at 4 C. Discard the solution after use
17. Air bubbles trapped under the glass coverslip can lead to uneven probe distribution which can cause signal intensity
References
1. Wolff AC, Hammond ME, Hicks DG et al (2007) American Society of Clinical Oncology/
College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 131:18–43
2. Wolff AC, Hammond ME, Hicks DG et al (2013) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer:
American Society of Clinical Oncology/College of American Pathologists clinical practice guide- line update. J Clin Oncol 31:3997–4013.
doi: 10.1200/JCO.2013.50.9984
3. Petersen BL, Sorensen MC, Pedersen S et al (2004) Fluorescence in situ hybridization on
formalin-fi xed and paraffi n-embedded tissue:
optimizing the method. Appl Immunohistochem Mol Morphol 12(3):259–265
4. Müller S, Matthiesen SH, Nielsen KV (2009) Preparation of FFPE tissue slides for solid tumor FISH analysis. In: Kumar GL, Rudbeck L (eds) Education guide. Immunohistochemical (IHC) staining methods, 5th edn. Dako North America, Carpinteria, CA
5. Sommerlad C, Mehraein Y, Giersberg M et al (2002) Formalin-fi xed and paraffi n-embedded tissue sections. In: Rautenstrauss BW, Liehr T (eds) FISH technology. Springer, Berlin, Heidelberg
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Thomas S.K. Wan (ed.), Cancer Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1541, DOI 10.1007/978-1-4939-6703-2_12, © Springer Science+Business Media LLC 2017
Chapter 12
Cytoplasmic Immunoglobulin Light Chain Revelation and Interphase Fluorescence In Situ Hybridization in Myeloma
Sarah Moore , Jeffrey M. Suttle , and Mario Nicola
Abstract
The cytogenetic analysis of plasma cell myeloma (PCM) allows stratifi cation of patients so that prognosis may be determined and appropriate therapeutic options can be discussed. Owing to the patchy nature of the disease in the bone marrow (BM), the low proliferative activity of plasma cells and the cryptic nature of some PCM-associated cytogenetic changes, karyotypic analysis in this disease should be augmented with targeted interphase fl uorescence in situ hybridization (FISH). Immunofl uorescent revelation of cytoplas- mic immunoglobulin light chains, together with interphase FISH (cIg-FISH), allows the identifi cation of plasma cells within a sample so that they may be scored preferentially. This is particularly useful in situa- tions where there are only a small percentage of plasma cells in a sample. Where an underlying myeloid disease is suspected the cIg-FISH-negative cells can be scored separately. Two methods are provided in this chapter: the technique for cIg-FISH in fresh PCM BM samples and a procedure for use in fi xed cytogenetics preparations.
Key words Plasma cell myeloma , Cytoplasmic light chains , Fluorescence in situ hybridization , cIg- FISH , Prognosis , Plasma cell
1 Introduction
Plasma cell myeloma ( PCM ) is an incurable malignancy characterized by the accumulation of monoclonal plasma cells and, in some patients, leads to cytopenias, bone resorption (lytic bone lesions, hypercalcemia), the production of a monoclonal protein (parapro- tein), and renal impairment [ 1 ]. It has a heterogeneous clinical course with a corresponding range of genetic and other prognostic markers. PCM can advance from the premalignant condition of monoclonal gammopathy of undetermined signifi cance (MGUS) in which the monoclonal protein is present, but there are <10 % plasma cells in the bone marrow . End-organ damage is not evident in this stage. Frank PCM can also be preceded by smoldering myeloma (SM ) and this is an indolent, asymptomatic form of the
disease with 10–30 % plasma cells in the bone marrow. In turn, PCM can evolve into an even more aggressive entity known as plasma cell leukemia ( PCL ) in which end-organ damage is evident and there are circulating malignant plasma cells.
PCM is an incurable disease with a mean survival of 3–4 years, but it is highly heterogeneous in that some patients die very early while others will remain alive up to 10 years later [ 1 ]. It is this large range in clinical outcomes that has driven the search for prognostic disease markers. These began with the Durie-Salmon system [ 2 ], which is mostly a measure of tumor burden and which considers the severity of anemia, calcium level, kidney function, presence or absence of bone lesions, and the quantity of abnormal proteins. It is a staging system and lacks prognostic strength. The International Staging System (ISS) [ 3 ] measured the peripheral blood ( PB ) β 2-microglobulin and albumin levels and was revised (ISS-R) [ 4 ] to include acquired genetic changes in the risk assessment. These acquired changes remain the best indicator of disease aggressive- ness despite their lack of consideration of constitutional genetic changes and other factors such as age and comorbidities [ 5 – 8 ].
The treatment for PCM is highly dependent on the stage of the disease and the risk stratifi cation [ 9 , 10 ]. For some elderly patients with advanced disease palliative care may be appropriate, while for others the aim is to prolong survival while maintaining quality of life. Unfortunately genomic studies of PCM are not straightforward; plasma cells are diffi cult to culture, perhaps because PCM is a malignancy affecting a more differentiated class of cells with a low proliferative rate, but also because plasma cells may die in culture. Particularly in the early stages of the disease the malignant plasma cells are anchorage dependent and rely on inter- action with the bone marrow stroma and extracellular matrix [ 11 ].
Cytogenetic culture (i.e., without a ‘feeder layer’) often results in growth of only karyotypically normal cells. As the disease pro- gresses plasma cells are less reliant on their environment, may switch on autocrine cytokine production or constitutive activation of signaling pathways, and are free to proliferate in a stromal- independent manner [ 11 ]. This latter situation is commonly associated with increased tumor burden, disease progression, and ability to give rise to karyotypic abnormalities in culture. To increase the number of patients in whom cytogenetic abnormalities are detectable at all stages of PCM it is necessary to examine nondividing cells and this is achievable by interphase FISH. Since the proportion of malignant plasma cells may be low (particularly in MGUS and SM ) it is recommended [ 1 , 12 , 13 ] to use plasma cell selection, either real or virtual, prior to FISH analysis. Plasma cell selection may be undertaken by fl ow sorting or by bead separa- tion, but these procedures are expensive and not available to most laboratories. Common to all malignant plasma cell populations is high surface expression of CD138 and cytoplasmic expression of Ig- Kappa or Ig- Lambda light chains. CD138 (Syndecan- 1 ) may be
a less useful marker since it is lost from the cells surface over time [ 14 ]. Cytoplasmic revelation of immunoglobulin light chains (by staining with goat anti-human kappa or lambda light chain conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA)), together with 2-color interphase FISH (cIg-FISH) allows the identifi cation of plasma cells within a sample so that they may be scored preferentially [ 15 – 17 ]. Immunostaining can be enhanced by the application of a secondary anti-goat immuno- globulin also conjugated with AMCA.
Recommendations for the FISH probes to be used are made according to their demonstrated prognostic utility. To this end it is considered useful to use dual fusion translocation probes for MMSET: IGH-FGFR3 to detect the cryptic t(4;14)(p16;q32) and IGH-MAF to detect t(14;16)(q32;q23) ; and deletion probes for TP53 (del17p). These probe sets identify very high-risk genetic features. Laboratories are encouraged to add to these probe sets as is required with, for example, probes for hyperdiploid PCM (which would include an odd-numbered chromosome such as chromo- some 15 or 19 with a probe for 5q) and a deletion 1p/gain 1q probe set for identifying changes associated with disease progres- sion. The IGH-CCND1 dual fusion translocation probes may also be considered since this translocation is seen in almost all cases of IgM PCM. It may also help to distinguish this type of PCM from lymphoplasmacytic leukemia (Waldenstrom’s macroglobulinemia).
Some laboratories use IGH break-apart probes to identify patients with IGH rearrangements and refl ex to IGH-FGFR3 , IGH-MAF , and IGH-CCND1 in positive cases. This can provide cost savings in terms of probe use.
The cIg-negative cells can also be examined in the event that an underlying myeloid disease is suspected. Myelodysplastic syndrome can arise post-therapy for PCM [ 18 ] and demonstra- tion of 5q- or 7q- in the cIg-negative cell population would be instructive.
The cIg-FISH utilizes immunofl uorescence for the immuno- globulin light chains that are produced by B-cells. The two meth- ods provided later are for different purposes. The fi rst method incorporates the use of paraformaldehyde as a fi xative. It is most useful when fresh PCM samples are received by the laboratory and can be preceded by a density gradient separation step to enrich for mononuclear cells (MNC). The MNC can then either be trans- ferred to a microscope slide by cytospin or spread onto a slide in the manner of blood fi lm preparation. This technique may be the more robust of the two methods.
The second method is applicable to cytogenetic preparations and it is recommended that an additional, preferably direct harvest is performed and set aside for this purpose. This technique fi ts well into the routine operation of a diagnostic cytogenetics laboratory.
These techniques have similar steps and an overview is pro- vided in Fig. 1 .
2 Materials
Universal precautions should be used throughout the procedure and personal protective clothing (laboratory gown, safety glasses, and gloves) should be worn at all times. For all chemicals the mate- rial safety data sheet (MSDS) should be consulted and chemicals should be used under conditions appropriate to their possible harm. All solutions are made with reverse osmosis (RO) water ( see Note 1 ).
1. 10-mL sterile disposable pipettes.