DIAGNOSI E TERAPIA DELLE LEUCEMIE
LINFOBLASTICHE ACUTE
e
DELLE SINDROMI LINFOPROLIFERATIVE CRONICHE,
DEI LINFOMI
E DEL MIELOMA MULTIPLO
Giovanni Martinelli, MD
Istituto di Ematologia e Oncologia Medica
L. & A. Seràgnoli, Università degli Studi di Bologna
ALL
DANNO GENETICO
ANLL o
AML
L’analisi molecolare:

Identifica gruppi di pazienti con diversa prognosi

Anomalie genetiche non citogeneticamente identificate o
caratterizzate

Significato clinico diverso della persistenza di MMR

Livelli di malattia minima residua assai piccoli.

Suggerisce la risposta alla chemioterapia

Meccanismi molecolari sconosciuti.
•
Geni non di Fusione
Si definiscono geni non di fusione gli oncogeni associati
a traslocazioni cromosomiche specifiche, generati dalla
ricombinazione tra due geni.
Danno origine a m-RNA neoplastici, il cui prodotto e’
spesso over espresso ed e’ oncogeno anche se solo
uno dei due geni e’ funzionante.
Un tipico esempio e’ l’oncogene Myc associato alla
traslocazione t(8;14)
Istituto di Ematologia ed Oncologia Medica “Seràgnoli”, Bologna
GENI NON DI FUSIONE
A
C
R
B
R
R
C
C
Geni di Fusione
Si definiscono geni di fusione gli oncogeni associati a traslocazioni
cromosomiche specifiche, generati dalla ricombinazione tra due
geni.
Danno origine a m-RNA chimerici il cui prodotto e’ espresso ed e’
neoplastico se entrambi i due oncogeni sono funzionanti.
Un tipico esempio e’ l’oncogene BCR-ABL associato alla traslocazione
t(9;22) ed alla Leucemia Mieloide Cronica, oppure l’oncogene PMLRAR alfa associato alla t(15;17) ed alla Leucemia Acuta a
promielociti.
Istituto di Ematologia ed Oncologia Medica “Seràgnoli”
GENI DI FUSIONE
R
R
C
R
C
C5’
C3’
Acute Lymphoid Leukemia
FREQUENCY OF PRINCIPAL TRANSLOCATION AND FUSION GENEs IN ALL
19%
3%
4%
5%
25%
1%
5%
4%
6%
28%
none 19%
random 25%
BCR-ABL4%
TEL-AML1 28%
MLL fusions 6%
E2A-PBX1
E2A-HLF 1%
MYC 5%
HOX11-LYL1-LMO-TAL:T cell 14q11/TCR ad translocation 4%
HOX11-LYL1-LMO-TAL:T cell 7q35/TCR B translocation 3%
ALL
20%
14%
25%
41%
none 20%
random 14%
TF 25%
RTK 41%
Principali alterazioni molecolari e fusion
gene e loro frequenza nelle ALL
•none
19%
•random
•BCR-ABL
•TEL-AML1
•MLL fusions
•11q23 (AMLL1/Htrx/Hrx/ALL-1)
• E2A-PBX
25%
4%
28%
6%
13 (1% )
1%
Istituto di Ematologia ed Oncologia Medica “Seragnoli”, Bologna
Estimated Frequency of Specific
Genotypes of ALL in Children and
Adults.
Transformation of Hematopoietic Cells in the Pathogenesis of ALL.
Mechanism of Transcriptional Repression by TEL-AML1.
The Retinoblastoma (RB) and p53 Tumor-Suppressor Network.
Kaplan–Meier Analysis of Event-free Survival According to the Subtype of Leukemia in
467 Children with ALL Who Were Enrolled in Three Consecutive Treatment Protocols at
St. Jude Children's Research Hospital from 1991 to 1999.
Influence of Host Germ-Line and ALL Blast Genotypes on the Probability of Cure and of
Adverse Events.
Analisi molecolare: Microarray
Principali alterazioni molecolari e fusion
gene e loro frequenza nelle ALL
•random
•BCR-ABL
•TEL-AML1
•MLL fusions
•11q23 (AMLL1/Htrx/Hrx/ALL-1)
• E2A-PBX
•none
25%
4%
28%
6%
13 (1% )
1%
19%
Istituto di Ematologia ed Oncologia Medica “Seragnoli”, Bologna
Is ALL Ph+ homogenous?
INDIVIDUAL VARIABILITY?
Deletions on the
derivative
chromosome 9q+
Struttura dei geni ABL e BCR e sonde utilizzate in FISH a 3 colori e in D-FISH
(Oncor)
Sinclair et al., Blood 2000; 95:738-744
chr.9
LMX2
NEK6
PSMB7
NR5A1
NR6A1
RPL35
GOLGA1
PPP6C
HSPA5
SIN1_HUMAN
PBX3
ANGPTL2
SLCA8
RPL12
STXBP1
SH2D3C
CDK9
FPGS
ENG
AK1
SIAT7D
DPM2
ZNF297B
LMX1B
C9orf15
C9orf16
LCN2
CIZ1_HUMAN
DNM1
GOLGA2
SLC27A4
ODF2
GLE1L
SPTAN1
SET
SH3GLB2
CRAT
PPP2R4
CCBL1
ENDOG
PMX2_HUMAN
PTGES
DYT1
USP2O
FREQ
ASS
FUBP3
PRDM12
RRP4_HUMAN
cen
Prostaglandin E synthase
tel
LMX2
NEK6
PSMB7
NR5A1
NR6A1
RPL35
GOLGA1
PPP6C
HSPA5
SIN1_HUMAN
PBX3
ANGPTL2
SLCA8
RPL12
STXBP1
SH2D3C
CDK9
FPGS
ENG
AK1
SIAT7D
DPM2
ZNF297B
LMX1B
C9orf15
C9orf16
LCN2
CIZ1_HUMAN
DNM1
GOLGA2
SLC27A4
ODF2
GLE1L
SPTAN1
SET
SH3GLB2
CRAT
PPP2R4
CCBL1
ENDOG
PMX2_HUMAN
PTGES
DYT1
USP2O
FREQ
ASS
FUBP3
PRDM12
RRP4_HUMAN
ABL
LMX2
NEK6
PSMB7
NR5A1
NR6A1
RPL35
GOLGA1
PPP6C
HSPA5
SIN1_HUMAN
PBX3
ANGPTL2
SLCA8
RPL12
STXBP1
SH2D3C
CDK9
FPGS
ENG
AK1
SIAT7D
DPM2
ZNF297B
LMX1B
C9orf15
C9orf16
LCN2
CIZ1_HUMAN
DNM1
GOLGA2
SLC27A4
ODF2
GLE1L
SPTAN1
SET
SH3GLB2
CRAT
PPP2R4
CCBL1
ENDOG
PMX2_HUMAN
PTGES
DYT1
USP2O
FREQ
ASS
FUBP3
PRDM12
RRP4_HUMAN
tel
LMX2
NEK6
PSMB7
NR5A1
NR6A1
RPL35
GOLGA1
PPP6C
HSPA5
SIN1_HUMAN
PBX3
ANGPTL2
SLCA8
RPL12
STXBP1
SH2D3C
CDK9
FPGS
ENG
AK1
SIAT7D
DPM2
ZNF297B
LMX1B
C9orf15
C9orf16
LCN2
CIZ1_HUMAN
DNM1
GOLGA2
SLC27A4
ODF2
GLE1L
SPTAN1
SET
SH3GLB2
CRAT
PPP2R4
CCBL1
ENDOG
PMX2_HUMAN
PTGES
DYT1
USP2O
FREQ
ASS
FUBP3
PRDM12
RRP4_HUMAN
cen
FBXW3
IGLL1
ZNF70
VPREB3
MMP11
SMARCB1
SLC2A11
MIF
GSTT2
DDT
GSTT1
CABI_HUMAN
GGTLA1
tel
chr.22
SMARCB1: SWI/SNF related, actin dependent
regulator of chromatin subfamily B member 1
GSTT1:Glutathione S-transferase theta 1
Principali alterazioni molecolari e fusion
gene e loro frequenza nelle ALL
•random
•BCR-ABL
•TEL-AML1
•MLL fusions
•11q23 (AMLL1/Htrx/Hrx/ALL-1)
• E2A-PBX
•none
25%
4%
28%
6%
1%
1%
19%
Istituto di Ematologia ed Oncologia Medica “Seragnoli”, Bologna
11q23 and MLL
MLL positive leukemias
t(1;11)
t(11;19)
t(4;11)
t(11;22)
t(6;11)
t(X;11) (q13;q23)
t(9;11)
t(X;11)(q24;q23)
MLL
t(10;11)
t(11;17)
del 11 (q11q23)
del 11 (q21q23)
Breakpoints in MLL
5 6
7 8
10
9
11
12
13
centromere
telomere
Zn fingers
A-T hooks MT
MLL
fusion point
ENL
MLL
ENL
der 11 chromosome
MLL and Fusion Patners in Leukemia
ENL
MLL
AF10
AFA
CALM
AF17
AF6q21
ELL
AF19
Eps15
AF5q31
AFX
ABI
MSF
ENN
AF3q21
AMLL1/Htrx/Hrx/ALL-1
AT
PHD
MTase
BP??
Chr. 8 and t(8;14) and BURKITT LUKEMIA
TRANSLOCATIONS OF c-Myc Locus
Reciprocal chromosomal translocations in Burkitt's
lymphoma, a solid tumour of B lymphocytes
involves chr.14 and
chr 8
chr.2
chr 22.
The genes for making the heavy chains of antibodies (CH) are located on chromosomes
14, whereas those for making the light chains are on chromosomes 2 and 22.
Struttura dell’anticorpo
Patologia dell’ sintesi Ig
Struttura dell’anticorpo
Le Ig possono essere suddivise in classi o sottoclassi sulla base delle catene
pesante
ISOTIPI
—IgM
m
—IgA
a1 a2
—IgG
g1, g2, g3, g4
—IgD
d
—IgE
e
Configurazione geni Ig germinale
IgH: Cromosoma 14
Igk: Cromosoma 2
Igl: Cromosoma 22
Riarrangiamento catena
leggera
Mapping of cloned germline and
rearranged antigen receptor
gene segments
Genomic structure of selected
antigen receptor loci
Cloning of the V(D)J recombinase genes.
The RAG proteins and the RAG
cleavage reaction
(A) Schematic of RAG-1 and
RAG-2,
illustrating
known
mofits and the putative RAG-1
DDE active site
(B) Summary of the RAG
cleavage reaction.
V(D)J recombination and the
non-homologous
end-joining
pathway
Pre-antigen
DH
JH
Antigen
Mature-B
VH
Plasma cell
IgD
DHJH
Pro-B
IgM
Pre-B
VKJK
Ig
IgM
Immature-B
Maturazione del B linfocita
Activated-B
Memory-B
Antigene interection with ”naive” B cell
Secrezione di Ig a bassa affinità
Partecipazione reazione CG
Selezione Ag-dipendente
 Proliferazione
 Ipermutazione somatica
 Selezione per: > affinità vs < affinità e/o autoreattività
 Switch isotipico
T-cell dependent Bcell immune response
CD40 on B cell
CD40L: on T cell
(CD154)
Proliferazione
Survivall
Memory
differentiation
Activation-Induced Deaminasi (AID)
 Somatic hypermutations (SHM)
 IgV Gene conversion (IGC)
 Class switch recombination (CSR)
Deaminazione G to U:
 G to U
 Repair with Uracil-DNA-glycosylase (UNG)
casistica
complessiva*
gruppo mutato
(≥2%)**
gruppo germline
(<2%)**
70
p<0.0001
VH1
23 (18.5%)
1 (1.4%)
22 (42.3%)
VH2
1 (0.8%)
1 (1.4%)
0 (0%)
VH3
70 (56.5%)
46 (63.9%)
24 (46.1%)
VH4
26 (21%)
22 (30.6%)
4 (7.7%)
VH5
2 (1.6%)
1 (1.4%)
1 (1.9%)
VH6
1 (0.8%)
0 (0%)
1 (1.9%)
VH7
1 (0.8%)
1 (1.4%)
0 (0%)
Percentuale
60
50
40
Gruppo
mutato
30
Gruppo
germline
20
10
0
Totale
124
72 (58%)
52 (42%)
VH1
VH2
VH3
VH4
VH5
VH6
VH7
Famiglie IgVH
Distribuzione delle famiglie VH nel gruppo con mutazioni nella
regione IgVH  2% e nel gruppo germline con livello di
mutazioni in IgVH <2%
•Gruppo Mutato
Gruppo Germline*
VH1-69
1 (6.7%)
14 (93.3%)
VH3-11
2 (25.0%)
6 (75.0%)
VH3-21
3 (42.9%)
4 (57.1%)
VH4-34
12 (100%)
0 (0%)
VH3-23
9 (81.8%)
2 (18.2%)
VH3-30
6 (66.7%)
3 (33.3%)
14.71 ± 3.90
25
•Gruppo Germline
19.02 ± 4.69
20
HCDR (aa)
Gruppo Mutato*
P<0.0001
15
10
5
GERMLINE
Rappresentazione delle
sottofamiglie VH
maggiormente espresse
nei gruppi mutato e
germline
MUTATI
Lunghezza della regione
ipervariabile CDR3 nei gruppi
mutato e germline.
Funzioni di sopravvivenza
Gruppo Mutato**
Gruppo
Germline**
Significatività
statistica
1,1
55,7 : 44,3
65.6 (32-87)
54,8 : 45,2
67 (50-87)
56,7 : 43,3
68 (32-85)
p = 0,886
p = 0,865
1,0
Gruppo mutato
,9
19,7%
32,1%
11,6%
57,4%
67,9%
61,5%
,8
sopravvivenza cumulata
Maschi:Femmine
Età mediana alla
diagnosi
STADIO RAI
•Basso
Casistica
complessiva*
p = 0,004
27,9%
15,4%
52%
p < 0,0001
9,8%
0%
24%
45.9%
34,6%
73,2%
•Intermedio
11,5%
0%
•Alto
STADIO BINET
45,9%
84,6%
•A
mut_unmut
,7
26,9%
unm
,6
unm-troncata
,5
24%
m
Gruppo germline
,4
m-troncata
0
20
40
60
80
100
•B
•C
Necessità di terapia
follow up(mesi)
p < 0,0001
p=0,0074
Comparazione delle caratteristiche cliniche e della sopravvivenza
fra i gruppi mutato e germline.
1,0
PFS
P <0,0001*
P =0,0012**
,8
disease-free survival
Quantità
Relativa
RNA ZAP70
,6
,4
,2
0,0
Gruppo
ZAP70 <2
Gruppo
ZAP70  2
-,2
0
Gruppo
Gruppo
Mutato
Gruppo
Germline
Espressione relativa del gene
ZAP70 nei gruppi mutato e
germline
40
80
120
160
200
mesi
Grafico di Kaplan-Meier relativo
al PFS nei gruppi con livello di
espressione di ZAP70 superiore
a 2 e inferiore a 2
BASI MOLECOLARI DELLE
SINDROMI
IMMUNOPROLIFERATIVE: MM
MM: UN MODELLO DI PATOGENESI
Normal
Plasma
Cell
MGUS
Primary
Ig translocations
Smouldering
Myeloma
Intramedullary
Myeloma
Extramedullary
Myeloma
Myeloma
Cell Line
Secondary
(Ig)
translocations
Karyotypic instability, 13q/13q14 deletions/monosomy
N-Ras, K-Ras,
or FGFR3 mutations
p53 mutations
p16 methylation/ p18 deletion
Istituto “Seràgnoli” - Bologna
14q32
IgH
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
BCL-1/
CCND1/
PRAD1,
myeov
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
4p16
MMSET,
FGFR3
BCL-1/
CCND1/
PRAD1,
myeov
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
4p16
16q23
MMSET,
FGFR3
BCL-1/
CCND1/
PRAD1,
myeov
c-maf
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
6p21
4p16
16q23
CCND3
MMSET,
FGFR3
BCL-1/
CCND1/
PRAD1,
myeov
c-maf
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
6p21
4p16
~ 20%
of pts
with 14q
translocations
16q23
CCND3
MMSET,
FGFR3
c-maf
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
6p21
4p16
16q23
CCND3
~ 20%
of pts
with 14q
translocations
c-maf
~ 10-15%
of pts
with 14q
translocations
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
6p21
4p16
16q23
CCND3
~ 20%
of pts
with 14q
translocations
~ 10-15%
~ 8%
of pts
with 14q
translocations
of pts
with 14q
translocations
Istituto “Seràgnoli” - Bologna
14q32
IgH
11q13
6p21
4p16
16q23
~ 20%
~ 4%
of pts
with 14q
translocations
of pts
with 14q
translocations
~ 10-15%
~ 8%
of pts
with 14q
translocations
of pts
with 14q
translocations
Istituto “Seràgnoli” - Bologna
IgH locus
reg
coding
oncogene
reg
coding
TRANSLOCATION
IgH locus
reg
oncogene
coding
TRANSCRIPTIONAL UPREGULATION
Istituto “Seràgnoli” - Bologna
MM: UN MODELLO DI PATOGENESI
Normal
Plasma
Cell
MGUS
Primary
Ig translocations
Smouldering
Myeloma
Intramedullary
Myeloma
Extramedullary
Myeloma
Myeloma
Cell Line
Secondary
(Ig)
translocations
Karyotypic instability, 13q/13q14 deletions/monosomy
N-Ras, K-Ras,
or FGFR3 mutations
p53 mutations
p16 methylation/ p18 deletion
Istituto “Seràgnoli” - Bologna
MICROARRAY ANALYSIS
Istituto “Seràgnoli” - Bologna
MICROARRAY ANALYSIS
Tutti i pz affetti da MM di nuova
diagnosi overesprimono
una delle cicline D
Istituto “Seràgnoli” - Bologna
MECCANISMI DI UP-REGOLAZIONE
DELLE CICLINE D NEL MM
t(11;14)
? (unknown)
t(4;14)
t(14;16)
Cyc D2
Cyc D1
?
?
t(6;14)
Cyc D3
?
Multiple Myeloma
Istituto “Seràgnoli” - Bologna
LA FAMIGLIA DELLE CICLINE D
Genomic location
Protein size, kD
CCND1
CCND2
CCND3
11q13
12p13
6p21
33
34
33
high
low
Expression in
none
hematopoietic tissue
Istituto “Seràgnoli” - Bologna
Cell cycle progression:
growth
factors
P
P
DP
pRB
E2F
HDAC
pRB/E2F-mediated
transcriptional
repression of genes
required for G1/S
transition
early G1
late G1
S
Istituto “Seràgnoli” - Bologna
Cell cycle progression:
growth
factors
cyc D
P
P
DP
pRB
E2F
HDAC
pRB/E2F-mediated
transcriptional
repression of genes
required for G1/S
transition
early G1
late G1
S
Istituto “Seràgnoli” - Bologna
Cell cycle progression:
growth
factors
cyc D
CDK4/6
P
P
DP
pRB
E2F
HDAC
pRB/E2F-mediated
transcriptional
repression of genes
required for G1/S
transition
early G1
late G1
S
Istituto “Seràgnoli” - Bologna
Cell cycle progression:
growth
factors
cyc D
P
CDK4/6
P
P
P
P
DP
pRB
E2F
HDAC
pRB/E2F-mediated
transcriptional
repression of genes
required for G1/S
transition
early G1
P
P
pRB
P
DP
E2F
phosphorilation by
cdk4/cyc D and
cdk2/cyc E relieves pRB
repression of E2F
transcription factors
late G1
S
Istituto “Seràgnoli” - Bologna
Cyclin D1 and CDK inhibitors
Cyclin D gene family members
Genomic location
Protein size, kD
Expression in
hematopoietic tissue
CCND1
CCND2
CCND3
11q13
12p13
6p21
33
34
33
none
low
high
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
CDC25
CAK
2. dephosphorylation
Thr14
3. phosphorylation
Tyr15
Thr161
cdk4/6
GROWTH
FACTORS
cyclin D1
1. cyclin D1 sinthesis
and association
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
Passage of the Restriction point:
D
P
CDK4
P
DP
P pRB
E2F
HDAC
pRB/E2F-mediated
transcriptional
repression of genes
required for G1/S
transition
early G1
P
E
P
pRB
P
DP
E2F
CDK2
P
phosphorilation by
cdk4/cyc D and
cdk2/cyc E relieves pRB
repression of E2F
transcription factors
late G1
S
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
+
+
Raf 1
MEK/MAPK
RAS
+
PI3K
Ral-GDS
+
Akt/PKB
-
+
ERK/MAP
GSK3b
+
CYCLIN D1
+
FAK
integrins
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
+
b-catenin + TCF
+
GSK3b
Wnt
CCND1/BCL-1/PRAD1 gene:
alternative splicing
A/G
polymorphism at
the splice donor
region (nt.870)
Ex.1
Ex.2
Ex.3
if nt.870 is A:
preferentially this
splicing pattern
Ex.5
Ex.4
if nt.870 is G:
preferentially this
splicing pattern
Intron 4
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
Cyclin D1 isoforms
Transcript “a”
Ex.1
Ex.2
Ex.3
Ex.4
“cyclin box”
(nt.312-630)
Ex.1
Ex.2
Ex.3
Ex.5
3’ UTR
“destruction box”
(PEST- rich region)
Ex.4
In.4
3’ UTR
Transcript “b”
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
TRASLOCAZIONE t(11;14)(q13;q32)
q
p
Cromosoma 11
BCL-1/CCND1/PRAD1
Cromosoma 14
q
p
IgH
BCL-1
+
tel.
switch
region
IgH locus
Ea
Chr.11
cen.
Chr.14
Istituto “Seràgnoli” - Bologna
t(11;14),
OVERESPRESSIONE
CICLINA D1:
SIGNIFICATO
PROGNOSTICO?
Istituto “Seràgnoli” - Bologna
Fonseca et al. “Multiple myeloma and the translocation
t(11;14)(q13;q32): a report on 13 cases” Br J Haematol,
1998
Sonoki et al. “Expression of PRAD1/cyclin D1 in plasma
cell malignancy: incidence and prognostic aspects” Br J
Haematol, 1998
Hoechtlen-Vollmar et al. “Amplification of cyclin D1 in
multiple myeloma: clinical and prognostic relevance” Br J
Haematol, 1998
Pruneri et al. “Immunohistochemical analysis of cyclin D1
shows deregulated expression in multiple myeloma with
the t(11;14)” Am. J. Pathol. 2000
FATTORE PROGNOSTICO SFAVOREVOLE??
Fonseca et al. “Myeloma and the t(11;14)(q13;q32);
evidence for a biologically defined unique subset of
patients” Blood. 2002;99:3735-41
• DISEGNO DELLO STUDIO:
336 pz. (ECOG 9486/9487) trattati con chemioterapia
convenzionale, analizzati in FISH per la presenza della
traslocazione t(11;14)(q13;q32)
•
RISULTATI:
-
53/336 pz. (16%) con evidenza di t(11;14)
-
pts positivi per t(11;14): sopravvivenza più lunga
OS: 49.6 vs. 38.7 mesi (log-rank P > .2)
PFS: 33.0 vs. 27.1 mesi (log-rank P > .2)
Istituto “Seràgnoli” - Bologna
Moreau et al. “Recurrent 14q32 translocations
determine the prognosis of multiple myeloma, especially
in patients receiving intensive chemotherapy” Blood.
2002;100:1579-83
• DISEGNO DELLO STUDIO:
168 pz. (IFM) trattati con diversi protocolli di chemioterapia ad
alte dosi, analizzati in FISH per le principali traslocazioni
14q32
• RISULTATI:
- 26/168 pz. (15.5%) con evidenza di t(11;14)(q13;q32)
- OS attesa a 80 mesi significativamente più lunga per i pazienti
t(11;14)-positivi (87.5 vs. 55.4 mesi; P=0.055)
Istituto “Seràgnoli” - Bologna
Soverini et al. “Cyclin D1 overexpression is a favorable
prognostic variable for newly diagnosed multiple
myeloma patients treated with high-dose chemotherapy
and single or double autologous transplantation” Blood,
2003;102:1588-94
• SCOPO DELLA RICERCA
Analizzare l’overespressione della ciclina D1 in termini di:
- frequenza;
- correlazione con alterazioni chr.11;
- associazione con delezione/monosomia chr.13 (D13);
- significato clinico e prognostico.
Istituto “Seràgnoli” - Bologna
• PAZIENTI E METODI
BM da 74 pazienti affetti
da
MM di nuova diagnosi
ed
arruolati nel protocollo “Bologna
96”
quantificazione
dell’mRNA per
ciclina D1 mediante realtime RT-PCR
(74 pts)
citogenetica
convenzionale (CC) ed
eventualmente FISH
per chr. 11 e 13
(46/74 pts)
Istituto “Seràgnoli” - Bologna
CORRELAZIONE TRA LIVELLI DI CICLINA D1
E ALTERAZIONI DEL CHR.11
t(11;14)
(n = 9)
+11
(n = 9)
No 11q abn.
(n = 28)
Normal
(n = 10)
Istituto “Seràgnoli” - Bologna
CORRELAZIONE TRA
OVERESPRESSIONE DELLA CICLINA D1
E D13
D13
38% dei pazienti
ciclina D1-pos
41% dei pazienti
ciclina D1-neg
nessuna associazione
Istituto “Seràgnoli” - Bologna
CORRELAZIONE TRA OVERESPRESSIONE DELLA
CICLINA D1 E CARATTERISTICHE CLINICOLABORATORISTICHE ALL’ESORDIO
Cyc D1 pos
Cyc D1 neg
No. patients
32 (43%)
42
Median age
55
Sex (M:F)
21:11
p
51.5
n.s.
31:11
n.s.
Durie and Salmon stage:
I
3
11
II
4
8
III
25
23
n.s.
Istituto “Seràgnoli” - Bologna
Cyc D1 pos
Cyc D1 neg
32 (43%)
42
IgA
IgG
17
26
8
10
BJ
7
6
k
20
28
l
12
14
No. patients
p
M component:
n.s.
Light chain:
n.s.
Median M component concentration:
IgA (g/dL)(range)
3.90 (1.00-11.17)
4.20 (2.55-8.40)
IgG (g/dL)(range)
3.80 (2.20-4.20)
3.65 (2.56-7.80)
BJ (g/24hrs)(range)
3.40 (1.90-6.40)
n.s.
4.25 (1.90-18.60)
Istituto “Seràgnoli” - Bologna
Cyc D1 pos
No. patients
32 (43%)
Cyc D1 neg
p
42
50 (10-100%)
40 (10-100%)
n.s.
median b2-m (mg/L)(range)
2.80 (1.20-16.00)
2.20 (1.10-6.90)
n.s.
median CRP (mg/L)(range)
3.60 (0.10-62.50)
3.10 (0.10-70.20)
n.s.
0.90 (0.70-1.40)
1.10 (0.70-3.00)
n.s.
median BMPC (%)(range)
median creat (mg/dL)(range)
Istituto “Seràgnoli” - Bologna
OVERALL SURVIVAL
Probability of survival (%)
1.0
0.9
0.8
0.7
0.6
Cyc D1 pos
0.5
0.4
Cyc D1 neg
0.3
0.2
0.1
0
6
12
18
24
30
36
42
48
54
months
Median OS
Cyc D1 pos
not reached
after 48
Cyc D1 neg
p
43
0.6
Istituto “Seràgnoli” - Bologna
TIME TO DISEASE PROGRESSION
Probability of progression (%)
1.0
0.9
Cyc D1 neg
0.8
0.7
0.6
Cyc D1 pos
0.5
0.4
0.3
0.2
0.1
0
6
12
18
24
30
36
42
48
54
months
Median TTP
Cyc D1 pos
41
Cyc D1 neg
p
26
0.02
Istituto “Seràgnoli” - Bologna
EVENT-FREE SURVIVAL
Probability of survival (%)
1.0
0.9
0.8
0.7
0.6
0.5
Cyc D1 pos
0.4
0.3
0.2
Cyc D1 neg
0.1
0
6
12
18
24
30
36
42
48
54
months
Median EFS
Cyc D1 pos
33
Cyc D1 neg
p
24
0.055
Istituto “Seràgnoli” - Bologna
CICLINA D1 vs. D13
nei 18 pts con D13:
Median TTP
Median EFS
D13 pos/Cyc D1 pos
(n=9)
D13 pos/Cyc D1 neg
(n=9)
41
26
D13 pos/Cyc D1 pos
(n=9)
D13 pos/Cyc D1 neg
(n=9)
41
23
Istituto “Seràgnoli” - Bologna
CONCLUSIONI (I):
L’overespressione della ciclina D1:
• è un’alterazione frequente (43%) nei pazienti
affetti da MM alla diagnosi;
• è strettamente correlata ad alterazioni del
cromosoma 11:
-
t(11;14),
-
trisomia 11.
Istituto “Seràgnoli” - Bologna
CONCLUSIONI (II):
L’overespressione della ciclina D1:
• E’ un fattore prognostico favorevole:
identifica
un sottogruppo di pazienti
che presentano
TTP ed EFS più lunghi
in seguito a
chemioterapia ad alte dosi
ed autotrapianto.
Istituto “Seràgnoli” - Bologna
QUESITI APERTI:
L’overespressione della ciclina D1 è in grado di
controbilanciare la monosomia/delezione del
chr.13?
Istituto “Seràgnoli” - Bologna
t(4;14)(p16;q32)
- cariotipicamente “silente”
- descritta solo nel MM e MGUS
- frequenza nel MM = 20%ca.
Istituto “Seràgnoli” - Bologna
t(4;14) translocation in Multiple Myeloma (MM)
Region 14q32 = locus IgH
L1 VH1
Ln VHn
(n=ca.300)
DH
(n=ca.30)
Cg1
JH
(6 genes + 2 ps.)
S
Ce2
S
MM
S
Ca1
Cm
Cd
Cg
Cg3
S
Cg2
Cg4
S = Class Switch Recombination (CSR)
Like many tumors of the B-cell lineage, multiple myeloma (MM) shows recurrent rearrangements of
the immunoglobulin heavy-chain (IGH) locus at 14q32 and molecular studies have identified FGFR3,
CCND1, CCND3, and MAF genes as targets of primary translocations in this malignancy.
unico caso, nel MM, in cui si forma
un
gene di fusione (IgH-MMSET),
che
rende la t(4;14) rilevabile mediante RT-PCR
IgH
3
4
IgH
4
5
IgH
5
6
6
5
6
MMSET
MMSET
MMSET
- RT
- I step PCR
- II step PCR
Istituto “Seràgnoli” - Bologna
Translocations involving locus IgH
are observed in 60-80% of patients with MM
4p16
11q13
16q23
cr.14q32
6p21
6p25
• In patients with MM a number of recurrent translocations involving chromosome 14 at band q32
have been recently identified, the most common being the t(11;14) and the t(4;14).
• Both these chromosomal abnormalities may help to identify patients at different risk of death;
• The t(4;14) has been recently reported to be associated with an unfavorable clinical outcome.
4p16 Region
ca. 100Kb
FGFR3
tel
LETM1
MM
MMSET
cen
The t(4;14) affects at least two potential oncogenes, MMSET on der(4) and Fibroblast Growth
Factor Receptor 3 (FGFR3) on der (14); the role of both these genes in the pathogenesis of MM
has not bee fully elucidated.
MMSET = Multiple Myeloma SET domain protein
Type I
5’UT
Polyadenilation Segnals
ORF 1911bp = 647aa
3
4
5
6
7
PHD fingers
NLS
N
HAT
Type II
5’UT
11
8 9 10
1
HMG
2
NLS
3
HAT
17
18 19
4
SET
C
ORF 4094bp = 1365aa
3
4
5
6
7
8
9 10 12 13
14 15 16
20 21 22 23
24
M.Chesi et al., Blood (1998)
MMSET/IgH = fusion gene
and alternative spicing
IgH
3
4
IgH
4
5
IgH
5
6
- RT
- I step PCR
- II step PCR
6
5
6
MMSET
MMSET
MMSET
Region 14q32 = locus IgH
MM
L1 VH1
Ln VHn
(n=ca.300)
DH
(n=ca.30)
Cg1
JH
(6 genes + 2 ps.)
S
Ce2
S
S
Ca1
Cm
Cd
Cg
Cg3
S
Cg2
Cg4
S = sequenze di DNA ripetitivo, non codificante,
associate alla Class Switch Recombination (CSR)
TRANSLOCATION = CSR abherrant OR
mechanismo involving regions near S
4p16 Region = genic cluster
MMSET
tel
cen
TACC3
FGFR3
TACC1
FGFR1
LETM1
MMSET/WHS1/NSD2
cr.4p16
t(4;14) MM
NSD3
t(8;11) AML
cr.8p11
TACC2
FGFR2
MMSET
cr.10q26
cr.5q35
FGFR4
NSD1/ALL1/MLL
t(5;11) AML
MMSET = Multiple Myeloma SET domain protein
Type I
5’UT
Polyadenilation Segnals
ORF 1911bp = 647aa
3
4
5
6
7
PHD fingers
NLS
N
HAT
Type II
5’UT
11
8 9 10
1
HMG
2
NLS
3
HAT
17
18 19
4
SET
C
ORF 4094bp = 1365aa
3
4
5
6
7
8
9 10 12 13
14 15 16
20 21 22 23
24
M.Chesi et al., Blood (1998)
MMSET: FUNCTIONALS DOMAIN
PHD fingers
NLS
N
hath
HMG
1
2
NLS
3
hath
SET
4
C
dominio di 140aa. molto conservati, descritto in fattori trascrizionali
regioni
probabile
conservate,
coinvolgimento
tipiche di proteine
nell’espressione
di originegenica
nucleare;
e proteine
dello
sviluppo,
molto
importante
per
il
mantenimento
regione
che
facilita
l’interazione
con
il
DNA;
segnali
di localizzazione
ruolo
attraverso
nella
crescita
la modificazione
e nel differenziamento
dellanucleare
cromatinacellulare
ell’espressione
coinvolti nello sviluppo.
correla dei
congeni
la PROLIFERAZIONE
cellulare
QUINDI
1. domini tipici di proteine nucleari coinvolte in
- rimodellamento della cromatina
- meccanismo regolatorio epigenetico
2. deleto nella Wolf-Hirshhorn Syndrome (severi difetti di crescita,
ritardo mentale, difetti scheletrici etc.)
3. overespresso nel MM per effetto della t(4;14)
MMSET: fattore trascrizionale?
Derivatives t(4;14)
der(14):
tel.
TACC3 - FGFR3 - LETM1 - MMSET
Sm
Cm
3`E
cen.
der(4):
tel.
JH
5`E
Sm
MMSET
cen.
AIM
In the present study we investigated the frequency and the prognostic relevance of the t(4;14) in a series of 63 patients
with de novo MM, who were randomized to receive either a single autotransplant (Tx-1) or double autotransplants (Tx-2)
as primary therapy for their disease.
- frequency and prognostic signifiance t(4;14)
in our casistic
- molecular variability of t(4;14)
For this purpose we analyzed:
(1) the presence of t(4;14) by RT-PCR of the hybrid transcript between MMSET and the IgH locus;
(2) the overexpression of FGFR3 by Real-time RT-PCR;
(3) the frequency of potentially activating point mutations in the FGFR3 translocated coding region, by direct
sequencing of RT-PCR products.
(4) The frequency of del 13 and t(4;14)
PATIENTS & METHOD
63/out of 123 patients enrolled in protocol BO’96
37 Tx1
23 Tx2
3 no Tx
- RT-nested PCR per IgH/MMSET (diagnosis + LFU)
- Real-Time PCR per FGFR3 (diagnosis)
- RT-PCR & direct sequencing for FGFR3 point mutation (diagnosis + LFU)
63 patients
17 MMSET/IgH + (27%)
Sex (M/F)
Age (years, median, range)
Durie-Salmon stage (no, %)
I
II
III
Isotyp (no, %)
IgG
IgA
Bence Jones 0
High chain (no., %)
k
l
Lytic bone disease (no, %)
0
1
2
>3
Calcium (mg/dl)
Hb (g/dl)
Plts
B2 mycroglobulin (mg/L)
C reactive protein (mg/L)
Creatinine (mg/dl)
Plasmacells (%)
46 MMSET/IgH – (63%)
12/5
51 (42-61)
28/18
55 (40-62)
4 (24%)
4 (24%)
9 (52%)
8 (17%)
5 (11%)
33 (72%)
11 (65%)
6 (35%)
26 (65%)
10 (22%)
10 (22%)
7 (41%)
10 (59%)
32 (70%)
14 (30%)
7 (41%)
2 (12%)
2 (12%)
6 (35%)
9.1 (8.2-12-3)
11.4 (6.4-14.5)
211 (157-300)
2.1 (1.2-6.9)
0.38 (0.01-6.6)
1.1 (0.3-3.0)
50 (10-90)
9 (20%)
4 (9%)
3 (6%)
30 (65%)
9.1 (5-13)
12.3 (5.6-15-5)
212 (59-416)
2.4 (1.1-16)
0.31 (0.01-70)
1.0 (0.6-1.7)
50 (10-100)
THERAPY RECEIVED
63 patients
17 MMSET/IgH + (27%)
No Tx
Tx1
Tx2
0
11 (65%)
6 (35%)
46 MMSET/IgH – (63%)
3 (7%)
26 (56%)
17 (37%)
RESPONSE TO TRANSPLANT
63 patients
46 MMSET/IgH – (63%)
17 MMSET/IgH + (27%)
N. Evaluables
17/17
Progression
0
No response
2 (12%)
Partial response
11 (65%)
Complete response (IF+) 3 (17%)
Complete response (IF-) (?) (6%)
43/46
1 (2%)
5 (12%)
15 (35%)
7 (16%)
15 (35%)
p = 0.05
GLOBAL SURVIVAL
100
80
MMSET/IgH60
MMSET/IgH+
n.s
40
20
0
0
12
Median Follow-up (months):
MMSET/IgH+: 36 (16-50)
MMSET/IgH-: 45 (13-72)
24
36
48
60
72
EVENT FREE SURVIVAL
100
80
60
40
MMSET/IgH-
20
p= 0.01
MMSET/IgH+
0
0
12
Median, range (months):
MMSET/IgH+: 23 (6-29)
MMSET/IgH-: 32 (6-72)
24
36
48
60
72
TIME TO DISEASE PROGRESSION in MMSET+ patients
MMSET/IgH+
100
p= 0.01
80
MMSET/IgH60
40
20
0
0
12
Median, range (months):
MMSET/IgH+: 23 (6-29)
MMSET/IgH-: 32 (6-72)
24
36
48
60
72
t (4;14): PROGNOSTIC SIGNIFICANCE
- Moreau et al. (Blood, 2002):
FISH - CD138+
168 pz. (high dose chemotherapy )
13% t(4;14)+
OS & EFS less than (p<0.0001)
- Fonseca et al. (Blood, 2003):
FISH - BM (PC)
351 pts. (conventional chemotherapy )
13% t(4;14)+
OS & PFS less than (p<0.001)
t(4;14) ASSOCIATED TO BAD THERAPY
t(4;14) & del(13)
38 patients
26 MMSET/IgH –
12 MMSET/IgH +
del (13)
5 (42%)
7 (29%)
NO del (13)
7 (54%)
17 (71%)
p = 0.2
t(4;14) & del (13)
del(13)
3/38 patients
t(4;14)
7/38 patients
del(13) + t(4;14)
5/38 patients
mortality*
relapse*
0
1/3 (33%)
0
4/7 (57%)
3/5 (60%)
4/5 (80%)
* = by 3 years (number; percentual)
CONCLUSIONS-1
- 27% of patients studied has t(4;14)
- t(4;14) = worst rensponse to transplant
- EFS & TTP shorter in t(4;14) carring patients
t(4;14): longitudinal study: MMSET type of transcript is conserved
through the therapy
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
7
X
IgH/ex4
D
LFU
X
IgH/ex5
D
LFU
X
X
X
X
X
X
3
3
X
nv
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
nv
X
X
X
X
X
X
8
12
X
X
X
8
alter.splic.
ov. FGFR3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
no
X
X
no
no
X
no
3
5
13
mut. FGFR3
D
LFU
no
no
no
no
no
X
X
X
no
no
X
nv
no
no
no
no
no
no
no
no
X
X
no
no
no
X
no
no
no
no
no
no
no
no
3
4
FU
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
al study: alternative spiced MMSET transcript are associated with
longest clinical outcome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
7
X
IgH/ex4
D
LFU
X
IgH/ex5
D
LFU
X
X
X
X
X
X
3
3
X
nv
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
nv
X
X
X
X
X
X
8
12
X
X
X
8
alter.splic.
ov. FGFR3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
no
X
X
no
no
X
no
3
5
13
mut. FGFR3
D
LFU
no
no
no
no
no
X
X
X
no
no
X
nv
no
no
no
no
no
no
no
no
X
X
no
no
no
X
no
no
no
no
no
no
no
no
3
4
FU
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
t(4;14):
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
7
X
IgH/ex4
D
LFU
X
IgH/ex5
D
LFU
X
X
X
X
X
X
3
3
X
nv
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
nv
X
X
X
X
X
X
8
12
X
X
X
8
splic. alter.
ov. FGFR3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
no
X
X
no
no
X
no
3
5
13
mut. FGFR3
D
LFU
no
no
no
no
no
X
X
X
no
no
X
nv
no
no
no
no
no
no
no
no
X
X
no
no
no
X
no
no
no
no
no
no
no
no
3
4
FU
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
t(4;14): alternative splicing & survival
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
X
X
X
7
X
nv
IgH/ex4
D
LFU
X
IgH/ex5
D
LFU
X
X
X
X
X
X
3
3
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
nv
X
X
X
X
X
X
8
12
X
X
X
8
vari
mut. FGFR3
ov. FGFR3
D
LFU
D
LFU
X
no
no
X
no
no
X X= 83%no
alive X
X 47mX(28-55m.)
X
X
no
no
X
X
nv
X
no
no
X
no
no
X
X
X X= 45%no
alive no
X
X
X
no
40mno
(16-58m.)
X
X
X
X
X
X
no
no
X
X
X
no
X
no
no
no
X
X
no
no
no
no
no
no
X
X
no
no
no
3
5
13
3
4
FU
status
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
V
M
M
M
M
M
M
V
RC
V
V
V
M
V
V
V
V
FGFR3: overexpression & point mutations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
IgH/ex4
D
LFU
X
- Nakazawa et al. (2000)
X GenetXCytogenet.:
Cancer
7/45nv
(15.6%) t(4;14)+
X
-Keats et al X
(2002)
Blood:
nv
31/208 (14.9%)
X t (4;14)X
X(2002) X
- Santra et al
Blood:X
X
X
30/172 (17.4%) t (4;14)
X
X
IgH/ex5
D
LFU
X
X
X
X
X
X
X
X
X
X
X
X
der(14):
X
X
TACC3 - FGFR3 - LETM1 - MMSET Sm
tel.
X
X
X
X
X
7
8 12
7 3
vari
ov. FGFR3
D
LFU
X
X
X
X
1/7 (14.2%)
X
NO overFGFR3
X
X
8/31
(26%)
X
X
NO over FGFR3
X
X
X
X
X
10/30 (32%)
X
X
NO over X
FGFR3
X
X
X
X
X
X
X
X
no
X
X
no
Cm
3`E
cen.
no
X
X
no
3
3
5
13
mut. FGFR3
D
LFU
no
no
no
no
no
X
X
X
no
no
X
nv
no
no
no
no
no
no
no
no
X
X
no
no
no
X
no
no
no
no
no
no
no
no
3
4
FU
status
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
V
M
M
M
M
M
M
V
RC
V
V
V
M
V
V
V
V
FGFR3: overexpression & point mutations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
IgH/ex4
D
LFU
X
- Nakazawa et al. (2000)
X GenetXCytogenet.:
Cancer
7/45nv
(15.6%) t(4;14)+
X
-Keats et al X
(2002)
Blood:
nv
31/208 (14.9%)
X t (4;14)X
X(2002) X
- Santra et al
Blood:X
X
X
30/172 (17.4%) t (4;14)
X
X
IgH/ex5
D
LFU
X
X
X
X
X
X
X
X
X
X
X
X
der(14):
X
X
TACC3 - FGFR3 - LETM1 - MMSET Sm
tel.
X
X
X
X
X
7
8 12
7 3
vari
ov. FGFR3
D
LFU
X
X
X
X
1/7 (14.2%)
X
NO overFGFR3
X
X
8/31
(26%)
X
X
NO over FGFR3
X
X
X
X
X
10/30 (32%)
X
X
NO over X
FGFR3
X
X
X
X
X
X
X
X
no
X
X
no
Cm
3`E
cen.
no
X
X
no
3
3
5
13
mut. FGFR3
D
LFU
no
no
no
no
no
X
X
X
no
no
X
nv
no
no
no
no
no
no
no
no
X
X
no
no
no
X
no
no
no
no
no
no
no
no
3
4
FU
status
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
V
M
M
M
M
M
M
V
RC
V
V
V
M
V
V
V
V
FGFR3: overexpression & point mutations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
tot
IgH/ex3
D
LFU
X
X
X
X
X
X
X
X
IgH/ex4
D
LFU
X
- Nakazawa et al. (2000)
X GenetXCytogenet.:
Cancer
7/45nv
(15.6%) t(4;14)+
X
-Keats et al X
(2002)
Blood:
nv
31/208 (14.9%)
X t (4;14)X
X(2002) X
- Santra et al
Blood:X
X
X
30/172 (17.4%) t (4;14)
X
X
IgH/ex5
D
LFU
X
X
X
X
X
X
X
X
X
X
X
X
der(14):
X
X
TACC3 - FGFR3 - LETM1 - MMSET Sm
tel.
X
X
X
X
X
7
8 12
7 3
vari
ov. FGFR3
D
LFU
X
X
X
X
1/7 (14.2%)
X
NO overFGFR3
X
X
8/31
(26%)
X
X
NO over FGFR3
X
X
X
X
X
10/30 (32%)
X
X
NO over X
FGFR3
X
X
X
X
X
X
X
X
no
X
X
no
Cm
3`E
cen.
no
X
X
no
3
3
5
13
mut. FGFR3
D
LFU
no
no
no
no
no
X
X
X
no
no
X
nv
no
no
no
no
no
no
no
no
X
X
no
no
no
X
no
no
no
no
no
no
no
no
3
4
FU
status
+43m
+37m
+37m
+40m
+41m
+16m
+25m
+42m
+45m
+55m
+49m
+58m
+28m
+52m
+40m
+34m
+50m
V
M
M
M
M
M
M
V
RC
V
V
V
M
V
V
V
V
CONCLUSIONS-2
- t(4;14) and MMSET was not lost during disease progression
- which is the role of aberant transcripts?
- not all the t (4;14) positive patients over-express FGFR3
CONCLUSIONS-3
-prognostic significance
-therapeutic implications
PROTOCOL MM’02
- patients enrolled:
126 (at 15/07/03)
- samples “for molecular byology”:
126 (at diagnosis)
91 (follow up)
- CD138+:
95 (at diagnosis)
24(post 4° month)
PROTOCOL MM’02: t(4;14)
25 patients at diagnosis
9 patients: t(4;14)+
(36%)
16 patients: t(4;14)-
6 patients: del13
2 patients: NO
1 patients: n.v.
2 patients: del13
14 patients: NO
PROTOCOL MM’02:
genic expression profile
Studiare contemporaneamente
l’espressione genica di migliaia di geni
GENIC EXPRESSION PROFILE IN MM
- Zahn et al: “Global gene expression profiling of multiple myeloma, monoclonal gammopathy
of undetermined significance, and normal bone marrow plasma cells.”
Blood (2002)
- Claudio et al. “Molecular compendium of gene expressed in multiple myeloma”
Blood (2002)
- De Vos et al. “Comparison of gene expression profiling between mulitple myeloma
and normal plasmacells”
Oncogene (2002)
- Underhill et al. “Gene expression profiling reveals a highly specialized genetic program
of plasma cells”
Blood (2003)
- Shaughnessy JD and Barlogie B “Interpreting the molecular biology and clinical behavior of
multiple myeloma in the context of global gene expression profiling”
Immunol. Rev. (2003)
PROTOCOL MM’02:
genic expression profile
- CD138+: selezione magnetica
(95 patients at diagnosis; 24 patients post 4° months)
- RNA extraction
- vetri MWG 10K
Abstract
In patients with MM a number of recurrent translocations involving chromosome 14 at band q32 have been recently identified,
the most common being the t(11;14) and the t(4;14). Both these chromosomal abnormalities may help to identify patients at
different risk of death; in particular, the t(11;14) predicts for good prognosis, whereas the t(4;14) has been recently reported
to be associated with an unfavorable clinical outcome. The t(4;14) affects at least two potential oncogenes, MMSET on der(4)
and Fibroblast Growth Factor Receptor 3 (FGFR3) on der (14); the role of both these genes in the pathogenesis of MM has
not bee fully elucidated.
In the present study we investigated the frequency and the prognostic relevance of the t(4;14) in a series of 63 patients with
de novo MM, who were randomized to receive either a single autotransplant (Tx-1) or double autotransplants (Tx-2) as
primary therapy for their disease.
For this purpose we analyzed (1) the presence of t(4;14) by RT-PCR of the hybrid transcript between MMSET and the IgH
locus; (2) the overexpression of FGFR3 by Real-time RT-PCR; (3) the frequency of potentially activating point mutations in the
FGFR3 translocated coding region, by direct sequencing of RT-PCR products.
Overall, the t(4;14) was detected by RT-PCR in 17/63 patients (27%); of these 17 patients, 13 had both MMSET/IgH fusion
gene and FGFR3 overexpression, while 4 patients had MMSET/IgH but did not overexpress FGFR3. This finding further confirms
the possible discrepancy between MMSET/IgH positivity and FGFR3 overexpression.
Comparison between t(4;14) positive and t(4;14) negative patients revealed that both groups were well balanced with respect
to the most common presenting features of MM. In 36 patients, for whom material was available, FISH analysis for the
detection of 13q deletion and/or monosomy was performed.
Results showed that t(4;14) positive patients were more likely to carry del(13) than t(4;14) negative patients (46% vs. 29%,
respectively). On an intention-to-treat basis, the probability of attaining stringently defined complete remission following either
Tx-1 or Tx-2 was significantly lower for t(4;14) positive patients in comparison with t(4;14) negative patients (6% vs. 35%,
respectively; p = 0.05). With a median follow-up of 40 months, no difference in overall (OS) was detected between the two
groups. At the opposite, in comparison with the t(4;14) negative subgroup, patients carrying the t(4;14) had significatly lower
event free survival (EFS) (23 vs 32 months, respectively; p=0.01) and duration of remission (23 vs 32 months, respectively;
p=0.01).
In summary, 27% of our MM patients carried the t(4;14). In this cohort of homogeneously treated patients, the t(4;14)
predicted for lower response to high-dose therapy. Longer follow-up is required to assess the influence of this abnormalities on
OS. In a subgroup of six patients carrying t(4;14), point mutations were detected in the FGFR3 coding region, thus suggesting
a possible constitutive FGFR3 activation.
t(4;14)(p16;q32)
- kariotipicamente “silente”
- described only in MM e MGUS
- frequency in MM = 20%ca.
A subset of multiple myeloma harboring the t(4;14)(p16;q32) translocation lacks FGFR3 expression
but maintains an IGH/MMSET fusion transcript
Madhumita Santra, Fenghuang Zhan, Erming Tian, Bart Barlogie, and John Shaughnessy Jr From the Donna and Donald
Lambert Laboratory of Myeloma Genetics at the Myeloma Institute for Research and Therapy, University of Arkansas for Medical
Sciences, Little Rock, AR
Like many tumors of the B-cell lineage, multiple myeloma (MM) shows recurrent
rearrangements of the immunoglobulin heavy-chain (IGH) locus at 14q32 and molecular
studies have identified FGFR3, CCND1, CCND3, and MAF genes as targets of primary
translocations in this malignancy. The multiple myeloma (MM) specific t(4;14)(p16;q32) not
only results in the activation of FGFR3 but also the creation of a chimeric fusion transcript
between IGH and MMSET. Given the transcription-activating nature of 14q32 translocations,
microarray profiling of global gene expression has emerged as a powerful method for
identifying IGH-associated rearrangements in B-cell malignancies. We have previously used
this strategy to identify a novel 14q32 translocation involving the cyclin D3 gene (CCND3) as
well as all known 14q32 translocations in MM. Using a combination of gene expression
profiling reverse transcriptase-polymerase chain reaction (RT-PCR) and interphase fluorescence
in situ hybridization (FISH) we present evidence that nearly 20% of newly diagnosed cases of
MM harbor the t(4;14)(p16;q32), and approximately 32% of these cases, while expressing the
IGH/MMSET fusion transcript, lack FGFR3 expression.
Blood, 2003, Vol.101 pp. 2374-2376
t(4;14): PROGNOSTIC SIGNIFIANCE
La disponibilità di fattori prognostici “genetici” è fondamentale
per:
- identificare sottogruppi di pazienti che potrebbero condividere
il meccanismo patogenetico
- proporre eventuali terapie “mirate”
Aim
- studio molecolare t(4;14)
- frequenza e significato prognostico t(4;14)
nella nostra casistica
FIBROBLAST GROWTH FACTOR
RECEPTORS (FGFR):
famiglia di recettori ad attività tirosin-chinasica
coinvolti in una grande varietà di processi
mitogenici e morfogenici: sviluppo embrionale,
angiogenesi, riparazione tissutale..
Istituto “Seràgnoli” - Bologna
FIBROBLAST GROWTH FACTOR
RECEPTORS (FGFR):
4 membri: FGFR1, FGFR2, FGFR3, FGFR4,
con distribuzione (almeno in parte) tessuto
specifica, in grado di riconoscere con affinità
variabile almeno 9 diversi FGFs.
Istituto “Seràgnoli” - Bologna
FGFR3:
• glicoproteina di 125 kDa, espressa
principalmente a livello del SNC, del polmone,
del rene e delle cartilagini;
• il gene che la codifica è stato isolato nel 1990
da una libreria di cDNA ottenuta dalla linea
cellulare K562
(Keegan et al,
Proc. Natl. Acad. Sci., 1991).
Istituto “Seràgnoli” - Bologna
FGFR3 = Fibroblast Growth Factor Receptor 3
- is a tyrosin-kinase receptor
- mutazioni nella linea germinale causano una grave forma di nanismo
- espressione assente nel tessuto ematopoietico dell’adulto sano
in MM:
- could be over-expressed
- in alcuni casi sono presenti le stesse mutazioni che causano il nanismo
FGFR3 GENE AT 4p16.3:
16,5 kb
coding region
1
2
6
7 (IIIa) 8 (IIIb) 9 (IIIc)
10
18
19
IIIa: secreted form
alternative splicing
IIIb: epithelial form
IIIc: mesenchimal form
Istituto “Seràgnoli” - Bologna
s-s
s-s
s-s
II
s-s
III
II
III
s-s
s-s
I
I
OUT
Ig-like
Domains
cell surface
Trans-Membrane
Domain
IN
Split Tyrosine
Kinase Domain
Istituto “Seràgnoli” - Bologna
FGF + heparan-sulfate
FGFR3
P
Ras
P
GRB2
2 Sos
P
Shc
dimerization and
transphosphorilation
Raf-1
GDP GTP
exchange
P
MAPK*
K*
P
GRB2
80K-H
*
§
o MEK
o ERK
P
Sos
MAPK §
kk
NUCLEUS
Jun
Fos
DNA
Istituto “Seràgnoli” - Bologna
5. Hypochondroplasia
(Asn540Lys)
4. Muenke coronal
craniosynostosis
(Pro250Arg)
1. Achondroplasia
(Gly375Cys; Gly380Arg)
FGFR3 mutations
2. Thanatophoric Dysplasia
type I and II
(Tyr373Cys; Arg248Cys;
Lys650Glu; Thr807Cys)
3. Crouzon syndrome
with acantosis
nigricans (Ala391Glu)
Istituto “Seràgnoli” - Bologna
SUBSEQUENT SELECTION OF
SOMATIC MUTATIONS:
Ex.7
Ex.10
Ex.13
Ex.15
Ex.19
cod.248 TM domain cod.540 cod.650 cod.807
mutations
constitutively activated
FGFR3
Istituto “Seràgnoli” - Bologna
EFFECTS OF
FGFR3
ECTOPIC EXPRESSION
ON MURINE B9 MM CELLS:
(Plowright EE et al, Blood 2000; 95:992-997)
FGFR3
phosphorilation
STAT3
P
?
Bcl-xL
DECREASED APOPTOSIS
ENHANCED PROLIFERATIVE
RESPONSE TO IL-6
Istituto “Seràgnoli” - Bologna
In multiple myeloma, t(4;14)(p16;q32) is an
adverse prognostic factor irrespective of FGFR3
expression.
Keats et al. Blood 2003;101:1520-29
Istituto “Seràgnoli” - Bologna
Clinical and biologic implications of recurrent genomic
aberrations in myeloma
Fonseca, et al Blood 2003;101:4569-4575
Recurrent 14q32 translocations determine the prognosis
of multiple myeloma, especially in patients receiving
intensive chemotherapy
Moreau et al. Blood 2002;100:1579-1583
Istituto “Seràgnoli” - Bologna
Chr. 12 and t(12;21)
Tel-AML1
Chr. 1 and t(1;19)
E2a- PBX
The genes for making the heavy chains of antibodies (CH) are located on
chromosomes 14, whereas those for making the light chains are on
chromosomes 2 and 22. These genes are expressed exclusively in B
lymphocytes, because only these cells have the necessary transcription factors
to switch on their expression. In most (over 90%) of Burkitt's lymphoma cases,
a reciprocal translocation moves the proto-oncogene c-myc from its normal
position on chromosome 8 to a location close to the antibody heavy-chain genes
on chromosome 14. In other cases, c-myc is translocated close to the antibody
genes on chromosome 2 or 22. In every case, c-myc now finds itself in a region
of active gene transcription, and it may simply be the overproduction of the cmyc product (a transcription factor essential for cell division) that propels the
lymphocyte down the pathway towards cancer.
Virus and Lymphoma
Figure 1 | Schematic representation of the HCV genome and encoded viral
proteins. The boxed area corresponds to the single open reading frame of the
hepatitis C virus (HCV) genome. The stem–loop structures represent the 5' and 3'
non-translated (NTR) regions, including the internal ribosome-entry site (IRES) and
3'X regions. The function and molecular mass (in kDa) of the gene products after
polyprotein processing are shown. Core (C)–E1, E1–E2, E2–p7 and p7–nonstructural protein 2 (NS2) junctions are cleaved by a cellular signal peptidase(s) to
yield structural proteins. The NS2–NS3 metalloproteinase undergoes autocatalytic
cleavage, which releases the mature NS3 serine protease. NS3 cleaves the remainder
of the NS polypeptide. The two regions that have extreme sequence variability in
E2, known as hypervariable regions 1 and 2 (HVR1 and HVR2), are indicated. A
region in NS5A, known as the interferon (IFN)-sensitivity-determining region
(ISDR), has been linked to the response to IFN- therapy in some strains of HCV.
Both NS5A and E2 have been implicated as antagonists of IFN (for review, see Ref.
34). ARFP/F, alternative reading-frame protein/frameshift protein; LDLR, lowdensity lipoprotein receptor; RdRp, RNA-dependent RNA polymerase.
Cenni di terapia delle ALL: nuovi approcci
CICLO A1
PREDNISONE
60 mg/sqm p os dal +1 al +5
ENDOXAN
200 mg/sqm e.v. dal +1 al +5
Giorno +6: PAUSA
RITUXIMAB
375 mg/sqm e.v. g +7 (CD20 > 20%)
DEX
10 mg/sqm p. os. dal +8 al +12
MTX
500 mg/sqm p.c. g +8
IFOSFAMIDE
400 mg/sqm ev dal +8 al +12
ARA-C
120 mg/sqm ev gg +11 e +12
VM-26
60 mg/sqm ev gg +11 e +12
Rachicentesi :gg +8
(MTX 12 mg)
G-CSF 5 mcgr/Kg sc dal +14 a
PMN > 1000/mmc
CICLO B1 (DAL +28)
RITUXIMAB
375 mg/sqm e.v. g +28 (CD20> 20%)
DEX
10 mg/sqm p. os. dal +29 al +33
VCR
1 mg ev g +29
MTX
500 mg/sqm p.c. g +29
ENDOXAN
200 mg/sqm ev dal +29 al +33
ADRIAMICINA
25 mg/sqm ev gg +32 e +33
Rachicentesi :gg +29
( MTX 12 mg)
G-CSF 5 mcgr/Kg sc dal +35 a
PMN > 1000/mmc
CICLO A2 (DAL +49)
RITUXIMAB
375 mg/sqm e.v. g +49 (CD20> 20%)
DEX
10 mg/sqm p. os. dal +50 al +54
MTX
500 mg/sqm p.c. g +50
IFOSFAMIDE
400 mg/sqm ev dal g +50 al g +54
ARA-C
120 mg/sqm ev gg +53 e +54
VM-26
60 mg/sqm ev gg +81 e +82
G-CSF 5 mcgr/Kg sc dal +56 a PMN > 1000/mmc
Rachicentesi :gg +50
(MTX 12 mg)
CICLO B2 (DAL +77)
RITUXIMAB
375 mg/sqm e.v. g +77 (CD20> 20%)
DEX
10 mg/sqm p. os. dal +78 al +82
VCR
1 mg ev g +78
MTX
500 mg/sqm p.c. g +78
ENDOXAN
200 mg/sqm ev dal g +78 al g +82
ADRIAMICINA
25 mg/sqm ev gg +81 e +82
G-CSF 5 mcgr/Kg sc dal +84 a PMN > 1000/mmc
Rachicentesi :gg +78
(MTX 12 mg)
CICLO A3 (DAL +98)
RITUXIMAB
375 mg/sqm e.v. g +98 (CD20> 20%)
DEX
10 mg/sqm p. os. dal +99 al +103
MTX
500 mg/sqm p.c. g +99
IFOSFAMIDE
400 mg/sqm ev dal g +99 al g +103
ARA-C
120 mg/sqm ev gg +102 e +103
VM-26
60 mg/sqm ev gg +102 e +103
G-CSF 5 mcgr/Kg sc dal +105 a PMN > 1000/mmc
Rachicentesi :gg +99
(MTX 12 mg)
CICLO B3 (DAL +119)
RITUXIMAB
375 mg/sqm e.v. g +119 (CD20 >20%)
DEX
10 mg/sqm p. os. dal +120 al +124
VCR
1 mg ev g +120
MTX
500 mg/sqm p.c. g +120
ENDOXAN
200 mg/sqm ev dal g +120 al g +124
ADRIAMICINA
25 mg/sqm ev gg +123 e +124
G-CSF 5 mcgr/Kg sc dal +126 a PMN > 1000/mmc
Rachicentesi :gg +120
(MTX 12 mg)
CONSOLIDAMENTO
SETTIMANA 21 (G +147) E 24 (G+ 168)
RITUXIMAB
375 mg/sqm e.v.
RADIOTERAPIA (dopo la fine della chemioterapia)
SNC+: 24 Gy
Bulky mediastinico (> 7.5 cm): 36 Gy
Malattia residua dopo CHT: 36 Gy
Localizzazioni extranodali: decisione libera
RAS inhibitors
Extracellular signals
Inactivation
GTPase activating
proteins (GAPs)
RAS
GDP
(inactive)
RAS
GTP
(active)
Activation
Exchange factors
(GEFs)
RAS effectors: PI3K, MAPK...
Effects: cellular growth, proliferation, differentiation
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
Post-translational RAS modifications:
s
c
Ras
fully activated
RAS
Ras
palmitoyl-PP
C
4. Palmitoylation
CH3
S
farnesyl
3. Methylation
Ras
Ras
CAAX
SH
Farnesyl
transferase
1. Isoprenylation
farnesyl-PP
C
Ras
S
farnesyl
CAAX-protease
C
S
AAX
2. Proteolysis
farnesyl
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
Posttranscriptional modification of Ras
P P
cell membrane
F(or GG)
Ras
SCH 66336 PHASE II
R 115777 PHASE II
C-S 4. Palmytolation
Me
Ras
C-S-F(or GG)
PPMTase
SAM
CaaX
aaX
3. Methylation
Ras
FTase I
or
GGTase
Ras
FPP
GGPP
Ras C-S-F(or GG)
C-S-F(or GG)
a
a 1. Prenylation
X
2.Proteolysis
microsomal
membrane
RAS
PI 3- kinase
RalGDS
Raf
PDK
PKB/Akt
?
Caspase-9
Mek
Rho
Erk
Bad
GSK3b
Forkhead
?
Rac
Fas ligand
?
Cyclin D1
Ets
p21
Istituto di Ematologia e Oncologia Medica “Seràgnoli” - Bologna
Fos
mTOR inhibitors
translation
nutrient transporter
turnover
autophagy
stationary
phase (G0)
transcription
TOR
tRNA and
ribosome
biogenesis
actin organization
Protein Kinase C
signaling
TOR controls a Large and Diverse Set of Growth-related Readout Green arrows indicate activation; red bars indicate repressionShown is a composite of yeast and mammalian
readouts.
RAPAMICIN
RADICICOL
TRAIL inhibitors
Targeting the :
• Tyrosine kinase activity of BCR-ABL
• FTI (Farnesyl-transferase activity)
• Tumor Necrosis Factor-related Apoptosis-inducing
Ligand (TRAIL or Apo-2L)
• Src pathway –TK pathway
• Proteasome activity