Early Molecular Events in Lymphomagenesis

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Early Molecular Events in Lymphomagenesis Powered By Docstoc
					Early Molecular Events in
   Lymphomagenesis
   The Follicular Lymphoma model




          Bertrand NADEL
Lab Genomic Instability & Human Hemopathies
             nadel@ciml.univ-mrs.fr


         Centre d’Immunologie de Marseille-Luminy
             INSERM-CNRS-Université de la Méditerranée
                     13288 Marseille, France
«To date, only a small fraction of the [cancer] genes
  has been analyzed, and the number and type of
  alterations responsible for the development of
      common tumor types are still unknown »  


    Velculescu, Kinzler and Vogelstein Science 314, 268 (2006)
        The multi-hit model of oncogenesis

   « Theoretical considerations have suggested that a handful of
 mutations, perhaps as few as 3, may be sufficient for developing
                            colorectal cancer…




   … However, [we recently] estimated that ~100 nonsynonymous
mutations and that as many as 20 of the mutated genes in individual
     cancers might play a causal role in the neoplastic process »


        Velculescu, Kinzler and Vogelstein PLoS Comput Biol. 2007 3:2239
       The multi-hit model of oncogenesis


Colorectal:       15-20 functional hits out of ~100 “passenger” hits
     Selection/counter selection “Darwinian” process
lymphoma:          ~5-7 hits ??

   Oncogene       « passenger »   « causative »   TS




   « negative »



                                                               ACUTE
                                          Pre-clinical phase
                                                               TUMOR
         oncogenesis: what does it take?

              oncogenes/TS          ~4 biological functions


                Alteration 1


                Alteration 2           Self renewal
Combination                                                   Functional
     A                                                        subtypes
                Alteration 3       Differentiation arrest


                Alteration 4       Apoptosis /Survival
                                                               Clinical
                    ??         ?
                                                            Significance?
                                   Adhesion  & motility


                    ??         ?       Ion transport


                    ??                 Metabolism
                               ?
                                         How long does it take?




                                                                                                                M Greaves  Nature Reviews
                                                                                                                Cancer 2006 ,6:193



  Marculescu et al 2002, V(D)J‐mediated translocations in        Fasching K et al Presence of clone‐specific antigen receptor gene
                   Most major hits are NOT causative/not sufficient to allow
 lymphoid  neoplasms:  a  functional  assessment  of             rearrangements at birth indicates an in utero origin of diverse types of early

                   malignant progression
 genomic instability by cryptic sites J Exp Med 195:85           childhood acute lymphoblastic leukemia. Blood. 2000 95(8):2722
   Marculescu  et  al  2003,  Distinct  t(7;9)(q34;q32)           Fischer et al Screening for leukemia‐ and clone‐specific markers at birth in
and/or
 breakpoints  in  healthy  individuals  and  individuals  with
 T‐ALL Nat Genet 33:342
                                                                 children with T‐cell precursor ALL suggests a predominantly postnatal origin.
                                                                 Blood. 2007 110(8):3036
   Dik  et  al  2007,  Different  chromosomal  breakpoints        Eguchi‐Ishimae et al NOTCH1 mutation can be an early, prenatal genetic event
                   Mutation rate is very slow
 impact  the  level  of  LMO2  expression  in  T‐ALL  Blood      in T‐ALL. Blood. 2008 111(1):376‐8.
 110:388
Non-Hodgkin’s Lymphomas                    (NHL)
epidemiology in brief
                                                   Mortality from NHL (France, Crude rate all ages)
                                                                                          Males


 •   7th cause of cancer
      th
                                                                                        Females



 •   7th cause of death by cancer
      th



 •   Steady increase of 3-4%/year
 (1970-2000)
 (1970-2000)
                                                   Chronologic
 •   Plateau at 11-18 new cases/100 000            Tendencies
                                                            (males)

     ~10 000 new cases/year         in France
                                    in France
                                                                                        Incidence




                                                                                         Mortality




           RISK FACTORS
        LARGELY UNKNOWN
     NHL: a Real Public Health Issue


                 Identify        Identify
Identify risk
                « at-risk »   « patients » at
  factors
                Individuals    early stages



  PREVENTION

                                         THERAPY

                              monitor MRD & Remission
FOLLICULAR LYMPHOMA                       in brief:

Clinical features
• 2nd most common adult lymphoma
• indolent but disseminated at
  presentation
• not curable with available treatments

Biological features
• germinal center B-cell
  malignancy
• highly dependent on
   microenvironment cross-talk


Etiology:
• initiated in the bone
  marrow through t(14;18)




                                                        From Küppers (2005)
                                                      Nature Rev. Cancer, 5:251
Follicular Lymphoma and t(14;18)(q32;q21) translocation




                            18q21: BCL2

                                                                                                         der18


   14q32: IGH
                                              balanced
                                            translocation                                  BCL2 deregulation

                                                                            der14

                                    Bcl2                                        IGH

                1      2                               3                                                      der14




                            Bcl2                                     block apoptosis
                    Adapted from Atlas of Genetics and Cytogenetics in Oncology and Haematology http://www.infobiogen.fr/
                                      The GC “checkpoint”

  BCL2+                              BCL2 DOWN-REGULATION              BCL2+




                                        BCL2
                                        t(14;18)

                                             RESCUE AND ACCUMULATION
adapted from Küppers (2005) Nature
                                                OF CC/CB-LIKE CELLS
        Rev. Cancer, 5:251
t(14;18)(q32;q21) and FL Lymphomagenesis




  • t(14;18) is considered to be the initial step
    of FL lymphomagenesis


           BUT: NOT SUFFICIENT


  • t(14;18) found in blood of ~70%
    HEALTHY INDIVIDUALS at low frequency

                     requires a “2nd hit”
           (e.g. gains 18q, +7, +8; loss 6q, 1p, mut EZH2..)
  The etiology of Follicular Lymphoma : the Classical View
“when a resting B cell that carries the BCL2 translocation undergoes blast
transformation in response to antigen, failure to switch off the BCL2 gene
may contribute to development of a lymphoma” (Harris et al., Blood 94 REAL classification)


                                             2nd HIT
 t(14;18)
                                                                  CSR
                                  Ag
                                            SHM

         Immature
           B cell




                         naive
                         B cell
                         t(14;18)+

                              ??
Bone marrow             periphery


                Healthy t(14;18)+       progression (or not)
                                                                      LYMPHOMA
                    carriers               to disease ??
         t(14;18)(q32;q21) and FL Lymphomagenesis




t(14;18)                     • immunomodulation t(14;18) frequency
                                i.e. t(14;18) in HCV+ patients
                                BUT: t(14;18) with a-viral treatment

      Immature
                             • t(14;18) with age
        B cell                 BUT: persistence /expansion clones
                             not accumulation of new translocations

                 naive
                 B cell
                 t(14;18)+
                                      naïve B cells??
                                      naïve B cells??
                      ??
                                             or
Bone marrow      periphery
                                    memory B cells??
                                     memory B cells??
                                    (encountered Ag)
                                     (encountered Ag)
Q1: are t(14;18)+ Cells in Healthy Individuals Naïve B Cells ?

                 PB
          10ml
                                            Experiment 1:
                      10-6   determine if t(14;18)+ cells have undergone
                                 Class Switch Recombination (CSR)
                                       (naïve devoid of CSR)
Experiment 1: determine if t(14;18)+ cells have undergone CSR -STRATEGY

                         PB
                10ml
                                              10-5       Determine switch across the breakpoint
                                                            on translocated allele (BCL2/Cγ)




                       Bcl2                                                              IgH
                                        J6     iEμ       Iμ       Sμ         Cμ     Cδ       Iγ Sγ1-4   Cγ1-4   Iα Sα1-2 Cα1-2
              1 2             3     N




BCL2/JH Short range PCR ~ 0,1 to 0,6 Kb



BCL2/Cµ Long range PCR                   ~ 7,5 to 8 Kb                                       Class switch
                                                                                            recombination

                                             Bcl2
                                                             J6        iEμ        Iμ Sμ/Sγ1 Cγ1    Iα Sα1-2 Cα1-2
                              1 2                    3   N




BCL2/Cγ Long range PCR                                        ~ 3,5 to 11 Kb
                                                                  RESULTS - 1

                                  4x10-5
        Bcl2


            t(14;18) frequency
                                  3x10-5J
                                           6     Cohort Sμ     Cμ
                                               iEμ Iμ 1: retrospective healthy individual samples
1 2                              3    N

                                  2x10-5


                                  1x10-5       BCL2/Cµ

                                 <1x10-6


                                                                                                    6 individuals with highest
t(14;18)+                        BCL2/JH                              BCL2/Cµ                           t(14;18) frequency
t(14;18)-



  6/6                                                     2/6
                                              RESULTS - 1


        Bcl2
                                                              Bcl2
                       J6   iEμ   Iμ   Sμ    Cμ                              J6    iEμ    Iμ Sμ/Sγ1 Cγ1
1 2            3   N                                    1 2          3   N




                            BCL2/Cµ                                               BCL2/Cγ




t(14;18)+      BCL2/JH                            BCL2/Cµ                                BCL2/Cγ
t(14;18)-



  6/6                                  2/6                               5/6




 Most individuals carry BCL2/Cγ, but not BCL2/Cµ
                                        RESULTS - 1




ID     Bcl2/Sμ             Bcl2/Sγ




                                               Average detection ratio
N=6    Detection (ratio)   Detection (ratio)
                                                                                           9/50
99      no       (0/10)     yes       (1/5)                              20%




                                               (% positive PCR)
102     yes       (1/5)     yes       (1/5)
103     yes      (1/10)     no       (0/10)
109     no       (0/10)     yes      (1/10)
                                                                         10%
120     no       (0/10)     yes      (2/10)                                      2/55
127     no       (0/10)     yes      (4/10)

        2/6      (2/55)     5/6      (9/50)
Mean   33%        3,6%     83%         18%
                                                                               BCL2/Cµ   BCL2/Cγ




Most individuals carry BCL2/Cγ, but not BCL2/Cµ
BCL2/Cγ are more frequent AND more abundant than BCL2/Cµ
Q1: are t(14;18)+ cells in Healthy Individuals naïve B cells ?



                     CONCLUSION 1:

        Most t(14;18)+ cells in healthy individuals
                  ARE NOT naïve B cells




        Q2: are t(14;18)+ cells in Healthy Individuals
                      MEMORY B cells ?
Experiment 2: determine if t(14;18)+ cells are memory B cells - STRATEGY

Cohort 2: 10 anonymous
healthy blood bank donors

                  PBMCs
          250ml




                                                    Cell sorting

                               naive B cells           memory B cells
         Total PBMCs
                              (CD19+ CD27-)            (CD19+ CD27+)

             BCL2/JH
          Short range PCR




        t(14;18) frequency       <1.1x10-6                   1.4x10-4
                                                RESULTS - 2



                      3.6x10-4

                      3.2x10-4
t(14;18) frequency


                      2.8x10-4                                                        Total Frequency

                      2.4x10-4                                                        Memory B cells

                      2.0x10-4                                                        Naïve B cells
                      1.6x10-4

                      1.2x10-4

                      8.0x10-5

                      4.0x10-5

                     <1.0x10-6
                                 12   21   18   11   15    13   14     24   99   20
                                                 Healthy donors n=10




                        Most peripheral t(14;18)+ are memory B cells
                                         RESULTS - 2




                                                                                   memory B cells
  3 x 10-4                                                                         (CD19+ CD27+)
                               Clone A    Clone B   Clone C   Clone D    nd

2.5 x 10-4


  2 x 10-4


1.5 x 10-4


  1 x 10-4


  5 x 10-5


        0
             #11   #14   #13      #12       #21     #18       #15       #24   99   #20




        T(14;18) frequency variations in the peripheral blood of healthy
             individuals result from clonal expansion in the GC
Experiment 3: define the Immunophenotype of t(14;18)+ memory B cells
                            – STRATEGY -

                                                                          IgD+/CD27-                         IgD+/CD27+                       IgD-/CD27+
                                                                              Naive                            Memory                           Switched
A.                                                             B.           IgD+/CD27-                           IgD+/CD27+
                                                                             B Bcells
                                                                            Naïve cells                        B cells                        Switched memory cells
                                                                                                                                             Memory BBcells
                        Peripheral B cells CD19+                                                                                                    IgD-/CD27+
                                                                                                                Memory Bcells
            104
                   23.6%                           23.1%                                                                          97.4%       98.9%
                    Switched memory
                          Bcells            IgM memory
            103                                Bcells



            102




            101                                    Naïve
     CD27




                                                   Bcells

                                                   50.4%                                     99.7%.
            100
                  100       101       102    103         104        100    101   102   103       104   100     101   102    103       104   100       101   102   103   104

                  IgD

                                                                                                                           Cell sorting



                          BCL2/JH
                   Short range PCR



                                                               Detection ratios                         Detection ratios                          Detection ratios
                                                 RESULTS - 3



                                          IgD+/CD27-     IgD+/CD27+      IgD-/CD27+
         Total
                                             Naive         Memory         Switched
        PBMCs
                                            B cells        B cells      Memory B cells

                                                          36/103
                                 40%
         % positive replicates




                                 30%



                                 20%              8/55
                                       16/165
                                                                      9/115
                                 10%



                                 0%
                                                 5/10    10/10        6/10




     In all healthy individuals, most t(14;18)+ are in the sIgM fraction

Unexpectedly, most t(14;18)+ in healthy individuals express a surface IgM
                     PARADOX:

   LR-PCR on retrospective cohort shows CSR on the
                 translocated allele

BCL2/JH PCR on blood bank donors shows NO CSR on the
                  expressed allele



         Q3: Is this real or is this an artifact ?
Experiment 3: check the genotype of t(14;18)+ memory B cells – STRATEGY


                                                                          IgD+/CD27-                      IgD+/CD27+                        IgD-/CD27+
                                                                             Naive                          Memory                            Switched
  A.                     Peripheral B cells CD19+               B.          IgD+/CD27-                        IgD+/CD27+                          IgD-/CD27+
                                                                             B Bcells
                                                                            Naïvecells                      B cells
                                                                                                             Memory Bcells                  Switched memory cells
                                                                                                                                           Memory B Bcells
              104
                     23.6%                         23.1%                                                                      97.4%        98.9%
                     Switched memory
                           Bcells           IgM memory
              103                              Bcells



              102




              101                                  Naïve
       CD27




                                                   Bcells

                                                   50.4%                                  99.7%.
              100
                     0
                    10       101        2
                                       10    103
                                                         10 4         0
                                                                     10     1
                                                                           10    2
                                                                                10    3
                                                                                     10       104   100     101   102   103       104    100       101   102   103   104

                    IgD



Short range PCR                             BCL2/JH



                                            BCL2/Cµ

Long range PCR

                                            BCL2/Cγ



                                                                          Detection ratios            Detection ratios                  Detection ratios
                                                     RESULTS - 4


                                        IgD+/CD27-     IgD+/CD27+     IgD-/CD27+
                                           Naive         Memory        Switched
                                          B cells        B cells     Memory B cells

                               4x10-5

                                                                                      BCL2/Cµ
         t(14;18) frequency




                               3x10-5
                                                                                      BCL2/Cγ

                               2x10-5




                               1x10-5



                              <1x10-6
                                          1/4 0/4      3/4 4/4      0/4 3/4



The IgD+/CD27+ Memory B cell fraction accounts for most of the peripheral
 t(14;18)+ B cells

At least half of the sIgM t(14;18)+ B cells have CSR on the translocated allele
                       ALLELIC PARADOX:

•   Functional allele:           Mostly CSR- (sIGM/D)
•   Translocated allele:         Mostly CSR+



                    3 B cell subsets in the periphery:

        - Naïve B cells (60-70%):           sIgM, but CD27-/CSR-
        - Switch memory B cells (10-20%):   CD27+/CSR+, but sIgG/A/E
        - IgM memory B cells (10-20%):      sIgM/CD27+, but CSR-



        Peripheral t(14;18)+ cells do not match any
          physiological peripheral B cell subset …
… but this allelic paradox is a hallmark of the
          FOLLICULAR LYMPHOMA

                   • Immunophenotype
                      Harris 1998 Blood 84:1361 REAL classification
                       - usually sIg+ (IgM>IgG>IgA)

                   • Fiber FISH
                      Vaandrager 1998 Blood 8:2871
                        - 56% (13/23) sIgM
                        - 74% (17/23) BCL2-Cγ (bias to γ1)

                   • LR-PCR
                      Akasaka 1998 Genes Chrom. Cancer 21:17
                       - 78% (41/52) BCL2-Cγ

                   • LR-PCR
                      Our collection of 30 FL patients
                       - 88% (23/30) BCL2-Cγ (bias to γ1)
             Downstream CSR in sIgM+ Follicular Lymphoma
                      (Vaandrager 1998 Blood 8:2871)
                                                              IgH
                      VDJ   iEμ    Iμ   Sμ        Cμ    Cδ         Iγ Sγ   Cγ        Iα Sα    Cα
Expressed allele
 sIgM

                                                                                       ds CSR

                             VDJ        iEμ   Iμ       Sμ     Cμ    Cδ       Sγ/Sα     Cα
 sIgM + ds CSR


                                          Inactivation of Sµ
                                        (deletions, mutations)



                                   Bcl2
                                                  J6    iEμ    Iμ Sμ/Sγ Cγ       Iα Sα       Cα
translocated allele         1 2           3   N

   CSR
t(14;18)+ expanding FL-like B-cells in PBMC from healthy individuals:
       A reappraisal of the current model of FL pathogenesis
                   Roulland (2006) J.Exp.Med 203:2425
Bone marrow                Peripheral Blood


                                                                                                                                              Secondary
     t(14;18)                                           Germinal Centre
                                             Ag
                                                                                                                                           Lymphoid Organs
                                         presentation                                     sIgG
                                                                    Mantle zone
                                                                                                              Release
                                                         Light zone
                                                                              Apoptosis
                                                                                                                                                     2nd HIT
                                                  Dark zone                                           sIgM


                            Mature                                  FDC
Pre-B      Immature B                                         FTH
                            Naïve B                                                                                                   Homing in
                                                                               BCL2
             CD19+20+      CD19+20+                                           Rescue                                                  activated
CD19+20+
                IgM+         IgM+/D+                                                                                                  follicles?
  IgM-
             [t(14;18)+]   [t(14;18)+]

                                                                                       sIgM                      "FL-like"                    Ag
     V(D)J                                                                           selection               CD19+27+ IgM+ > IgG+           Recall ?
                                                 SHM                  CSR                                    [CSR+ SHM+ t(14;18)+]
 Recombination


                                                                                                                                     Trafficking ?

                BM
             Niches ?
                                                                                                 Accumulation of secondary events and progression
                                                                                                                                       to disease?
  t(14;18) found in blood at variable frequencies:
 is variability associated with malignant progression ?


                          Environmental Risk
                                Factors




                                                         ?
                                       « at risk »           Patient
  Healthy           « Healthy »      Individuals?
Individuals         Individuals
  1 cell in       1 cell in100,000
  1 million                           Progression (or not)

                                                     Years
       Epidemiological features

     • Are there at risk individuals ?
• Do such groups display higher t(14;18)?
  Agricultural exposure & lymphomagenesis


Specific association between pesticide exposure and risk of NHL limited
to t(14;18)-positive cases (Chiu et al., Blood, 2006)




Do exposed farmers display higher t(14;18) frequencies in blood ?

 What is the molecular connection between pesticide exposure,
    t(14;18) translocation, and malignant progression ?

            Is t(14;18) in blood a biomarker of risk?
Farmers exposed to pesticides present a dramatic
         increase of t(14;18) frequency
Pesticide exposure: a genotoxic effect?

                            Genotoxic stimuli inducing
                            breaks on the BCL2 gene ?



              t(14;18)
                                                Accumulation of distinct
                                                       breaks
    pro‐B          Im‐B              naive

              t(14;18)



    pro‐B          Im‐B              naive

                                                Increased polyclonal pool
               t(14;18)
                                                 of t(14;18)+ naive B-cells

      pro‐B          Im‐B               naive


      Bone marrow                      blood
Clonal expansion of GC-activated t(14;18)+ cells




            Immunogeno/phenotyping:
   sCD27+, sCD10+/-, sIgM+>sIgG+, SHM+, CSR+
    Is this clonal expansion connected to high risk of
                malignant transformation?

                 Bone marrow                      blood                                                                           germinal centers
                                                                                         Y
                         t(14;18)                                         Ag             YY              T
                                                                                                                                  Apoptosis
                                                                                                                               Differentiation

               pro‐B            Im‐B                  naive              primed                                                   Genomic
                                                                                                                                 instability
                                                                                              cb/cc


                                                                                         Dissemination
                                                                                         & trafficking                           Secondary
                                                                                                                                  lesions
                                                                                          (BM, SLO)



                                                               Pre-clinical phase (up to 20 years)                            OVERT FL



          4 causative steps of FL progression:
•   Ectopic BCL2 expression in the GC
•   Arrest of differentiation as GC-like B-cells with ongoing AID activity
•   Accumulation of additional hits, partly due to AID-mediated genomic instability
•   Dissemination and trafficking (BM, SLO)
1- Are circulating t(14;18)+ cells from
  exposed farmers expressing BCL2?
BCL2 expression level is similar to FL cells
  2- Are t(14;18)+ cells “frozen” GC B-cells
         with ongoing AID activity ?


AID off                        AID on
                                         CSR
                               SHM

                   identical                      intra-clonal
                     clones                         variation
                                                      (ICV)


Clonal expansion               Clonal expansion
ICV in expanding clones: AID is “on”
ICV in expanding clones: AID is “on”
3- Are t(14;18)+ cells undergoing AID-mediated
             genomic instability?




                          In DLBCL, sustained AID activity
                          In DLBCL, sustained AID activity
                          is associated with aberrant SHM
                          is associated with aberrant SHM
                                 and CSR, including:
                                  and CSR, including:

                         - Large intra-Sµ deletions
                         - Large intra-Sµ deletions
                         - Sµ tandem duplications
                         - Sµ tandem duplications
                         - insertion of DNA fragments
                         - insertion of DNA fragments
                         from distinct chromosomes
                         from distinct chromosomes
AID-mediated genomic instability




                                ~30%
                            aberrant CSR
Sµ/γ insertions: markers of AID-mediated genomic
     instability leading to disease progression

      Exposed individual          DLBCL patient
                               (Lenz 2007 JEM 204:633)
             CONCLUSION:
Ectopic BCL2 expression in the GC

Arrest of differentiation as GC-like B-cells with
ongoing AID activity

Accumulation of additional hits, partly due to AID-
mediated genomic instability

Extensive trafficking & dissemination in SLO/BM




 Expanded t(14;18)+ clones
constitute bona-fide precursors at
various stages of tumor progression
        Where do we stand in the connection between
              t(14;18) and progression to FL?

  Bone marrow                      blood                                                                           germinal centers

                                                                               Y
          t(14;18)                                                             YY               T
                                                               Ag
                                                                                                                            Apoptosis
                                                                                                                            Differentiation
pro‐B              Im‐B                   naive                primed                                                     Genomic             “DARWINIAN”
                                                                                     cb/cc
                                                                                                                        instability             evolution:
                                                                                                                                              not ineluctable
                                                                           Dissemination                             Secondary
                                                                           & trafficking                              lesions?
                                                                            (BM, SLO)


                                               Pre-clinical phase (up to 20 years)                                       OVERT
                                                                                                                           FL
                      Environmental Risk
                            Factors




                                                         ?
                                     « at risk »             Patient
  Healthy       « Healthy »        Individuals?
Individuals     Individuals       Additional hits?
  1 cell in   >1 cell in100,000
  1 million    Markers of AID-
              mediated genomic
                  instability
                                    Progression (or not)

                                                     Years
     Lab B NADEL Genomic Instability & Human Hemopathies
        Centre d’Immunologie de Marseille-Luminy (CIML)                               With the
                                                                                      support of…
Bertrand Nadel
Mustapha Faroudi
Jean-Marc Navarro
Melanie Bonnet
Marie Loosveld
Ester Morgado
Dominique Payet
Stephanie Sungalee
Sandrine Roulland
Lydie Pradel
Emilie Mamessier
Marie-Amélie Goujart
Stéphanie Gon

Collaborators

 CIML                  UPRES EA 3889,   CRLCC F. Baclesse,   La Conception Hospital
 Marseille             Rennes           CAEN                  Marseille
 C Schiff              K Tarte          P. Lebailly          J Hardwigsen
 S Mancini             T Fest           AC. Gac
 A Anginot             P Ame-thomas     M. Briand            NCI, NIH
 J Tellier             C Meynard        Y. Lecluse           Bethesda, MD, USA
                                                             E Jaffe
 E Vivier              Hôpital Purpan   U918,
 E Tomasello           Toulouse         ROUEN
 C Luci                S Valitutti      C. Bastard
                       P Brousset       P. Ruminy
                       C Laurent        H Tilly

				
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