chest#wall,#aSer#modified#radical#mastectomy#(MRM),#is#rouBnely#administered#using#three4dimensional#conformal#radiotherapy#(3D4CRT)#with#relaBvely#low#risks# for# late# toxicity.# More# recently,# intensity4modulated# radiotherapy# (IMRT),# has# been# rouBnely# implemented# in# the#clinic.##

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3D5CRT$ IMRT$ Proton5therapy$

PROS$ Low#risks#for#late#toxicity# High#conformal#dose#distribuBons##

Low#entrance#dose#to#health#Bssue;#Homogeneous#dose#distribuBon;#High#conformal#dose#distribuBon;#

steep#fall4off#to#zero#dose#distally#to#the#target.#

CONS$ Low#conformal#dose#distribuBons##PotenBal#carcinogenic#effects#of#low#dose#

to#the#lung#and#contralateral#breast#/#

Breast#Cancer#–#Proton#therapy#Proton$beam$5$Accelerated$Par2al$Breast$Irradia2on$(PB5APBI)$is#one#technical#development#being#invesBgated#to#achieve:#

•  saBsfactory#local#control#of#breast#cancer#with##•  reduced#toxicity#to#normal#organs#through#the#use#of#hypofracBonaBon.##

Proton#beam#RT#is#characterized#by#its#unique#dose#distribuBon#involving#a#Bragg#peak#effect.#It#delivers#a#high#dose#to#a#limited#region#followed#by#rapid#fall4off,#thereby#enhancing#normal#Bssue#sparing#while#conserving#dose#homogeneity.##

[1]#Bush#DA#et#al..#A#technique#of#parBal#breast#irradiaBon#uBlizing#proton#beam#radiotherapy:#comparison#with#conformal#X4ray#therapy.#Cancer#J#2007;13:114–8.#[2]#Kozak#KR,#et#al.#Dosimetric#comparison#of#proton#and#photon#three4dimensional,#conformal,#external#beam#accelerated#parBal#breast#irradiaBon#techniques.#Int#J#Rad.#Onc.#Biol#Phys#2006;65:1572–8.#[3]#Taghian#AG,#et#al.#Accelerated#parBal#breast#irradiaBon#using#proton#beams:#iniBal#dosimetric#experience.#Int#J#Radiat#Oncol#Biol#Phys#2006;65:1404–10.#[4]#Wang#X,#et#al.#External4beam#accelerated#parBal#breast#irradiaBon#using#mulBple#proton#beam#configuraBons#

•  excellent#results#in#terms#of#planning#target#volume#(PTV)#coverage##•  dose#homogeneity#•  significantly#reducing#the#volume#of#irradiated#non4target#breast#Bssue#

Dosimetric$comparisons$between$PB5APBI$and$3DCRT5APBI$[154]$have$shown$that:$$

PB5APBI$achieved##

MCF7 human breast cancer cell line MDA-MB-231 human metastatic breast cancer cell line

MCF10A human mammary non-tumorigenic cell line

Intra Operative Electron RadioTherapy Exclusive ! 23 Gy 10 Mev

Boost !9 Gy 10 MeV

MCF7 MCF10A

(LuminalB, Er+/Pr+) BasalB, Er-/Pr-

MDA-MB-231

(BasalB, Er-/Pr-)

Western Blot

Microarray

Immunofluorescence

Morphologic and Clonogenic Assay

Luminex

Genotyping

MCF7

9 GY- 23 Gy

Senescence radioinduced and cell death following both

treatments

MCF10A 9 GY- 23 Gy

Radioresistent cell fraction with 9 Gy

Cell death with 23 Gy

Surviving Fraction:0 Surviving Fraction:0 Surviving Fraction:9.6%

In addition, by Microarray experiments we described the involvement of known genes related to ionizing radiation and introduced novel ones able to activate molecular networks that could contribute to guide cell fate decision and in cancer care

able to provide the opportunity to explore molecular target-directed interventions

Hadrontherapy

1.In vitro approach with cell culture models

2.In vivo approach with mouse models

Biomarkers discovery by Proteogenomic technologies

Microarray

Immunofluorescence

Morphologic and Clonogenic Assay

Luminex

Genotyping

Evaluation of morphology and cell viability in terms of reproductive capacity, by clonogenic survival assay of treated cells

Comparative differential gene expression analysis beween treated and untreated cells by Microarray

Analysis of γ-H2AX foci formation after treatment, as a marker of sensitive early cell response to the presence of DNA double-strand breaks

Evaluation# of inflammatory response induced by treatment and able to influence cell fate decision

Analysis of genetic alterations that could regulate radio-sensitivity and cell survival/death balance

Pre4Clinic#study#

To#consider#all#necessiBes#that#a#preclinical#model#requires,#Monte#Carlo#calculaBons#must#give#a#considerable#contribuBon#to#characterize#the#beam's#dosimetry#simulaBng#the#physics#characterisBcs#of#the#beam,#the#beam#line#and#the#irradiaBon#setup.#

#Preclinical$models$are#a#crucial#component#of#research#in#radiaBon#therapy#and#in#nuclear#medicine.#Small4animal#irradiaBon#systems#must#mimic#the#clinical#applicaBon#of#radiaBon#therapy#as#closely#as#possible.##

Aim$of$this$experiment$is#manly:#•  to#analyze#molecular)mechanisms)induced)by)hadrons)

in#preclinical#models#in#order#to#study#the#behavior#of#healthy#Bssue#exposed#to#radiaBon##

•  to# achieve# a# highly) conformal) dose) to) the) target)volume,#while#sparing#the#surrounding#healthy#Bssues.#

#

In# addiBon,# we# will# compare# in# vivo# cell# response# to#treatment#in#mouse#with#or#without#BC#cell#inoculaBon#in#order# to# analyze# the# influence# of# tumours# on# healthy#Bssue#in#radiaBon#treatment#cell#response.##

BALB/c. BALB/c nude. NU/NU Nude C57BL/6 Mouse, CD1 General multipurpose model

In vivo Mouse models

Ideal for tumor biology and xenograft research. The animal lacks a thymus, is unable to produce T-cells, and is, therefore, immunodeficient.

MCF7

MCF10A

MDA-MB-231 Injection of tumour cells$

Leg injection to test the device $

Breast injection

Experimental Design part 1

Cellular models MCF-7 MCF-10

MDA-MB231

Injection of tumor cells

Housing at Animal House UniCT#

Transfer to LNS #

Cells growth

MCF7

MCF10A

MDA-MB-231

Leg injection for testing the device $

Breast injection for Experimental protocol

When tumour reach 10mm about 15 days#

Caliber #

Monitoring of tumor growth for 72H (Acute response)

15 days (tardive response)

Day 2 Hadrontherapy Treatment#

Day 3 Come back to Animal House#

Day 1 Transfer to LNS #

Experimental Design part 2

1) Dissection and removal of tumour a n d h e a l t h y t i s s u e f o r anatomopathology and proteogenomic analisys

2) Removal of hearth, lung, liver, kidney to analise toxicity#

Geant4#applicaBon:#Hadrontherapy#&#Dicom#

Hadrontherapy$applica2on$can#be#used#to#provide#a#good#source#of#informaBon#about#how#to#build#and#opBmize#the#experimental#setup#for#preclinical#applicaBons.##For#example,#the#geometrical#dimensions#of#the#system,#posiBon#of#small#animal,#source#surface#distance#SSD#etc.,#are#variables#that#can#be#managed#with#Geant4#simulaBons.#

DICOM$images$will#be#given#as#input#to#hadrontherapy#applicaBon#to#study#in#more#detail#the#damage#to#healthy#Bssue#surrounding#the#tumoral#area.##Thus,#Monte#Carlo#simulaBons#will#allow#us#to#understand#how#well#we#can#deliver#dose#to#targets#in#a#small#animal#subject,#and#to#esBmate#accurately#the#dose#released#to#the#Organ#at#Risk#(OAR),#main#goal#of#this#project.#

It#will#be#developed#a#new#Geant4#applicaBon#that#will#be#based#on#the#following#advanced#Geant4#example:#

PETherapy$

Geant4:#PETherapy#The#new#Geant4#PETherapy$applicaBon$will#be#developed#to#perform:#•  preliminary#dosimetric#studies##•  preliminary#RBE#analysis#and#that#will#compared#with#pathologist#biological#results.##

And#to#achieve:#•  the#best#irradiaBon#condiBon#(e.g.#holder,#size#of#the#housing,#etc.)#•  more#knowledge#about#the#dose#distribuBon#inside#the#target#(that#will#upload#in#GEANT4#geometry#using#the#real#DICOM#TC#images#of#animals)#and#in#the#surrounding#healthy#Bssue.#

##In#parBcular,#since#not#all#quanBBes#involved#in#radiobiological#and#medical#physics#studies#are#measurable#experimentally#

(e.g.#LET),#hence#tools#such#as#Geant4#and#biophysical#modelizaBon#of#ion#acBon#at#the#subcellular#level#are#necessary#to#model# experimental# beam# lines# and# predict# parBcle# behaviour# in# the# cellular# environment# compensaBng# for# the#disconBnuous#dose#delivery#pamern#inherent#to#radiaBon,#whether#administered#externally#or#internally.#

Experimental#setup#

The#animal#housing:#

•  will#be#made#of#Polymethyl#methacrylate#(PMMA)#

•  will#be#design#to#allow#an#easy#placement#of#tumor#region#respect#proton#beam.#

The#irradiaBon#will#be#performed#inside#CATANA#facility#at#LNS#4#Catania.##So,#it#will#be#possible#to#use#a#modulated#clinical#beam#to#deposit#an#uniform#dose#to#enBre#tumour#volume.#

Since#breast#cancer#cells#will#be#implanted#subcutaneous,#a#set#of#tantalum#clips,#that#will#be#implanted#around#breast#cancer#cells,#will#be#used#to#perform#a#precise#posiBoning#of#animals.#

cancer#

The#hadrontherapy#treatment#was#made#of#only#one#fracBon#and#the#prescribed#dose#will#be#of#about#24#Gy*.#

*The#prescribed#dose#esBmated#by#means#of#a#bibliographic#revision.#

Dosimetry#

Usually,#the#absolute#dosimetry#will#be#performed#by#using#a#Markus#chamber#but#due#the#limle#dimension#of#the#PTV,#it#will#needed#to#use#specific#dosimetric#tools#for#the#measuring#of#the#dose#released#by#this#limle#beam#field.#

Radiochromic#film#are#present#the#following#features:#

•  SpaBal#resoluBon#of#µm#

•  Self#developing#•  24D#detector#•  Tissue#equivalent#•  Dose#rate#indipendent#(about#5%)#•  Easy#to#use##

The#previous#features#permit#their#use#as#in)vivo)dosimetry.##

FUTURE PERSPECTIVE Validation of identified biomarkers

TARGETED THERAPY Hadrontherapic treatments combined with drug molecules targeted in vivo.

0 3 6 9 12 15 18 21 24 27 30 33 36

Data Integration

Pathways analysis

Comparison between in vivo and in vitro data

Irradiating in vivo mouse models

Irradiating in vitro cell models

Preliminary irradiament on murine model

Setup of the device

Protocol approval by the Ethics Committee

Simulations of irradiation region

2015 | 2016 | 2017