Development of concurrent plasma cell FISH
and immuno-fluorescent staining for use in the
diagnosis of multiple myeloma.
Dave Wallace - Cytogenetics
Cytogenetic analysis in multiple myeloma
 Chromosomal abnormalities in myeloma are nearly
universal.
 Typically complex, with >10 abnormalities in 50%
cases and >20 abnormalities in 10% cases.
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(4;14).
 Whilst the IgH rearrangement t(11;14) is
associated with a good prognosis.
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.
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(4;14)(p16.3;q32) can be
detected
Cytogenetic analysis of multiple myeloma
at The Christie Hospital
Number of patients referred for myeloma testing per year to The Christie cytogenetic unit
50
45
40
Number of patients
35
30
25
20
15
10
5
0
1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Year of patient referral
The number of samples The Christie cytogenetics unit received
for multiple myeloma analysis per year
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.
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 antikappa light chain immunoglobulins as the preferred
method.
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.
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
Co-immunofluorescence/FISH using light chain
antibodies
 The specificity of the light chain antibodies for
malignant plasma cells was investigated using coimmunofluorescence/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.
Co-immunofluorescence/FISH using the light
chain antibodies
Laboratory case 06.1895, a confirmed myeloma with hyperdiploidy
including trisomy 7.
Co-immunofluorescence/FISH using the light
chain antibodies
A: Light chain antibodies,
7 centromeres and 7q31
B: Light chain antibodies
C: 7 centromeres
A
B
C
D
D: 7q31
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
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 colocalisation.
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.
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
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(4;14) (the IgH/FGFR3 fusion) in
five cases of myltiple myeloma.
 p53 and t(4;14) were chosen as these particular
probes would probably be used in a myeloma FISH
diagnostic testing regime.
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
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
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
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
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
Co-immunofluorescence/FISH using light chain
antibodies
 FISH was partially successful in most of these
cases. In only one case was the entire sample’s
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.
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.
Acknowledgements
 Nick Telford
 The entire Christie Hospital cytogenetics unit.