1. Francesco Forconi Ematologia e Trapianti Università di Siena
Orvieto, Palazzo Coelli
21 Novembre 2009
Immunogenetica della LLC: implicazionui patogenetiche e prognostiche mediante analisi del gene IGHV1-69
Francesco Forconi
Ematologia e Trapianti
Università di Siena

2. IGHV-D-J rearrangement
IGH Variable region
IGHV-D-J rearrangement
Hypervariable Region
HCDR1
HCDR2
HCDR3
HFR1
HFR2
HFR3
N Region
IGHV
IGHD
IGHJ
51 IGHV genes
27 IGHD genes
6 IGHJ genes

3. Organi linfoidi secondari/ Marginal Zone
Midollo Osseo
Organi linfoidi secondari/ Marginal Zone
IgD
IgM
IgG
UM-CLL
40 %
M-CLL
60%
Antigen
MUTAZIONI SOMATICHE
IgM
IgM Ig
IgD
Nell’HCL, solo un piccolo gruppo di pazienti ha i geni IGHV non-mutati, ed inoltre nell’HCL, solo una minoranza di pazienti ha una cattiva prognosi e non risponde alla terapia convenzionale con analoghi purinici. Ig
Cellula B immatura
Cellula B naive
Cellula B memoria

4. Sopravvivenza e stato mutazionale dei geni IGHV
17 anni
293 mesi
9 anni
95 mesi
Damle, R. N. et al. Blood 1999;94:
Hamblin, T. J. et al. Blood 1999;94:

5. phosphorylation of p72Syk intracellular [Ca(2+)](i)
Antigen
BCR
IgD
IgM
IgG
UM-CLL
40 %
ZAP70 +
M-CLL
60%
ZAP70 -
phosphorylation of p72Syk
intracellular [Ca(2+)](i)
Rapid disease progression
Slow disease progression
Nell’HCL, solo un piccolo gruppo di pazienti ha i geni IGHV non-mutati, ed inoltre nell’HCL, solo una minoranza di pazienti ha una cattiva prognosi e non risponde alla terapia convenzionale con analoghi purinici.

6. Shared sequence “stereotypic” characteristics of the HCDR3 suggest antigen selection of the leukemic clones
Not only is there conservation of IGHVDJ rearrangements, but these can also be combined with particular IG light chain variable and joining gene rearrangements, strongly suggesting that there may be common (super)antigens binding to subsets of B cells found in CLL. While this could reflect the selective stimulation of the B cell of origin, there also exists the possibility that antigenic drive continues following transformation.
However, the question of whether such conserved sequences could be detected in significant numbers among normal B cells remained. Analysis of “non-CLL” sequences from the databases found very few,18,19 but deposited sequences are not generally derived from naïve unmutated B cells, the real comparator for U-CLL.

7. Top 10 in CLL
Murray et al BLOOD, 2008 (111).

8. Selective stimulation of the B cell of origin (?)
Antigenic drive continuing following transformation (?)
Are Stereotypes CLL-specific?

9. 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

10. 51p1-IGHJ6 rearrangements expressed in the normal B cell repertoire
Figure 2. Stereotypic 51p1-IGHJ6 sequences detected in normal B cells and in CLL. Sequences were assigned to known subsets or to new subsets (prefix S). Dark grey bars: % of normal B-cell sequences assigned to subsets. Light grey bars: % of CLL sequences assigned to subsets. For each subset, code, IGHD gene and reading frame are indicated.

11. Comparison of the HCDR3 sequences of CLL and normal B cells in the 51p1-IGHJ6-derived subset 5.
Figure 3. Comparison of the HCDR3 sequences of CLL and normal B cells in the 51p1-IGHJ6-derived subset 5. Amino acid sequences of the HCDR3 of each normal sequence aligned to the closest CLL HCDR3 are represented. Dashes indicate homology to the germline IGHV1-69, IGHD3-10 in reading frame (RF) 3 and IGHJ6 genes at the top of the figure. Identical N1 and N2 amino acids between different sequences are highlighted in shades of grey.

12. G6-positive (IGHV p1-expressing) B- cells are part of the conventional resting naïve B-cell population.
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).

13. 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

14. HCDR3 driven clustering to identify prognostic subsets
How does antigenic stimulation would continue following transformation?
HCDR3 driven clustering to identify prognostic subsets
Stamatopoulos, K. et al. Blood 2007;109:

15. Subset 1
CRO
AVIANO

16. IGHV4-39 and transformation to Richter Sydrome
p<.001
No IGHV4-39
IGHV4-39
IGHV4-39/stereotypic HCDR3
IGHV4-39/no stereotypic HCDR3
No IGHV4-39/stereotypic HCDR3
No IGHV4-39/no stereotypic HCDR3
Events/N
5-year risk SE
IGHV4-39
6/20
35.4%
13.5%
No IGHV4-39
33/733
5.6%
1.1%
5-year risk p
IGHV4-39/stereotypic HCDR3
68.7%
.003
IGHV4-39/no stereotypic HCDR3
No IGHV4-39/stereotypic HCDR3
9.9%
.005
No IGHV4-39/no stereotypic HCDR3
4.2%
Rossi, Clinical Cancer Research 2009

17. Stamatopoulos, K. et al. Blood 2007;109:259-270
IGHV1-69 & progression
Stamatopoulos, K. et al. Blood 2007;109:

18. HCDR3 length in CLL
HCDR3 length in 1-69

19. 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

20. p66Shc levels and clinical behavior of U-CLL and M-CLL
Capitani et al, submitted 2009

21. Siena
Emanuele Cencini
Roma
Elisa Sozzi
Dimitar Efremov
Nagaja Capitani
Luca Laurenti
Cosima Baldari
Giovanni Del Poeta
Novara
Modena
Davide Rossi
Roberto Marasca
Gianluca Gaidano
Torino
Aviano
Marta Coscia
Riccardo Bomben
Massimo Massaia
Valter Gattei
Southampton
Bellinzona
Kathy Potter
Andrea Rinaldi
Freda K. Stevenson
Francesco Bertoni
Salonicco
Niguarda Milano
Kostas Stamatopoulos
Silvio Veronese
Marco Montillo
“The research was funded partly by a Clinical Research Grant awarded by the European Hematology Association"