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

AdhesionApoptosis

Matrix

Proliferation Signal transduction

Cytoskeleton TranscriptionPER1PER1CBX4CBX4CHD2CHD2COPEBCOPEBCREMCREMEPC1EPC1JMJD1CJMJD1CMAFMAFNR4A2NR4A2NR4A3NR4A3SERTAD1SERTAD1ZBTB10ZBTB10ZBTB24ZBTB24ZNF198ZNF198ZNF331ZNF331BCL10BCL10

GJA1GJA1

TNSTNS

VCAM1VCAM1

LIFRLIFR

CD69CD69DUSP2DUSP2DUSP8DUSP8GADD45AGADD45AGADD45BGADD45BING3ING3JUNDJUNDMOAP1MOAP1PPP1R15APPP1R15A

FN1FN1COL12A1COL12A1COL1A2COL1A2COL3A1COL3A1COL4A1COL4A1COL4A2COL4A2FBN1FBN1LAMB1LAMB1SPARCSPARCCDH11CDH11

AXUD1AXUD1FOXP1FOXP1RHOBTB3RHOBTB3CAV2CAV2PLEKHC1PLEKHC1BTG1BTG1CLK1CLK1HECAHECAJUNJUNRGC32RGC32TOB1TOB1

CALD1CALD1STK17BSTK17BMKNK2MKNK2HIPK1HIPK1PTP4A1PTP4A1PDE4DPDE4DMAP3K8MAP3K8ITPKBITPKBSEPT10SEPT10TJP1TJP1IRS2IRS2

TPM1TPM1Dlc2Dlc2MGAT4AMGAT4AMYLIPMYLIPNFIBNFIBWASPIPWASPIP

PP Piccaluga et al. J Clin Invest 2007

Gene expression profiling (GEP) allowed the identification of PTCL/nos-associated molecular signatures

Looking for molecules responsible for cell cycle deregualtion in PTCL/nos

0

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p<0.0001

Score 1

Score 2

Score 3

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PP Piccaluga et al. J Clin Invest 2007

Proliferation

AXUD1AXUD1FOXP1FOXP1RHOBTB3RHOBTB3CAV2CAV2PLEKHC1PLEKHC1BTG1BTG1CLK1CLK1HECAHECAJUNJUNRGC32RGC32TOB1TOB1

AXUD1AXUD1FOXP1FOXP1RHOBTB3RHOBTB3CAV2CAV2PLEKHC1PLEKHC1BTG1BTG1CLK1CLK1HECAHECAJUNJUNRGC32RGC32TOB1TOB1

Clinico-pathological score: Age, PS, LDH, Ki-67

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, encoded by CDKN1B, is a member of a family of cyclin-dependent-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.

P27 - CDKN1B

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

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.

Methods

Gene expression results

diseased normal

Diseased/Normal0,01

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diseased normal

Diseased/Normal0,01

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CDKN1B

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PTCL/NOS Normal T-cells

PTCL/NOS Normal T-cells

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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.

-4 0 4

CDKN1B expression is inversely related to MKI67 and CCNE1 expression.

Hierarchical clustering of PTCL/nos (blu), and Normal Tissues (red) according to the expression of CDKN1B, MKI67 and CCNE1.

H...

Selected Gene Tree: GT_HC_PTCL-JCI_60...Selected Condition Tree:HC_PTCL-JCI_60_p27...Branch color parameter:Normal vs PTCLs

Colored by: PTCL-JCI_60 (Default Interpretation)Gene List: p27 pathway (9), 213523_at selected

HC_PTCL-JCI_...

Normal vs PTCLs

C...

2...T...

2...T...

2...M...

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Gene expression results

Ki-67 (MKI67)

Ki-67 (MKI67)

Cyclin E1 (CCNE1)

Ki-67 (MKI67)

Ki-67 (MKI67)

P27 (CDKN1)

H...

Selected Gene Tree: GT_HC_PTCL-JCI_60...Selected Condition Tree:HC_PTCL-JCI_60_p27...Branch color parameter:Normal vs PTCLs

Colored by: PTCL-JCI_60 (Default Interpretation)Gene List: p27 pathway (9), 213523_at selected

HC_PTCL-JCI_...

Normal vs PTCLs

C...

2...T...

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Immunohistochemestry results

A B

p27 expression in PTCL/nos (A) and normal T-cells (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

-79 C/T

G(258)C; T(326)G; T(356)C

A(4149)C

Direct sequencing did not indicate somatic mutations, but rather known polymorphisms of CDKN1B (blu and green), not related to its expression.

p27 seems to be regulated in PTCL/nos as in normal T-lymphocytes. Whether its co-expression with Ki-67 in few cells in a strict minority of cases might represent a pathologic phenomenon deserves future investigations.

Conclusions

AcknowledgementsThis 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.