Lymphoproliferative disorders

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Lymphoproliferative disorders Powered By Docstoc
					      Lymphoproliferative disorders




AM1
      Aims
      • lymphoreticular system – a reminder
      • lymph nodes
         – functional histology
         – lymphadenopathy
             • non-neoplastic
             • neoplastic
             • lymphoma as an example of the impact of new technology on
               Dx
         – pathology of thymus and spleen (for you to do)




AM2
      The lymphoreticular system
      • organs and tissues associated with the proliferation,
        development and deployment of white blood cells

      • an important part of the immune system

      • primary lymphoid organs
          – thymus and bone marrow
          – produce mature immune response cells regardless of presence of
            antigen

      • secondary lymphoid organs
          – spleen, lymph nodes
          – mucosa associated lymphoid tissue (MALT) – tonsils, adenoids,
            Peyer’s patches
          – produce mature immune cells as a reactive process

      • The lymphatic system ‘filters’ extracellular fluid

AM3
      Cells involved in the immune response
      • T cells and their derivatives
      • B cells and their derivatives
      • Antigen presenting cells
         – macrophages
         – dendritic cells




AM4
                                    Antigen
                                                          Mounting the immune
      Cytotoxic T cell activation     B cell activation
                                                          response


                                                          PA    processed antigen
                                                          CD    cluster of differentiation
                                                          MAC   membrane attack complex
                                                          Tc    cytotoxic killer cell




                                                                Plasma cells
                                                                synthesise
                                                                antibodies




AM5
                                Thymus H&E

      Thymus
 • Pre-T cells, formed in the
   red bone marrow, pass to
   the thymus in infancy and
   childhood, where they
   mature
 • This organ is at its
   maximum size by age 10-
   12, after which it
   atrophies (involution)
 • still produces some T
   cells even in the very
   elderly
                                C    capsule
 • SCID
                                S    septa
                                Cx   cortex
AM6                             M    medulla
                                        Thymic cortex



  Mt     mitotic cells (lymphoblasts)
  BM     basement membrane
  E      endothelial cells
  M      macrophages



  Thymocyte maturation occurs
  by the cells moving deeper into
  the cortex during which time TCR
  genes are rearranged and specific
  surface markers are acquired




AM7
                             Thymic medulla

  E epithelial component
  H Hassall’s corpuscles                      E
  keratinised (degenerate)
  epithelial cells


  The epithelial mesh is
  easier to see in the
  medulla




AM8
Diagram of splenic anatomy
  • the spleen is the largest mass of bodily lymphoid tissue
  • produces lymphocytes in the new-born, phagocytes that remove old
    RBCs and foreign bodies, blood reservoir
  • the white pulp largely consists of B lymphocytes, but there are no
    afferent lymphoid vessels nor lymph sinuses
  • B cells proliferate in the white pulp




AM9
       Histology of the spleen




            • There is T cell white pulp and B cell white pulp.
              This makes up 5-20% of the spleen.
            • The cells are able to migrate, divide and mature
              in the spleen
AM10
Anatomy of the lymphatic system
• Lymphatic capillaries (blind ended) – similar
  to veins, but with more valves. In skin they lie
  close to veins, around the viscera they follow
  arteries and form plexi

• Lymphatic ducts and trunks carry lymph to the
  brachiocephalic veins

• Lymph drains into venous blood via the right
  lymphatic duct and the left lymphatic duct
  (thoracic duct)

• Lymph nodes occur throughout the trunk,
  neck and lower chin
                                                     spleen
                                                     thymus

AM11
       The lymphatic system

       • ‘swollen glands’ in the neck usually refers to swollen
         (cervical) lymph nodes.

       • common areas where lymph nodes can be easily felt,
         especially when enlarged:
          –   inguinal and iliac (groin)
          –   axilla (armpits)
          –   supraclavicular (above the clavicle)
          –   cervical (neck)
          –   occipital (back of the head just above hairline)



AM12
       Lymph node histology

       • cortex
          – nodules of B-lymphocytes
            (follicles or germinal centres)


       • paracortex
          – region containing T-cells


       • medulla
          – medullary cords and sinuses that
            drain into the hilum



AM13
       Plan of a lymph node




AM14
       Lymph node histology
• germinal centre
       – region rich in B-cells (CD20 or
         CD79α)
       – B-cells are activated in response to
         the entry of antigens
       – dendritic reticulum cells trap
         antigens (antigen-presenting cells)
       – antigen presented to naïve B-cells
         with the assistance of TH cells
       – induces B-cell transformation and
         maturation (or apoptosis)
       – plasma cells are the final product –
         found in the medulla




AM15
       Lymph node histology

 • paracortex
       – T-cell rich region
       – mostly CD4+ (TH)
       – antigen presenting cell

       – can use CD3 to identify
         them




AM16
       Lymph node histology

  • medulla
       – sinuses lined with
         macrophages – clears
         lymph fluid
       – cords contain plasma
         cells – antibody
         production




AM17
                             Lymph node H&E

       C capsule
       Cx cortex
       M medulla
       H hilum
       F follicles
       P paracortical zone
       MC medullary cords
       T trabeculae
       S sinus



AM18
       Lymphocyte domains in
       the lymph node

       F follicle with (light coloured
       germinal centre)
       P paracortex
       MC medullary cords




AM19
 Lymph node




   CD3




 CD79a




AM20
       Lymphadenopathy

       •   = enlargement of the lymph node
            – palpable or visible
            – local or widespread
            – frequently biopsied for Dx

       •   causes
            – local or systemic infection
                 • inflammatory or reactive response

            – neoplastic disorders
                 • primary (lymphoma)
                 • secondary (metastasis)              Burkitt’s lymphoma




AM21
       Inflammatory and reactive responses
   • Infection                           Caseous necrosis of LN (TB)
        – bacterial, viral, fungal
        – tuberculosis


   • Autoimmune
        – rheumatoid arthritis
        – systemic lupus erythematosis
        – tuberculosis


   • other reactive changes
        – drugs
        – foreign material (e.g.
          silicone…)



AM22
       reactive responses


   • cell proliferation (hyperplasia) and LN enlargement
        – B-cell response – germinal centre (and marginal zone) hyperplasia

        – T-cell response – paracortical hyperplasia

        – macrophage response – sinus hyperplasia (sinus histiocytosis)

        – mixed response – most common




AM23
Reactive tonsil (ICC +ve control)




    CD3




 CD79a
AM24
  Non-neoplastic lymphadenopathy
   non-specific reactive hyperplasia
   • acute lymphadenitis – entry of pyogenic bacteria – acute inflammation
   • granulomatous lymphadenitis – e.g. mycobacterium
   • necrotising lymphadenitis – e.g. cat scratch fever
   • sinus histiocytosis

   specific reactive hyperplasia
   • paracortical hyperplasia – infectious mononucleosis (EBV)
   • toxoplasmosis
   • drugs and other foreign bodies
AM25
Neoplastic disorders of the lymphoreticular system

       • Haematological
          – lymphoma, leukaemia, myeloproliferative diseases


       • Solid tumours
          – melanoma, breast, head and neck cancers




AM26
       Lymphoid neoplasia



       • Abnormal proliferation and/or survival of a lymphoid cell
           – acquired genetic changes
           – clonal expansion
           – selective advantage in a given microenvironment


       • Characterised by
           – expansive growth
           – genetic changes
           – clonality



AM27
 Classifiying lymphoid neoplasia

 • Approx. 8% of all cancers

       – Lymphoma
          • solid masses
          • may involve bone marrow

       – Leukaemia
          • bone marrow
          • blood




AM28
       Lymphomas
       • Nodal (60%)
       • Extranodal (40%)




AM29
       Lymphomas
          • Hodgkin’s lymphoma (disease) (30%)
             • characterised on morphology – Reed-Sternberg cells

          • Non-Hodgkin’s lymphoma (70%)
             • characterised by certain clinical symptoms




AM30
       Lymphomas
          • Hodgkin’s disease (lymphoma) (30%)
             • neoplastic proliferation of large, activated lymphoid cells
             • T or B cells (Reed-Sternberg cells)

          • Non-Hodgkin’s lymphoma (70%)
             • neoplastic proliferation of lymphoid cells (T or B cells) or sometimes
               histiocytes
             – B cell (85%)
             – T cell (10%)
             – NK cell (<5%)




AM31
       Lymphomas
       • Usually present with a group of involved nodes
       • spread to other lymphoid organs including other groups of
          lymph nodes, spleen and bone marrow
       • peripheral sites may be involved eventually including liver
          and skin
       • Non-Hodgkin’s lymphoma may arise in other organs (leading
          to complicated classification)


AM32
       Clinical assessment
       •   history, duration of symptoms
       •   presence of pain
       •   associated symptoms – fever, malaise, weight loss
       •   examination
           – distribution of lymphadenopathy
           – lymph nodes are firm or rubbery, discrete or matted
           – presence of hepatosplenomegaly




AM33
       Clinical investigation
                        • Excision or incision biopsy required if a
                          haematological disorder is suspected
                            – fine needle aspiration cytology may be
                              useful for solid tumours
                            – risks of node biopsy (e.g. damage to
                              accessory nerve) should be appreciated
                        • Specimens should be sent ‘dry’ to
                          laboratory
                            – Will allow samples for imprint cytology or
                              microbiological culture




AM34
       Time for a break




AM35
                       • First described by Sir
                         Thomas Hodgkin in 1832

Hodgkin's lymphoma 1   • Can present at any age but
                         most commonly seen in 3rd
                         and 4th decades

                       • Male : Female ratio 2:1

                       • Usually presents as
                         painless lymphadenopathy
                         in superficial lymph nodes
                         involving
                          – Cervical nodes (60-70%)
                          – Axillary nodes (10-15%)
                          – Inguinal nodes (6-12%)

AM36
                       •   Constitutional symptoms
                           include fever (night sweats),
                           pruritus, unexplained weight
                           loss, alcohol-induced pain

Hodgkin's lymphoma 2   •   Splenomegaly occurs in 50%
                           patients (also hepatomegaly
                           and some bone marrow
                           involvement)

                       •   may be cutaneous
                           involvement as a late
                           complication (c. 10%)

                       •   Diagnosis confirmed by
                           histological examination of
                           involved node

                       •   Reed-Sternberg cells are
                           diagnostic of the disease

AM37
 Hodgkin's lymphoma 3
  • Staging investigations
       –   Chest X-ray
       –   Bone marrow trephine biopsy
       –   Abdominal and chest CT scan
       –   Staging laparotomy - often not required


  • Staging of Hodgkin's disease
       – Stage I     Confined to one lymph node region
       – Stage II    Disease confined to 2 or more nodal regions on one side of diaphragm
       – Stage III   Disease involving nodes on both sides of diaphragm
       – Stage IV    Extra-nodal disease - usually liver or bone marrow




AM38
Hodgkin's lymphoma 4
       • Generally a good response to treatment

       • Treatment
          – Stage I & II: radiotherapy
          – Stage III & IV: chemotherapy and radiotherapy


       • Survival
          – Stage I: 90% at 5 years
          – Stage IV: 60% at 5 years

AM39
       Lymphomas
          • Hodgkin’s disease (lymphoma) (30%)
             • neoplastic proliferation of large, activated lymphoid cells
             • T or B cells (Reed-Sternberg cells)

          • Non-Hodgkin’s lymphoma (70%)
             • neoplastic proliferation of lymphoid cells (T or B cells) or
               sometimes histiocytes




AM40
       Non-Hodgkin’s lymphoma

       • group of diverse lymphoid cell neoplasms
       • presents as enlarged lymph nodes within groups
       • biopsy is usual first step to Dx
       • conventional histology, ICC and molecular
         diagnostics used to identify origin of neoplastic cell
       • variable response to therapy:
          – indolent but respond poorly to therapy
          – aggressive clinical course but respond well to treatment



AM41
       Origins of Non-Hodgkin’s lymphoma

       • B and T cells
       • Mostly from B cells in lymph nodes
       • Can arise from MALTs, but these are less likely to
         become disseminated
          – gut, salivary gland, lung
       • mycosis fungoides T cell lymphoma arising in the
         skin




AM42
       Classification of NHLs
       • Different lymphomas arise in/differentiate
         towards different normal counterparts
       • WHO Classification of Lymphomas
       • Identifies disease entities based on biological
         principles (>40 types)
       • Uses all available information:
          –   Morphology
          –   Immunophenotype (cell of origin; oncoproteins)
          –   Growth pattern (follicular or diffuse)
          –   Lymphocyte maturation
          –   Genetic alterations (gene translocations, clonality)
          –   Clinical behaviour (site; indolent or aggressive)

AM43
                                                     marginal zone
                                                     lymphoma
                                                     memory B cell


                    naïve cell
   lymphoblastic mantleB cell
    B lymphoblast                follicular B cell
                                  follicular
   lymphoma       lymphoma       lymphoma




                                                           myeloma
                                                           plasma cells

       Bone marrow

       B cell differentiation and lymphoma
AM44
  Immunocytochemistry in classification

   lymphoblastic    mantle cell        follicular    marginal zone
    B-lymphoblast                                   Memory B-cell    myeloma
     lymphoma       lymphoma         lymphoma         lymphoma


                                  CD20
       TdT
                                    IgM
                                     IgG/A
                                                                     IgG/A
                        IgD
                                    Bcl-6

       CD10                         CD10
                     CD5
AM45
       information slide
       •   CD20
            – marker of B cell lineage, from pre-B to mature B cells but not plasma cells
       •   TdT
            – Terminal deoxynucleotidyl transferase is a 58kD nuclear DNA polymerase that
              catalyses the polymerisation of deoxynucleotides. TdT is expressed in immature
              B cells
       •   IgM, IgG, IgA, IgD
            – alternative immunoglobulin (Ig) class variants are expressed depending on the
              maturation and activation state of the B cell
       •   Bcl-6
            – marker for germinal centre B cells
       •   CD10
            – not restricted to B cells, but is a useful marker for follicular lymphoma
       •   CD5
            – mostly expressed in T cells, but may appear in B cells of the mantle zone



AM46
Combinatorial diversity: rearranging the Ig gene

       V(ariable)         D(iversity)       J(oining)   C(onstant region)

                                                         Germline DNA



                                                        VDJ
        V        D    J                     C           recombination



             V        D      J          C               Exon splicing




               CDR1          CDR2           CDR3    Ig heavy chain protein

       CDR = complementarity determining region
AM47
           Polyclonal           Monoclonal




       Polymerase chain reaction (PCR)
       technology can be used to assess clonality


AM48
                                                       Results for gel run on 11/11/02


                                                       Key:
                                                       L = DNA ladder
                                                       T1 = control: monoclonal
                                                       lymphoma
                                                       T2 = negative control
                                                       (polyclonal)
                                                       T3 = control: monoclonal
                                                       lymphoma
        L   T1   T2   T3   A1   A2 A3   A4   B1   B2
                                                       A – C = group efforts




       B3   B4   C1   C2   C3   C4 D1   D2   D3   D4

AM49
       Genetic abnormalities
       • Gene translocation is common cause of lymphoma
          – promoters or fusion proteins




AM50
  Translocations: translocation of heavy chain enhancer or promoter elements to sites
  flanking functional genes will result in inappropriate overexpression of those genes.
  Depending on the gene product, this may result in immortalisation (Bcl-2) or
  deregulated proliferation (oncogenes such as c-myc)

  e.g. t(14;18), IgH & Bcl-2:
                                                                     MBR

 Ch18 : Bcl-2                   5’                                                      3’

                                           J                   C
 Ch14 : IgH                     5’                                                    3’
                                               Enhancer

 t(14;18)               5’                                                          C        3’
                                                                   Enhancer

AM51
               Translocations
   • Involving 2 non-Ig genes leading to
     formation of a novel fusion protein with
     oncogenic function (eg. t(2;5), Alk-1 & NPM)



 NPM     N     DD                                   C

 Alk-1 N         EC       TM        TKD                 C


                 DD                    TKD P
 NPM-Alk
  Fusion                                     P
                 DD                    TKD

AM52
       Detecting Translocations
       – Fluorescent In Situ Hybridistion (FISH)
         t(11;18)
       API2-MALT1




AM53
       Viral Lymphomagenesis

       • EBV
         – classical Hodgkin’s lymphoma
         – Burkitt’s lymphoma
         – postransplant lymphoproliferative disease


       • HTLV-1
         – adult T-cell lymphoma/leukaemia


       • HHV8/KSHV
         – primary effusion lymphoma
         – plasmablastic lymphoma




AM54
              Hodgkin’s lymphoma




                CD15




       CD30     CD20               EBV LMP-1
AM55
                                                       TNM staging
                                                       Tumour size
                                                       Nodes involved

  Metastasis                                           Metastatic spread


  • distant spread of a malignant tumour from its original site

  • lymph node surgery may be used as both a diagnostic and
    staging procedure
  • staging may be achieved by a full regional lymph node
    dissection




AM56
       Lymph node assessment


       • provides useful prognostic information but does not
         necessarily increase survival
       • also associated with significant complications (e.g.
         lymphoedema, sensory disturbances)
       • many patients have no evidence of metastatic spread
       • node dissection can be associated with unnecessary
         morbidity


AM57
       Sentinel lymph node biopsy (SLNB)

       • The sentinel lymph node is the first draining node
         from a tumour

       • dye or radioisotope injected next to a tumour
          – patent blue dye - blue node
          – technetium nanocolloid – ‘hot’ node with gamma probe
          – blue dye and isotope in combination


       • SLNB is an accurate predictor of overall node status
          – malignant melanoma and breast ca


AM58
       SLNB – a panacea?

       • Advantages
         – associated with few complications
         – reduced morbidity as node-negative patients will not require
           lymph node dissection


       • Disadvantages
         –   dye tracing
         –   skills
         –   labour-intensive
         –   node groups (e.g. PCa)



AM59
       Imprint cytology
       • Motomura et al., 2000. Intraoperative sentinel lymph
         node examination by imprint cytology and frozen
         sectioning during breast surgery.
         Br J Surg. 87(5):597-601.




AM60
       Sentinel lymph node
       HMB45




                         Malignant
                         melanoma
AM61
       Summary



       •   short review of the lymphoreticular system
       •   structure and function of lymph nodes
       •   examples of non-neoplastic lymphadenopathies
       •   neoplastic lymphadenopathies
           – Hodgkin’s lymphoma
           – Non-Hodgkin’s lymphoma
       • diagnostic strategies in the investigation of neoplastic
         lymphadenopathies
       • discuss the use of sentinel node biopsy to assess metastasis



AM62
       Further reading
       •   Chapter 10, Kierzenbaum (2002)
       •   Capters 9 and 22, Underwood (2004)
       •   Chapter 23, Bancroft and Gamble (2002)


       •   Harris et al. (2001). New Approaches to Lymphoma Diagnosis. Hematology,
           2001:194
       •   Kearney et al. (2005). Molecular cytogenetics in haematological malignancy:
           current technology and future prospects. Chromosoma, 114: 286
       •   Isaacson & Du (2005). Gastrointestinal lymphoma: where morphology meets
           molecular biology. The Journal of Pathology, 205: 255
       •   Thomas et al. (2004). Part I: Hodgkin's lymphoma - molecular biology of
           Hodgkin and Reed-Sternberg cells. Lancet Oncology, 5: 11




AM63