Några glimtar ur professor Bertil R.R. Perssons Några glimtar ur professor Bertil R.R. Perssons translationella gärning under 4 decennier vid translationella gärning under 4 decennier vid vad som nu är Institutionen förvad som nu är Institutionen förvad som nu är Institutionen förvad som nu är Institutionen förKliniska Vetenskaper, LundKliniska Vetenskaper, Lund

presenterade av hans vänpresenterade av hans vänpresenterade av hans vänpresenterade av hans vänoch forskarkollegaoch forskarkollegaLeif G. SalfordLeif G. Salford

Forskningens Dag 1983 Bertil R.R. till höger i bild med hyperthermiposter (skymd)

GLIOMA THERAPY

THE HERBERT OLIVECRONA LECTURESLECTURESKAROLINSKA HOSPITALDECEMBER 6-7 2002

Grows like anoctopus.Sends migrating“ ill ll ”“guerilla-cells”into the surroun-di lding normal brain

Salford

Less than 2 per thousandLess than 2 per thousandare cured from a GBM!are cured from a GBM!

Out of more than 1100 patients treated in the pDept of Neurosurgery, Lund University Hospital during a 18-year period only 3 adultsHospital during a 18 year period, only 3 adultssurvived >10 years. They were 22, 32 and 38years old at operation One had a recurrenceyears old at operation. One had a recurrence after 12 years and the other two live happily

i h d lwithout tumour 42 and 38 years later.Due to a strong immune system?

Salford 02

Due to a strong immune system?

Examples of new therapies against glioblastomasExamples of new therapies against glioblastomastested in the Laboratory for Experimental Neuro-Oncology:

* Magnetically responsive microspheres for drug targeting in braintumours by the use of external magnetic gradients

* Modification of tumour cell metabolism and interference with DNArepair by Chlorpromazine or Methoclopramide and BCNU

* M difi ti f th li htl lk li t li H f t ll b* Modification of the slightly alkaline cytosolic pH of tumour cells by the use of salicylate

* Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives* Radioactive Thallium (Tl 201) which accumulates in the tumour cells* Electroporation in vivo in the brain parenchyma with or without

concomitant hydrophilic cytotoxin treatment (e.g. Bleomycin)* Boron Neutron Capture Therapy (BNCT)

I t till ä kli tInte tillräckligtbra för tumör-behandling menbehandling mendirekt efter denkonungsliga träffen rekom-menderade prof.P t tPersson patentstagande för meto-dens utnyttjandedens utnyttjandesom kontrastmedeför MRI.Forskningssamar-bete rivstartade

Examples of new therapies against glioblastomasExamples of new therapies against glioblastomastested in the Laboratory for Experimental Neuro-Oncology:

* Magnetically responsive microspheres for drug targeting in braintumours by the use of external magnetic gradients

* Modification of tumour cell metabolism and interference with DNArepair by Chlorpromazine or Methoclopramide and BCNU

* M difi ti f th li htl lk li t li H f t ll b* Modification of the slightly alkaline cytosolic pH of tumour cells by the use of salicylate

* Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives* Radioactive Thallium (Tl 201) which accumulates in the tumour cells* Whole body hyperthermia 42oC for 30 min x 3/week for 2 weeks* Electroporation in vivo in the brain parenchyma with or without

concomitant hydrophilic cytotoxin treatment (e.g. Bleomycin)* B N t C t Th (BNCT)* Boron Neutron Capture Therapy (BNCT)

Ethyl-NitrosoUrea to pregnant ratEthyl NitrosoUrea to pregnant ratOffspring develop transplantable CNS tumours

(RG2 N32 N29)(RG2, N32, N29)ENU

Stereotacticimplantationp

In vitro culture SalfordSalford

The rat glioma models RG2, N32, N29Stereotacticimplantation in in the brain (caudate n.)in the brain (caudate n.)

In vitro cultureof rat glioma cells

3 weeks later,a tumour (diam. g4-6 mm) giving symptoms, has developed

Salford 99Salford 99

developed

N tNot efficient

Examples of new therapies against glioblastomasExamples of new therapies against glioblastomastested in the Laboratory for Experimental Neuro-Oncology:

* Magnetically responsive microspheres for drug targeting in braintumours by the use of external magnetic gradients

* Modification of tumour cell metabolism and interference with DNArepair by Chlorpromazine or Methoclopramide and BCNU

* M difi ti f th li htl lk li t li H f t ll b* Modification of the slightly alkaline cytosolic pH of tumour cells by the use of salicylate

* Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives* Radioactive Thallium (Tl 201) which accumulates in the tumour cells* Electroporation in vivo in the brain parenchyma with or without

concomitant hydrophilic cytotoxin treatment (e.g. Bleomycin)* Boron Neutron Capture Therapy (BNCT)

Cytotoxin (eg bleomycin)Cytotoxin (eg bleomycin)not penetrating cell wall

Cell

SalfordSalford

ELECTROPORATION in vivo800 V

Salford 00Salford 00

POST ELECTROPORATIONPOST ELECTROPORATIONafter minutes the cell wall closes and the cytotoxin is trapped in the cell

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Adjuvans in immunotherapyAdjuvans in immunotherapyOpens the tumour cell wall to allow p

for leakage of tumour antigens to b id tifi d b th i tbe identified by the immune system

Engström et al. Lund, Sweden

ELECTROPORATION for immunotherapy in vivoImmunogenic material enclosed in the tumourcell behind intact cell membranecell behind intact cell membrane

Salford 00

ELECTROPORATION in vivo800 V

Salford 00Salford 00

Spread of tumour cell content,Spread of tumour cell content, stimulating the immune defence system

Tumour cell

SalfordSalford

Experimental set upExperimental set upp pp pCatrin Bauréus KochCatrin Bauréus Koch

Intraperitoneal injection of Pulse Generator forPEF treatementIntraperitoneal injection of

Radiation sterilized IFNsecreting N29 tumour cells

Contra lateral reference fN29 tumour Impedance spectrometry

ZOK Aditus Lund SwedenTreated TumourTreated N29 tumour

Examples of new therapies against glioblastomasExamples of new therapies against glioblastomastested in the Laboratory for Experimental Neuro-Oncology:

* Magnetically responsive microspheres for drug targeting in braintumours by the use of external magnetic gradients

* Modification of tumour cell metabolism and interference with DNArepair by Chlorpromazine or Methoclopramide and BCNU

* M difi ti f th li htl lk li t li H f t ll b* Modification of the slightly alkaline cytosolic pH of tumour cells by the use of salicylate

* Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives Photodynamic therapy (PDT) with laser and hematoporphyrin derivatives* Radioactive Thallium (Tl 201) which accumulates in the tumour cells* Electroporation in vivo in the brain parenchyma with or without

concomitant hydrophilic cytotoxin treatment (e.g. Bleomycin)* Boron Neutron Capture Therapy (BNCT)

Neutron capture in 10BNeutron capture in B

4He SlhPHe10 m

510Bn

Slh3800 barn

(10000 ggr > än H)

Prompt gamma

7Li

5 mBn

Thermal neutrons

(10000 ggr > än H)

Energy (2 3 MeV)

Thermal neutronsCaptured by 10B Energy

Energy (2.3 MeV)very restricted spread

N l i i llNuclear reaction in tumour cell

4He

10B7Li7Li

Bimodal radiotherapyBimodal radiotherapy

10B ti t dThermal 2.10B activatedIn the tumourneutrons

Inactive 10B 1.

administeredintravenously

B k hi lBoron uptake vs. histology

Histology

and boron uptake

overlay

Strike in the guerilla nestCrister Ceberg et. al.

Rat glioma with a contralateralmetastasis (diameter 0 2 mm)metastasis (diameter 0.2 mm)6 hours after boron infusion.

Forskningsreaktorn i Studsvik

B t ål i itiBestrålningspositionen

The Rausing Laboratory for Experimental Neurosurgeryand Radiation Physics, Lund University Hospital

Leif G. Salford

BRain GliomaI

Electro- EffectsBoronNe tron

ChemicalNe ro Stem Spinal

Bertil Persson

Immuno-GeneTumour

ImmunoTherapy.

Visse

porationand -fusionfor genetransduction

of EMFon theBrain, BBB d

NeutronCaptureTherapy

Neuro-Anatomy

cells

Epilepsy

CordInjuryGroup

Sayer

TherapyTranslationalresearch in

VisseDarabiJanelidzeHegardtEsbjörnsson

transductiontreatment oftumours,neurological

BBB andtumourdevelopm.

Munck af RydeliusAl-Khaledicollab.with

dept of Tumor Immunol, 35 researchers and

EsbjörnssonEnell

gdiseases

EngströmBauréus Koch

MalmgrenBrun

RosensköldBrun

y

Bexell

PI: ONil

researchers and staff involved5 dissert.PI: LG Salford

2 dissertPI:

Bauréus KochGrafström2 dissertPI:

1 dissert

PI: J

2 dissert

PI: PI: Gk b

PI: JB NilssonB Widegren P Siesjö B Persson EberhardtC Ceberg Skagerberg Bengzon

19801980Human IFN-Daily localyInjections for3 months4 ti t4 patients

SalfordSalfordSjögren et. al.

illR ti “Guerilla” cellnests

BrainResectioncavity

nests

The T lymphocytesActivation when T-lymphoc tes o tside the brain are

T lympho

The T-lymphocytesmust be activated in order to pass the

cytes outside the brain are presented to tumour cellswhich produce immunelympho

- cytesp

Blood-Brain Barrierwhich produce immune stimulating products such as Interferon-

Immunisation against inoculated experimental brain tumours

N32 brain tumour cells N32 brain tumour cellstransfected with IFN-gamma genes

Day 0Day 1 8 15

Stereotaxicinoculation of 5000 cells

VACCINATION

Day 1,8,15or 3,10,17

I

Day 21 DEAD CURED!Immuneresp

BRIGTTThe Brain Immuno Gene Tumour Therapy project The Rausing Laboratory, Lund University and Lund University Hospital,

Lund, Sweden

Neurosurgery and the Leif G. Salford (PI), Peter Siesjö, Gunnar Skagerberg, R i l b C li L Ch l O SRausing laboratory Carolina Larsson, Charlotte Orre, Susanne

Strömblad, Catarina Blennow, Lena-Liebera-Jannert, Edward Visse, Shorena Janelidze, Anna Darabi, Maria L K i E ll A it L GLarsson, Karin Enell, Anita Larsson, Gunnar Gunnarsson

Tumour immunology and Bengt Widegren (Lab Chief), Hans Olov Sjögren, Genetics Xiaolong Fan Johan Rebetz Anna EdqvistGenetics Xiaolong Fan, Johan Rebetz, Anna EdqvistMedical Radiation Phys. Bertil Persson, Catrin Bauréus Koch, Gustaf GrafströmClinical Genetics Nils Mandahl Neuropathology Elisabet Englund Annette Persson Arne BrunNeuropathology Elisabet Englund, Annette Persson, Arne BrunDiagnostic Neuroradiology Elna-Marie Larsson, Cecilia Petersen, Lars StenbergNeurology Anna Rydelius, Eva AskNeuropsychology Åsa Lilja, Christina ElfgrenNeuropsychology Åsa Lilja, Christina ElfgrenOncology Per Malmström, Sara Kinhult

Normal cells havestopped dividing

BRIGTTIFN-gamma

Surgery>80%

Resected cells95% glioma 5% normal Normal cells

divide 1/1 d

stopped dividingGlioma cellscontinue anddominate the>80%

resect.divide 1/1 d. Glioma cells 1/4

dominate theculture (50%)

2-3 monthsKaryotyping

l l

IMMUNISATIONIrradiation

regularly

Lymph nodesActivated T-cells

IMMUNISATION3-4 months postop.

(repeated every 3 weeks,

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(repeated every 3 weeks,4 - 10 times)

IntradermalIntradermal immunisation

Gene-modified brain tumour cells for immunisation against malignant brain tumours (the BRIGTT study)

Antigen-specific T-cells (T) pass actively through the BBB and localize the migrated “guerilla” cells. (M: monocytes)

BrainT T

TT TT

TTT M

LymphM

T

T

T Tnodes. T

T

CapillaryTBlood Brain Barrier T

M TT

T

Brain tumouri d ispecimen, during

immunisation periodT l h t bT lymphocytes brown (CD3 antibody)

BRIGTT 2000-2005 Goals: Results:Goals: Results:to study whether vaccination with

l ll iautologous tumour cells expressing gene-sequences for human interferon-gamma 1 is safe for the patients YES !1. is safe for the patients YES !2. gives rise to an immunological

YES !response YES !3. adds any beneficial effect to

conventional therapy (tumor growth, ?l d i l)prolonged survival)

BRIGTT Status Jan 2005BRIGTT Status Jan 2005# pat # immunis. age (op) survival after diagn

4 10 52 21 months DZ4 10 52 y 21 months DZ67 y 12 GF58 y 19 5 EK58 y 19,5 EK64 y 24,5 IC

1 10 + 4 52 y 26 5 GA1 10 + 4 52 y 26,5 GA3 8 62 y 10 AMN

67 y 13 BP67 y 13 BP66 y 12 AO

1 6 65 y 10 KN1 6 65 y 10 KN

Mean age 61 4y Mean survival time 16 4 m (169%)Mean age 61,4y Mean survival time 16,4 m (169%)(c.f. not imm. pats 60,3y 9,7 m)

BRIGTT Non-imm. pats Jan 2005BRIGTT Non imm. pats Jan 2005# pat # immunis. age (op) survival after diagn

11 0 62 811 0 62 869 659 559 556 657 1757 1751 1766 6

age non-imm imm.>60y 6,8m 13,6

66 659 1666 5

<60y 12,5m 22,3

surv >18m66 562 956 14

surv >18m0/11 4/9

56 14Mean age 60,3y. 9,7 months

100Patient Survival

65 t l

80

9065

55

5865

62

controlspatients

60

70

(%) 65

69

55

65

66

40

50

Sur

viva

l (

62

61

57

67

20

30

S

50

55

63

52

57

0

1057

5952

63

00 100 200 300 400 500 600 700 800

Days after operationAge in graphmean age controls = 59.6age range controls = 50-69

mean age patients = 61age range patients = 52-67

BRIGTT FutureBRIGTT Future Continue BRIGTT, add 11 patientsInclude patients younger than 50 y.Utilize h-TERT cell selection? Less delayyAdd other genes? TGF-2 antisense, IL-4 etcAllogeneic cells instead of autologous? No delayAllogeneic cells instead of autologous? No delay Tumour cells direct from resected material?C bi ith l ti iCombine with oncolytic virusesCombine with electropermeabilizationCombine with BNCTCombine with Stem Cell therapypyCollaboration with other centres

MD h.c. Bertil R.R. Persson, Vår tids Carl von Linné,

Midnattsexpedition för infångande av Aspleniumruta muraria å enda dittillsruta muraria å enda dittillskända lokala lokalen.

Vem stod bakom dennaVem stod bakom dennabragd?

Bertil R R PerssonBertil R.R. PerssonPhD

Bilden visar det historiska ögonhistoriska ögon-blicket då BertilR.R. Persson är på väg att emotpå väg att emot-taga insigniernapå sin upphöjelsetill MD h ctill MD h.c.(se pilen)

Prof, general Merritt from USAF, Brooks AFBase, San AntonioProf, general Merritt from USAF, Brooks AFBase, San Antonio

EMF – BBB studier startade 1988 vid Lab. för Exp. Neuroonkologi, USiL

© Salford et al

Världens största biologiska experimentVärldens största biologiska experiment25% av jordens befolkning frivilliga25% av jordens befolkning frivilligaförsöksförsöks kaninerkaninerförsöks försöks -- kaniner kaniner

On-line publication 2003012920030129

I i t J 2003In print June 2003

10

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7.75

met

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SAR

3.25

5.5

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t/m

d0 0.5 1 1.5 2

1SAR=1mW/kg

dmeter 1.85 meter

SAR = 1mW/kg1.85 metres awayf th bilfrom the mobilephone

d

© Salford et al

1010

Eb d( )

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met

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Eb d( )

7.75

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3.25

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t/m

( )

SAR

3.25

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Vol

t/m

0 50 100 150 200 2501

SR=1mW/kg

0 50 100 150 200 2501

SAR=1mW/kg

dmeter

190 meterdmeter

190 meter

d © Salford et al

?NMTNMT GSMGSM 3G3G

?

CWCW 217 Hz217 Hz DifferentDifferentmicrowaves!microwaves!microwaves!microwaves!

The Lund group has shown in a large material thatThe Lund group has shown in a large material that both NMT and GSM open the Blood-Brain Barrier in the RAT brain The 3G technique sendsin the RAT brain. The 3G technique sends microwaves of a different character, and it is quite possible that the biological effect of 3G differs frompossible that the biological effect of 3G differs from that of NMT och GSM. We want to test this hypothesis in our animalWe want to test this hypothesis in our animal model.

© Salford et al

TACK BERTIL !Polfarare också i dePolfarare också i demagnetiska fälten

Vännen Leif

O k å IKVL ill t k i hö tOckså IKVL vill tacka sin oerhört upp-skattade professor sen många decen-

nier Bertil R.R. Persson, MD h.c.(med en omöjlig figur av O R v g v )(med en omöjlig figur av O.R. v.g.v.)

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