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The Identification of Kappa and Lambda in Multiple Myeloma Using Automated In-Situ Hybridisation Techniques

Multiple myeloma is the second most frequent B-cell malignancy in Western countries. Current guidelines suggest that light chain clonality needs to be demonstrated to confirm diagnosis of multiple myeloma. Immunohistochemistry of kappa and lambda light chains can often give difficulties in interpretation, especially in cases of recurrent or residual myeloma. In-situ hybridisation techniques have gone some way to solve these problems. However, these techniques have been expensive and time consuming for a routine diagnostic laboratory. The introduction of automation has allowed Hammersmith Hospital Molecular Diagnostic Laboratory to carry out routine in-situ hybridisation analysis of multiple myeloma bone marrow trephine biopsies to confirm diagnosis and is now proving to be an invaluable tool. Since introducing this technique eight months ago, 29 cases have been diagnosed on bone marrow trephines that would otherwise have presented difficulties.

Introduction

Multiple myeloma (MM), also known as plasma cell myeloma, is a B-cell malignancy with a median age at presentation of 60–65 years. MM is the second most frequent B-Cell tumour in Western countries. Generally treated by different regimens of chemotherapy, the outcome for MM patients has not changed dramatically for a long time. In patients who do not show significant improvement to conventional chemotherapy regimens, high-dose therapy with autologous stem cell transplantation (ASCT) is the treatment of choice.1

The current guidelines on management of MM in the United Kingdom advocates performing bone marrow trephine biopsy (BMTB) or a clot biopsy along with the conventional bone marrow aspiration (BMA) at diagnosis. The guidelines suggest that BMTB may be more reliable in assessing the extent of plasma cell (PC) infiltration. The guidelines further suggest that clonality and abnormal phenotype need to be demonstrated in cases with <10% PCs in the marrow by flow cytometry on the marrow aspirate or clonality can be assessed through immunohistochemistry on BMTB.2

An increase in plasma cells can be found in many other pathological conditions. However in MM, the cell clone produces an excess of immunoglobulin heavy and/ or light chains (M proteins). These light chain proteins are designated kappa or lambda. There are other known causes of monoclonal gammopathy, which include:

  •         Heavy Chain Disease
  •         Waldenstrøm macroglobulinemia
  •         Monoclonal Gammopathy of Undetermined Significance (MGUS)
  •         Plasmacytoma.

These must be differentiated from MM for a correct diagnosis.3 & 4

Discussion

In most cases, with significant infiltration, MM is a straightforward diagnosis. However, bone marrow infiltration in MM is highly variable.

Immunohistochemistry (IHC) is extremely important for a diagnosis, especially

  •         to discriminate from MGUS
  •         identify small numbers of tumour cells after therapy
  •         distinguish from other neoplasms in morphologically unusual cases.

The presence of clear cut light chain restriction with a kappa/lambda ratio more than 10:1 (or the reverse) confirms diagnosis of MM.5

IHC with kappa and lambda antibodies is used to identify MM, however these antibodies are notoriously difficult technically, particularly in BMTB specimens following decalcification, which can ruin antigenicity of tissue, producing a weak reaction in plasma cells and a high background staining.6, 7

There are also additional difficulties in interpretation due to:

  •         uptake of polytypic immunoglobulin by dead or damaged cells
  •         extracellular immunoglobulin, which obscures patterns of cellular staining

This can make diagnosis with only IHC very difficult.

The introduction of In-Situ Hybridisation (ISH) techniques for kappa and lambda probes on BMTB specimens has solved these problems.

ISH provides direct molecular demonstration of a myeloma clone in terms of light chain mRNA expression producing unequivocal evidence of presence of monotypic plasma cells, which serves as a surrogate marker for presence of clonal plasma cells, even in cases where plasma cell levels are only marginally raised.8, 9 In all situations where a low volume of clonal plasma cells is present in the marrow, there is often an admixture with non-clonal normal plasma cells. Interpretation is greatly influenced by the distribution of plasma cells. In such cases, on CD138 immunostain, neoplastic plasma cells are seen are seen as microclusters in addition to singly scattered normal plasma cells.10 However, these features need to be confirmed by appropriate light chain restriction studies. With highly reliable and informative techniques such as ISH, one sees microclusters of monotypic plasma cells in the background of singly scattered polytypic plasma cells.

For such critical analysis, ISH has the advantage of:

  • being free of problems associated with detection of extracellular target and its uptake by other cells.
  • easier to interpret than IHC
  • equivalent or indolent cases of myeloma (such as smouldering myeloma, light chain only secretion and MGUS) can be fully characterised.

The sensitivity and specificity of ISH technique allows an objective assessment of disease response following therapy.8

Historically, ISH techniques have been time consuming and expensive; needing specialised equipment and clean RNA free glassware that is not always available in a routine IHC laboratory.

Hammersmith Hospital Molecular Diagnostic Laboratory only carried out the ISH technique in batches when absolutely necessary, as it took approximately two days lab time and seconded a biomedical scientist from essential routine work.11

The introduction of automated stainers with ISH capability has now allowed the introduction of ISH techniques routinely, where kappa and lambda IHC staining is inadequate, to make a definitive diagnosis, where previously it may have been ambiguous.

Methodology

Sample Preparation
BMTBs fixed in acetic acid-zinc-formalin (AZF) for 24 hours and washed in distilled water for 30 minutes before decalcification in Gooding and Stewart’s Fluid (formic acid based decalcification solution) for approximately 6 hours.

The BMTBs are then routinely processed overnight and embedded in paraffin wax.11

Sections are cut on a clean dedicated rotary microtome at 1µm and picked up onto poly-L-lysine coated slides.


Controls

In addition to kappa and lambda test sections, two further sections are taken from the BMTB. One section for negative control and one for positive control.

The negative control used is an oligonucleotide probe, which has no homology to human sequences but produced identically to other probes.

The positive control is an oligonucleotide probe, which hybridises with the Poly (A) tail of mRNA, ensuring there is viable mRNA in the test section.


ISH Technique

This technique incorporates Fluorescein conjugated kappa (PB0645) and lambda (PB0669) probes identified by a polymer based detection system with Diamino-benzidine (DAB) carried out on the Bond-max™ automated staining system.

Probes and detection system (supplied by Leica Microsystems) specifically for use with the Bond-max.

Test and control slides are loaded onto the Bond-max as per manufacturer guidelines for staining overnight. (N.B. The standard Bond-max ISH protocol involves a two hour probe hybridisation step). See Table 1 for full protocol.

Step

Temp

Time

Dewax

72 °C

30 min

Alcohol

Room

X3

Bond Wash Solution

Room

5 min

Enzyme pretreatment

37 °C

10 min

Bond Wash Solution

Room

X3

Probe hybridisation

37 °C

12 hrs

Bond Wash Solution

Room

X3

Peroxide block

Room

5 min

Bond Wash Solution

Room

X3

Anti-FITC

Room

15 min

Bond Wash Solution

Room

X3

Post primary

Room

8 min

Bond Wash Solution

Room

X3

Polymer

Room

8 min

Bond Wash Solution

Room

X3

DAB

Room

10 min

Deionized water

Room

X3

Haematoxylin

Room

5 min

Deionized water

Room

X3

Table 1. ISH Staining Protocol

Figure 1. In-Situ Hybridisation staining of multiple myeloma in a bone marrow trephine biopsy.

a = kappa probe X100,
b = kappa probe X200,
c = lambda probe X100,
d = lambda probe X200.

Conclusion

With this technology we have achieved good, clean, crisp, dark staining of plasma cells with very little or no background staining as shown in the following images (Figure 1).

These images are taken from a primary case of MM in a BMTB with a clear lambda restriction, stained with kappa (a & b) and lambda (c & d) probes using Bond-max automation. It can clearly be seen on higher magnification, clusters of plasma calls staining strongly positive for kappa and lambda with a higher than 10:1 ratio.

This standardised automated technique also allows reproducible and auditable staining, which is becoming an important requirement for laboratory accreditation.

In the last eight months, since fully integrating automated ISH technique into our department, we have had 190 BMTB specimens for suspected myeloma or suspected residual myeloma. Out of these, 29 cases could not have been diagnosed without the aid of ISH kappa and lambda probes.

References

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  2. Smith A, Wisloff F, Samson D. UK Myeloma Forum; Nordic Myeloma Study Group; British Committee for Standards in Haematology. Guidelines on the diagnosis and management of multiple myeloma 2005. Br J Haematol 2006; 132 (4): 410–51.
  3. George ED, Sadovsky R. Multiple myeloma: recognition and management. Am Fam Physician 1999; 59 (7): 1885–94.
  4. Thiele J, Arenz B, Klein H, Vierbuchen M, Zankovich R, Fischer R. Differentiation of plasma cell infiltrates in the bone marrow. A clinicopathological study on 80 patients including immunohistochemistry and morphometry. Virchows Arch A Pathol Anat Histopathol 1988; 412 (6): 553–62.
  5. Kremer M, Quintanilla-Martinez L, Nahrig J, von Schilling C, Fend F. Immuno-histochemistry in bone marrow pathology: a useful adjunct for morphological diagnosis. Virchows Arch 2005; 447 (6): 920–37. Epub 2005 Oct 18.
  6. Mullink H, Henzen-Logmans SC, Tadema TM, Mol JJ, Meijer CJ. Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies. J Histochem Cytochem 1985; 33 (11): 1103–9.
  7. Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J Clin Path 1974; 27 (1): 14–20.
  8. Akhtar N, Ruprai A, Pringle JH, Lauder I, Durrant ST. In situ hybridization detection of light chain mRNA in routine bone marrow trephines from patients with suspected myeloma. Br J Haematol 1989; 73 (3): 296–301.
  9. Togel F, Kroger N, Korioth F, Fehse B, Zander AR. Molecular methods for detection and quantification of myeloma cells after bone marrow transplantation: comparison between real-time quantitative and nested PCR. J Hematother Stem Cell Res 2002; 11 (6): 971–6.
  10. Ng AP, Wei A, Bhurani D, Chapple P, Feleppa F, Juneja S. The sensitivity of CD138 immunostaining of bone marrow trephine specimens for quantifying marrow involvement in MGUS and myeloma, including samples with a low percentage of plasma cells. Haematologica 2006; 91 (7): 972–5.
  11. Naresh KN, Lampert I, Hasserjian R, Lykidis D, Elderfield K, Horncastle D, Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol. J Clin Pathol 2006; 59 (9): 903–11.