Phenotypic Heterogeneity of Subclinical Adult TCell Leukemia

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Phenotypic Heterogeneity of Subclinical Adult
T-Cell Leukemia
Sapandeep K. Singh, MDTexas AM Health Science
Center CIS School in Hypersensitivity and
Allergic Diseases August 21-24, 2008
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Background Cases

• CASE 1
• 60yo woman who presented with cryptococcal
  meningitis and was found to have CD4 lymphopenia
  in the absence of HIV.
• CASE 2
• 55yo woman with a pruritic rash and chronic
  sinusitis, found to have elevated IgE levels and
  eosinophilia.

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Background Cases

• CASE 1
• 60yo woman who presented with cryptococcal
  meningitis and was found to have CD4 lymphopenia
  in the absence of HIV.
• CASE 2
• 55yo woman with a pruritic rash and chronic
  sinusitis, found to have elevated IgE levels and
  eosinophilia.

All POSITIVE for a clonal TCR gamma gene
rearrangement
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T-cell development Background

• Thymic T-cell development results in alpha/beta
  or gamma/delta receptors on CD4 and CD8
  T-cells.
• The core of TCR/CD3 complex is a heterodimer
  composed of either alpha and beta chains, or
  gamma and delta chains.

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T-cell receptor Background

• Each TCR chain is encoded by multiple rearranging
  gene segments.
• The gamma chain genes are the first to rearrange.
• Even though most T-cells then go on to rearrange
  the alpha chain genes, resulting in 95 of
  peripheral T-cells expressing alpha/beta
  receptors, the gamma chain remains rearranged in
  the T-cell genome.
• This pattern of TCR gene rearrangement is
  preserved in T-cell neoplasms and therefore
  provides a molecular marker for clonality.

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TCR gamma gene rearrangement Background

• TCR gamma chain genes have been mapped to
  chromosome 7p15, while the beta chain genes are
  at 7q35 and both alpha and delta chain genes are
  at 14q11.
• The TCR gamma chain locus, which undergoes VJC
  rearrangement, consists of
• Six functional V gamma genes, five pseudogenes,
  and three ORFs.
• Three J gamma 1 and two J gamma 2 gene segments,
  preceding the C gamma 1 and C gamma 2 genes,
  respectively.

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TCR gamma gene rearrangement Background

• A polyclonal population of lymphocytes will have
  a heterogenous admixture of cells, each with a
  different V-J rearrangement.
• Conversely, a monoclonal population, derived from
  a single transcribed cell, will produce a single
  homogenous population of cells with an identical
  gene rearrangement.
• Polymerase chain reaction (PCR) assays can be
  used to identify
• lymphocyte populations derived from a single
  cell by detecting unique V-J rearrangements
  (using a cocktail of amplifiers) within these
  receptor loci.
• Identification of a clonal TCR gamma chain gene
  rearrangement using PCR, can thus be a marker for
  clonality.

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Patient 1 History A 60 year old Caucasian
female with a history of recurrent sinusitis and
Candida vaginal infections presented with
cryptococcal meningitis and was found to have
CD4 lymphopenia (CD4 count 18 and 24). Despite
treatment with amphotericin B followed by oral
diflucan she continues to have CD4 lymphopenia
(CD4 count 31) and remains on oral diflucan for
persistent cryptococcal antigen positivity.
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Patient 2 History A 54 year old African American
female was referred for evaluation of pruritic,
erythematous rash, hypereosinophilia (4186
eosinophils/ µL) and an elevated IgE level (1330
U/mL). Workup was negative for atopy and
parasites.
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TCR gamma gene rearrangement Cases

• Questions
• Why do these patients have a clonal TCR gamma
  gene rearrangement?
• How are these cells affecting these patients
  immunologic clinical symptoms?
• How can we characterize this clonal population of
  T-cells? (i.e. What type of cells are these
  T-cells?)

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TCR gamma gene rearrangement Cases

• Hypotheses
• The clonal populations functional phenotype
  accounts for these patients clinical
  presentation.
• In patient 1, the clonal population may directly
  or indirectly lead to CD4 lymphopenia.
• In patient 2, the clonal population may lead to
  a TH2 predominance causing elevated IgE levels
  and eosinophilia.

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METHODS
PBMCs isolated by density centrifugation.
T-cells divided into TCR a/b CD4 or CD8
subpopulations using negative selection by
magnetic cell sorting and verified by flow
cytometry.
Stimulated CD4 or CD8 T-cells with and without
anti-CD3 and anti-CD28 beads for 24hrs and
evaluated for proliferation and cytokine
expression
Performed TCR? gene rearrangement PCR assay on
separate subpopulations
Quantified (using ELISA) IL-4, IL-5, IL-10,
IL-13, TGF-ß and IFN-? levels in stimulated and
unstimulated sub-populations
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Methods

• TCRg Gene Rearrangement
• IdentiClone TCRg PCR assay, which uses multiple
  DNA primers to target conserved genetic regions
  within the TCRg chain gene, was used to test the
  CD4 and CD8 subpopulations for clonality.
• Positive and negative controls are used for
  quality control.

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Methods

• Capillary Electrophoresis
• Beckman Coulter CEQ 8000 Genetic Analysis
  System, a differential fluorescence detection
  instrument, was used to resolve differently sized
  amplicon products.
• Differential fluorescence detection primers are
  conjugated with fluorescent dyes corresponding to
  different targeted regions. This results in high
  sensitivity, single base resolution, differential
  product detection, and capability for
  quantification.
• Criteria for defining a positive peak Products
  generated that fall within the valid size range
  and are at least three times the amplitude of the
  third largest peak in the polyclonal background.
  

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ResultsPBMC TCRg Clonality Sample Data for
Patient 2
T cell clonal peak in PBMC
Three times the amplitude of the third highest
peak of polyclonal background
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ResultsCD4 TCRg Clonality Sample Data for
Patient 2
No discernible third highest peak of polyclonal
background.
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ResultsCD8 TCRg Clonality Sample Data for
Patient 2
Clonal peak for a/b CD8 T-cells
Three times the amplitude of the third highest
peak of polyclonal background
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Cytokine ELISA Sample Data for Patient 2

• The ELISA cytokine profiles of the patients PBMC
  supernatants under stimulated and nonstimulated
  conditions were within the range of the three
  normal controls. The patients ELISA cytokine
  results did not display a Th1 or Th2 cytokine
  profile.

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Conclusions

• Patient 1
• This study is the first to demonstrate a clonal
  T-cell phenotype in the context of idiopathic
  CD4 lymphopenia and raises the possibility that
  T-cell clonality may contribute to the
  pathogenesis of this disorder.
• Patient 2
• This study is the first to demonstrate a clonal
  a/b CD8 T-cell population in the context of a
  clinical Th2 phenotype of hypereosinophilia and
  elevated IgE levels.
• Summary
• In conclusion, a clonal T-cell population is
  characteristic of T-cell leukemia. However,
  neither of these patients displayed overt
  manifestations of T-cell leukemia. We
  hypothesize that subclinical T-cell leukemia may
  present as a paraneoplastic immunophenotype.

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Support

• KSF (2007 AAAAI Summer Research Fellowship
  Medical Student Grant 2008 Chrysalis Project)
• DGG and SKS (T32 AI053831)
• DPH (ROI AI36936).

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TCR gamma gene rearrangement clonesSpecial
thanks

• David Huston, MD
• Dalia Galicia, MD
• Kevin Farnam, MS
• Atoosa Tavana

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