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 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 anti- kappa 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 AB Laboratory case , 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 co- immunofluorescence/FISH. Light chain antibodies were used together with the Vysis FISH probe for chromosome 7, on case a confirmed myeloma with hyperdiploidy including trisomy 7.

Co-immunofluorescence/FISH using the light chain antibodies Laboratory case , a confirmed myeloma with hyperdiploidy including trisomy 7.

Co-immunofluorescence/FISH using the light chain antibodies AB C D A: Light chain antibodies, 7 centromeres and 7q31 B: Light chain antibodies C: 7 centromeres 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 signal2 FISH signals3 FISH signals Case : antibody +ve0694 Case : antibody -ve1936 Case : antibody +ve01090 Case : antibody -ve4913

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.

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 Cells counterstained with DAPI Laboratory case , 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 AB

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 Laboratory case A : FISH for p red signals B : FISH for IgH/FGFR3 – 1 red, 1 green, 1 fusion AB

FISH for p53 and IgH/FGFR3 on lymphoprep seperated cells Laboratory case A : FISH for p53 – 2 red signals B : FISH for IgH/FGFR3 – 2 red signals, 2 green signals AB

FISH for p53 and IgH/FGFR3 on lymphoprep seperated cells Laboratory case A : FISH for p53 – ?3 red signals B : FISH for IgH/FGFR3 – 2 red signals, 2 green signals AB

FISH for p53 and IgH/FGFR3 on lymphoprep seperated cells Laboratory case A : FISH for p53 – 2 red signals B : FISH for IgH/FGFR3 – 2 red signals, 2 green signals AB

FISH for p53 and IgH/FGFR3 on lymphoprep seperated cells Laboratory case A : FISH for p53 – 1 red signal B : FISH for IgH/FGFR3 – 2 red signals, 2 green signals AB

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.