Presentation on theme: "Francesco Forconi Ematologia e Trapianti Università di Siena"— Presentation transcript:
1Francesco Forconi Ematologia e Trapianti Università di Siena Orvieto, Palazzo Coelli21 Novembre 2009Immunogenetica della LLC: implicazionui patogenetiche e prognostiche mediante analisi del gene IGHV1-69Francesco ForconiEmatologia e TrapiantiUniversità di Siena
3Organi linfoidi secondari/ Marginal Zone Midollo OsseoOrgani linfoidi secondari/ Marginal ZoneIgDIgMIgGUM-CLL40 %M-CLL60%AntigenMUTAZIONI SOMATICHEIgMIgMIgIgDNell’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.IgCellula B immaturaCellula B naiveCellula B memoria
4Sopravvivenza e stato mutazionale dei geni IGHV 17 anni293 mesi9 anni95 mesiDamle, R. N. et al. Blood 1999;94:Hamblin, T. J. et al. Blood 1999;94:
5phosphorylation of p72Syk intracellular [Ca(2+)](i) AntigenBCRIgDIgMIgGUM-CLL40 %ZAP70 +M-CLL60%ZAP70 -phosphorylation of p72Sykintracellular [Ca(2+)](i)Rapid disease progressionSlow disease progressionNell’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.
6Shared sequence “stereotypic” characteristics of the HCDR3 suggest antigen selection of the leukemic clonesNot 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.
8Selective stimulation of the B cell of origin (?) Antigenic drive continuing following transformation (?)Are Stereotypes CLL-specific?
9IGHV1-6914 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 CLL30% delle UM-CLL227/259 (88%) cases >98% homology to germline allelesDal 47% al 55% delle CLL stereotipateNella CLL mediana dei casi 1-69 è 69 anniN=214
1051p1-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.
11Comparison 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.
12G6-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).
13Are 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
14HCDR3 driven clustering to identify prognostic subsets How does antigenic stimulation would continue following transformation?HCDR3 driven clustering to identify prognostic subsetsStamatopoulos, K. et al. Blood 2007;109:
19SummaryBy investigating the IGHV1-69-J6 repertoire we can observe that “CLL-specific” HCDR3 are present in the normal individualsThe 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
20p66Shc levels and clinical behavior of U-CLL and M-CLL Capitani et al, submitted 2009
21SienaEmanuele CenciniRomaElisa SozziDimitar EfremovNagaja CapitaniLuca LaurentiCosima BaldariGiovanni Del PoetaNovaraModenaDavide RossiRoberto MarascaGianluca GaidanoTorinoAvianoMarta CosciaRiccardo BombenMassimo MassaiaValter GatteiSouthamptonBellinzonaKathy PotterAndrea RinaldiFreda K. StevensonFrancesco BertoniSaloniccoNiguarda MilanoKostas StamatopoulosSilvio VeroneseMarco Montillo“The research was funded partly by a Clinical Research Grant awarded by the European Hematology Association"