Development of concurrent plasma cell FISH and immuno-fluorescent staining for use in the diagnosis of multiple myeloma.

1
Development of concurrent plasma cell FISH and
immuno-fluorescent staining for use in the
diagnosis of multiple myeloma.
Dave Wallace - Cytogenetics
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Cytogenetic analysis in multiple myeloma

• Chromosomal abnormalities in myeloma are nearly
  universal.
• Typically complex, with gt10 abnormalities in 50
  cases and gt20 abnormalities in 10 cases.

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Cytogenetic analysis in multiple myeloma

• Specific chromosomal abnormalities can provide
  prognostic information.
• Deletions of chromosome 13 and p53 (17p13.1) are
  associated with a poor prognosis, as is the IgH
  rearrangement t(414).
• Whilst the IgH rearrangement t(1114) is
  associated with a good prognosis.

4
The difficulties of cytogenetic analysis in
multiple myeloma

• Due to their terminally differentiated state,
  B-cells are slow to divide leading to low numbers
  of metaphase chromosomes.
• A low percentage of plasma cells within a sample
  means it is necessary to score a high number of
  cells during analysis.

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The difficulties of cytogenetic analysis in
multiple myeloma

• Particularly, FISH analysis is not advised
  without a method of plasma cell enrichment or
  plasma cell identification, as low numbers of
  diseased cells may be indistinguishable from
  false positive FISH results.
• However, FISH analysis is desirable as
• Large numbers of cells can be analysed
• Cryptic rearrangements including the
  prognostically significant IgH translocation
  t(414)(p16.3q32) can be detected

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Cytogenetic analysis of multiple myeloma at The
Christie Hospital

• The number of samples The Christie cytogenetics
  unit received for multiple myeloma analysis per
  year

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Methods of plasma cell FISH

• The European myeloma network recommends either
• plasma cell separation to enrich the sample and
  hence be more likely to be scoring plasma cells
  when carrying out FISH analysis.
• or plasma cell identification to allow FISH
  analysis to be carried out only on known plasma
  cells.

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Plasma cell identification

• It was decided, given the difficulties and costs
  involved in plasma cell enrichment, that plasma
  cell identification was more suitable for our
  purposes.
• Such identification can be performed using plasma
  cell specific antibodies. The European myeloma
  network recommends a mix of anti-lambda and
  anti-kappa light chain immunoglobulins as the
  preferred method.

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Density centrifuged cell suspensions

• Experiments attempting to carry out fluorescent
  antibody labelling of fixed cell suspensions
  proved unsuccessful.
• Cell suspensions were freshly prepared using
  density centrifugation to separate the
  mononuclear cells (including plasma cells) from
  the remaining bone marrow sample.
• Slides were prepared using the cell suspension
  and cells were fixed to the slide with an acetone
  wash.

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Treatment using fluorescent light chain antibodies
A
B

• Laboratory case 06.1770, a confirmed case of
  multiple myeloma.
• A Light chain antibody treated sample under
  fluorescence
• B Under brightfield

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Co-immunofluorescence/FISH using light chain
antibodies

• The specificity of the light chain antibodies for
  malignant plasma cells was investigated using
  co-immunofluorescence/FISH.
• Light chain antibodies were used together with
  the Vysis FISH probe for chromosome 7, on case
  06.1895 a confirmed myeloma with hyperdiploidy
  including trisomy 7.

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Co-immunofluorescence/FISH using the light chain
antibodies
Laboratory case 06.1895, a confirmed myeloma with
hyperdiploidy including trisomy 7.
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Co-immunofluorescence/FISH using the light chain
antibodies
A Light chain antibodies, 7 centromeres and
7q31 B Light chain antibodies C 7
centromeres D 7q31
A
B
D
C
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Co-immunofluorescence/FISH using light chain
antibodies

• Specificity of the light chain antibodies for
  abnormal plasma cells was checked by scoring the
  FISH patterns of 100 fluorescent and 100
  non-fluorescent cells, for two separate myeloma
  cases.

1 FISH signal 2 FISH signals 3 FISH signals
Case 06.1770 antibody ve 0 6 94
Case 06.1770 antibody -ve 1 93 6
Case 06.1895 antibody ve 0 10 90
Case 06.1895 antibody -ve 4 91 3
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Co-immunofluorescence/FISH using light chain
antibodies

• FISH results showed the light chain antibodies to
  be highly specific for the malignant plasma cells
  present in both cases examined.
• 90 or more of antibody labelled cells were shown
  to be abnormal by FISH with at least a proportion
  of the remainder probably skewed by signal
  co-localisation.

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Co-immunofluorescence/FISH using light chain
antibodies

• The 7 centromere and 7q31 FISH carried out on
  these samples was successful.
• However, one of these two cases also had c-myc
  rearrangement and amplification. FISH for c-myc
  was not successful on this sample, with very high
  background fluorescence and weak FISH signals.

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FISH for IgH/c-myc on lymphoprep seperated cells
compared to standard cultured fixed cells
A
B
Cells counterstained with DAPI Laboratory case
06.1895, a confirmed myeloma with
hyperdiploidy including trisomy 7, as well as
c-myc rearrangement and amplification. A
Cultured and fixed cells B Lymphoprep separated
cells
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Co-immunofluorescence/FISH using light chain
antibodies

• In order to test the possible extent of the
  problem with FISH background fluorescence when
  carried out on lymphoprep separated cells, we
  carried out FISH for p53 and t(414) (the
  IgH/FGFR3 fusion) in five cases of myltiple
  myeloma.
• p53 and t(414) were chosen as these particular
  probes would probably be used in a myeloma FISH
  diagnostic testing regime.

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FISH for p53 and IgH/FGFR3 on lymphoprep
seperated cells
A
B
Laboratory case 06.1725 A FISH for p53 - 2
red signals B FISH for IgH/FGFR3 1 red, 1
green, 1 fusion
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FISH for p53 and IgH/FGFR3 on lymphoprep
seperated cells
A
B
Laboratory case 06.1929 A FISH for p53 2
red signals B FISH for IgH/FGFR3 2 red
signals, 2 green signals
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FISH for p53 and IgH/FGFR3 on lymphoprep
seperated cells
A
B
Laboratory case 06.1943 A FISH for p53 ?3
red signals B FISH for IgH/FGFR3 2 red
signals, 2 green signals
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FISH for p53 and IgH/FGFR3 on lymphoprep
seperated cells
A
B
Laboratory case 06.1972 A FISH for p53 2 red
signals B FISH for IgH/FGFR3 2 red signals, 2
green signals
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FISH for p53 and IgH/FGFR3 on lymphoprep
seperated cells
A
B
Laboratory case 06.2135 A FISH for p53 1
red signal B FISH for IgH/FGFR3 2 red
signals, 2 green signals
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Co-immunofluorescence/FISH using light chain
antibodies

• FISH was partially successful in most of these
  cases. In only one case was the entire samples
  background fluorescence high enough to mask the
  FISH signals.
• However, several of the samples showed a high
  proportion of cells to exhibit such background
  and therefore allowed imaging only of select
  cells.

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Thoughts and conclusions

• A protocol for combined FISH and
  immuno-fluorescence using antibodies for light
  chain immunoglobulins has been developed.
• However, FISH on lymphoprep separated cells often
  shows high background and weak signals.
• Whilst it was possible to capture images from
  most of the experimental samples, the high volume
  of unsuitable cells within some samples meant
  that diagnostic scoring would be very time
  consuming and, in some cases, impossible.
• Further investigation will be necessary if this
  technique is to be used diagnostically within The
  Christie cytogenetics service.

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Acknowledgements

• Nick Telford
• The entire Christie Hospital cytogenetics unit.