Immunogenetica della LLC: implicazionui patogenetiche e prognostiche mediante

analisi del gene IGHV1-69

Francesco Forconi

Ematologia e Trapianti

Università di Siena

Orvieto, Palazzo Coelli21 Novembre 2009

IGHV-D-J rearrangement

HFR1 HFR2 HFR3

HCDR1 HCDR2 HCDR3

IGHV IGHJIGHDN Region

Hypervariable Region

IGH Variable region

51 IGHV genes 27 IGHD genes 6 IGHJ genes

Antigen

IgIgMIgM

IgD

Ig

MUTAZIONI SOMATICHEMUTAZIONI SOMATICHE

Cellula B memoria

Cellula B immatura Cellula B naive

Midollo Osseo Midollo Osseo Organi linfoidi secondari/ Marginal ZoneOrgani linfoidi secondari/ Marginal Zone

IgMIgD

IgG

UM-CLL40 %

M-CLL60%

Sopravvivenza e stato mutazionale dei geni IGHV

Hamblin, T. J. et al. Blood 1999;94:1848-1854

95 mesi

293 mesi

Damle, R. N. et al. Blood 1999;94:1840-1847

17 anni

9 anni

Antigen

IgMIgD

IgG

UM-CLL40 %

M-CLL60%

ZAP70 + ZAP70 -

•phosphorylation of p72Syk •intracellular [Ca(2+)](i)

•Rapid disease progression •Slow disease progression

Shared sequence “stereotypic” characteristics of the HCDR3 suggest antigen selection of the leukemic clones

Top 10 in CLL

Murray et al BLOOD, 2008 (111).

● Selective stimulation of the B cell of origin (?)

● Antigenic drive continuing following transformation (?)

● Are Stereotypes CLL-specific?

IGHV1-69● 14 alleli di cui i più frequenti IGHV1-

69*01, *02, e *12 (riconosciuti dall’anticorpo anti-51p1 G6)

● Infrequente nella popolazione B del sangue periferico da analisi molecolari (Lipsky: <1%)

● 13% di tutte le CLL

● 30% delle UM-CLL

● 227/259 (88%) cases >98% homology to germline alleles

● Dal 47% al 55% delle CLL stereotipate

● Nella CLL mediana dei casi 1-69 è 69 anni

N=214

51p1-IGHJ6 rearrangements expressed in the normal B cell repertoire

Comparison of the HCDR3 sequences of CLL and normal B cells in the 51p1-IGHJ6-derived subset 5.

• 4.8% of all B-cells. • CD27-negative, indicative of naïve B cells. • IgM+ IgD+ CD23+ CD5- CD38+ (as in G6-ve naïve B-cells). • A small percentage of CD5+ B cells, not found in the memory B-cell subset. • CD38 expression was similarly high in naïve and G6-positive populations. • IgK (65%) : IgL (35%) comparable to normal B cells and 51p1+ve CLL (data not shown). • Absence of activation markers (CD25 and CD69).

G6-positive (IGHV1-69 51p1-expressing) B- cells are part of the conventional resting naïve B-cell population.

Are Stereotypes CLL-specific?

● by focusing only on the IGHV1-69-derived sequences combined to IGHJ6 in age-matched normal subjects, we have found “Stereotypic” sequences of several of the major subsets described in CLL and of new potential subsets in > 33% sequences cloned from normal donors.

● it is possible that this conserved sequences are a likely source of transformation to U-CLL and that they derive from the naïve B-cell repertoire.

● Little similarity in the HCDR3 junctional amino acids between cases of CLL and little similarity within normal B cells

How does antigenic stimulation would continue following transformation?

HCDR3 driven clustering to identify prognostic subsets

Stamatopoulos, K. et al. Blood 2007;109:259-270

Subset 1

CROAVIANO

Events/N 5-year risk SE

IGHV4-39 6/20 35.4% 13.5%

No IGHV4-39 33/733 5.6% 1.1%

p<.001 No IGHV4-39

IGHV4-39

Rossi, Clinical Cancer Research 2009

No IGHV4-39/stereotypic HCDR3

IGHV4-39/stereotypic HCDR3

No IGHV4-39/no stereotypic HCDR3

IGHV4-39/no stereotypic HCDR3

5-year risk p

IGHV4-39/stereotypic HCDR3 68.7%.003

IGHV4-39/no stereotypic HCDR3 0

No IGHV4-39/stereotypic HCDR3 9.9%.005

No IGHV4-39/no stereotypic HCDR3 4.2%

IGHV4-39 and transformation to Richter Sydrome

IGHV1-69 & progression

Stamatopoulos, K. et al. Blood 2007;109:259-270

HCDR3 length in CLL

HCDR3 length in 1-69

Summary● By investigating the IGHV1-69-J6 repertoire we can observe that “CLL-

specific” HCDR3 are present in the normal individuals

● The subsets with different clinical behavior may rely on (super)antigen stimulation. However, it remains to be demonstrated that stimulation occurs through specific CDR3 interaction.

● Lack of different behavior (CLL progression and overall survival) between stereotyped and non stereotyped UM-CLL using IGHV1-69 point to antigen stimulation via CDR3-independent antigen.

● Clinically, mutational status keeps being confirmed as the relevant tool to stratify progression risk in CLL

●

p66Shc levels and clinical behavior of U-CLL and M-CLL

Capitani et al, submitted 2009