Transcript Diapositiva 1

P27 REGULATION IN PERIPHERAL
T-CELL LYMPHOMA NOT
OTHERWISE SPECIFIED
Gazzola A, Agostinelli C, Righi S, Rossi M, Sista MT, Zinzani PL,
Pileri SA, Piccaluga PP
Department of “L. and A. Seràgnoli”, Hematology and
Ematopathology Sections, Molecular Pathology Laboratory, S.
Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
Peripheral T-cell lymphoma not
otherwise specified (PTCL/nos)
PTCL/nos accounts for about half of peripheral T
cell lymphomas in Western Countries.
This is a heterogeneous category of tumours,
both on morphologic, phenotypic grounds.
In particular, these tumours are not still clearly
defined by genetic analyses. Many alteration
were found, but no single genes were
demonstrated to have a pathogenetic role.
Cellular programs de-regulated in
PTCL/nos
Cytoskeleton
Matrix
FN1
COL12A1
COL1A2
COL3A1
COL4A1
COL4A2
FBN1
LAMB1
SPARC
CDH11
Transcription
TPM1
Dlc2
MGAT4A
MYLIP
NFIB
WASPIP
Apoptosis
Adhesion
CD69
DUSP2
DUSP8
GADD45A
GADD45B
ING3
JUND
MOAP1
PPP1R15A
Proliferation
AXUD1
FOXP1
RHOBTB3
CAV2
PLEKHC1
BTG1
CLK1
HECA
JUN
RGC32
TOB1
PER1
CBX4
CHD2
COPEB
CREM
EPC1
JMJD1C
MAF
NR4A2
NR4A3
SERTAD1
ZBTB10
ZBTB24
ZNF198
ZNF331
BCL10
GJA1
TNS
VCAM1
LIFR
Signal transduction
Gene expression
profiling (GEP) allowed
the identification of
PTCL/nos-associated
molecular signatures
CALD1
STK17B
MKNK2
HIPK1
PTP4A1
PDE4D
MAP3K8
ITPKB
SEPT10
TJP1
IRS2
PP Piccaluga et al. J Clin Invest 2007
Looking for molecules responsible for
cell cycle deregualtion in PTCL/nos
Clinico-pathological score:
Age, PS, LDH, Ki-67
Proliferation
1
.8
Cum. Survival
AXUD1
FOXP1
RHOBTB3
CAV2
PLEKHC1
BTG1
CLK1
HECA
JUN
RGC32
TOB1
.6
Score 1
Score 2
.4
Score 3
.2
p<0.0001
0
0
20
40
60
80
100
120
140
160
180
Time
PP Piccaluga et al. J Clin Invest 2007
P Went et al, J Clin Oncol 2006
Proliferation control turned out to be strongly affected. High
proliferation index was associated to unfavourable prognosis.
p27 in PTCL/nos
P27 - CDKN1B
p27, encoded by CDKN1B, is a member of a family of cyclindependent-kinase inhibitors (CDKIs). CDKIs family binds to
specific CDKs or CDK-cyclin complexes and inhibits kinase
activities, resulting in arrest of cell cycle (G1 phase). Alterations of
p27 result in loss of normal cell cycle control contributing in
neoplastic transformation.
p27 has been found to be deregulated in many different cancer
types. Down-regulation is associated with hyper-proliferative
status, up-regulation, in some instances, with drug resistance.
Aim of study
To identify possible determinants of abnormal
proliferation in PTCL/nos and in particular to analyze
expression and molecular structure of p27 in
PTCL/nos cases
Methods
Gene expression analysis
28 PTCL/NOS and 20 samples of normal T-cells (resting,
activated, CD8+, CD4+) were studied by HG U133 2.0 plus
Affymetrix microarray
Immunohistochemical analysis on tissue-microarray
Expression of p27, cyclin E (CCNE1), Ki-67 was evaluated
on tissue micro-arrays (TMAs) containing 98 PTCL/nos
cases
Molecular Analysis
Direct sequencing of 81 PTCL/nos was performed
The primers used for PCR, components, cycling conditions and sequencing were kindly
provided by Center for Human Genomics, Wake Forest University School of Medicine,
Wiston-Salem, North Carolina.
Gene expression results
100
CDKN1B
10
Expression level (raw data)
Normalized expression value
350
1
0,1
300
250
200
150
100
50
0
Diseased/Normal
0,01
diseased
PTCL/NOS
PTCL/NOS
Normal T-cells
normal
Normal T-cells
Y-axis:
PTCL28-Normal, Interpretation by Diseased-Normal
Colored by: diseased
Gene List: CDKN1B (1)
Expression values of CDK1NB, in PTCL/nos cases vs. samples
of normal tissues. Median values are indicated by bars. No
significant alterations in p27 expression was shown in
PTCL/nos cases compared to normal tissues.
HC_PTCL-JCI_...
Gene expression results
HC_PTCL-JCI_...
C...
2...
T...
2...
T...
2...
M...
Ki-67 (MKI67)
2...
2...
M...
Ki-67 (MKI67)
T...
2...
C...
2...
Cyclin E1 (CCNE1)
2...
T...
M...
Ki-67 (MKI67)
C...
2...
2...
M... Ki-67
M...
2...
2...
C...
M...
(MKI67)
CDKN1B
expression is
inversely related to
MKI67 and CCNE1
expression.
P27 (CDKN1)
2...
Normal
C... vs PTCLs
T_HC_PTCL-JCI_60...
Colored by: PTCL-JCI_60 (Default Interpretation)
-4
0 Gene List: p27
4 pathway (9), 213523_at selected 2...
C_PTCL-JCI_60_p27...
ormal vs PTCLs
M...
2... (blu), and Normal Tissues (red) according
Hierarchical clustering of PTCL/nos
M...
to the expression of CDKN1B, MKI67
and CCNE1.
2...
C...
Immunohistochemestry results
p27 expression in PTCL/nos (A) and normal T-cells (B)
A
B
Immunohistochemestry results
IHC confirmed physiological inverse relation between p27 and Ki67
expression (p<0.02). Conversely, in almost a third of cases, we
appreciated an apparent non-physiological balance between the two
molecules. However, the two proteins were indeed co-expressed by a
limited number of cells in 5 cases only.
p27 >30
p27 <30
Ki67 >80
1
4
Ki67 20-80
36
38
Ki67<20
9
10
TOTALE
46
52
In theese 5 cases we excluded an aberrant expression of CCNE1, which
was reported to overcome p27 function in Ki67+/p27+ neoplastic cells.
Sequencing results
C(-838)A
G(258)C; T(326)G;
T(356)C
A(4149)C
-79 C/T
Direct sequencing did not indicate somatic mutations, but rather
known polymorphisms of CDKN1B (blu and green), not related
to its expression.
Conclusions
p27 seems to be regulated in PTCL/nos as in normal Tlymphocytes. Whether its co-expression with Ki-67 in
few cells in a strict minority of cases might represent a
pathologic phenomenon deserves future investigations.
Acknowledgements
This work was supported by grants from:
 BolognAIL;
 AIRC;
 FIRB/RFO (Professor Pileri and Professor Zinzani);
 Fondazione Cassa di Risparmio in Bologna;
 Fondazione della Banca del Monte e Ravenna;
 Progetto Strategico di Ateneo 2006 (Dott. Piccaluga).
The Authors have no conflict of interest to disclose.