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 Adhesion Apoptosis Matrix Proliferation Signal transduction CytoskeletonTranscription PER1CBX4CHD2COPEBCREMEPC1JMJD1CMAFNR4A2NR4A3SERTAD1ZBTB10ZBTB24ZNF198ZNF331BCL10 GJA1TNSVCAM1LIFR CD69DUSP2DUSP8GADD45AGADD45BING3JUNDMOAP1PPP1R15A FN1COL12A1COL1A2COL3A1COL4A1COL4A2FBN1LAMB1SPARCCDH11 AXUD1FOXP1RHOBTB3CAV2PLEKHC1BTG1CLK1HECAJUNRGC32TOB1 CALD1STK17BMKNK2HIPK1PTP4A1PDE4DMAP3K8ITPKBSEPT10TJP1IRS2 TPM1Dlc2MGAT4AMYLIPNFIBWASPIP 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 PP Piccaluga et al. J Clin Invest 2007 Proliferation AXUD1 FOXP1 RHOBTB3 CAV2 PLEKHC1 BTG1 CLK1 HECA JUN RGC32 TOB1 AXUD1 FOXP1 RHOBTB3 CAV2 PLEKHC1 BTG1 CLK1 HECA JUN RGC32 TOB1 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 U 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 PTCL/NOSNormal T-cells Normalized expression value 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.

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. Gene expression results Ki-67 (MKI67) Cyclin E1 (CCNE1) Ki-67 (MKI67) P27 (CDKN1)

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 >30p27 <30 Ki67 >80 14 Ki Ki67< TOTALE 4652 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

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.