V kterém roce byla získána Nobelova cer\a
Molekulární diagnostika
Vrozené genetické choroby - DNA změny vrozené (monogenní onemocnění, chromozomální poruchy, multifaktoriální poruchy)
Onkologické choroby - DNA změny získané
Diagnostika patogenu - DNA cizorodá
Přímá-nepřímá diagnostika
Molekulární diagnostika
Detekce prenašeču patologické alely Prenatální diagnostika Stanovení genetického rizika
Včasné stanovení diagnózy
Včasné stanovení oportunních patogenů
Diferenciální diagnostika
Minimální zbytkové onemocnění
Včasná detekce relapsu choroby
Molekulární diagnostika
•  Specif icita a senzitivita
•  Rychlost výsledku
a možnost správné a rychlé terapeutické intervence
i
Podle klasické Omranovy teorie z roku 1971 všechny společnosti procházejí třemi stádii souvisejícími s procesem modernizace:
1. Stadium moru a hladomoru
2.Stadium poklesu frekvence výskytu pandemií infekčních nemocí
3.Stadium rozvoje degenerativních a civilizačních chorob (novotvary a nemoci oběhové soustavy)
Omran, A.R.: The epidemiologie transition: a theory of the epidemiology of population change. Milbank memorial Fund Quaterly, 1971, 29
Původ hematoonkologických
onemocnění
Systémová klonální onemocnění, vznik neregulovaným dělením jediné nádorově transformované buňky
nižší počet genetických změn nutných pro vznik, někdy stačí narušení kontroly buněčného dělení
změna zahrnující cca 10 6-10 7 bp se muže projevit bez viditelné změny morfologie chromozomu
Dělení hematoonkologických
onemocnění
Stem Cell
Myeloid stem «N
3>
udali
9
;.vT:)hůid sr&mcéll
/Myeloid blast \
Red blood cells      Plareleis
A) Podle charakteru
■  Difúzni (leukémie)
■  Ložisková (lymf omy)-s nádorovými ložisky v lymfatické tkáni
B) Podle postižené krevní vývojové řady
■  myeloidní prekurzorova buňka    (CML nebo AML)
-nádorová choroba vlastního krvetvorného systému
■  lymf oidní prekurzorova buňka (CLL nebo ALL nebo lymfomy)
-nádorová onemocnění imunitního systému
LympiKüd Mast
O e
While blood celľs
Hematoonkologická diagnóza
1.  Specifické markery pro daný sub/typ
2.  Nespecifické (nezávislé) markery
3.  Diferenciální diagnostika
4.  Stratifikace podle rizika
5.  Minimální reziduálni choroba
6. Detekce oportunních patogenu
Molekulárne biologické vyšetření oportunních patogenu
IHOK FN BRNO
•CM V
•HHV6
•HSV-1,-2
•EBV, VZV
•BK virus, JC virus
•Respirační viry (RSV, PIV1-3, AĎV1-7, Flu-A, -B
•Adenoviry
•Detekce mutací vedoucích k rezistenci na léčbu Q>CM
•Pneumocystis jirovecii
•My kozy                                                                        15
^Tftrs week's citation Classic
Nov/eU P C & Hungerfard D A. A minule chromosome in human chronic granulocytic leukemia. Science 141:1-497, I960,
fSctioul of Medicine. Universiiv of Pennsylvania, and Insihure lor Cancer Research. Philadelphia, PA]
COHUMBEBS FESBUARY 15. I?SJ
This abstract descibed seven patient' (five m^le. tw,o female) *Yi(h chronic (rírtyl^íytic leuJt^mia (CGI) in ^rioni a íimifir miriuts ífir<?nio5c*me was found in rbfr rteoplaaric eŕlls in eich cas* The finding suggested a causal jŕlarŕeinsriip bf!vi*eí> the chromosome abnormality and CGL [The SCI* indicates 1hat this briei "paper" has been cued rn ever 510 publications since 19b0 J
Pet« C. Nowell
Pathology VMedicti
Tilinucr ť hrflnnrMiňlí Ľlininic ííťjrtukiĹ|H iu  [.rjtiLflllil
In rfwn d^ iTiiií fur in\fJip.,l*J 'rive miilíi. Lwn ľtmi(*i,rr j minule rh.ŕ«rtíUTMmt ha.* Oírn il*nr>fJ rfpfj^inf btc Ľ<r ihc fair hn.i!!r%l uu lovurnc-Y in Ihm (hriJlruřiuKU; í «níJÍíííHiníl of «My ní shjunc gmíluIttyErc lCükrJmii ťulLU(Ot fíCÍTt ptŕipíltral hlfpüj, Nň jbrrtUTnuliLv iji oh^írviiJ in Irw nik uľ ŕu-iir er** <if utni*- g(jnitlwj-;if kuVi-mdü in >.Il:Iu «r c E tu (too ľ-1 utjIc Iculc/iua in ľJuldŕcŕi Thíir hive h«f- ^MfiLl r«cm rfpanu of ŕhŕultuŕrtolri* a*r»ŕrYi;ilhLiri in i rtu rtthŕf (M '.i-'^ ol hniitijn IijijL(-jtíIj |jncljiling Ewa oľ ;tit '.ci.en cilyüy  KpLgfoii rtrrr   Nuinell
J!*J     HliAfC-l foŕlj,    J-      ŕ*W,     fdnriT    /niť.
IS, MS i ľHMIil, hil no. Kfics ha* upnrdrňl in »nich. Lhiŕc »ji 1 tOrVuHrfíl (h-Hlpf Ur.ii;ul oľ -j f_rl :iilj.r Ivpv of Iculeniii. t*IL uf lh* \i*t flt<* <*r> *€*( <it*-i-'ifttd Iron-i pvriphíril MihkI tjnJ kinu nuímvr in Drrr invluTKL- I. pmwrY "i CciIeuJC
ívr M-T: THrwfi. mJ pn«»d ľ<w cyl»
l(itii,'j.1 (HirTiiniliTMi hj !i ľLLLMľľi Uc-.[lupe J air-dryirif lt-.h-'iiitilt IMrorh-EJiJ. í-T J.. ŕrJ'Jwŕ. {\l( krinirflt. in prijivl. The piilimlv '.ai'iciJ IrOfll aVifflrdwnrlfc híirŕJteJ    ííiyíí   1cj    fiK nsiiílh'    lrr-iicd
Cllíi Oľ Itvtral yesrr Jlirjlimi in Irrminií rn^lpWimic  .ľri-iv   All   ■*<•«*  ijvlIi■-iLli■ -ä-1 F
^f40«.ŕd J ^iíTiil.ir fniniiue [IiEximcviDfnc:, :inJ none ihrnwcd any ďhcr f[ti;.irni Oť riíiil.ir ch IPrtiOWnií íhinp. In inml oľ h h c cj«í, cíILl vHih noŕiťiil chrarno-^mH
u.crc ;il^S OriiŕrvcJ, 'ITiu, i"iŕ minnk ň IKH 3 pni oľ Ihí iKipmnl íhŕOnK^amt onnslLlultSn of ^lioh r-hj iviJiuk.
T hr (inJinfi MiEgcii a ciuhiil íclnhon-thip M*Kn 1hc chrDmDVHnc iKftOfŕTiJilily pKcri.cil   ,iíhJ    chucn«   (rjnulcV) Lit   ItU-
■AnlL
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Imtimtf lor Catwtr Kcita'ih
cells, and he looked at lbem Our first casts, of acule leulemla, were unrtwaiding. Then Dave spotted a small chromosome in cells from two male patients with chronic granulocytic leukemia (CGL) These findings were published with caution [because the Edinburgh group had found no abnormality in CGI4) and with the suggestion that the "minute" chromosome might be m allered V. Subsequent cases, including women and using an improved ,Jair-drying" techniques led Dave to assign the minute chromosome cor-rectly to the larger pair of G group auto-mbered 31 and later changed, i, to no. 22. The additional re being readied for publita-ftichards asked us lo present National Academy of Seihe was organizing at Penn. published in Science, led to on [and citationj of Ihe first iiiitent chromosome abnor-isia. The Edinburgh group ^sted the name "Phi lade I' 'SO me."
de was frustrating. Cyfoge-CCL proved of some diagnostic value and provided clonal evolution in tumor helping to ««plain clinical m. But since other consis-' changes in neoplasia were nificance in tumorigenesis nd the term "epiphenome-used. With the advent of ndtng techniques in the lecular genelit techniques itory changed dramatically, igenetic alterations were iany tumors and are now f u I for exploring oncogene iromosome studies remain oking at genomic changes, nise of the Ph chrrtmosome of specific genomic alter-in human tumorigenesis is fruition.
r, ^116-17. 1155. (Cited 7p riratl.J adina 1he chrDUDDHHim of cf Hi In
«n 1evhDť>t?3
tumaít leukatniLj.
»Olime pr«ptrttL4nt of kjkocvtcS ill*: MSMtud P S.
ffmenisancl nAU'ítfnťMj
igůfl      ítieticB  1J2,1»í97
'í
Chronická myeloidní leukémie -CML
1845 první dokumentovaný klinický popis choroby
1960 publikace   Ph chromozomu u CML pacientů
1970 izolace nového myšího onkogenního viru Abelson
1973 odhalení reciproké translokace t(9;22) jako příčiny Ph chromozomu
1980-1983 klonování v-Abl onkogenu, c-Abl protoonkogenu, jeho lokalizace na chromozomu 9 a demonstrace, že Abl kóduje protein-tyrozin kinázu
1986 klonování BCR-ABL cDNA z CML buněk
1996 imatinib mesylate   inhibuje BCR-ABL tyrozin kinázu a CML buněčný růst in vitro
2001 imatinib (Glivec) jako selektivní inhibitor je potvrzen v terapii CML
30 - 40 cycles of 3 steps :
Step 1 : denaluration 1  minut 9A °C
TTT" n "r-TTf rTTTTTTTTTTTTTrr-rr
n^f^TTlTnTrnTr^rrr^^                       y       Step 2 : annealing
.-»            v in   nrlli I  Mil   <>
3" liuiLLLUilill 5

45 seconds 54 ÖC
forward and reverse primers !!!
5"
I      \
I
35'S^^
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-      J'^JJlLLLJMlLiUiiljUjIiiliiliLs1
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35th cycle 2 36 = 68 billion copies
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Enzymatic Amplification of ß-Globin
Genomic Sequences and Restriction Site
Analysis for Diagnosis of Sickle Cell Anemia
fcmdaU K. Saiki. Stephen Scharf. Fred Fakxma. Kary E. MuUis Glenn T. Hont. Henry A, ErILch. řfommn, Arnheidi
Recent advances in «ewruiiriant SM A technology have made possible- the molecular analysis and ppmatal diagnc-n* of lcveraJ numac, cenené diseases, heiaL DNA obtained by imiitOíHliesis « chorionic viUiii sarnnlini tin 1* analyieJ by reiiriclion elYZVDte JÍBÍ5IÍM. «with sobvc-quent electrophoresis. SouLhem tniAa-fer, and specific hyciidizaiifln í» cloned Brne «r otijůnueleotide prob«.   With
Thli disease mulls from hsmoiyfjosily jf UM tieide-celL allele IĚS) ai lit p> itobiA (BÜ loco*. The S allele differs from ihe »iH-type »Utk IBA> by «itali-itilwn uf an A in lbs wjld-lype in a T at the vee*íid posiiioň üi the si**h tödön of the e chain jene. resullinp. h Ihe replacement oí s * [manne «id hv » ™hflc '" <h* enpresMtl protein. Kw the gnniul djaj.-nwis Si sktle od anemia. DNA ob-
Abitracl   ľ™ new ne»*0* "'f" ""^ '* »««*"'* ů ™P«Í *"* * W* K""1*«
ŕrtiWKií dKunoiirf rfiJŕrjirUí r<í(™»Mii. 7V>nr «wtw '*« prmtr-mtimitJ
(iuvntótlf anrjrfířírurion o/ JpeYttK P-n'rHNU rortfí JeoťíŕlCfí "■ («toniie DÍM. wuJir'jMr m f*' tifowHvS tittrtair ii».ŕM JŇneíl ti/ra/erf D WA rapi«- '* Wie „eond r«*,«,». tne Jv™,ŕr »f* U1 ^ Ě1 <•"•<■" '^ .frrtn-iVrf iy'">""'""
(-JHfajiKtaur Jôrejríiifl n/ a™ tnít'tvbrl'il aríUínuŕlMNÍÍ ŕ™(* M>™rJ«r<' ut joint™ W i*e i/njriSM ŕ-JcJocJín i«rur/IÍM. í*' h-Jiŕtf&in ře-oryne f« ** delfmínf« i" O« r*W ŕ ^y M HMipfM «MMWim UI/lífflMlfr íf« ŕ**" ' mic/ORťům tí penrtiilrr D^A.
polyraůipliit ONA mlíttcs United frenetically to * specific Jiná« loci", set-re^aijún analysis myli be carried «ji wilh restnciion fTairfleei lenilh polymorphisms lUFLP's» found to be: Softe mau™ by tiimiiunis DNA in™ family members <í. .TV
Many ůf live henujslohmopattiies, twweťíi. can br iítictted l>y íňůre direct melhůíls in which anály» of ibx feuui akmciä «iflicieni íof ciisioin. Fores-ample. Uie diísneaii of Kydre.pi felilii tlwTuQayíoi>s íi.ihalaiwrrtiaŕ íai be m*d* hy dociwťienunt the atfltnee of *ny (t^lfthin jenei by hybrnüiiiinn with m a-alubin probe IM1. Homeiysoiity fi» ceriiin U-tbid^«mia illeies can >e fie-teniuňcd i a Süülhcni tcMifer espeí^ TiKňia by Tisine oliarniocltůtidí prohd licit form stíWe JupleK* ^ilh lile i»*f-m*J p-ajobin t^rw: se<n^:nfie bot fcym oniubic hvbňtls with iBKific muiaaii (Ě. 71-
Sickle c*U anemia cín alio he Jiaj-iioved by dbeci undyns ní fe<»l O^A.
laincd ty íminiMenliesil« chorem«: vJ-Lüi sampliflS íim he treated with i t» íinccioa cndünudeM* ifor e^iJüplfi. Due ] ssvt Út lít thai recosniiti a scnutnce iJieJtdi by rlie p1 mutamm till). Uli; generates i1-- ami iť^petifit ntiTfictLon rragraeii^ ihai íarv h= n> solyerl by Svumem inmiCtr and hytwid-ixarioťi w^iii a ä-globm probe.
We have developed a pracedure: tct the deieeiLon of Uw uetlc ceJl mutiiion ■hit is very raoid and ii at leaji im! orders «ľ magnitude um wtisui« ihau irandart SuUlhern. blorrinj. There air !Wü special feature! tg rhiipcolocOI. The jint is a method for arnpufyini ipeciik: p-ajobin niJ A sequencei «Till the uae of oliícmucleůddie preperi and DMA Polymerase Hi). The yetajid is Ihe analysis. ■A the S-Slobui jenoivpr by loluiiLm hy-hTidiiatiOfl of the aoielited DMA wnh a speciíe oliiiomKKeitide probe: and sub-seíivent ilijeuion wiih a Kstnctiim en-donocleasc Iŕ/Í. These Vw lechnKnJ« Lncruiae tbt ipetd and innsiliviiy, and
[essen the complexity oi rweiiaial laiagnox mi for rick le cell anemia; they may also be jcneralLy applicahJe t? üw ■-:-u:.n.|.; ůf other Benenc disease! .-i:d in the. es$; of DNA jifobci for inCřtiioiti disease J i-.r^-'-
SequHKX im Uli liö n i- a hT ^nm e chain reaction- We use a iwu-itep pnace-dure for dereieanunj the |š-ĚHHlin fctiio-Lyec of honĽĽi fjcnomic IJ--A samplea. Fir«h a snsall portion ^ lee |S-sbťAňi tcnese4uenec spanniroilhepolyrhurniiJA IMe I fcsínčiiůfi sile Jnucnc-iliu of llic [J* allele issmplirkd. rfrai. the pcfscn;c or abjavoe of the Dde I r«ifi™»n ii« im lbeampii/ved DNA wtnple a deiermirtet by solution hybnditatiůn wťdi an end-labeled complimentary ubgcmcf followed by resiricuon cndůniicícase diatt-tion, saeclrtíhůKsis, ami auiotadiop*
phy-
Tie &-sJt*iti jene sesmenn "as arririo-rted by the iKtyrnerese chain reaufaH (PCRJ procedure of Mullia and Falomu ill} in whidi we used Iwo. 20-iiSí ůlbjů-luieleoüde pfiowrs lhát run« me rejioa tu he amplifled- One juiffltr. fC«. ii eůnaplemeniary to the (^Hltand and the ^iher, PCOJ, is complemciitiry to ±e i-Kirand HPHe.   I>. The ajinealiiii ef K.1H M (be (+r*eaod oj dtnaiured i=-nooúc- DNA followed by eiKnuon «nth ihe btleaow ffajmenc offjcAenc^ira coli DMA polymerase I arad deosyciiKleotkki tnplBiphate» rcaults in the syntht sLi of a C - ■:'.: and fraginc ii: c jni.nnin? the large* sequence. Al the same tune, a sinuJar rracnon occurs with PC03, creaiiru a new l-r-j-strand- Since these ae*ly sy»-tlTevccd DNA iirands are Jiemsel«» remplate for the PCR primen. repealed cycles of denatnration. pnmer ajtriealicvl* ami eniension result n Ihe e<fonenii»l ^cumulation Of ihe Llů-base pairieRŠoa deľincd by the primers.
,\n eiample of ihe degree uť 'E«** tone ampliFjcatiDn achieved by ihe CCP metluKl is: shown m Fie, 1A. Sampicaioi DN A 11 iitl were amplified for ÍŮ eye*0 andafffiouon ofcacri larr.ijlc. cquivale"! to » ni oi the ůíipnal DNA. ™»s st*-jecutd to alltiline jti eleclroprioresiij™ umtferred to a nylon filter. ITie filui vnj then Ityrmdurd with a ^P-lalw]*1 40-bate oJiiouueleoiide urthe, Ei*. which is complic«ti[riry i# the lanp wqueoct HFirj. 1AI huf ~H is (he W1 pníůen. The rsmlti. afters ;-boutai*5' railiöftraEiut eKposure. snow lhal a Ě*ř ment ^lybriditilUJ "nth the P,3* (««•
Tb. .uihon m i He tel*«™ ^"iSS Si™!. t™r—. .-J«™ «* ■■■< jj£ d», cnormiy ■* 5*iibcm Oiŕwt*. L*1 ^"^
iqiEKit, «OL S*
Science. 230 (4732): 1350-1354. 1985
Kompetitivní PCR a vyznačení bodů ekvivalence

1: Biotechnology (N Y). 1992 Apr;10(4):413-7.
Simultaneous amplification and detection of specific DNA sequences. Higuchi R, Dollinger G, Walsh PS, Griffith R.
Roche Molecular Systems, Inc., Emeryville, CA 94608.
We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube. This enhancement requires the addition of ethidium bromide (EtBr) to a PCR. Since the fluorescence of EtBr increases in the presence of double-stranded (ds) DNA an increase in fluorescence in such a PCR indicates a positive amplification, which can be easily monitored externally. In fact, amplification can be continuously monitored in order to follow its progress. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample throughput.
1: Biotechnology (N Y). 1993 Sep; 11(9): 1026-30.                                                                    Related Articles. Links
Kinetic PCR analysis: real-time monitoring of DNA amplification
reactions.
Higuchi R. Fockler C. Dollinger G. Watson R.
Roche Molecular Systems, Inc., Alameda, CA 94501.
We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.
1: Nucleic Acids Res. 1993 Aug ll;21(16):3761-6.                                                                  Related Articles, Links
Allelic discrimination by nick-translation PCR with fluorogenic probes. Lee LG. Connell CR. Bloch W.
Applied Biosystems, Division of Perkin-Elmer, Foster City, CA 94404. Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.
ABI PRISM 7700 (Perkin
Elmer/ABI) první dostupný RQ-PCR systém s laserem /l 996
Light Cyder (Roche Molecular Biochemicals) extrémní rychlost, kapiláry
5700SĎS (Perkin Elmer/ABI) halogen, CCĎ /l998
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			Standard Curve										
34													
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30													
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28													
													
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24													
													
22									*				
													
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	5	3       3,5       4       4,5       5       5,5							6       6,5			;	7
			Log CO										
Slope: -3.778070 Intercept: -14.51 EGE1 Correlation:   0.SSSSG8
[p^i      3
Leukemia (2003)17, 2318 2357
■S 2003 Nature Publishing Group All rights reserve: 0887 6924/03 $25.00
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LEADING ARTICLE
Standardization and quality control studies of'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual dis detection in leukemia - A Europe Against Cancer Program
Detection of minimal residual disease (MRD) has proven to         (EAC) program. Four phases were scheduled: (1) trai
provide   independent   prognostic   information   for   treatment         optimization.   (3)   sensitivity   testing   and   (4)   patient
stratification in several types ol leukemias such as childhood        testing. During our program, three quality control rour
acute lymphoblastic leukemia (ALL), chronic myeloid leukemia         large series o1 coded RNA samples were performed inc
(CML) and acute promyelocyte leukemia. This report focuses on         balanced randomized assay, which enabled final valic
the accurate quantitative measurement of fusion gene (FG)        the EAC primer and probe sets. The expression level of
TSl
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Control Gene Standards:	
Product	Reference Hune
CGRŮ-01	ABL
CGRŮ-02	B2M
CGRŮ-03	GUS
CGRŮ-04	TBP
Fusion Gene Standards:
Product	Reference Hune
FGRS-01	AML1-ETO
FGRS-02	CBFB-MYH11 A
FGHS-03	CBFB-MYH11 D
FGFIS-04	CBFB-MYH11 E
FGFIS-05	PML-RARA boří
FGF1S-0Ě	PML-RARA bcrí
FGFIS-07	PML-RARA bcr3
FGFIS-Oa	E2A-PBX1
FGF1S-Ü9	BGR-ABL e1 a2 jn-bcr
FGHJS-10	BGR-ABL b3a£ M-bcr
FGHJS-11	TEL-AM LI e4*11
FGHJS-12	SIL-TAL
FGHS-13	MLL-AF4e10a4=HS411 typa
FGHJS-14	MLL-AF4 e9e5 = MV411 type
FGHJS-15	MLL-AF4 e11 aS = ALL-PO typa
FGF1S-1Ě	MLL-AF9 typa A
FGHJS-17	MLL-AF9 type B
FGHS-1S	MLL-AFĚ
FGHJS-19	MLL-DUP
FGFIS-20 New	MLL-ENL ex9
FGFIS-21 New	MLL-ENL exIO
FGFIS-22 New	MLL-ENL exil
FGFIS-23 New	MLL-AF9 «9
FGFIS-24 New	MLL-ELL ax9
FGFIS-25 New	MLL-ELL ax10
FGF1S-2Ě New	MLL-AFIp ax11
Bibliography
i.J. Gsbertet d. Stercfcfctzaltriand quBlry control studies d
"real-ma" quanHIaoVe reraa^lranscrlplaseportfiieraEecriElri reaction
(RQ-PCRioTlLElongene transcripts 1or .ti řtnal lestbat crease
detection In leukaula - ŕ. Buq:« AgEŕisJ Cancer Program.
In Prší-, Laments.
I.E. Ballard at at. Evalualon or canddata control genes for dagnosls
Výhody RQ-PCR ve srovnání s end-point PCR
*  amplifikace je průběžně monitorována během reakce
*                                             s ampl ikony (snížení nebezpečí kontaminace následné PCR)
(30 min-2 hod)
*  široký dynamický rozsah 10-10 10 kopií, menší spotřeba testovaného materiálu (RNA),
*           specif ita, senzitivita a
*  cena reakce srovnatelná
Nevýhody RQ-PCR ve srovnáni s end-point PCR
t vysoká pořizovací cena přístroje (stále ještě)
t vyšší nároky na technickou zručnost a interpretaci výsledku
t problémy s RNA kontaminovanou gDNA
Ee.org
.FRED NOBEL
:e in Physics          Nobel Prize in Chemistry          Nobel Prize in Medicine          Nobel Prize in Literature          Nobel Peace Prize          Prize in Economics
The Nobel Prize in Physiology or Medicine 1990
"for their discoveries concerning organ and cell transplantation in the treatment of human disease"
	jj£
i^iŕ	
WLc	fc
j*>	^M
* -By	M
Joseph E.	Murray
<D 1/2 of the	prize
USA	
Brigham ant	i Women's
Hospital	
Boston, MA,	USA
b. 1919
E. Do n nail Thomas
<Dl/2.of the prize
USA
Fred Hutchinson Cancer Research Center Seattle, WA, USA
b. 1920
F^l Printer Friendly
SjP Comments B. Questions
EE3Tell a Friend

The 1990 Prize in:
Medicine
® Prev. year     Next year ©
The Nobel Prize in Physiology or Medicine 1990
Press Release Presentation Speech
Joseph E. Murray
Autobiography Nobel Lecture Interview Banquet Speech
E. Do n nail Thomas
Autobiograph/ Nobel Lecture Other Resources
Blood 1959; 14:720-736
Homografts of Bone Marrow in Dogs After Lethal Total-Body Radiation
Btj E. DoNľfALL Thomas, Charles A. Ashley, Harry L. Lochte, Jr.. Alfred Jargtzii III, Ono D. 5ahlej< and Joseph W. Fehrfbef:
11FE-SAVING HOMOCfiAFTS of bone marrow after lethal whole-body j irradiation usually succeed in rodents1 but fail in dugs.'-" Recently we reporteil a successful and life-saving homograft of marrow after 1,200 roentgens of total-body irradiation in a beagle,* Success was attributed to the following procedures; (1) use of a donor and a recipient with a close genetic relationship (litter-mates), (2) use of a supralethal dose of irradiation (1200 r
n vet ,i three dav period), and (3) splenectomy and administration of ACTH hi the recipient prior to irradiation. It was felt that splenectomy, *\CTH and the supra Lethal dose of x-ray were necessary to inhibit vigorous immune reactions that nurmally make Ibumotranapldntatioii impossible in the canine. The intensity of host-donor reactions may have been further reduced by the use of a female litter-mate as the marrow demur.' Studies with skin grafts have indicated an occasional relative histo-comoatibility between litter-mates in this species,4
The present report describes studies on 27 dogs. An attempt is made to evaluate the relative importance of the procedures tinted above- In addition a follow-up account >s given of the subsequent clinical course and autopsy findings in the dug previously reported (dog 1, tabic 1),
Methods
Th* animals Studied war be*ifei, ň u/relcs lu \H months iJd, rvlutiwly puivbred but ■u* inhrd.   Tvŕcnty.fivĽ   wüne   male»:   Iwu  Vmu:  fťiiul»   f 17   ;iikJ   tri,   lablľ   1).   TllĽ   tlojs
w«r« purchased Írom outlying farms and confined in n kxut vetťrinitiy kmncl  in tu 14
ílays   brfnhTR   use-.   IJo^p   Ich   bt   irradiated   were   treatrd   fur   wunus   with   Vernňple*.*   lnl-
itiuiiuanon against distemper and hepatitis was attempted Ivy adniinirtration of a formalm-Idlled virus preparation (í ml. of Virtwun ĽJ-H*}. Irradiation w,™ given with 3 General Electric M Hindi Unit, operating at £50 lev. arid ID Ma. with ,1 half-value layer of 2.2 mm, or copper, and 11 tniptt-snurer distnnee of ion em. "flic conditions used to uehiive uniform
vťtiůlif-bťrcly iiradiatjuii   WLie previously   deKXJlx'd.'  A   důee uf -100 j  iiK'aiured   iti  uir  wu*
gi™n on (lie hrst day. Don 14 received 2ÜÖ r en the second day. All other dogs received
4O0 r on  tfie strand day.  In tlw?  l'3m: of anliiuilx rcuľivinjj   iiMtfe than   StKI r  aji  jdditiunal
From the Mary Iihujeob Harnett Hinpital (affiliated with Columbia Universit?}, Coapífatowa, N. V.
Supported in part li> J research jj^uit {C-2fHH} frum Hi* United Stit« PobÜc Health Sendee and hy the Atomic Energy Cmiuidsritm, Cuoitrat't  A.T( 30.1) .SOUS.
A portion, of this materia] was presented to the meeting of the American Society ior Clinical Investigation, Atlantic City, May 4? ]^>H and Ih> the mee-ting «f the American Collect of Physicians, Atlantic City, May 1, 195*.
Submitted  Aug.   4,   I9SS;   aĽtejJled   für pnhlleatüm  New.   [S,   I^ŤJS,
*Fitman-Moore Company, Indianapolis, Lid.
"ill
U Thomas DE p r vnf transplantace pes Blood 1962.pdf - Adobe Reader
Soubor   Úpravy   Zobrazení   Dokument   Nástroje   Okna   Nápověda
Qi 43*      I ♦ *H'13 j« ®
\d ä
Hledat
MARROW TRANSPLANTS IN IRRADIATED DOCÍS
219
							
WBC							
lO4-				Bi>	DOG 401		
				^*	15.5	x  10*	MARROW
					\ l		CELLS
							
							
						^     >v	
I03-					T 1250 R		lx|                       / i \                      / \ \                    í \ \                 f i L                / i \               í 1   \            ti
I02-	:						L i* "X \ \ \ \ v \DOG 407 (NO MARROW)
Ký<	"     D	D	D	D	D     D		\-------.x + G     D     G        DD
		METHOTREXATE			1.0 >	mgm.	1                 ..J--------------L------------1--------------1--------------1------------
-10   -8    -6    -4   -2     0     2     4     6     8     10    12    14
DAYS
Fig- 1-—White blood cell counts and Methotrexate administration in two dogs irradiated with 1250 r. Dog 401 received homologous marrow on the day after radiation and was alive and well 201 davs later. Dog 407 received no marrow and died on the 12th dav.
...wl   <lw.   m,.n----------:-    y.^Tl..!,.-   -I.    ....+„,■„■, ,      [f   ,'.■    »liinniir    4-Vi.if    ,1    ■ ■ i i,.. ir ., ■ ľ.£ijJ_ljUUUlĽJiUlJ-
In the fall of 1955, Thomas and colleagues began infusing patients with donor bone marrow harvested from fetal and adult cadavers, from ribs removed at surgery and from hipbones using an aspiration needle. The collected marrow was passed repeatedly through a stainless-steel screen and broken into a smooth cellular suspension. Fat was removed using centrifugation. The marrow cells were suspended in tissue-culture fluid and serum. Unused cells were frozen in glycerol and stored at -80 C.
d první ublikace trampi human 1959.pdf - Adobe Reader
Soubor   Úpravy   Zobrazení  Dokument   Nástroje   Okna   Nápověda
♦ ^Q]'8 i t 0
1* í ME 4
•T ■» 4 Hl   i  S T 9  II  130 17 19 2t a 25 27 2» JI SBSr^tTSSOIIgSttBttffll
C^mwmw             DAtS POST RADIATION                           WEEKS
Fig. 3a.   Hematologic Events in Case 2 The small rertanffle laheled "Cn*" indicates the administration of a midline air dnse of
8,50x11,00"
J Clin Invest 1959; 1709-1716
1958 On October 7 the marrow donor was taken and under general anesthesia 25 marrow aspirations were performed on the sternum, tibiae and anterior and posterior iliac crests. This marrow was transferred immediately to Hanks' solution containing heparin and passed through stainless steel screens. The marrow was frozen in 15% glycerol and stored at - 80oC. On October 12 this marrow was thawed, deglycerolized and administered to the irradiated recipient 3-year-old girl with end-stage leukemia This time, a large dose of radiation and marrow frort twin resulted in a successful transplant. The patient did well for six months until her leukemia returned.
5UFRALETHAL WHOLE BODY IRRADIATION AND ISOLOGOUS MARROW TRANSPLANTATION IN MAN-f
Jív E. ĽONNALL THOMAS, HARRY L LOCHTE, Jb., JOE H, CANNON, OTTO D, SAHLEÄ um JOSEPH W. ťEKREBEE
(Franc Ihr Mary InHtginr Basseil Hvipitai [affifatttS with Columbia University),
Cooperttüum, N. Y.)
(Submitted fůr publication May 5, 195*; accepted June 19, 1S59J
Infusions of normal marrow Will prevent death from marrow i: i lLi : re in animals that have received lethal doses of tutal body irradiation, doses oľ t lie order of 1,000 rucntgcns (r.) (1), Hyanal-Qgy similar infusions might bt txpťctŕd to be use-rful iu treating postradíatíve marrow failure in man. Two patients with leukemia requiring treatment by radiation Have been given 850 r. and ljL40 r., re-apectively. Each patient had an identical twin to serve as donor of normal isologous marrow. In these twins it was possible to study the problem.-- of lethal irradiation and marrow restoration Free from the immunologic complications of humo-transplantation. It wa£ ftlw possible to observe the effect of lethal irradiation upon leukemia in man.
METHODS
The methods «f attaining, preparing, storing and infusing bone marrow have been described previously (2, 3, A"). The two Co* units used to administer whole body irradiation hava atso been described (5), All Mood transfusions were of freshly obtained blood drawn into plastic bags containing 50 ml. of i í per cent etliylene-:i:"i eneteli •■..i.Mií acid (EDTA) and 0.7 per cent NaCI.' A platelet transfusion (one unit of platelets) represented the platelet concentrate from 5O0 ml. of blood obtained hy difierential centrifugarion. in plastic bags i fů). Platelet counts were performed with a phase microscope by the method of Brecher and Cronkite {7). Reticulocytes were counted by LUe method of Brecher (&). Hemoglobin was determined  by  the tyamnethemaglobin  mcĽh.ŕ":    [9).
CASE REPORTS
Cast I; íD. C. Number 793S9}. This colored female aged twů years and eleven :.r ■ -1 ľ li =-  was :idni:ii(-.-! to the
* Supported \:y ;± grant from the John A. Hartford Foundation, Inc.; Research Grant C-2&i3 from the United .Stales Public Hearth. Service; and by Contract AT (30-1) -2O05 from the United States Atomic Energy Commission.
Ť Presented in part before tne national meeting of the American Society for Clinical Investigation, May, 1959.
1 Fenwal EDTA Blood-Pack, Ethicon, Somerrilleh M.J.
Mary Imogene Bassett Hospital for the first time on October 4, 1958. She was one of identical twins with a history of normal development She was seen at tlie Harriet Lane Home of the Johns Hopkins Hospital, Baltimore, Maryland on June tl, 1953 because of swell' ing of the eyes and lips, diagnosed as angioneurotic edema, which responded to Benadryl®, She was admitted to the Harriet Lane Home on Jtdy 25, 1958 because of nigt '. Sweats, vaulting, mild latiguc and a low-grade fever. At that time her white bloud cell count was ■! 7.■"■'■< pčť tu. mm.r and the ■: I i iL litíti e i;±! showed 57 per cent blaat cells_ The hematocrit was 20 per tent. A bone mar raw study showed hypocel tularity and numerous blast cell*. A diagnosis, of acute [eulcenu'a was made JnitiaJly the patient was treated with o-msreaptopurine, 2.j mg, per day, On August 29, 195& Prednisone® was started at 30 nig. per day and increased to 40- ing. per day on September p. On September -it\ ů mercaptopuriiie was discontinued and Methotrexate® was started at 2.5 mg. per day. There was no sign of rcmiiBJQu, and the patients persistent anemia required transfusions of 250 ml of whole blood on August 14, August 27 arid September 14. Because uf failure to secure a remission on chemotherapy x.'l because the patient had Rn identical twin, it was decided to transfer her to the Mary Imogene Basse« Hospital fur whole body irradiation and marrow transplantation:.
Her physical examination on admission showed resemblance to Gushing syndrome. There were sbotty cervical and inguinal nodes and one smalt left axillary node, The abdomen was. protuberant, and the spleen extended 2 cm. below the left costal margin. The liver was not enlarged.
Laboratory data shewed a normal urinalysis, The he-jnoglobin was 4.b Gm. jjer cent The white blood cell count was 3,&00 per tu. mm. with 19 polymorphonuclear leukocytes, 2 band forms, 2 blast cells. 50 lymphocytes, 5 atypical lymphocytes, 1 young lymphocyte and 21 monocytes. There were 2Ů nucleated red cells per lufl white cells. The platelet cotint was 13,000 per cu. mm. and the reticulocyte count 27 per cent. A chest X-ray w*s no-rmal.
The patient was: continued on Prednisone®, 40 mg. daily. She had been receiving Methotrexate^ 7.5 mg. daily. This drug was discontimied on October 9n Tne patient was placed in strict isolation. She was observed for infection, and frequent cultures showed no significant organisms..     Whole   body   irradiation   was   started   on
1700
DOGS AROUND THE WORLD ARE HAPPY ABOUT THE NEWS!
2008
Stem Cells Now Curing Dogs of Leukemia
WS U to offer bone marrow transplants to sick pets It's a big "give-back" to dogs for cancer help
The bone marrow or stem cell transplant, a procedure that every year saves tens of thousands of lives and won for the Seattle physician who pioneered it the 1990 Nobel Prize in Medicine, appears poised to come full circle and finally become more widely available to those who first made it all possible.The WSU transplant program, which will be a partnership with a private business based in North Carolina, is intended to make the procedures available to pet owners for about $15,000 to $20,000 per dog. Dogs suffering from lymphoma will be able to receive the same type of medical treatment as their human counterparts, as North Carolina State University becomes the first university in the nation to offer canine bone marrow transplants in a clinical setting. Dr. Steven Suter, assistant professor of oncology in NC State's College of Veterinary Medicine, received three leukophoresis machines donated by the Mayo Clinic in Rochester, Minn. Leukophoresis machines are designed to harvest healthy stem cells from cancer patients. The machines, once used for human patients, are suitable for canine use without modification, as bone marrow therapy protocols for people were originally developed using dogs.
ABK PRISM
194     196      198
Detekce buněčného chimérismu po HSCT
HBI     lG:10ľD/
lij    ZGitfflV
BB      1 R : 10ZD / ■ I      ZR: 102P/
Dye/Sample Peak
|      IG, 61
IG,64
2G, 63
2G, 65
Peak Height
Peak Area
53936
PRISM
Gum-Scan» 3.1 M
Pagsioti
Pagei oil
HBI     3G : 102D * 11 OD /
BDI     8G:102^2D/
BBI     2G:102-48D - 99P ar /
HBI     6fi : 102-1140 /
BB       3 R : 102D . 11 OD /
I       8R:102-42D/
□ □       Stí : 102 48D i 99Par/
BB       fift : 10?'l 54Í1 /
Oye/Sample
3G, 42
3Ľ   4-1
8G, 93
SG, 94
an, 9 9
2G, 1 S 23, 16 2G, 20
6G, 50
6G. 51
Minutes
1S.02 IS ?h 15.91
16.14
Size
189.53 1 tfi 41 181.90
UK. Iii.
Peak Height
7134
419
51SS 6530
G070
Peak Area
3164
37007 47401
■H9fi0
4368
4472 4401 «"3
Hematologický relaps-příklad monitorovaní pacientu do HSCT
VH
Si
i_
O
U)
_o o
+-' _> (0
o
154 dní
R
RQ-PCR
FA RQ-PCR
1.8.2006
fontiiwriDftKřiDralKi frtfniliotuftfceJdonüi
MnirwMlierstí in cpeitfing rwm
Filtered nunusii gLVĚ.i tg íc-:ipk-Ti
BMT
Gleevec"
fimj.1..-:* iwiíi^ía;^

2001
Point mutations in the Abi kinase domain
(MAA^y
CV3J8I y (L3B7M)-
(H3KR)
vation loop
H 396
Catalytic
After Ohvashiki et al, 2004
Screening of ABL kinase domain mutations
RT-PCR: region ABL rearranged domain in BCR-ABL by Expand High Fidelity Enzyme (Roche) Primers: F-BCR-A (exon el2/el3) x R-ABL-A (exon a8)
typ b3a2___________________________________
j e11 |  e12 | el 3 | e141  a2  | a3
R-ABL-A
i a8 | a9 j
F-BCR-A
Conventional direct sequencing: by using a BigDye®Terminator v1.1 Seq.kit/AbiPrism 310 System/, exon4-exon8/   Primer: ABL-ALT Comparison with the GeneBank mRNA sequence XI6416
Primer
F-BCR-A
R-ABL-A
5-GAG CAG CAG AAG AAG TGT TTC AGA-3
5-CTC TAG CAG CTC ATA CAC CTG GG-3'
Sorre M.E. et al. 2001 Science     Soverini S. et al. 2004 Clin.Chem.
Reference (wt)
G    AGCAG    GT 530
GAT 540
G   A   C   A
GCT     GGAGC    CAAGTTCCCCAT     CA 570                                                     580
22/9 PB
Stdl - HUS 1F-ABL-5-23 06 2008; 0%
1 000
4 580   4 600   4 620   4 640
4 680
4 700   4 720   4 740   4 760
Sample - STY-F-ABL-08_10_2008; 32,6%(D) - 28,31%(I)
1 000 800 600 400 200

"3T5~~
-T.J
^esf
A...C..\.l.
330
4 580   4 600   4 620   4 640
5td2 - JOCH-F-ABL-08_10_2008; 88,89% 1 000
4 6!
0   4 680
4 700   4 720   4 740   4 760
800
600
400
200
4 580   4 600   4 620   4 640
4 680
4 700   4 720   4 740   4 760
Table 2: FDA-approved Indications of Tyrosine Kinase Inhibitors
Gleevec
(imatinib
mesylate)
Sprycel (dasatinlb)
Ta signa
{nilotinib)
Approved in May 2001
Treatment of newly diagnosed patients with Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in chronic phase (400 mg daity
Treatment of patients with Ph+CML in blast crisis, accelerated phase, at in chronic phase after failure of interferon-alpha therapy (600 mg daily)
Treatment of pediatric patients with Ph+CML in chronic phase who are newly d iagnosed or whose disease has retu rred after stem cell tra nspla nt or who are resistant to interferon-alpha therapy (260 mg/m^ daily or 340 mg/mJ daily)
Treatment of adult patients with relapsed or refractory Ph+ acute lymphoblastic leukemia {600 mg daily)
Treatment of adult patients with myelodysplastic/myeloproliferative diseases (MDS/MPD) associated with PDGFR {platelet-derived growth factor receptor) gene rearrangements {400 mg daily)
Treatment of adult patients with aggressive systemic mastocytosis (ASM) without the D816Vt-Kit mutation or withe-Kit mutational status unknown (100 mg daily or 400 mg daily)
Treatment of adult patients with hypereosinophilit syndrome (HES) and/or chronic eosinophilic leukemia (CEL) who have the FlPl LI -PDGFR alpha fusion kinase (mutational analysis or FISH demonstration of CHIC2 allele deletion) and for patients with HES and/or CEL who are FlPl Ll -PDGFR alpha fusion ki nase negative or u n known (100 mg daily or 400 mg da 11 y
Treatment of adult patients with unresectable, recurrent and/or metastatic dermatofibrosarcoma protuberans (DFSP) (800 mg daily)
Treatment of patients with Kit (CDl 17) positive unresectable and/or metastatic malignant gastrointestinal stromal tumors (GIST) (400 mg daily or 800 mg daily)
Approved in June 2006
Treatment of adults with chronic, accelerated, or myeloid or lymphoid blast phase CML with resistance or intolerance to prior therapy including imatinib
Treatment of adults with Ph+ ALL with resistance Or intolerance to prior therapy
Chronic phase, 100 mg daily
Advanced phases of disease, 70 mg twice daily
•   Approved in October 2007
•   Treat men t of c hruri it phase d r
d accelerated phase Ph+CML in adult patients resistant to or intolerant to prior therapy that included imatinib
Dose, 400 mg twice daily
Acute myeloid leukaemia (AML) and related   precursor neoplasms
AML with recurrent genetic abnormalities
AML with gene mutations
AML with myelodysplasia-related changes
Therapy-related myeloid neoplasms
AML, not otherwise specified
Myeloid sarcoma
Akutní myeloidní leukémie
heterogenní skupina onemocnění
různé chromozomální aberace
40-50% případu AML s molekulárním markerem
acute
myeloid
leukaemia
AMLl-ETO t(8;21)
CBFp-MYH inv(l
random
NUP98-HOXA9
tC7;ll)
FUS-ERG
t(ló;21)
MĹL-AF9
t(9;ll)
EVU PML-RARa    ZZ\
PLZF-RAR*   K3'V)
NPM-RARa
t{15;17),t(ll;17),t(5;17)
NPM-MLFl
t(3;5)
DEK-CAN
t{6;9)
Prognostické faktory AML
Fúzní geny s diagnostickým významem
-  PML/RARa ® dobrá prognóza
-  AML1/ETO ® dobrá prognóza
- CBFb/MYH ® dobrá prognóza
-  přestavby MLL genu ® špatná prognóza ■ změny genu s prognostickým významem
-  interní tandemové duplikace ITD-FLT3 genu
-  bodové mutace aktivační smyčky FLT3 genu
-  mutace CEBPa genu
-  mutace NPM1 genu
AML M2 s AMLl/ETO (53 let)
%EXPRESE
200 '
190
180
170
160
150
141
120 110 100 90 80 70 60 50 40 30
ill]
2.RI
l.W
H
3.K
CYTOGENETIKA
MYELOGRAM
Fi nwrYTfiMET														
														
	DNY	0	49	88	126	152	172	180	214	250	272	285	348	3881
	%EXP.- BM	149	11,74	21,77	0,00003	0,03	0,04	0,02	0,09	12,68	74	204	127	I33I
	%EXP.- PB												42	
														
%EXPRESE
252
AML M2 s AMLl/ETO (27 let)
l.W
3.K
CYTOGENETi:
MYELOGRA.
FLOWCYTO"
DNY            I	0	23	62	99	118	160	198	232	241	289
%EXP.-BlJ	252	1,19	0,0013	0,001	0	0	0,008	0,39	0,97	0,015
|%exp.-pb|		0,74			0	0				0,01
Fi niAirvTnMFT									
									
DNY	0	33	62	96	105	138	175	182	201
|%EXP.- BM	60	73,98	27,49	0,02	0,07	0,07		0	
|%EXP.- PB			18,69	0		0,02	0	0	
Detekce mutací metodou f ragmentační analýza CEQ 8000 Genetic Analysis ]d System (Beckman Coulter,CA)
| D GŕHf^lgif
t^úM MM sá\
\ď Study
■■Íj7 Fragments List ■Jf Results Set
60000 rö 50000 §i 40000 CD 30000 £, 20000 Q 10000 0
270.782, 1S673S.133 200000 -ř
rö 150000 -E
CG 100000
50000
0
279.669. 109014.797
100000 -ž
75000 -=
50000 -I
q    25000
0
296.334. 77247.358
70 _ 60 | 50
40
30 >> 20
10
000 -| 000 -I 000 -| 000 -| 000 -| 000 -j 000 - = 0 -E-
Analyses "j    Data   }   Reports J
290 Size (nt)
iReady
Nucleophosmin (NPMl) , B23, N038, Numatrin
•In humans, accumulating evidence that NPM is directly implicated in the pathogenesis of cancer
•Over-expressed in solid tumors of diverse histological origin, is involved in tumor progression
•In hematologic malignancies the locus NPM is lost or translocated - formation of oncogenic fusion proteins
•NPM gene contains 12 exons in maps to 5q35
•C-terminus contains a short aromatic stretch with two tryptophans at positions 288 and 290, which are crucial for NPM binding to the nucleolus (nucleolar localization signal)
•Is more highly expressed in proliferating cells
•Is involved in the apoptotic response to stress and oncogenic stimuli and can modulate the activity and stability of the oncosupressor protein p53
•Mutations are consistently heterozygous, are restricted to exon 12, except for 2 cases: exon 11(2007) and exon 9(2006), about 50 molecular variants of mutations to date in AML with >95% at nucleotide position 960
•Mutation A duplicates a TCTG tetranucleotide at positions 956 to 959   75-80% of adult NPMc+ AML cases
Falini et al., NEJM, 352, 254-266, 2005
mutations
.-y-třšřřř	**
NES
N LS
NoLS
			
1		1                1	1
homo-oligomerization domain
heterodimerization domain nucleic acid binding domain
B	tyPG                        sequence		protein
	wt	CTCTG.........GC AGT......GG AGG AAGTCTCTTAAC A A A AT AG	DLWQWRKSLM
	A	CTCTGT: T(. GCAGT......GGAGGAAGTCTCTTAACAAAATAG	D CLA  EE.3 RK
	e	CTCTÖCA i y GCAGT......GGAGGAAGTŮTCTTAAČAaAATA	DiCMA/EEvS RK
	c	CTCTGCGTGGCAGT......GGAGGAAGTCTCTTAACAAAATAG	DLCVA-'EE/S.RK
	D	CTCTG<".'■";T. .GCAGT......GGAGGAAGTCTCTTAACAAAATAG	Dl CLA   EE  S  RK
	E	CTCTG.........GCAGTCTCTTGCCCAAGTCTCTTAACAAMTA	D! WOSLAQVS RK
	F	CTCTG          GCAGTCCCTGGAGAAAGTCTCTTAACAAAATA	Dl ■.VOSLEKVS R K
Figure 1. Schematic presentation of the nucleophosmin íNPMUgene and oť normal and mutant NPM protein. A, Schematic representation of NPMl gene and normal ft'Pvf protein. 8, Examples of NPMl gene mutations (types A—f). Inserted nucleotides are in violet. All iN'P.Vf mutant proteins show mutations in at least one of the tryptophan residues iW) within the nucleolar localisation signal domain and share the same last 5 amino acid residues (VStBUf. SES indicates nuclear eaporf signal; ľJLS, nuclear localisation signal; t^oLS, nucleolar localisation signal; **, site of mutations in exon 12.
o
!
B
o
í
I 9-Mar-07
1C 16-Apr-07
2C 30-May-07
3C 25-Jul-07
HR 23-Apr-08
Rl 7-May-08
-BM PB
o      o       o       o
Date
I 28-Apr-07
HR 7-May-08
1C 11-Jun-07
—■-----BM
Date
O
CD       CD       CD       CD       CD
CD       CD       CD       CD
CO       CO       CG       CO CO       CD       CD       CD
CD       <JD       CO        CD
^       Q       Q       Q       Q -^-       ^-       CÓ
■=—       CN       CN
	1	1 28-Oct-07			
	p-L			■	
		------*,          ■			
u		v.			
				1	Rl 20-Jan-08
		s			
					1
		\		/ /	
		V.		/ /	
n ■				-------1--------1--------1     *-!-•     1--------1-----	
BM
PB
Date
The kinetics of changes of mutant signal in two representative patients with mutation A (A,B) and one patient with mutation B (C) during the clinical courses.
The number of NPM1-mutated copies in bone marrow (BM) and peripheral blood (PB) correlated with the treatment and disease status (I: induction chemotherapy; 1-, 2-, 3-C: first, second, and third consolidation chemotherapy;         Rl:         reinduction
chemotherapy; HR: hematological relapse, GE: genomic equivalent).
FLT3 ŕFMS-like tyrosine
kinase)                         STKl
řStem cell kinase ľ
flk2 rFetal liver kinase2
lokalizace 13ql2
je členem rodiny receptorových tyrozinových kináz, exprimován na hematopoetických kmenových buňkách a také na buňkách leukemických. Mutace dvou typů popsány:
1)  délkové v JM doméně (ITĎ, interní tandemové duplikace části genu) v exonu 14 (příp. 15)
2)  mutace v katalytické doméně TKĎ, kde kodóny Ď835 a 1836 jsou kódovány nt GATATC, tvořící reštrikční místo pro EcoRV (FLT3/Ď855)
Mutace jsou detekovány r AML s normálním karyol
představují nepříznivou prognózu
sekundami aberaci a nejsoi stabilní v průběhu follow-up.
FLT3 Mutations in AML
TK2
Internal tandem duplication (FĹT3HJD)
Point mutation in activation Loop (FĹT3ÍTKD)
Asp835, II6836
Reštrikční analýza - Detekce mutací D835 v katalytické doméně TKĎ v exonu 20 (17) genu FLT3
^\r\T\rv^
JM      Tftl
IK3
fcson 1/
K  D  I  K CGAGATATCATG
68  >p
KcoRV
»I«
-46 Up-
li L;
B
M    wild   Ml
l:-p
114 «0
4fi
Figure 1. Detection of D635 mutations in the FĹT3 gene, To detect D&35 mutations, we amplfied exon 17 by PCR, and then digested it with the EccR\' endonuclease (A). The amplified products of wild type were digested to 2 bands {68 bp and 46 bp) by the EcoRV. When amplified products contained D835 mutations, undigested bands (114 bp} were visualized on agarose gel electrophoresis. M indicates the molecular weight marker (Haetll digested pBR332 plasmid DNA) (B). The undigested bands were directly sequenced (C). In this sample, the first nucleotide G of DB35 was substituted with T, resulting in an Asp to Tyr amino acid change (DS35Y).
Yamamoto, Y., et al., Blood, 97, 2434-2439, 2001
CEBPA gene
Encodes transcriptional factor (CCAAT/enhancer binding protein alpha) playing crucial role in myeloid differentiation
Loss of CEBPA function leads to blocking of granulocytic differentiation of AM L
When mutated confers favorable prognosis for patients with CN-AML
HRM pre-amplification
PP1F/PP1R
PP2F/PP2R
CEBPA qene
Four independent real-time PCRs performed in       I volume each, selectively amplifying CEBPA gene within four overlapping fragments (PCR products C1-C4)
Reaction mixture C1-C4
• 5ul SensiMix HRM (Quantace, UK)
• 0.4ul EvaGreen Dye (Quantace, UK)
• 2x0.8ul specific primers (10uM)1
• 2ul distilled water
• 1 ul DNA template
1CEBPA primers (Pabst T. et al., Nature genetics 2001, Vol. 27, 263-270)
High Resolution Melt Analysis
Real-time program
•95°C/10min
• 95°C/15s ------1
• 65°C/40s         ■> x35
• 72°C/20s ------'
•53°C/1min
• 87°C/90s
• rising 0.1 °C to 97°C/2s
pre-amphfication
HRM results
poorly performed assays must be omitted
runs with low reproducibility of melting curves must not be included in analysis
particular wild type sample is set as a reference
normalized melting graphs and difference graphs are used to distinguish between mutation and wild type samples
only mutation positive samples are further sequenced
/I sequencing
G C    CCÁCGC    CC    G
GCCCAC'lCCCG
Polymori
hism1281G>T
/IAA/
Myeloproliferative Neoplasms
Chronic myelogenous leukaemia BCR-ABL positive
Chronic neutrophilic leukaemia
Polycythaemia vera
Primary myelofibrosis
Essential thrombocythaemia
Chronic eosinophilic leukaemia
Mastocytosis
-     .      :,	
m^\v>n	
I	Stí  SS M V
	S^N*      U ' *t
	
■	I'tf'"   1
2005 období objevu mutací JAK2 genu -
pracoviště D. Gary Gilliland (Boston), William Vainchenker (INSERM), Radek Skoda (Basel), Anthony Green (Cambridge) JAK2NÜW? (exon 14)
2006 Gary Gilliland skupina somatické mutace (exon 10) MPLWblbL a MPLWblbK u JAK2Nbľ7F negativních 5% PMF (primárních myelof ibróz) a 1% ET
2007 A. Green další 4 mutace v JA K2 gern (exon 12, delece, inzerce)
Clarifications on the precise pathogenetic role of these mutations as well as their importance as targets for small molecule therapy .
Award winners
25th September 2008: Casino Restaurants
Bern As the chairperson of the five-member jury, professor Andreas Tobler, M.D., Berne, past president of the Swiss Society for Hematology, presents the HEMATOLOGICAL MALIGNANCIES AWARD 2008 carrying a value of CHF 100.000 to professor of biomedicine Radek Skoda, M.D., Basel. The award was assigned to professor Skoda for a study on the pathogenesis of myeloproliferative disorders entitled "Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice"1 and published by his research team of Experimental Hematology.        i
A
Inactive JAK2
B
ActivatedJAK2
C
Active J AK 2
Inhibition of the JH1 domain kinase activity by the JH2 domain
Ligand occupies the receptor. JAKZ conformation changes, and JH2 no longer inhibits JH1
JH2 domain with the V617 mutation does not inhibit the JH1 kinase activity
The domains of JAK2 illustrating binding to the receptor and changes consequent to receptor binding and mutation in the JH2 domain. The V617F mutation of the JH2 domain of JAK2 results in constitutive kinase activation. Panel A: When no ligand in bound to the EPO, TPO, G-CSF or GM-CSF receptors, the kinase activity of the JH1 domain is inhibited by the JH2 domain and JAK2 is inactive. Panel B: When EPO binding to it receptor, the two strands of the receptor come closer together, JAK2 changes conformation, the JH1 kinase activity in no longer inhibited by JH2. Panel C: The JAK2 V617F mutation prevents JH2 from inhibiting JH1 and the kinase is active even when no ligand is bound by the receptor.
Bennett and Stroncek Journal of Translational Medicine 2006 4:41   doi: 10.1186/1479-5876-4-41
JAK2 mutation site identification
substituce 1849 £>T, cxon 14,   záměna val inu za fenylalanín v kodónu 617 (V617F)
JAK2exon14
codon617
5' - TTTGGTTTTAAATTATGGAGTATG' L    V    L   N    Y    G    V    C
mut-JAK2exon14
5' - TTTGGTTTTAAATTATGGAGTATG' L    V    L   N    Y    G    V    C
TCTGTGGAGACGAG -3 C    G    D    E
CTGTGGAGACGAG -3 C    G    D    E
TCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAAT TATGGAGTATGTGTCTGTGGAGACGAGA                                                              Q -,
Gen JAK2 (9p24), substituce 1849 G>T, exon 14,   záměna valinu za fenylalanin v kodónu 617 (V617F)
u 95% pacientů s PV (z toho cca 33% je homozygotních v důsledku LOH při mitotickém crossing-overu) (Pardanani 2008, Leukemia)
u 50% pacientů s ET a PMF (homozygotní stav je u těchto pacientů vzácný)
vzácně se mutace může vyskytnout u pacientů s MDS (5%) nebo s CMML (chronic myelomonocytic leukemia) (3%) i u jiných malignit myeloidní řady-RARS-T (ringed sideroblasts associated with marked thrombocytosis), JMML (juvenile myelomonocytic leukemia), aCML (atypical CML), de novo AML
potvrzen i souběžný výskyt V617F mutace s BCR-ABL mutací, přičemž výsledný fenotyp závisí na tom, který buněčný klon získá převahu (Hussein et al. 2008, Leukemia)
někteří autoři uvádí výskyt V617F mutace i u zdravých jedinců (Sidon et al. 2006, Leukemia), nebo u náhodně vybraných pacientů s jiným než hematologickým onemocněním (Xu et al. 2007, Blood).
pacient může nést V617F mutaci několik let předtím, než se u něj vyvine myeloproliferative onemocnění (Bellanné-Chantelot et al. 2008, Leukemia)
Detekce mutace V617F JAK-2
Metoda alellcke diskriminace pro JAK21617*
separace granulocytu z PB
izolace gĎNA
RQ - PCR s využitím fluorescenčně značených LNA
modifikovaných hybridizačních sond (Locked Nucleic Acids)
vyznačují se 100% alelickou diskriminací obou genotypů
citlivost detekující 10% příměs granulocytu nesoucích mutantní
alelu na pozadí zdravé populace
fluorescenční značení FAM pro sondu s WT sekvencí a JOE pro
sondu s MUT sekvencí
(Pekova, S. et al., Blood, 108, No 11, 313B, 2006 Veselovska et al. Leukemia Research, 32: 369, 2008)
Alelická diskriminace pro detekci JAK^ei7?
CT GAAAGTAGGAGAAAGT GCAT CTT TAT TATGGCAGAGAGAAT TT TCT GAACT AT T TATG GACAACAGT CAAACAACAAT TCT TT GT ACT TT T TT TT T TCCT T AGTCTTTCTTTGAAGCÄ GCMGTÄTGÄTGAGCMGCTTTCTCACAAGCÄTTTGGTTTTÄÄÄTTÄTGGAGTÄrGTTTC TGTGGAGACGAGÄGT AAGTAAAACT ACAGGCT T TCTAATGCCT TT CT CAGAGCAT CTGTT
i    mm    mm    MM    MM    MM    ■
T T TGT TT AT ATAGAAAAT TCAGT TT CAGGATCACAGCT AGGT GTCAGT GT AAACT ATAAT T T AACAGGAGTT AAGTAT TT TT GAAACT GAAAACACT GTAGGACT AT T CAGTT AT ATCTT
primer F:   5'- GAAGCAGCAAGTATGATGAGCAA - 3 primer R:   5'- ACTGACACCTAGCTGTGATCC - 3' JAK2 «t LNA sonda:     FAM - tcCacAgaCaCatAc - BHQ1 JAK2V617F LNA sonda: HEX - ctcCacAgaAacAtaCtc - BHQl
- PCR analýza
Norm	Fluoro.										
0,8 0,6		Cycling A.FAM/Sybr - No Markers Cycling A.JOE - Circles									
						/K					
											
											
0,1											
0,2					/						
											
0		Threshold									
											
	0                   '5                   '10		H 5	'20	'25	30	'35	40	45	50	Cycle
Cycling A.FAM/Sybr - No Markers Cycling A.JOE - Circles
35                   '40                    '45                   '50                   Cycle
Cycling A.FAM/Sybr - No Markers Cycling A.JOE - Circles
'35                   '40                    '45                   '50                   Cycle
A co dál?
Nové léky na bázi TK inhibitoru první, druhé a další ... a další... a další
Automatizace extrakce DNA/RNA
Robotizace míchání reakčních směsí PCR a titrace standardní DNA
Automatizace PCR vyšetření pacienta na bázi kartridge nebo čipu
A. RNA Isolation
Sample A
Sample B
*                   \
B. cDNA Generation
C.  Labeling o ť Probe
RevľrxĽ Tmnsíriptasř
t «v                         FluorrOťrnl
% T        Taj;s     T
f.

+
"Y>
D. Hybridization to Array
E. imaging
0 Saupk A > B
;   Sn«i|>k B > A
Sample A = B
	•          •
•	•
	• #
	•
...objevují se nové přístupy v monitorování
Genomic analysis
(clai-sjrJcíilEon ami r^pcmse)
Im.itin i h responsive
lmiitinih
■	IhM
HPPPP?	
SI	1 fr'a
	$w
,,   tn	í á
MKD monitoring
Hi-risk of iťLipsť
Tnirttiiiib unresponsive
Transplant
Bcr-íit>]
Umo
Goldman, J. et al.: Evolution of Management of CML, Tenth International Congress on Hematologie Malignancies
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