RRML Vol 18 Iunie 2010 Laboratory Diagnosis of Acute Leukemia

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

COURSE NOTESLaboratory diagnosis of acute leukemia

Diagnosticul de laborator al leucemiilor acute

Selicean I. Elena-Cristina*, Paiu Mariana

Institute of Oncology Prof. dr. I. Chiricu Cluj-Napoca, Medical Laboratory Hematology

Abstract

Acute leukemias are a heterogeneous group of hematopoietic malignancies, characterized by uncon-trolled growth of a leukemic clone, which is blocked in the maturation process. First classifications of acute leuk-emias were based solely on morphologic criteria, but the actual trend is to take into account biological andpathogenetic aspects. This review's purpose is to cover this complex subject, according to the latest WHO classi-fication, in respect with examination requirements and from the perspective of laboratory physicians.

Keywords: acute leukemia, morphology, immunophenotyping, cytogenetics, molecular biology

Rezumat

Leucemiile acute reprezint un grup heterogen de mbolnviri acute ale sistemului hematopoietic, ca-racterizate prin proliferarea necontrolatra a unei clone de celule leucemice blocat in maturaie. Dac primeleclasificri ale LA se bazau exclusiv pe criterii morfologice, tendina actual de abordare vizeaz aspecte biolo-gice si patogenetice. Lucrarea i propune abordarea acestui subiect complex, respectnd cerinele tematicilor deexamen pentru medici rezideni si specialiti, innd cont de clasificarea actual a OMS i abordnd diagnosticulLA din perspectiva medicului de laborator.

Cuvinte cheie: leucemie acut, morfologie, imunofenotipare, citogenetic, biologie molecular

*Corresponding author: Selicean Elena-Cristina,Tel. 0723 769479, E-mail: [email protected]

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Definiie, mecanisme i terminologie n LA

Leucemiile acute sunt un grup heterogende boli maligne ale sistemului hematopoietic,caracterizate prin proliferarea necontrolat aunei clone de celule imature, blocate n matu-raie, care nlocuiete esutul hematopoietic nor-mal i poate invada teritorii extramedulare.

Proliferarea se poate produce pornind dela o celul mieloid sau limfoid, LA fiind clasi-ficate n dou mari categorii: mieloblastice ilimfoblastice.

Termenul de LA mieloblastic include pro-liferri de linie mieloid granulocitar, monocitar,eritrocitar, megakariocitar, termenul alternativ pen-tru denumirea lor comun fiind LA non-limfoide.

Pacienii cu LA se prezinta cu anemie ,trombocitopenie, de obicei neutropenie i blatin sngele periferic.

Clasificarea FAB a LAPrima clasificare a LA, bazat exclusiv

pe criterii morfologice, a fost fcut de un grupde experi Franco-Americano-Britanic n 1976,ulterior revizuit.

Ultima versiune a clasificrii FAB (1985)include 8 tipuri de LAM i 3 tipuri de LAL.

Principalul criteriu de diagnostic estecantitativ: peste 30% blati din totalul de ele-mente nucleate non-eritroide n aspiratul medu-lar examinat la microscop, n coloraie tip Ro-manovski (May-Grnwald Giemsa). Urmtoareaetapa n vederea clasificrii pe criterii FAB estestabilirea apartenenei de linie a blatilor, tot pecriterii morfologice, eventual folosind coloraiicitochimice ajuttoare.

Clasificarea FAB a LAM se face nfuncie de seria/seriile mieloide care prolifereazi gradul de maturaie a clonei leucemice.

Astfel, leucemia acuta mieloid fr ma-turaie (M1), leucemia acuta mieloid cu matu-raie (M2) i leucemia acuta promielocitar(M3) sunt proliferri de serie granulocitar, cugrade de maturaie diferite.

i pentru celelalte tipuri FAB denumirea

Definition, mechanisms, terminology for AL

Acute leukemias are a heterogeneousgroup of hematopoietic system malignancies,characterized by uncontrolled growth of a cellclone which does not mature normally replacesnormal hematopoietic tissue and can invade ex-tramedular sites.

Proliferation may occur from a myeloidor lymphoid cell. ALs are classified into twobroad categories: myeloid and lymphoblastic.

The term of myeloid leukemia includesproliferation of granulocytic, monocytic,erythroid, megakaryocytic lineage, referred asnon-lymphoid leukemias.

Patients with AL present anemia, throm-bocytopenia, usually neutropenia and blasts inperipheral blood.

FAB classification of ALThe first classification of AL based

solely on morphological criteria was made in1976 by a group of experts of the French-Amer-ican-British group, and was later revised.

The latest FAB classification, released in1985, includes 8 types of AML and 3 types of ALL.

The main diagnostic criterion is quantit-ative: over 30% blasts of the total nucleatednon-erythroid cells in bone marrow smear ex-amined under a microscope in Romanovski(May-Grnwald Giemsa) stain. The next step inthe FAB classification is to establish lineage as-signment of the blasts by using morphologicalcriteria and cytochemical staining.

The FAB classification of AML is basedon the type of myeloid series that proliferates andthe degree of maturation of the leukemic clone.

Thus, acute myeloid leukemia withoutmaturation (M1), acute myeloid leukemia withmaturation (M2) and acute promyelocytic leuk-emia (M3) are proliferations of the granulocyticseries, with different degrees of maturation.

For all other FAB types, their name in-

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010 79

dicates lineage assignment: M4- myelomono-cytic, M5-monocytic (M5a-monoblastic withoutmaturation and M5b monoblastic with matura-tion), M6-erythroleukemia, M7-megakaryoblast-ic leukemia, M0 - acute myeloid leukemiawithout differentiation (Table 1).

FAB group classifies LAL in three cat-egories, based upon the morphological appear-ance of blasts (Table 2).

L1 and L2 are similar in terms of mor-phology and it has been proved that they do notrepresent clinically and biologically distinct en-tities. L3 has a morphological appearance highlysuggestive of lymphoblastic leukemia- Burkittslymphoma, which requires confirmation bydemonstrating a mature B phenotype.

indic apartenena de serie a clonei leucemice:M4 - este leucemia acuta mielomonocitar, M5 -leucemia acuta monocitar (M5a monoblasticfr maturaie i M5b - monoblastic cu matu-raie), M6 - eritroleucemie, M7 - leucemie acutmegakarioblastic, M0 - leucemie acut mieloi-d minim difereniat (Tabelul 1).

Grupul FAB clasific LAL, pe baza as-pectului morfologic al blatilor, n trei categorii(Tabelul 2).

Categoriile L1 i L2 sunt asemntoaredin punct de vedere morfologic. S-a dovedit c nureprezint nici din punct de vedere clinic i biolo-gic entitti distincte. L3 reprezint aspectul morfo-logic nalt sugestiv pentru leucemia limfoblastic -limfomul limfoblastic Burkitt, care necesit confir-mare prin imunofenotipare (fenotip B-matur).

Table 1. FAB classification of AML

FAB Blasts in BM Granulocyticcomponent

Monocyticcomponent Other

M1 90% from NEC, 3% MPO+ - 10% from NEC -

M2 30-89% from NEC >10% from NEC =5x 10 9/l -seric lysozyme raised-cytochemistry- specific aspect

Similar aspect with M2

Additional criteria :-monocytes in PB >=5x 10 9/l andseric/ urinary lysozyme three timesnormal values-monocytes in PB>=5x 10 9/l andcytochemistry specific for monocyticseries

M5M5a Monoblasts 80% fromthe monocytic series - 80% from NEC -

M5b Monoblasts= 50%

M7 -

lineage assignment to megakaryocytesdocumented by ultrastructuralcytochemistry (platelet peroxydase) orby immunophenotyping(megakaryocytic markers)

M0


Identificarea blatilor - criterii morfologice

Determinarea procentului de blati nmduva osoas se face prin numrarea a 500elemente nucleate.

Grupul FAB recunoate 3 tipuri de blati cucaractere morfologice distincte: limfoblast, mielo-blast de tip I - agranular i mieloblast de tip II - cugranulaii azurofile. National Cancer Institute Wor-kshop descrie un tip adiional, mieloblastul de tip IIIcu peste 20 granulaii azurofile n citoplasm.

Din punct de vedere practic i conformrecomandrilor International Working Group onMorphology este important descrierea blatilorca agranulari i granulari.

Blatii agranulari se ntlnesc n leuce-miile acute MPO-negative: LAL, LAM - M0,M6 i M7. n prezent se consider c morfologiaclasic ofer doar criterii orientative de identifi-care a acestor tipuri de blati i c sunt necesarestudii de ultrastructur i imunofenotip pentrucorecta lor ncadrare.

n ceea ce privete mieloblatii granulari,este important diferenierea lor de promielocitulnormal i displazic. Principala deosebire a blati-lor granulari de promielocitul normal este consi-derat zona clar perinuclear (zona Golgi) carac-teristic promielocitului. Excepia de la aceastregul o reprezint blatii granulari din LAM cut(8;21), care pot avea o mic zon Golgi distinct,dar fr alte caractere de promielocit.

Promielocitul displazic ntrunete, pe

Identification of blasts - morphologicalcriteria

The percentage of blasts in bone mar-row is determined by counting 500 nucleatedelements.

FAB Group recognizes 3 types of blastswith distinct morphological characters: lympho-blast, type I agranular myeloblasts and type IImyeloblasts with azurophilic granules. NationalCancer Institute Workshop describes an addi-tional type III myeloblast with more than 20azurophilic granules in the cytoplasm.

From a practical perspective and as re-commended by the International Working Groupon Morphology, it is important to describe theblasts as agranular and granular.

Agranular blasts are seen in MPO-neg-ative acute leukemias: ALL, AML-M0, M6, M7.Today it is considered that classical morphologyyields only indicative criteria for identifyingthese types of blasts and ultrastructural studiesand immunophenotyping are needed for theircorrect classification.

Regarding granular blasts, it is import-ant to differentiate them from normal and dys-plastic promyelocytes. The main differencebetween a granular blast and a normally appearingpromyelocyte is the clear perinuclear hof (Golgiarea), a promyelocytic feature. An exception tothis rule are the granular blasts of AML witht(8; 21), which may have a separate small Golgiarea, but no other characters of a promyelocyte.

Table 2. FAB classification of ALL

Blasts Cytoplasm Nucleus

L1 small scant Rather coarse chromatin,Nucleoli - indistinct or absent

L2 medium moderate, week basophilic Finely dispersed, evident nucleoli

L3 largeAbundant,

intense basophilic,sometimes vacuolated

condensedvariable visible nucleoli


Dysplastic promyelocytes display fea-tures belonging to the maturation stage, in addi-tion to abnormal patterns: uneven cytoplasmicbasophilia, poorly represented Golgi zone,hipergranularity or hipogranularity, irregular dis-tribution of granules.

The morphological identification ofthese forms is important for: MDS-differential diagnosis - where the per-

centage of blasts is crucial differential diagnosis of AML - agranulocytosis-

morphological aspects can be critical in certainstages

further laboratory investigation for a possiblet(8,21).

In AML-M3 malignant promyelocytesare counted as blasts. In the classical form, theyare hypergranular and may have multiple Auerrods. The morphologic microgranular/ hypo-granular variant requires cytogenetic confirma-tion (the presence of t(15, 17)). Prompt diagnos-is of APL is followed by establishment of specif-ic therapy and DIC prevention.

Other situations in which morphologicalassessment of blasts is crucial for diagnosis areproliferations of the monocytic compartment. Inthese cases the percentage of blasts results byadding the following: myeloid blasts, mono-blasts and promonocytes. Differential diagnosisof chronic versus acute myelomonocytic leuk-emia depends on the morphological differenti-ation of monoblasts and promonocytes frommore mature monocytes (Table 3).

lng caracterele aparinnd stadiului de matu-raie, aspecte anormale: bazofilie citoplasmaticneuniform, zona Golgi slab reprezentat, hiper-granularitate sau hipogranularitate, distribuieneregulat (n grmezi) a granulaiilor.

Identificarea acestor forme morfologiceeste important pentru: diagnosticul diferenial LAM - SMD - unde

procentul de blati este hotrtordiagnosticul diferential LAM - agranulocitoz,

unde aspectele morfologice pot fi decisive nanumite stadii

orientarea investigaiilor n vederea eventualeiidentificri a t(8;21).

n LAM-M3, promielocitele maligne suntnumrate ca i blati. n forma clasic acestea sunthipergranulare i pot prezenta corpi Auer multiplidispui n grmad de surcele. Varianta morfolo-gic microgranular/hipogranular necesit confir-mare citogenetic (prezena t(15;17)). Diagnosticulpozitiv de LA promielocitar este decisiv pentru in-stituirea terapiei specifice i poate reprezenta o ur-gena din cauza riscului crescut de CID.

O alt situaie n care evaluarea morfolo-gic a blatilor este crucial pentru diagnostic suntproliferrile de linie sau cu component monocita-r. n aceste cazuri procentul de blati este repre-zentat, n funcie de situaie, de nsumarea procen-telor urmtoarelor elemente: mieloblati, mono-blati i promonocite. Diagnosticul diferenial alLA de leucemia mielomonocitar cronic depindede deosebirea morfologic a monoblatilor i pro-monocitelor de monocitele mai mature (Tabelul 3).

Table 3. Morphological aspects of monocytic lineage

Nucleus Chromatin Cytoplasm Other

Monoblast Round/ oval delicate/ lacyevident nucleolus Basophilic, rare azurophilic granules large, 20-30m

Promonocyte Convoluted/indenteddelicate/ lacyevident nucleolus

Variable basophilia,variable azurophilicgranules

Monoblast- like, butdifferent nucleus shape

Immaturemonocyt e

Convoluted/indented

condensed,sometimesnucleolated

Intermediate basophilia betweenpromonocytes or blasts and maturemonocytes

Resembles monocytes, butmore immature and smaller

Monocyte Lobulated/indentedCondensed, withoutnucleoli

Grey, occasionally azurophilic granules,vacuoles large, 20-25m


Other important morphological charac-ters for the diagnosis and classification of AMLare dysplastic changes of hematopoiesis. Thus,the identification of more than 50% dysplasticelements in at least two myeloid lineages allowscategorization, according to recent WHO criter-ia, in the category of dysplasia related AML.Observation of abnormal eosinophils with large,basophilic granules guides investigations to-wards identification of inv(16) or t(16,16).

Cytochemical stains improve accuracyand reproducibility of lineage assignment inAL. The most useful cytochemical stains areperoxidase and non-specific esterase.

Myeloperoxidase is present in granulo-cytic and monocytic cells, absent in lymphocyticcells. Therefore MPO-stained myeloid cells(from granular blasts, promyelocytes to segmen-ted granulocytes) have a specific yellow-ish-brown color, proportional to the content ofMPO; monocytes are weakly positive; lympho-cytic cells are always negative. Interpretation re-quires experience in order to distinguish falsenegative reactions (for technical reasons) fromsituations with peroxidase deficiency (dysplasticfeature sometimes encountered in AML).

As per the FAB classification, peroxidasereaction allows differentiation in POX- positiveand POX-negative AL and differentiation of AML-M0 and AML-M1 (the percentage of POX-posit-ive blasts), aids the identification of the monocyticcomponent by specific reaction appearance (thinveil) and detects the presence of Auer rods morefrequently than the MGG stain.

In most laboratories where immun-ophenotyping is available, cytochemical stainswere abandoned.

The Perls reaction can be useful in ALfor identification of ringed syderoblasts as a dys-plastic feature.

The FAB classification represents the firstattempt to divide the heterogeneous group of acuteleukemias, where each of the subtypes does not rep-resent a distinct clinical and biological entity.

Alte caractere morfologice importante pen-tru diagnosticul i clasificarea LA sunt reprezentatede modificrile displazice ale seriilor mieloide. Ast-fel, identificarea a peste 50% elemente displazice ncel puin dou linii mieloide permite ncadrarea,conform criteriilor recente ale OMS, n categoria deLAM legat de displazie, iar evidenierea pe frotiu aunor eozinofile anormale, cu granulaii mari, bazo-file, orienteaz investigaiile n vederea identificriiinv(16) sau t(16;16).

Coloraiile citochimice mbuntescacurateea i reproductibilitatea identificrii delinie n LA. Cele mai utile coloraii citochimicesunt mieloperoxidaza i esteraza nespecific.

Mieloperoxidaza este o enzim din gra-nulaiile celulelor de linie granulocitar i mono-citar, absent n celulele de linie limfocitar. nconsecint celulele de linie granulocitar prezin-t n coloraia specific, o reacie glbui-maronieproporional cu coninutul de MPO; monocitelesunt slab pozitive; celulele de linie limfocitarsunt ntotdeauna negative. Interpretarea reactieimieloperoxidazei necesit experien pentru aputea distinge reaciile fals negative (din motivetehnice) de situaiile cu deficit de peroxidaz(caracter displazic ntlnit uneori n LAM).

Reacia mieloperoxidazei permite difere-nierea LA n MPO - pozitive i MPO - negative idiferenierea LAM - M0 de LAM - M1 (prin pro-centul de blati MPO - pozitivi), ajut la identifica-rea componentei monocitare prin aspectul specifical coloraiei (vl fin) i detecteaz mai frecvent de-ct coloraia MGG prezena corpilor Auer.

n majoritatea laboratoarelor n care estedisponibil imunofenotiparea, reaciile citochi-mice au fost abandonate.

In LA mai poate fi util coloratia cu al-bastru de Prusia - reacia Perls pentru identifica-rea sideroblatilor inelari ca semn de displazie.

Clasificarea FAB a LA reprezint primancercare de mprire a grupului heterogen alleucemiilor acute, fr ca fiecare din subtipuri sreprezinte o entitate distinct din punct de vede-re clinic i biologic.


Ulterior, clasificarea propus de Grupulde Studiu Cooperativ MIC (morfologie, imuno-logie i citogenetic) a deschis calea pentru re-cunoaterea semnificaiei modificrilor citoge-netice i respectiv a imunofenotipului n LA.

Urmtoarea clasificare propus i accep-tat pe scar larg a fost clasificarea OMS din2001, revizuit n 2008. Principiile pe care sebazeaz aceast clasificare sunt urmtoarele: procentul de blati necesar pentru diagnosticul

pozitiv de LA este de 20%; procentul de blati nu mai reprezint criteriu

de diagnostic pentru LA atunci cnd ntr-uncontext hematologic semnificativ se demon-streaz prezena unei mutaii citogenetice recu-rente;

include dou entiti provizorii caracterizateprin mutaii NPM1 i CEBPA;

introduce clasa LAM legat de SMD, avndca i criterii de diagnostic antecedentele deSMD, anomaliile citogenetice asociate SMD,displazia multilinial;

introduce clasa LAM post- chimioterapie;

Subsequently, the classification pro-posed by MIC Cooperative Study Group (mor-phology, immunology, cytogenetics) paved theway for the recognition of the significance ofcytogenetics and immunophenotype in AL.

The next classification proposed andwidely accepted was the WHO classification of2001, revised in 2008. The principles underlyingthis classification are: the percentage of blasts required for diagnosis

according to WHO criteria is 20% the percentage of blasts is not a diagnostic criterion

for AL, when there is evidence of recurrent cytogen-etic mutations in a significant hematologic context

includes two provisional entities characterizedby mutations in NPM1 and CEBPA

introduces MDS- related AML, diagnosis basedon MDS history, cytogenetic abnormalities as-sociated with MDS, multilineage dysplasia

introduces the post-chemotherapy AML class maintains FAB terminology for morphological

classification of AML in the category "not oth-erwise specified"

WHO 2008 classification of AL

I. Acute myeloid leukemiaI.1 AML with recurrent cytogenetic abnormalities

I.1.1 AML with t(8:21)(q22;q22) (RUNX1;RUNX1T1)I.1.2 AML with inv(16)(p13.1q22) or t(16;16)

(p13.1;q22)(FBMYH11)

I.1.3 AML with t(15;17)(q22;q12) (PML-RARA) (promyelocytic)I.1.4 AML with t(9;11)(p22;q23) (MLLT3-MLL)I.1.5 AML with t(6;9)(p23;q34); (DEK-NUP214)I.1.6 AML with inv(3)(q21;q26.2) or t(3;3)

(q21;q26);(RNP1-EVI1)

I.1.7 AML with t(1;22)(p13;q13) (RMB15-MKL1) (megakaryoblastic)

Provisional entities :AML with mutated NPM1 AML with mutated CEBPA

I.2 AML dysplasia relatedI.3 Myeloid neoplasia therapy relatedI.4 Myeloid sarcomaI.5 Myeloid proliferations Down syndrom related

I.5.1 Transitory abnormal myelopoiesis . I.5.2 AML Down syndrome associated


classifies ALL and ambiguous line leukemiason immunophenotypic and genetic criteria

correlates ALL with lymphoblastic lymphoma.

Imunophenotyping - role in diagnosis of AL

Technical ConsiderationsDetermination of cell surface antigen

expression may be done on cytological and his-tological preparations (imunocytochemistry,imunohistochemistry), but imunophenotypingby flow-cytometry became the most widely usedbecause it has many advantages.

menine terminologia FAB pentru clasificareamorfologic a LAM din categoria not other-wise specified ;

clasific LA limfoblastice i de linie ambigupe criterii de imunofenotipare i genetic;

coreleaz LAL cu echivalentul tisular de lim-fom limfoblastic.

Imunofenotiparea rolul n diagnosticul LA

Consideraii tehniceDeterminarea expresiei antigenelor de

suprafa celular se poate face pe preparate cito-

I.6 AML not otherwise specifiedI.6.1 AML with minimal differentiationI.6.2 AML without maturationI.6.3 AML with maturationI.6.4 Myelomnocytic ALI.6.5 Myeloblastic/ monocytic ALI.6.6 Acute erythroid leukemia

Pure erythroid leukemia Erythroleukemia (erythroid/ myeloid)

I.6.7 Acute megakaryoblastic leukemiaI.6.8 Acute basophilic leukemia I.6.9 Acute panmyelosis with myelofibrosis

II. Acute leukemia of ambiguous lineage II.1 AL undifferentiatedII.2 AL mixed phenotype with t (9;22)(q34;q11.2) BCR-ABL1II.3 AL mixed phenotype with t(v;11q23); MLL rearrangementsII.4 AL mixed phenotype B/myeloid, NOSII.5 AL mixed phenotype T/myeloid NOSII.6 AL mixed phenotype NOS-rare typesII.7 Acute lymphoblastic leukemia/ lymphoma NK cells

III. Acute lymphoblastic leukemia/ lymphomaIII.1 ALL/ LL B NOS III.2 ALL/LL B with recurrent cytogenetic abnormalities

III.2.1 t (9;22)(q34;q11.2) BCR-ABL1III.2.2 t(v;11q23) MLL rearrangementsIII.2.3 (12;21)(p13;q22) TEL-AML1 (ETV6-RUNX1)III.2.4 hyperdiployd III.2.5 hypodiployd t(5;14)(q31;q32); IL3-IGHIII.2.6 t(1;19)(q23;p13.3) E2A-PBX1 (TCF3-PBX1)

III.3 ALL/LL T


Currently available protocols for in vitrodiagnostic allow concurrent reading at up to 3-4wavelengths and perform a multiparametric, ob-jective, sensitive and rapid analysis of a largenumber of cells, with the ability to delineate andcharacterize distinct populations of cells in theanalyzed sample in terms of expression of sur-face and intracellular antigens.

This technique may be used in samplesof peripheral blood, bone marrow, and whenwarranted, cerebrospinal fluid, other fluids(pleural, pericardic, peritoneal), tumor masses.

Before being analyzed, the primarysample is subject to a preliminary process forseparating mononucleate cells in a concentrationgradient or for red cell lysis. Many laboratoriesprefer lysing protocols, because separationmethods are more laborious and cells could getlost during processing. For analysis of intracel-lular antigens the method involves a step con-sisting in permeabilisation of cell membranes.

The current strategy for demarcating cellpopulations from the sample ("gating) is plottingCD45 versus SSC (side scatter - side scattering ofa laser beam). Blasts have a low side scatter (be-cause they are weak granular) and express lowCD45 +, compared to other nucleated cells in thesample: lymphocytes intensely CD45 +, erythro-blasts CD45 negative, precursors of granulocyticseries high side scatter.Antibody panel selection

The use of minimal antibody panels fordiagnostic purposes is recommended, since theyoffer great diagnostic information and are repro-ducible in laboratories with different equipment.

The panel should help distinguishingbetween AML and ALL, establishing lineage as-signment (myeloid, monocytic, megakaryocytic,erythroid, namely B or T), highlighting possibleaberrant expression of antibodies and outlining acharacteristic immunophenotype (leukemia-as-sociated immunophenotype "LAIP")

The choice of fluorochrome depends on thetype of device; most devices can use fluorescein iso-thiocyanate, phycoerythrin, phycoerythrin-cyanin 5.

logice (imunocitochimie) sau histologice (imuno-histochimie), dar n practic s-a impus imunofe-notiparea prin citometrie n flux continuu, datori-t multiplelor avantaje pe care le prezint.

Metodele aplicate n prezent n scopdiagnostic permit citirea concomitent la 3-4lungimi de und i efectueaz o analiz multipa-rametric, sensibil i rapid a unui numr marede celule, oferind posibilitatea de a delimita icaracteriza din punct de vedere al expresiei anti-genelor de suprafa i intracelulare, populaiilecelulare din eantionul analizat.

Tehnica se poate aplica pe probe de sngeperiferic, mduv osoas, iar atunci cnd este justi-ficat, lichid cefalorahidian, alte lichide (pleural,pericardic sau peritoneal) sau mase tumorale.

nainte de a fi analizat, proba primar sesupune unui procedeu de separare a mononucleate-lor n gradient de concentraie sau unui procedeu deliz a hematiilor. Multe laboratoare prefer metodacu liz, deoarece metodele de separare sunt mai la-borioase i exist riscul de a pierde celule pe parcur-sul procesului de prelucrare. Pentru analiza antige-nelor intracelulare, metoda presupune i o etap depermeabilizare a membranei celulare.

Strategia actual pentru delimitarea popu-laiilor de celule din proba de analizat (strategia degating), pentru leucemiile acute, este CD45 versusSSC (side scatter- dispersia laterala sub unghiuri mari90o a fasciculului laser). Blatii au un grad redus dedispersie laterala a fascicului (deoarece sunt slab gra-nulari) i exprim CD45 dim+. Aceasta permite dife-renierea lor de celelalte celule nucleate din probaanalizat: limfocite CD45 +++, eritroblati CD45-,precursori neutrofilici i eozinofilici intensitatemare a fasciculului laser dispersat lateral.Alegerea panelului de anticorpi

n practic se urmrete utilizarea unorpaneluri minimale de anticorpi, care s permitobinerea unor date cu valoare diagnostic marei reproductibile n laboratoare cu dotri diferite.

Panelul trebuie s permit deosebirea LAMde LAL, stabilirea apartenenei la o linie (mieloid,monocitar, megakariocitar, eritrocitar, respectiv Bsau T), evidenierea expresiei aberante de anticorpi,


care s contureze un imunofenotip asociat leucemiei.Alegerea fluorocromilor depinde de tipul aparatului,majoritatea aparatelor pot utiliza izotiocianat de fluo-rescein, ficoeritrin, ficoeritrin - cyanin 5. Interpretare

Din raiuni practice s-a stabilit ca limitde pozitivitate expresia la peste 20% din celulelepopulaiei examinate a antigenelor de suprafa,respectiv 10% pentru markeri citoplasmatici.Diferenierea LAM de LAL

n principiu, blatii din LAM exprim anti-gene imature de linie neutrofilica i /sau mono-citar: CD13, CD15, CD33, CD64, CD117 i MPO.

Markerul cel mai sensibil i specificpentru limfoblatii de linie B este CD19, urmatde CD22c. Pentru limfoblatii de linie T, cea maimare sensibilitate o are CD3c.Caracterizarea imunologic a LAM MPO negative

Imunofenotiparea este esenial pentrudiagnosticul diferenial al LA MPO negative.

Expresia de CD13, CD33, MPO, cu CD19,CD79a, CD3c i TdT negative, este fenotip de LAM.

LAM - M0 se definete n citochimia cla-sic avnd sub 3% blati MPO +. Prezena MPOse poate demonstra mai bine prin imunofenotiparei prin microscopie electronic. n aceste condiii,imunofenotipul cu cel puin un marker mieloid po-zitiv (CD113, CD33 sau MPO), n absenta marke-rilor limfoizi (CD3, CD22a, Cd79a), a markerilormegakariocitari (CD41 i CD61) si eritrocitari(CD235), permite diagnosticul de LAM - M0. Potfi pozitivi: HLA-DR, CD34, CD117, CD65, mairar CD14 i CD15 i uneori markeri limfoizi expri-mai n mod aberant pe celulele mieloide (CD2,CD4, CD7 sau CD19).

Blatii din LAM M7 exprim markerimegakariocitari: CD41, CD42 i CD61.

n eritroleucemie blatii exprim CD235,iar, n absena acestui marker, imunofenotipul ca-racteristic este CD36+/CD64-/MPO-.Caracterizarea imunologic a celorlalte clasemorfologice de LAM

LAM-M1 i LAM-M2 se caracterizeaz

Interpretation For practical reasons, it was established

that the positivity limit is 20% for surface anti-gens, and 10% for cytoplasmic markers. Differentiation of ALL AML

AML blasts express immature neutro-philic and monocytic antigens: CD13, CD15,CD33, CD64, CD117, MPO.

The most sensitive and specific markerfor B lymphoblasts is CD19, followed byCD22c. In T lineage lymphoblasts the highestsensitivity is provided by CD3c. Immunological characterization of POX-negative AML

Immunophenotyping is essential for dif-ferential diagnosis of POX- negative ALs.

Expression of CD13, CD33 and MPO,together with the lack of expression of CD19,CD79a, TdT and CD3c demonstrates an AMLphenotype.

AML-M0 is defined as having less than3% MPO-positive blasts in classic cytochem-istry. The presence of MPO in turn can be betterdemonstrated by immunophenotyping and elec-tron microscopy. In these conditions, at least onepositive myeloid marker (CD113, CD33, orMPO), in the absence of lymphoid markers(CD3, CD22a, Cd79a) and megakaryocyticmarkers (CD41 and CD61), allows the diagnosisof AML-M0. HLA-DR, CD34, CD117, CD65,CD14 and CD15 may be positive and sometimeslymphoid markers (CD2, CD4, CD7, CD19) areaberrantly expressed by myeloid cells.

Blasts of AML-M7 express megakaryo-cytic markers: CD41, CD42, CD61.

In erythroleukemia blasts expressCD235 or the characteristic phenotype CD36 + /CD64-/MPO-. Immunological characterization of AML mor-phological classes

AML-M1 and AML-M2 are character-ized by the expression of CD13, CD33, MPO,CD34, and CD117, without expression ofCD79a, CD14, CD64.


Subtypes of monocytic lineage or withmonocytic component express CD13 and CD33,and are characterized as follows: AML-M4 can present two blast subpopula-

tions: monocytic cells with stronger CD45 ex-pression and CD117-/ CD64 +, while myeloidcells are CD117 +, CD64-;

In AML-M5 it has been proven that CD14 isnot highly specific, as blasts can be CD14 + ornegative, but CD64, CD11c, CD15, CD117 aregenerally positive.

Attempts to establish morphology - im-munophenotype correlations showed that, withinthe same FAB classes, there are different im-munophenotypes and the same immunopheno-type is found in different FAB classes.

However, there are two AML classescharacterized by recurrent cytogenetic abnormal-ities and specific morphology, which show strongassociation with certain immunophenotypes:

APL with t(15;1 7) is usually HLA-DRand CD34 negative, with MPO + ++, CD33 +,CD11b-, CD14-. This is particularly useful fordifferential diagnosis of APL hypogranularvariant and AML M4 and M5.

AML with t (8; 21) with M2-type mor-phology is characterized by MPO +, CD13 +,CD33 weakly +, HLA-DR +, CD34 + and ab-errant expression of CD19, frequently asyn-chronous expression of CD13 and CD33,which are weakly positive in comparison withMPO expression.

These immunophenotypes are not con-sidered of absolute diagnostic value but, togetherwith the morphology, they guide the genetic invest-igation towards specific recurrent abnormalities.ALL immunophenotyping

Immunophenotyping ALL has demon-strated that many cases with FAB L3 morphologyare malignancies with mature B cells and representthe leukemic phase of Burkitts lymphoma.

80% of cases of ALL have B precursorimmunophenotype. These blasts express a vari-ety of B and pan-B antigens, and typically donot express the surface immunoglobulin charac-

prin expresia de CD13, CD33, MPO, CD34 iCD117, fr expresie de CD79a, CD14 i CD64.

Leucemiile de linie monocitar sau cucomponent monocitar exprim CD13 i CD33,putnd fi caracterizate n plus astfel: n LAM-M4 se evideniaz dou subpopulaii

de blati, populaia monocitar cu expresie maiintensa de CD45 i CD117-, CD64 +; celulelemieloide sunt CD117+, CD64-.

n LAM-M5 s-a dovedit c CD14 nu este ex-trem de specific, blatii pot fi CD14+ sauCD14-, dar CD64, CD11c, CD15 i CD117sunt de obicei pozitive.

ncercarea de a stabili corelaii ntremorfologie i imunofenotip, a demonstrat c, ncadrul aceleiai clase FAB, ntlnim imunofeno-tipuri diferite i acelai imunofenotip se reg-sete n diverse clase FAB.

Totui, exist dou clase de LAM cuanomalii citogenetice recurente i morfologiespecific, la care s-a demonstrat o corelaie pu-ternic cu imunofenotipul, astfel:

LA promielocitar cu t(15;17) se pre-zint cu HLA-DR-, CD34-, MPO intens+,CD33+, CD11b-, CD14-. Acest imunofenotipeste util n special pentru diagnosticul diferenialdintre LA promielocitar, varianta hipogranula-r, i LAM - M4 sau M5.

LAM cu t(8;21) cu morfologie tip M2este caracterizat de imunofenotipul MPO+,CD13+, CD33 slab+, HLA-DR+, CD34+ i ex-presie aberant de CD19. Se ntlnete frecventexpresia asincron de CD13 i CD33, slab pozi-tive comparativ cu expresia MPO.

Aceste imunofenotipuri nu reprezint uncriteriu de diagnostic, dar, mpreun cu morfolo-gia, pot orienta investigaiile de genetic.Imunofenotiparea LAL

Imunofenotiparea LAL a demonstrat cmulte din cazurile cu morfologie FAB L3 suntneoplazii cu celule B mature care reprezint fazaleucemic a limfomului Burkitt.

80% din cazurile de LAL au imunofenotipB precursor. Aceti blati exprim o mare varieta-te de antigene B (tinere i pan-B) i nu exprim


teristic for B mature lymphocytes. They alsocontain TdT, a nuclear enzyme present in theprecursors of B or T lymphocytes but absentfrom mature lymphocytes.

15% of ALL have a precursor T profileexpressing young T, pan-T antigens and TdT.

Immunological classification of ALL is im-portant in terms of prognosis and therapeutic de-cision. Precursor B type leukemia has more favor-able prognosis, but in this group there still are pro-gnostic subgroups according to cytogenetic results.

ALL immunophenotype associated withrecurrent cytogenetic changes: B-ALL with t(1, 19)(q23, p13): CD9 +, CD10

+, CD19 +, CD20-or partially +, CD34-;B-ALL with t(4; 11)(q21, q23): CD10-, CD15

+, CD24-or partially +, CD34-;Leukemic blasts usually differ from nor-

mal blasts by aberrant antigen expression: asynchronous expressions (simultaneous ex-

pression of young and mature markers) expression of antigens which are specific for

other lines lack of expression of specific antigens antigen overexpression.

Thus, myeloid blasts may present asyn-chronous expressions of CD15 (a marker of ma-

imunoglobuline de suprafa care sunt caracteristi-ce limfocitelor B mature. De asemenea coninTdT, enzim nuclear prezent n precursori de li-nie B sau T, dar absent din limfocitele mature.

15% din LAL au profil precursor T. Limfo-blatii T exprim antigene T tinere, pan-T i TdT.

Clasificarea imunologic a LAL esteimportant din punct de vedere prognostic i alatitudinii terapeutice. Categoria precursor B areprognostic mai favorabil, dar i n cadrul acestuigrup exist subgrupe de prognostic, n funcie derezultatele citogenetice.

Imunofenotipurile LAL asociate cu mo-dificri citogenetice recurente sunt:B-LAL cu t(1;19)(q23;p13), imunofenotip: CD9+,

CD10+, Cd19+, CD20- sau parial +, CD34-B-LAL cu t(4;11)(q21;q23), imunofenotip

CD10-, CD15+, CD24- sau parial +, CD34-.Blatii din LA se deosebesc de obicei de

blatii din hematopoieza normal prin expresiiaberante de antigene:expresii asincrone (expresie concomitent de

markeri de celul tnr i matur);expresia de antigene specifice pentru alte linii; lipsa expresiei unor antigene specifice; supraexpresia unor antigene.

Astfel, blatii mieloizi pot prezenta ex-

Table 4. ALL immunophenotype

B-ALL classification according to Ig expressionPAX-5, CD19, CD20, CD22(s,cyt), CD24, CD79a (cyt), CD20 (part/dim/-), CD10 (CALLA), CD45dim, CD34, TdT

cyt surface Expression/ restriction of lightchain

Pro-B - - -Pre-T + - -Pre-T transitional + + (dim) -T-ALL classification according to tymocyte maturation stageCD2, CD3, CD4, CD5, CD7, CD8, CD1a, CD10, CD34, CD99, HLA-DR, Tdt

CD1a CD2 cCD3 sCD3 CD4 CD5 CD7 CD8 CD34Pro-T - - + - - - + - +Pre-T - + + - - +/- - - +/-Cortical + + + - + +/- + + -Medular - + + + +/- +/- + +/- -


turity) and CD117 (a marker usually present onyoung elements of the myeloid lineage) and theexpression of antigens specific for other lines:CD19, CD7, CD56

Lymphoblasts can express aberrant my-eloid antigens, B lymphoblasts sometimes ex-press CD20 and T lymphoblasts may expressasynchronous markers.

It is important to establish the leukemia-associated immunophenotype for each individu-al case at diagnosis. This may be useful later inthe follow-up and the diagnosis of minimal re-sidual disease, with the specification that blastsmay undergo immunophenotypic changes dur-ing evolution or post-therapy.

Diagnosis of acute leukemia with mixedphenotype is the result of immunophenotypingand applying the EGIL score. This score assignsvalues of 2, 1, 0.5 points for markers of B, T andmyeloid lineage, respectively (Table 5).

AL with mixed phenotype is furtherclassified according to recurrent cytogenetic ab-normalities seen in this group.

The role of cytogenetics in AL

Clonal cytogenetic abnormalities, bothnumerical and structural, are present in 60-80%of the AML and respectively 80% of the ALL.

Identification of genetic changes in ALat karyotype and molecular level allowed for:

presii asincrone CD15 (marker de maturitate) cuCD117 (marker prezent de obicei pe elemente ti-nere) i expresia de antigene specifice pentrualte linii: CD19, CD7 sau CD56.

Limfoblatii pot exprima n mod aberantantigene mieloide, limfoblatii B exprim uneoriCD20, iar limfoblatii de linie T exprim uneorimarkeri asincroni.

Pentru fiecare caz n parte este impor-tant stabilirea la diagnostic a imunofenotipuluiasociat leucemiei. Acesta poate fi util ulteriorpentru urmrirea n dinamic i pentru diagnos-ticul de boal minim rezidual, cu specificaiac imunofenotipul blatilor poate suferi modifi-cri pe parcursul evoluiei sau post-terapie.

Diagnosticul de leucemie acuta cu fenotipmixt se stabileste pornind de la imunofenotipare.Scorul EGIL atribuie valori de 2, 1, 0.5 puncte pen-tru markerii de linie B, T i mieloizi (Tabelul 5).Scorul peste 2 pentru dou din linii definete LAbifenotipic. Clasificarea OMS 2008 redefinetecriteriile pentru LA de linie ambigu, incluznd LAbifenotipice si biliniale n aceast categorie.

LA cu fenotip mixt sunt clasificate ncontinuare n funcie de anomaliile citogeneticerecurente ntlnite la acest grup.

Rolul citogeneticii n LA

Anomalii citogenetice clonale, att nu-merice ct i structurale, se ntlnesc la 60-80%

Table 5. EGIL scoring for biphenotypic leukemia

Lineage

Scoring B T myeloid

2 CD79c

CD3TCR MPO(lysozime)

1

CD 2CD19CD10CD20

CD2CD5CD8

CD10

CD13CD33

CDw65CD117

0.5 TdTCD24TdT

CD17CD1a

CD24CD25CD64


din LAM i respectiv la 80% din LAL.Identificarea modificrilor genetice din LA,

la nivelul cariotipului i la nivel molecular, a permis: ncadrarea LA n clase de risc - modificrile ci-

togenetice s-au dovedit a fi factor prognosticmajor i independent, cu recomandri diferiten ceea ce privete utilitatea i momentul optimal transplantului medular;

o mai buna nelegere a fenomenului leucemo-genezei cu perspective n ceea ce privete tera-piile medicamentoase.

n practic, analiza citogenetic stan-dard cu bandare G a cromozomilor este ncmetoda cea mai utilizat pentru a detecta ct maimulte dintre rearanjamentele de la nivelul cloneileucemice. Aceast tehnic necesit prezena decelule n diviziune n proba de analizat, analize-az un numr mic de metafaze i nu permite evi-denierea mutaiilor submicroscopice.

Tehnicile de biologie molecular utiliza-te n prezent pentru diagnosticul i clasificareaLA sunt hibridizarea fluorescent in situ(FISH) i reacia de polimerizare n lan(PCR). Acestea se utilizeaz pentru:demonstrarea clonalitii (de exemplu rearanja-

mente TCR T cell receptor); identificarea mutaiilor criptice, adic acele mutaii

care implic fragmente mici, submicroscopice;descifrarea cariotipurilor complexe.

Conducerea acestor investigaii este orien-tat de aspecte clinice, morfologice, citogenetice.Rolul biologiei moleculare este de a contribui ladiagnostic (vezi LA cu anomalii citogenetice recu-rente) i de a demonstra prezena unor anomalii im-portante din punct de vedere prognostic.

De asemenea, se tinde ca tehnica PCRs i gseasc locul n definirea noiunilor deremisiune complet i boal minim rezidualpentru ct mai multe tipuri de rearanjamente, cucondiia standardizrii metodelor utilizate.

Hibridizarea fluorescent in situ combi-n avantajele analizei citogenetice standard cutehnicile moleculare. Aceasta permite vizualiza-rea rearanjamentelor pe celule n metafaz i ninterfaz i analiza unui numr mai mare de ce-lule dect citogenetica clasic.

classification into prognostic classes- cytogen-etic changes were found to be significant inde-pendent prognostic factor, with different re-commendations regarding the usefulness andright moment for bone marrow transplantation;

a better understanding of the leukemogenesiswith achievements and promises regardingdrug therapy.

In practice, standard cytogenetic ana-lysis of chromosome G banding is still the mostused method to detect many rearrangements inthe leukemic clone. It is a laborious method, per-formed by trained personnel, requires the pres-ence of dividing cells in the analysis sample, ex-amines a small number of metaphases and leavesno evidence of submicroscopic mutations.

Molecular biology techniques currentlyused in the diagnosis and classification of LA arefluorescent in situ hybridization (FISH) andpolymerase chain reaction (PCR) used for: clonality demonstration (e.g. TCR - T cell re-

ceptor rearrangements); identification of cryptic mutations in classic cyto-

genetics, i.e. mutations involving small fragments;explaining complex caryotypes.

These investigations are guided by clinical,morphological and cytogenetic aspects. Molecularbiology finds its practical application in AL, whencontributing to diagnosis (see AL with recurrentcytogenetic abnormalities), or documents a signific-ant abnormality in terms of prognosis.

PCR technique also tends to find itsplace in the definition of complete remission andminimal residual disease for many types of re-arrangements, provided the methods used arestandardized.

Fluorescent in situ hybridization (Fluor-escent in Situ Hybridization) combines the ad-vantages of standard cytogenetic analysis withmolecular techniques. It allows identification ofclonal rearrangements in metaphase and in-terphase nuclei and analyzes more cells thanconventional cytogenetics.

Recommended PCR techniques useRNA as target and synthesize by reverse-tran-scription a DNA copy subsequently amplified


(RT-PCR). DNA is recommended as target onlywhen break points are located nearby, on a shortfragment of DNA.

Important genetic rearrangements in AMLTranslocation (15;17)(q22;q21) is always

associated with APL, a subtype of AML with clinicaland morphological distinctive characters. The fusionproduct of the PML gene on chromosome 15 withRAR-alpha gene on chromosome 17 triggers a path-way to block granulocytic maturation at the promyel-ocytic stage. This is a pathogenetic mechanism onwhich the therapy with ATRA (All Trans RetinoicAcid) specifically acts. No other AML has a thera-peutic response to ATRA. There are molecular vari-ants of APL which do not respond to ATRA therapy. AML with rearrangements involving CBF(Core binding factor)

t(8;21)(q22;q22) leads to the fusion geneof AML1 (CBF-alpha) gene on chromosome 21with the ETO (eight twenty one) on chromosome8. 90% are AML-M2 with Auer rods, dysgranu-lopoiesis, eosinophils in large numbers in bonemarrow, and favorable response to repeatedcycles of high dose cytarabine.

Inv (16) or t(16;16)(p13;q22) (CBF-beta-MYH11) have M4Eo morphology with high per-centage of immature eosinophils with primary(basophilic) granules and a favorable prognosis.

Tehnicile preferate de PCR sunt cele care uti-lizeaz ARN-ul ca int i sintetizeaz sub aciunea re-vers-transcriptazei (RT) o copie de ADN, care ulteriorse amplific (RT-PCR). ADN este recomandat ca intdoar pentru situaiile n care punctele de ruptur suntsituate n apropiere, pe un fragment scurt de ADN.

Rearanjamente genetice importante n LAMTranslocaia (15;17)(q22;q21) se aso-

ciaz ntotdeauna cu LAP, subtip de LAM cu ca-ractere clinice i morfologice distincte. Produsulde fuziune a genei PML de pe cromozomul 15cu gena RAR- de pe cromozomul 17 declane-az o cale de blocare a maturaiei pe linia granu-locitar la nivel de promielocit, mecanism pato-genetic asupra cruia acioneaz n mod specificterapia cu ATRA (all trans retinoic acid). Nici oalt LAM nu beneficiaz de aceast terapie.Exist variante moleculare de LAP care nu rs-pund la terapia cu ATRA.LAM cu rearanjamente ce implic CBF (Corebinding factor)

t(8;21)(q22;q22) se soldeaz cu fuziuneagenei AML1(CBF-) de pe cromozomul 21 cugena ETO (eight twenty one) de pe cromozomul 8.O proporie important (90%) sunt LAM-M2 cucorpi Auer, disgranulopoiez, eozinofile n numrmare n mduv, i rspuns favorabil la cicluri re-petate cu citarabin n doz mare.

Table 6 Cytogenetic prognosis classes in AML

FavorableInv (16) , t(16;16)(p13;q22), del(16q)t(8;21)(q22;q22)t(15;17)

Intermediate

Abnormalities of 11q 23 ex. t(11 ;19)(q23 ;p13.1)+8, +21, +22, +11,+13, +6t(6;9)del(5q), del(7q), del(9q), del(11q), del(12p), del(20q)-7, -y

Unfavorable

-5, -7t(v;11)(v;q23)Inv(3), t(3 ;3)Complex karyotype (>= 3 unrelated abnormalities)t(6 ;9), t(6 ;11), t(11 ;19)


LA cu inv (16) sau t(16;16)(p13;q22)(CBF- - MYH11) au morfologie M4Eo cu pro-cent mare de eozinofile imature cu granulaiiprimare (bazofile) i prognostic favorabil.LAM cu anomalii 11q23 cu implicarea genei MLL

Cea mai frecvent este t(9;11)(q23;q23)(MLL-AF9), deseori cu morfologie monoblasti-c i prognostic defavorabil. Mutaiile sunt crip-tice i se identific prin RT-PCR sau FISH.

Alte anomalii citogenetice ntlnite nLAM sunt trisomia 8, monosomia 5, 7, del(5q),del(7q); se descriu de asemenea LAM cu cario-tip normal sau complex.

Anomalii citogenetice n LAL Cea mai comun anomalie citogenetic

n LAL precursor B este hiperdiploidia cu 50de cromozomi. ntlnit cel mai frecvent la paci-eni de 2-10 ani, se asociaz cu numr mic sauintermediar de blati n sngele periferic. Frec-vent se ntlnete o copie n plus pentru cromo-zomii 4 i/sau 10. LAL cu hiperdiploidie cu 50de cromozomi au rspuns terapeutic foarte bun.

Modificrile citogenetice structurale nLAL B au prognostic intermediar sau defavora-bil. O excepie o reprezint t(12;21)(p12;q22)care nu este evideniabil de obicei prin citoge-netica clasic i trebuie identificat prin FISHsau PCR, cu prognostic favorabil.

Pacienii cu LAL precursor B cu t(9;22)(q24; q11) sau anomalii la nivelul 11q23 - maifrecvent translocaia t(4;11)(q21; q23), au prog-nostic defavorabil.

Strategia de investigare genetic aleucemiilor acute poate s difere, dar presupunentotdeauna o analiz citogenetic la debut, ur-mat, n funcie de caz, de studii de biologie mo-lecular, de exemplu:pentru cazurile de LAL B la copii se caut

translocaia criptic t(12;21)(p13;q22) ETV6-RUNX1 prin FISH sau PCR;

cazurile de LAM cu morfologie sugestiv pen-tru inv(16) sau t(16;16)(p13.1;q22) vor fi in-vestigate n acest sens;

AML with 11q23 abnormalities in-volving the MLL gene: the most common ist(9;11)(q23;q23) (MLL-AF9), often withmonoblastic morphology and unfavorable pro-gnosis. Mutations are cryptic for classical cyto-genetics and are identified by RT-PCR or FISH.

Other common cytogenetic abnormalitiesin AML are trisomy 8, monosomy 5, 7, del(5q),del (7q); there are also AML with normal karyo-type and AML with complex karyotype.

Cytogenetic abnormalities in LAL

The most common cytogenetic abnormalityin precursor B ALL is hyperdyploidy with 50 chro-mosomes, most frequently encountered in patients of2-10 years of age, associated with low or intermedi-ate number of blasts in peripheral blood. An addition-al copy of chromosomes 4 and/or 10 is frequentlyencountered. ALL with 50 chromosomes hyper-dyploidy has a very good therapeutic response.

Structural cytogenetic changes in BALL have an intermediate to unfavorable pro-gnosis. An exception is t(12;21)(p12;q22),which is usually not showed by classical cyto-genetics. It requires FISH or PCR for identifica-tion and has a favorable prognosis.

Patients with precursor B ALL with t(9;22)(q24;q11) or abnormalities on 11q23- most com-monly t(4;11)(q21;q23), have unfavorable prognosis.

Strategy for genetic investigation ofacute leukemia may vary, but always involves acytogenetic analysis at onset, followed if neces-sary by molecular biology studies, for example: for ALL B cases in children one should seek

cryptic translocation t(12;21)(p13;q22) ETV6-RUNX1 bu using FISH or PCR;

cases of AML with morphology suggestive forinv(16) or t(16;16)(p13.1;q22) should be con-firmed by genetic analysis;

in cases of Burkitts lymphoma, in whichMYC gene translocation from chromosome 8to chromosome 14 specifically occurs, thePCR technique is not recommended due to the


heterogeneity of break points and, as such,FISH is preferred;

identification of specific translocations forAPL in cases with uncommon morphology isessential for initiating treatment with ATRAand reduces the risk of DIC, extremely high inthese cases;

identification of BCR-ABL fusion gene (p190)with prognostic significance in ALL.

Current theory on leukemogenesis im-plies the existence and time sequence of at leasttwo mutations: class I mutation providing sur-vival and proliferation advantage and class IImutation affecting the differentiation, matura-tion and apoptosis.

Thus NPM1, FLT3-TKD and CEBP areclass II mutations with favorable prognosis, unlessFLT3-ITD mutation is associated. The latter is aclass I mutation with unfavorable prognosis.

Other mutations with unfavorable pro-gnosis are MLL-PTD (class II), overexpressionof BAALC gene (class II), ERG overexpression(class I), and WT1 mutation.

In conclusion, the diagnostic steps forLA currently involve conducting the followinginvestigations:automated blood cell count and blood film

morphology bone marrow aspiration biopsy- is absolutely

necessary until the number of blasts in peri-pheral blood is high

cytochemistry and immunophenotyping byflow cytometry

cytogenetic examination in all cases, RT-PCRand FISH for selected cases.

Other laboratory investigations essentialto describe the clinical-biological status of thepatient at onset and in follow-up are: coagula-tion studies, biochemical determinations: LDHand uric acid are usually high in patients withLA, liver and kidney function assessment beforestarting treatment, microbiologic investigationof different biological products for appropriateantimicrobial therapy, HLA and DNA typing forpotential candidates for allogenic transplant.

pentru cazurile de limfom Burkitt, n care n modspecific se produce translocaia genei MYC depe cromozomul 8 pe cromozomul 14, tehnicaPCR nu este recomandat datorit heterogenitiipunctelor de ruptur i se prefer FISH;

identificarea translocaiei specifice pentru LAP n ca-zurile cu morfologie necaracteristic este esenialpentru iniierea tratamentului cu ATRA i reducereariscului de CID, extrem de mare la aceste cazuri;

identificarea genei de fuziune ABL-BCR(p190) cu importan prognostic n LAL.

Teoria actual a leucemogenezei presu-pune existena i succesiunea n timp a cel puindou mutaii: o mutaie de clasa I, care oferavantaj de supravieuire i proliferare a clonei io mutaie de clasa II, care altereaz diferenie-rea, maturaia i apoptoza.

Astfel mutaiile NPM1, FLT3-TKD iCEBP - sunt mutaii de clasa II cu prognosticfavorabil, cu condiia s nu se asocieze cu mu-taia FLT3-ITD. Aceasta din urm este o mutaiede clas I cu prognostic defavorabil.

Alte mutaii cu prognostic defavorabil suntMLL-PTD (clasa II), supraexpresia genei BAALC(clasa II), supraexpresia ERG (clasa I), mutaia WT1.

In concluzie, etapele de diagnostic al LApresupun n prezent efectuarea urmtoarelor in-vestigaii:hemograma, frotiu sanguinpuncie - biopsie medular - se poate renuna

doar dac numrul de blati din sngele perife-ric este mare;

citochimie sau imunofenotipare prin citometrien flux;

citogenetic pentru toate cazurile, RT-PCR iFISH pentru cazuri selectatate.

Alte investigaii indispensabile pentru con-turarea tabloului clinico-biologic al bolnavului cu LAla debut i n evoluie sunt explorarea coagulrii, de-terminri biochimice (LDH i acid uric sunt de obiceimari la pacienii cu LA), explorarea funciei hepaticei renale nainte de iniierea tratamentului, culturi dinproduse biologice pentru terapie antimicrobianadecvat, tipizare HLA i ADN pentru pacienii po-teniali candidai pentru transplant allogen.


LAM M1 LAM M1 peroxidaza

Corpi Auer LAM M2 peroxidaza

LAM M3 LAM M3 varianta


LAM M4 LAM M4 cu eozinofile

LAM M55 LAM M6

LAL 1/ LAL 2 LAL 3


Abbreviations:

ABL BCR Abelson - breakpoint cluster region, AL acute leukemia,ALL acute lymphoblastic leukemia, AML acute myeloid leukemia, APL acute promyelocytic leukemia, ATRA all trans retinoic acid, BAALC Brain and Acute Leukemia, Cytoplasmic, CBF core binding factor, CD cluster of differentiation, CEBPA CCAAT/enhancer-binding protein, del deletion, DIC disseminated intravascular coagulation, DNA deoxyribonucleic acid, EGIL European Group for the Immunological

Characterization of Leukemias, ERG Ets -erythroblastosis virus E26 oncogene

homolog - related gene, FAB French - American - British, FISH fluorescent in situ hybridization, FLT3 FMS-like tyrosine kinase 3, HLA human leukocyte antigen, i inversion, ITD internal tandem duplication, M4Eo acute myeloid leukemia M4 with

eosinophilia, MDS myelodysplastic syndrome, MGG May Grunwald Giemsa, MIC morphology, immunophenotyping,

cytogenetics, MLL mixed lineage leukemia, MPO or POX Myeloperoxidase, MYH myosine heavy chain, NPM1 nucleophosmin 1, PCR polymerase chain reaction, PTD partial tandem duplication, RNA ribonucleic acid. RT-PCR reverse transcriptase polymerase chain

reaction, SSC side scatter, t translocation, TCR T-cell receptor, TdT Terminal deoxynucleotidyl transferase, TKD tyrosine kinase domain, WHO World Health Organization, WT Wilms tumour,

Abrevieri:

ABL-BCR Abelson - breakpoint cluster region, ADN acid dezoxiribonucleic, ARN acid ribonucleicATRA all transretinoic acid, BAALC Brain and Acute Leukemia, Cytoplasmic, CBF core binding factor, CD cluster of differentiation, CEBPA CCAAT/enhancer-binding protein, CID coagulare intravascular diseminat, del deleie, EGIL European Group for the Immunological

Characterization of Leukemias, ERG Ets -erythroblastosis virus E26 oncogene

homolog - related gene, FAB French - American British, FISH fluorescent in situ hybridization, FLT3 FMS-like tyrosine kinase 3, HLA human leukocyte antigen, i inversiune, ITD internal tandem duplication, LA leucemie acut, LAL leucemie acut limfoblastic, LAM leucemie acut mieloid, LAP leucemie acut promielocitar, M4Eo leucemia acut mieloid M4 cu eozinofile, MGG May Grunwald Giemsa, MIC morphology, immunophenotyping, cytogenetics, MLL mixed lineage leukemia, MPO mieloperoxidaz, MYH myosine heavy chaine, NPM1 nucleophosmin 1, OMS Organizatia Mondial a Sntaii, PCR polymerase chain reaction, PTD partial tandem duplication, RT-PCR reverse transcriptase polymerase chain

reaction, SMD sindrom mielodisplazic, SSC side scatter, t translocaie, TCR T-cell receptor, TdT Terminal deoxynucleotidyl transferase, TKD tyrosine kinase domain, WT Wilms tumour.


References

1. Paiu M. Examenul citologic al frotiului sangvin Editura Alma Mater, Cluj- Napoca 20092. Cucuianu A. Leucemiile acute. In Hematologie clinic-editor coordonator Petrov L. Casa Crii de tiin Cluj-Napoca 2009 3. Vardiman JW, Thiele J, Arber DA, Brunning RD,Borowitz MJ, Porwit A, Harris NL, Le Beau MM,Hellstrm-Lindberg E, Tefferi A, Bloomfield CD. The 2008revision of the World Health Organization (WHO) classi-fication of myeloid neoplasms and acute leukemia: ra-tionale and important changes. Blood 2009 114: 937-9514. Amy Heerema-McKenney, Arber DA. Acute MyeloidLeukemia. Hematol Oncol Clin N Am 23 2009: 6336545. Onciu M. Acute Lymphoblastic Leukemia. HematolOncol Clin N Am 23 2009 ;114: 937-9516. Dhner H, Estey EH, Amadori S, Appelbaum FR,Bchner T, Burnett AK, et al; European LeukemiaNet. Dia-gnosis and management of acute myeloid leukemia inadults: recommendations from an international expert pan-el, on behalf of the European LeukemiaNet. Blood 2010Jan 21;115(3):453-747. Fiona E. Craig, Foon KA. Flow cytometricimmunophenotyping for hematologic neoplasm. Blood2008; 111: 3941-39678. Marie C. Bn. Biphenotypic, bilineal, ambiguous ormixed lineage: strange leukemias! Haematologica;2009;94(7) : 891-8939. Frattini MG, Maslak PG. Strategy for IncorporatingMolecular and Cytogenetic Markers into Acute MyeloidLeukemia Therapy. J. Natl. Compr. Canc. Netw. 20086(10):995-1002

References

1. Paiu M. Examenul citologic al frotiului sangvin. Edi-tura Alma Mater, Cluj-Napoca 20092. Cucuianu A. Leucemiile acute. In Hematologie clinic-editor coordonator Petrov L. Casa Crii de tiin. Cluj-Napoca 2009 3. Vardiman JW, Thiele J, Arber DA, Brunning RD,Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hell-strm-Lindberg E, Tefferi A, Bloomfield CD. The 2008 re-vision of the World Health Organization (WHO) classifica-tion of myeloid neoplasms and acute leukemia: rationaleand important changes. Blood 2009 114: 937-9514. Amy Heerema-McKenney, Arber DA. Acute MyeloidLeukemia. Hematol Oncol Clin N Am 23 2009: 6336545. Onciu M. Acute Lymphoblastic Leukemia. HematolOncol Clin N Am 2009 114: 937-9516. Dhner H, Estey EH, Amadori S, Appelbaum FR,Bchner T, Burnett AK, Dombret H, et al; European Leuk-emiaNet. Diagnosis and management of acute myeloidleukemia in adults: recommendations from an internationalexpert panel, on behalf of the European LeukemiaNet.Blood 2010 115(3):453-747. Fiona E. Craig, Foon KA. Flow cytometric immun-ophenotyping for hematologic neoplasm. Blood 2008 111:3941-39678. Marie C. Bn. Biphenotypic, bilineal, ambiguous ormixed lineage: strange leukemias! Haematologica 200994(7): 891-8939. Frattini MG, Maslak PG. Strategy for IncorporatingMolecular and Cytogenetic Markers into Acute MyeloidLeukemia Therapy. J. Natl. Compr. Canc. Netw. 20086(10):995-1002

RRML Vol 18 Iunie 2010 Laboratory Diagnosis of Acute Leukemia

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