Leucemia Linfoblastica Acuta

(adulto)

Classificazione delle leucemie linfoidi acute, WHO 2008

Leucemie linfoblastiche acute a cellule «precursor» B

• Leucemia/linfoma linfoblastica/o B, NAS

• Leucemia/linfoma linfoblastica/o B con anomalie genetiche ricorrenti

• Leucemia/linfoma linfoblastica/o B con t(9;22) (q34;q11.2);BCR-ABL1

• Leucemia/linfoma linfoblastica/o B con t(v;11q23); MLL riarrangiata

• Leucemia/linfoma linfoblastica/o B con t(12;21)(p13;q22); TEL-AML1(ETV6-RUNX1)

• Leucemia/linfoma linfoblastica/o B con iperdiploidia

• Leucemia/linfoma linfoblastica/o B con ipodiploidia

• Leucemia/linfoma linfoblastica/o B con t(5;14)(q31;q32);IL3-IGH

• Leucemia/linfoma linfoblastica/o B con t(1;19)(q23;p13.3); E2APBX1 (TCF3-PBX1)

Leucemie linfoblastiche acute a cellule «precursor» T

• Leucemia/linfoma linfoblastica/o T

Neoplasie a cellule B mature

• Linfoma di Burkitt (include anche la rara variante leucemica, L3 FAB)

NB Esistono poi forme miste, indifferenziate, bifenotipiche, bilineari…

LLA: Esami diagnostici

Sangue periferico:

Emocromo con formula leucocitaria ed esame morfologico (ematologo esperto) mediante striscio periferico

blasti > 2=% (leucocitosi non sempre presente, neutropenia, piastrinopenia, anemia)

Immunofenotipo (citometria a flusso): linea linfoide (CD13-; CD14-), linea B (CD10+, CD19+, CD20+, CD22+)

o T (CD1a+, CD3+, CD4+, CD7+, CD8+). Altri antigeni: CD34, HLA-Dr, TdT

Citogenetica/FISH e biologia molecolare: cromosoma Philadelphia / traslocazione BCR/ABL (30 % circa nel paziente

adulto;

va eseguita in tutti i pazienti indipendentemente dall’età, per la possibilità di terapia con TKI)

Aspirato midollare:

Striscio con mielogramma per valutazione (ematologo esperto)della percentuale di blasti midollari e della riserva

emopoietica

Citochimica: non più consigliata (puo’ essre utile la perossidai: negativa)

Immunofenotipo (citometria a flusso: linea linfoide (CD13-; CD14-), linea B (CD10+, CD19+, CD20+, CD22+)

o T ( CD1a+, CD3+, CD4+, CD7+, CD8+). Altri antigeni: CD34, HLA-Dr, TdT.

Citogenetica/FISH e biologia molecolare: cromosoma Philadelphia / traslocazione BCR/ABL

Biopsia osteomidollare:

Raramente necessaria (aspirato midollare povero di cellule) e nel caso immunoistochimica

NB. In tutti i pazienti es. del liquor (morfologico ed immuofenotipico)

LLA: Diagnostica avanzata

• Identificazione forme Ph-like (altri geni di fusione: potrebbero giovarsi di terapia con TKI come le LAL Ph+)

• Mutazione di IKZ (valore prognostico nelle forme sia Ph+che Ph-)

• TAC total body (nelle forme T massa mediastinica, nelle Burkitt-like o L3 masse toracoa-addominali)

• NGS (identificazione di sottogruppi di mutazioni sensibili a nuovi farmaci)

LLA: Follow-up

• Valutazione della minimal residual disease

(MRD), dopo consolidamento nelle forme Ph-, al

termine della prima fase di terapia con TKI, in

genere 80-90 giorni nelle Ph+.

• MRD: Ph+ molecolare

• MRD: Ph- immunofenotipo

Leucemia Mieloide Acuta

984-993 LEUCEMIE MIELOIDI

LAM con traslocazioni citogenetiche ricorrenti:9896/3 LAM con t(8;21)(q22;22), AML1(CBFα)/ETO M29866/3 LA Promielocitica [LAM con t(15;17)(q22;q21) e varianti, PML/RAR-α] M39871/3 LAM con ipereosinofilia midollare [inv(16)(p13;q22) o t(16;16), cbfb/myh11] M4eo9897/3 LAM con anomalie 11q23 (MLL)

LAM con displasia multilineare 9895/3 LAM con/senza precedente sindrome mielodisplastica9920/3 LAM e sindromi mielodisplastiche correlate a terapie agenti alchilanti, epipodofillotossine, altri tipi

LAM non altrimenti classificate9872/3 LAM scarsamente differenziata M09873/3 LAM senza maturazione M19874/3 LAM con maturazione M29866/3 LA promielocitica M39867/3 LA mielomonocitica M49891/3 LA monocitica M59840/3 LA eritroide M69910/3 LA megacariocitica M79870/3 LA basofilica9931/3 Panmielosi acuta con mielofibrosi

9805/3 Leucemie acute bifenotipiche

9860/3 Leucemia mieloide, NAS

9861/3 Leucemia mieloide acuta, NAS

Classificazione WHO 1997

Emocromo con formula leucocitaria (ematologo esperto in morfologia)

• Blasti > 20% condizione necessaria e sufficiente

Di solito si associa anemia, neutropenia, piastrinopenia, non sempre è

presente leucocitosi (se presente >10.000/µL)

Può essere anche l’unico esame se paziente molto anziano e/o frail, nel quale si

decide esclusivamente terapia di supporto

Diagnosi di LAM Su sangue periferico:

• Striscio + Mielogramma

valutazione morfologica e quantitativa del midollo al microscopio ottico

(ematologo esperto in morfologia) per valutare la percentuale e la morfologia dei

blasti e la riserva emopoietica

• Citofluorimetria cellule fenotipo immunologico:

CD34, CD13+, CD14+, CD33+ (markers di differenziazione della linea mieloide)

Il pannello viene allargato ad altri antigeni anche allo scopo di identificare i LAIP da utilizzare

nello studio della MRD

• BOM:

solo in caso di aspirato non informativo

Diagnosi di LMA

Su agoaspirato midollare:

LAM: Citogenetica e biologia molecolare

• Citogenetica: ricerca di traslocazioni specifiche

utili a definire la prognosi

• Biologia molecolare: mutazione di FLT3, NPM1

e mutazione biallelica di CEBPa (definizione del

rischio nella citogenetica normale secondo le

raccomandazioni ELN e NCCN)

• FISH in casi selezionati

Leucemia promielocitica, FAB M3 APL

La traslocazione t(15;17) coinvolge il gene che codifica per il recettore nucleare a dell'acido retinoico RAR sul cromosoma 17 ed il gene PML (promielocitica) sul cromosoma 15 11 t(15;17)(q22;q11).

Fondamentale per la diagnosi e la terapia la

biologia molecolare con il monitoraggio del gene

ibrido PML-RARalpha, che va eseguito al termine

del consolidamento e poi ogni 2-3 mesi

LMA: Diagnostica avanzata

• Congelamento di cellule patologiche all’esordio

(utile per studi futuri)

• NGS (possibilità di sottogruppi sensibili a farmaci

in grado di inibire specifiche mutazioni)

LMA: Follow up

• RC: valutazione morfologica

• MRD: solo in studi clinici

• MRD: “mandatory” nella LAP

• Different clonal neoplastic disorders of hematopoietic stem cells

• Heterogeneous biologic and clinical characteristics

• “Rich marrow” (dysplastic, ineffective myelopoiesis due to excess

of apoptosis) and “poor peripheral blood “ (variously combined

cytopenias: anemia, leucopenia, thrombocytopenia)

• Low-to-high risk of leukemic transformation

• Very variable prognosis

Myelodysplastic Syndromes: a lot of directions

Myelodysplastic Syndromes

Increased apoptosis

Ineffective hemopoiesis

Lower-risk MDS: 75%

Apoptosis

Proliferation

Myelodysplastic Syndromes

Reduced apoptosis

Genetic evolution

Leukemic transformation

Higher-risk MDS: 25%

Proliferation

Apoptosis

Diagnosis and Evaluation

No specific clinical feature that distinguishes MDS from other causes of anemia (or other cytopenias)

Lab evaluation often prompted by signs or symptoms of the underlying cytopenias

– Fatigue, pallor, cardiac failure (anemia)– Infections (neutropenia)– Bleeding, ecchymoses, petechiae (thrombocytopenia)

Minimum work-up (1)

• Detailed patient’s history of transfusion need, professional toxic exposure and chemotherapic or radio-therapic treatments*, as well as severe co-morbidities

• Complete blood count, a peripheral blood smear examination with differential leukocyte count and a bone marrow aspiration with cytogenetics and morphologic evaluation, including Perls staining

• Bone marrow biopsy in order to assess marrow architecture, cellularity (hypoplastic MDS), fibrosis (primary myelofibrosis) and percentage of blasts

SIE, SIES and GITMO Guidelines, Santini et al. Leuk Res 2010* Secondary/Therapy-related MDS

• Serum erythropoietin determination in patients with symptomatic anaemia

• Iron status evaluation, i.e. serum ferritin and transferrin saturation in patients who are transfusion dependent or who start transfusion therapy

• DEB test in patients younger than 30 years who are possible candidates for high-dose chemotherapy or allogeneic HSCT, in order to exclude a Fanconi anaemia-associated MDS that is contraindicating chemotherapy

• HLA typing in patients eligible for HSCT, and those with an hypoplastic bone marrow (HLA-Dr15), in order to further support decision on immunosuppressive therapy

Minimum work-up (2)

SIE, SIES and GITMO Guidelines, Santini et al. Leuk Res 2010

Malcovati et al, European LeukemiaNet Guidelines, Blood 2013

B-2

* Prothrombin time

*

Malcovati et al, European LeukemiaNet Guidelines, Blood 2013

*

*

* and monocytes

301

530 529

0

100

200

300

400

500

Hb < 8 gr/dl Hb da 8 a 10 gr/dl Hb > 10 gr/dl

22% 39% 39%

FISM data: Cytopenias in 1361 MDS patients

232

339

786

0

100

200

300

400

500

600

700

800

<50 51-100 >100

17% 25% 58%

161213

371

573

0

100

200

300

400

500

600

<500 501-1000 1001-2000 >2000

12% 16% 28% 44%

Anemia

Thrombocytopenia

Neutropenia

• Idiopathic

• Cytopenia/Dysplasia

• Undetermined

• Significance

• Might be MDS, but might not

• Needs follow-up, no treatment!

A definitive diagnosis

of MDS may be not

immediate!

ICUS / IDUS

Tefferi and Vardiman, N Engl J Med, 2009

• It demonstrates the clonality of the disease, resolving problems of differential diagnosis

• It allows to identify specific genomic regions where genes involved in the pathogenesis of the disease are located

• It contributes to recognize specific biological and clinical entities

• It is probably the most important prognostic factor

• It may be helpful for monitoring the effects of therapies applied

Clinical relevance of detecting chromosomal abnormalities at diagnosis

Malcovati L, Educational Book, EHA 2012

Recurring chromosomal abnormalities in

t - MDS / t - AML

Normal

8%Balanced

4%

Other

12%

Abnl 5

22%

Abnl 7

30%

Both 5/7

24%

SingleDel(11q)-Y

Schanz et al, J Clin Oncol 2011

Prognostic relevance of cytogenetic abnormalities in MDS

FAB WHO• Not described Uni/Multilineage dysplasia

• Not described MDS with isolated del(5q)

• RAEB-T (BM blasts 20-30%) > AML

• CMML > MDS/MPD

• del5q as unique chromosomal abnormality

• Macrocytic anaemia, slight leucopenia, normal to elevated platelet

• Small, hypolobated, mononuclear, spheronuclear megakaryocytes

• Predominantly middle-aged to older women

• < 15% blasts in blood and marrow

• Refractory anaemia, erythroid dysplasia/ hypoplasia, transfusion dependence

• Indolent course, 10–15% acute myeloid leukaemia, median survival > 5 years

• Interstitial, variable size 5q13-33 deletions (CDB: “common deleted band” 5q 31-32) in hematopoietic stem cells

• Marked hematological and cytogenetic response to lenalidomide

5q- “syndrome”: a distinct form of MDS in a subset of patients with isolated del 5q

Evidence suggests that haploinsufficiency of genes encompassed in or around the CDB

5q32–33 leads to the development of 5q– syndrome

Disease complexity and heterogeneity in MDS with del(5q)

MDS del(5q)

• There is a general belief that MDS with del(5q) as an isolated cytogenetic abnormality has a favorable prognosis

• This is probably due to confusion about an old definition of the term “5q-syndrome”, that should be reserved only

to a distinct form of MDS in a subset of patients with isolated del 5q and well defined clinical and morphological

characteristics (see above)

• Today, there is increasing evidence that del(5q) MDS is heterogeneous with respect to clinical, pathological,

molecular and prognostic findings

Platelet count

Karyotype

complexity

Extend of

deletion Transfusion

status

Age/Sex

TP53

mutationsErythroid

hypoplasiaBM Blast count

WHO

morphology

The FAB had also already arbitrarily

categorized CMML into MDS-like and

MPD-like groups, using a white blood

count of 13x109/L as a cut-off to

differentiate the two entities.

Chronic Myelo-Monocytic Leukemia

Refractory anaemia with ring sideroblasts (RARS) and RARS with marked thrombocytosis (RARS-T):

provisional entity in the WHO 2008 classification characterized by high proportion of JAK2V617F and SF3B1 mutations

Cazzola et al, Blood, 2013

RARS

RARS-T

International Prognostic Scoring System (IPSS)

Punteggio RischioSopravvivenza

mediana(anni)

0 Basso 5,7

0,5-1,0 Intermedio-1 3,5

1,5-2,0 Intermedio-2 1,2

2,5 Alto 0,4

Modificata da Greenberg P, et al. Blood 1997;89:2079-2088.

* ANC <1500/mm3, Hb <10 g/dl, PLT <100.000/mm3

Punteggio

Variabili prognostiche 0 0,5 1,0 1,5 2,0

Blasti midollari <5 5-10 – 11-20 21-30

CariotipoNormale o-Y o del 5qo del 20q

Altre anomalieAnomalie

complesseo del cr.7

Citopenie* 0-1 2-3

Effect of comorbidity on survival of MDS patients

Overall Survival Risk of Non-Leukemic Death

Della Porta et al, Haematologica 2009

Italian registry for MDS:

Presence of comorbidities (n = 388)

• CIRS, Cumulative Illness Rating Scale.

142

99

7770

0

20

40

60

80

100

120

140

160

Grade 0–2 n. 1 n. 2 n. > 2

20% 18%25%37%

Pa

tie

nts

, n

Comorbidities

63% with (at least one) comorbidities degree > 3 (CIRS)

5

12

19 18

3634

29

14

0

5

10

15

20

25

30

35

40

< 60 anni 61-70 71-80 > 80 anni

MDS indipendente MDS correlabile

22

5

149

1510

38

54

0

10

20

30

40

50

60

%

RA/RARS RCMD CMML RAEB

MDS indipenenti MDS correlabili

55

27

15

39

0

10

20

30

40

50

60

%

IPSS low-int1 IPSS int2-high

MDS indipendente MDS correlabile

Dati su 167 decessi in pazienti con SMD

Bejar, Haematologica 2014

Impact of mutations on survival of MDS patients

0 5 10 15 20 25 30 35 40Time from randomization (months)

0

10

20

30

40

50

60

70

80

90

100

Pe

rcen

tage s

urv

ivin

g

CCR

AZA

Difference in median OS was 9.4 months

24.4 months

15 months

50.8%

26.2%

100 200 300 400 500

1.0

0

0.25

0.50

0.75

0

0

Duration, months

Pro

po

rtio

n

su

rviv

ing

Non-chelated (n = 336)

Chelated (n = 264)

Lyons RM, et al. Blood. 2012;120:abstract 3800.

Fenaux et al, Blood 2011

Park et al, Blood 2008

Fenaux P, et al. Lancet Oncol. 2009;10:223-32.

Impact of novel treatments on survival of MDS patients

Erythropoietin

Chelation therapy

P < 0.0001 Lenalidomide

Azacitidine