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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