Radiobiologia delle Radiobiologia delle particelle pesantiparticelle pesanti

Angelica FacoettiAngelica FacoettiFondazioneFondazione CNAOCNAO

CorsoCorso teoricoteorico--praticopratico sull’adroterapiasull’adroterapia: : l’altal’alta tecnologiatecnologia applicataapplicata allaalla clinicaclinicaCNAO, Pavia, 17CNAO, Pavia, 17--18 18 MaggioMaggio 20132013

• Interazioni iniziali

Radiaz. indirett. ionizzanti (raggi X, raggi γ) 10-24 - 10-4 s

Radiaz. dirett. ionizzanti (elettroni, protoni) 10-16 - 10-14 s

RadiobiologiaRadiobiologia

“studio degli effetti delle radiazioni “studio degli effetti delle radiazioni ionizzanti sulla materia vivente”ionizzanti sulla materia vivente”

Radiaz. dirett. ionizzanti (elettroni, protoni) 10 - 10 s

• Stadio fisico-chimico 10-12 - 10-8 s

• Danno chimico (radicali liberi) 10-7 s - ore

• Danno biomolecolare (proteine, acidi nucleici) ms - ore

• Effetti biologici precoci (morte cellulare) ore - settimane

• Effetti biologici tardivi (induzione di neoplasie,

effetti genetici) anni, secoli

E. Fokas et al, 2009E. Fokas et al, 2009

X-ray

Protons

Carbonions

RBE (Relative RBE (Relative BiologicalBiological EffectivenessEffectiveness))

Schardt & Elsasser, 2010

• Dose level• Measured endpoint• Particle charge and velocity• Dose rate or fractionation• Energy/LET of the particle

BUT, RBE BUT, RBE isis a a complicatedcomplicated radiobiologicalradiobiologicalconceptconcept thatthat dependsdepends on:on:

• Energy/LET of the particle• Cell/tissue type• Oxygen concentration• Cell cycle phase• Etc…

…and…and itit givesgives a a greatlygreatly simplifiedsimplified picturepictureofof the high LET the high LET radiationradiation effectseffects!!! !!!

RBE RBE isis greatergreater forfor lowerlower dosesdoses

� Protons have radiobiological properties that are close to thoseof photons and electrons (RBE does not change very muchwith the depth and a significant increase appears only in the distal part of the Bragg peak) so that their main advatagerelies on the superior dose distribution compared to low LET radiations

7

� Carbon ions offer an even larger efficacy for tumourtreatments due to the enhanced biological effect at the end of their range in the tumor.

RBE of C ions changes along the SOBPRBE of C ions changes along the SOBP

Variation of RBE as a function of depth in the 290 MeV carbon-ion beam at HIMAC-Chiba (Japan). The RBE is given at the four depths in water that are indicated.

The SOBP is 6 cm thick.

IAEA-TECDOC-1560

RBE RBE in vivoin vivo

RBE values based on the dose for 50% complication probability for the radiation tolerance of the rat spinal cord after 1, 2, 6, and 18 fractions of carbon ions. Spread-out Bragg peak (125 keV/mm), plateau (13 keV/mm)

Schardt & Elsasser, 2010

AimAim

• to confirm the uniformity of biologic doses in PTV

positions with in vitro and in vivo experiments

• to estimate biologic RBE values for in vitro and in• to estimate biologic RBE values for in vitro and in

vivo endpoints in order to evaluate whether the

new carbon beam works in safe and optimal

conditions for the clinic.

Radiother Oncol. 2004

MicroscopicMicroscopic understandingunderstanding ofof RBERBE

Belli et al.

Low LETLow LET High LETHigh LETioni C (15 MeV/u)

Formation of fluorescent γ-H2AX clusters in irradiated human fibroblasts at 10 min postirradiation with 2 Gy of gamma rays or 0.5 Gy of 176 keV/mm iron ions

Modificato da: JAEA R&D, 2007; Cucinotta and Durante, 2006

ClusteredClustered DNA DNA DamageDamage

It has been proposed that Carbon ions radiation produces multiple lesions within a few nm in DNA molecules, so-called clustered DNA damage.

http://jolisfukyu.tokai-sc.jaea.go.jp/fukyu/mirai-en/2008/6_5.html

clustered DNA damage. This densely localized damage might distort the tertiary structure of DNA and consequently interfere with the binding of repair enzymes to the damage site.

The The optimaloptimal LETLET

Diagram illustrating whyradiation with a LET of 100 keV/µm has the greatest RBE for cell killing, mutagenesis, or oncogenic trasformation.

Fot this LET, the averageseparation between ionizingevents coincides with the events coincides with the diameter of the DNA doublehelix (i.e. about 2 nm).

Radiation of this quality ismost likely to produce a double strand break from onetrack for a given absorbeddose.

Radiobiology for the radiologist, 7th edition

The The largerlarger sizesize and the and the longerlonger persistancepersistance ofof foci foci observedobservedafterafter C C ionsions comparedcompared toto γγ--raysrays can can bebe due due toto multiple and/or multiple and/or

complexcomplex DSB DSB whichwhich are are difficultdifficult toto repairrepair

Antonelli F et al, 2005; Desai N et al, 2005

Comparison between H2AX phosphorylation-dephosphorylationkinetics after irradiation with 1 Gy of γ-rays (closed circles) or

carbon ions (open circles).

1.7 Gy of iron ions fixed 5 h after

irradiation.

ChromosomalChromosomal aberrationsaberrations inducedinduced bybyCarbonCarbon ionsions

Chromosomal damage (mFISH) in first cycle cells after exposure to 0.2 Gy X-rays or 1 carbon ion per nucleus (0.2 Gy). Fraction of aberrant cells.

Fournier C et al, 2012

1 hit C ions per nucleus

Examples of Examples of karyotypeskaryotypes ((mFISHmFISH) with ) with clonalclonal complex aberrations occurring in the progeny of complex aberrations occurring in the progeny of cells exposed to Carbon ionscells exposed to Carbon ions

Fournier C et al, 2012

5 hits C ions per nucleus

Fournier C et al, 2012

FACTORSFACTORS THATTHATDETERMINEDETERMINE RBERBE

((BASICBASIC RADIOBIOLOGICALRADIOBIOLOGICAL((BASICBASIC RADIOBIOLOGICALRADIOBIOLOGICALPROPERTIESPROPERTIES OFOF CHARGEDCHARGED

HADRONSHADRONS))

CellCell cyclecycle phasephase radiosensitivityradiosensitivity

The The oxygenoxygen effecteffectThe presence or absence of molecular oxygen

dramatically influences the biologic effect of X-rays.

Basic clinical radiobiology, Joiner & van der Kogel

The ratio of the dose required to kill the cells with the oxygen divided by the amount to kill cells without oxygen is referred to as the oxygen enhancement ratio (OER).

E. Hall, Radiobiology for the Radiologist

OxygenOxygen--fixation fixation hypothesishypothesis

OER OER isis significantlysignificantly reducedreduced forfor ionionirradiationirradiation

Hirayama E et al, 2005

The OER decreases with increasing LET

Basic clinical radiobiology, Joiner & van der Kogel

CARBONCARBON IONSIONS AND TUMOUR AND TUMOUR CELLS CELLS GENETICGENETIC BACKGROUNDBACKGROUNDCELLS CELLS GENETICGENETIC BACKGROUNDBACKGROUND

p53p53The p53 tumor suppressor limits cellular proliferation by inducing cell cycle arrest and apoptosis in response to cellular stresses such as DNA damage

x

About 50% of cancers harbormutation in the TP53 gene,

P53-dependent apoptoticpathway

mutation in the TP53 gene, which result in a decreasedpropensity to undergoapoptosis. These tumurs are more resistant to treatment, bothby X-rays or chemotherapy

• Bcl-2 is an anti-apoptotic protein• Bcl-2 overexpression has been associated with the resistance to conventional photons and chemotherapeutic agents

High LET radiations High LET radiations render Bclrender Bcl--2 cells more 2 cells more render Bclrender Bcl--2 cells more 2 cells more

vulnerablevulnerable

Hamada N et al, 2008

TakingTaking intointo considerationconsideration thatthat BclBcl--22overexpressionoverexpression andand pp5353 mutationsmutations occuroccur ininmoremore thanthan halfhalf ofof tumorstumors,, highhigh--LETLET heavyheavy ionsionsappearappear toto effectivelyeffectively killkill aa widewide varietyvariety ofofappearappear toto effectivelyeffectively killkill aa widewide varietyvariety ofofradioresistantradioresistant tumorstumors..

!

Human lungadenocarcinoma (A549)

Human lung squamouscell carcinoma(EBC-1)

Cell migratoryactivity

Invasive activity

Akino Y et al, 2009

X- rays C ions

Particle Irradiation Suppresses Metastatic Potential Particle Irradiation Suppresses Metastatic Potential of Cancer Cellsof Cancer Cells

Ogata et al, 2005LM8 osteosarcoma cells

X- rays C ions

Ogata et al, 2005

Therapeutic effects of cells irradiated with X-ray or carbon ion on experimental pulmonary metastasis from mice inoculated intravenously

“About 90% of the cells irradiated even at 0.1 Gy could be destroyed compared with unirradiated cells. On the other hand, sublethal X-ray irradiation promoted migration of endothelial cells, and the capillary- like tube structure in three dimensional culture progressed even after 16 Gy irradiation.”

BYSTANDERBYSTANDER EFFECTSEFFECTS AND AND HADRONTHERAPYHADRONTHERAPYHADRONTHERAPYHADRONTHERAPY

Morgan WF & Sowa MB, 2005

� Radiation-induced bystander effect represents a paradigm shift in the understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the final biological consequences of exposure to low doses of radiation.

� There is evidence that cells that are not directly hit by radiation, whether nuclear or cytoplasm, but in the vicinity of one that does, contribute to the biological response of the of one that does, contribute to the biological response of the cell population

� The progeny of non-targeted cells shows an increase in genomic instability as evidenced by an increase in delayed mutations and chromosomal aberrations many generations afterwards

Irradiated cells communicate Irradiated cells communicate to neighboring nonto neighboring non--irradiated irradiated bystander cells by transmitting bystander cells by transmitting an an irradiation signal irradiation signal substance, which induces substance, which induces

Iwakawa, M et al, 2008

substance, which induces substance, which induces radiation effect on bystander radiation effect on bystander cells as well.cells as well.

Iwakawa, M et al, 2008

The expression profile of the bystander cells was completely different from that of the irradiated cells.

CANCERCANCER STEMSTEM CELLSCELLS AND AND HADRONTHERAPYHADRONTHERAPYHADRONTHERAPYHADRONTHERAPY

CancerCancer stemstem cellscells“A small subset of cancer cells within the tumor mass, which constitutes a reservoir of self-sustaining cells with exclusive ability of self-renewal and tumor maintenance” (from the Cancer Stem Cell Workshop of

the American Association for Cancer Research in 2006)

In the radiotherapy

An anticancer therapy can cure a

tumour only if all cancer stem cells are

killed (without producing serious side

effects in surrounding normal tissues)

In the radiotherapy

context, a cancer

stem cell is a cell

which, when left

after irradiation to

its natural

environment, has

the capacity to

cause a tumour

recurrence.

Baumann M et al, 2008

Since cancer stem cells appear to be responsible for driving and Since cancer stem cells appear to be responsible for driving and maintaining tumor growth in many tumors, it is critical to maintaining tumor growth in many tumors, it is critical to understand the mechanisms by which these cells resist commonly understand the mechanisms by which these cells resist commonly used therapies such as chemotherapy and radiotherapyused therapies such as chemotherapy and radiotherapy

� A high frequency of CSCs are believed to be quiescent, and this would make them more resistant to cycle active agents, including radiation.

� Their residence in a microenvironmental niche may provide them with both direct physical contacts (eg, cell-cell or cell-stroma) and growth factor/cytokine signaling that may provide

CSC: CSC: knownknown (???) (???) mechanismsmechanisms ofofradioresistanceradioresistance

X

Xstroma) and growth factor/cytokine signaling that may provide additional survival signals in response to the stresses inducedby radiation

� Increased potential of defense against ROS mediated by high levels of free-radical scavengers

� The capacity to recover and repair sublethal damage between irradiation fractions

X

XX

SummarySummary

Durante M & Loeffler JS, 2010