Cancer Treatment Today

A recent workshop was conducted to agree on guidelines for use of FISH in myeloma. These recommendations arose from a workshop organised for the European Myeloma Network, held at the Royal Marsden Hospital, London on March 11th 2005. 31 European laboratories were represented at the meeting.

These recommendations are intended to apply only to newly diagnosed cases of myeloma or frank relapse cases. The use of FISH to monitor response to high dose therapy, or to study diseases such as MGUS or primary amyloidosis where only a small proportion of the plasma cells may belong to the abnormal clone is still considered to be a research tool, and different criteria may need to be used.

The purpose of the workshop was to agree rules for FISH in myeloma but consideration was also given to conventional cytogenetic studies. It was agreed that these should not be discouraged but that, especially in a multi-centre setting, full cytogenetic studies were often impracticable due to the poor quality of samples (see below) and the poor ratio of number of man-hours required for the analysis to the number of patients on whom an abnormal result is obtained.

It was felt very strongly that much still needs to be learned about the significance of chromosome abnormalities in myeloma. For this reason, FISH results should not yet be used to make treatment decisions, except in the context of a clinical trial.

1. Material for FISH. All laboratories, particularly those involved in multi-centre studies, confirmed that there are major problems with the quality of the bone marrow aspirates received for FISH studies; these frequently contain drastically fewer plasma cells than the corresponding smear used for morphological assessment. It is difficult for clinicians to accept that a normal FISH result from a patient that had 80% plasma cells on the morphology slide could be meaningless, but that is the reality. Modern haematologists should accept that diagnosis and management depends on a multidisciplinary approach so that it is important to ensure that there is suitable material for all necessary tests, and morphology is not supreme. Clinicians should therefore be encouraged to send part of the first draw of the aspirate for FISH studies, and should certainly be told that the needle must be repositioned for further aspiration, rather than simply continuing to withdraw marrow blood from the initial puncture site.

Even if these measures are put in place, many samples will still have relatively low plasma cell percentages. For this reason it is not acceptable to report FISH results in myeloma without either concentrating the plasma cells or employing some means of plasma cell identification so that only these cells are scored.

2. Timing of samples. It is important that aspirates are processed as soon as possible if FISH is to be acceptable in myeloma. Processing, either by purification or by simultaneous staining of cytoplasmic immunoglobulin with FISH (cIgFISH) is time-consuming. It is therefore strongly recommended that marrow aspirates are not performed on a Friday.

3. To purify or not. The benefits and disadvantages of plasma cell purification versus cIgFISH primarily apply to associated studies: In general the expense of purification is best justified in the context of plasma cell banking. Johannes Drach reported on a comparison of the two methods which showed no major difference in the results obtained and therefore we recommend that each laboratory chooses the method that is most suitable for their circumstances.

4. Choice of purification method. All laboratories at the meeting that had experience of plasma cell purification were using Miltenyi Biotec CD138 magnetic bead separations. One laboratory also had experience of the StemCell Technologies system and reported that there were differences in yield and purity. However, these should not affect the FISH results therefore the choice of purification method can be left to the individual laboratory. Density gradient separation is generally recommended prior to purification, rather than red cell lysis on the grounds of cost; the majority of mature neutrophils will be lost in the former procedure which reduces the quantity of beads necessary for purification. It is stressed that the purified sample MUST be checked for the proportion of plasma cells, as poor initial samples can lead to relatively low proportions in the final cell suspension. Either morphology or immunostaining (flow) can be used to assess the final plasma cell percentage.

5. Slide making for purified plasma cells The purified plasma cell suspension can either be put directly on to slides by cytospin, can be fixed directly in 3:1 methanol:acetic acid and either dropped on to slides or stored as a frozen cell suspension, or can be treated with 0.075M KCl and fixed with methanol acetic acid as for standard cytogenetic preparation. The last method produces bare nuclei which can either be dropped on to slides immediately or stored at -20oC until required. (NB one group had had problems with long term storage of some fixed cell suspensions, so individual laboratories should check the reliability of storage). It was not considered that any of these methods would significantly affect the final result.

6. Simultaneous plasma cell identification + FISH. It is recommended that immunostaining for light chains is used to identify the plasma cells. This gives a much stronger signal than CD138 and is also more likely to identify only the malignant clone if there is contamination with normal plasma cells, although it was stressed that the level of such contamination is extremely low at diagnosis.

7. Slide making for cIgFISH cIgFISH can be used on marrow aspirate smears but only if these are very fresh. It is therefore recommended that wherever possible the cells are subjected to red cell lysis or density gradient centrifugation and the resultant suspension fixed in 3:1 methanol:acetic acid. This fixed suspension can then be dropped directly on to slides or stored at -20oC until required.

8. Cut-off levels for a positive result. Myeloma FISH is known to be particularly difficult1, thought to be due to the additional problems posed by the paraprotein. Ideal control material is difficult to come by. For these reasons the workshop recommends relatively conservative uniform cut-off levels. These are roughly based on the levels found in a number of laboratories using the mean +3SD of 5-10 controls. In practice, there was considerable concern that results just above the actual mean+3SD were artefactual. Thus the following levels are recommended: For dual fusion or break-apart probes 10% For numerical abnormalities or single fusion results with dual fusion probes 20%

Any laboratory setting up myeloma FISH should ensure that their results are compatible with these cut-off levels.

Laboratories with very low mean+3SD for deletions may wish to consider results in the 10 – 20% range to be borderline for their own records but they should not be reported to clinicians as positive.

9. Control probes It is recommended that a control probe be used in all experiments where deletions are expected. This probe should be of the same type as the probe under test; i.e. a centromere probe is not a suitable control for a locus-specific probe. It is not recommended that 13q14 and p53 probes are used to control for each other because of the difficulty of interpreting cases where both are deleted. It is not felt necessary to use control probes for break-apart, dual fusion or trisomy probes as residual non-plasma cells can be used to assess hybridisation efficiency for these.

10. Number of analysts Provided the previous recommendations are followed all or most of cells being scored will be plasma cells and most of the important abnormalities are likely to be present in the majority of these. Thus a single experienced analyst is considered adequate to examine the majority of cases. However, results should always be checked where there is an equivocal signal pattern or where purified plasma cells make up less than 30% of the cells. Smaller labs are recommended to use 2 analysts with a third to check any results with a discrepancy of >5%.

11. Number of cells to score It is recommended that 100 cells be scored wherever possible. In exceptional circumstances an abnormal result in as few as 20 confirmed plasma cells is acceptable if at least 15 are abnormal.

12. Abnormalities to test for It is recommended that all labs should test for deletion 13, t(11;14) and t(4;14) and include p53 deletion wherever possible. Where material is limited it is often possible to re-probe slides to increase the number of results obtained. (In practice, several labs are testing for more than this but these four abnormalities are thought to be most practical in a diagnostic setting). Chromosome 13 results must not be reported as normal in the absence of an indication of the ploidy of the sample, as most near tetraploid karyotypes will have deletion of two copies of 13 and therefore give an apparently normal result. The simplest way to obtain this information is to use the results from the t(4;14) and t(11;14) probes; near tetraploid cases are likely to have at least two copies of each of the 4 and 11 probes, although they may not have four IgH signals. Some laboratories prefer to use an IgH break apart probe to decide whether or not to use the t(11;14) and t(4;14) probes. If this is done, they need to remember also to test all cases that do not have an IgH rearrangement but have two copies of the 13q14 probe for t(4;14) and t(11;14) to establish ploidy status.

13. Probes to use 13q There was no consensus on which locus to use to detect deletion 13, apart from that it should be in band 13q14. Those who had been using a 13q14 and 13q34 probe confirmed published results that ~90% of deletion cases have lost the whole chromosome. Results of array CGH experiments in Ulm show that in the few cases with deletion rather than monosomy 13 there is no consistent minimal region of deletion. Two groups had a comparison of different 13q14 probes, one had <1% discrepancy between RBI and D13S319 in more than 1100 cases and the other had ~1% discrepancy between RBI, D13S319 and D13S25 in over 350 cases. It was therefore agreed that any of these 3 probes is acceptable for testing for 13q deletion. Abbott/Vysis, Qbiogene and Cytocell commercial 13q14 probes are known to be useful in testing for 13q deletions.

IgH translocation probes Many labs had experience of the Abbott/Vysis probes for IgH break-apart and specific translocations. These were all considered acceptable as they cover large areas on the partner chromosomes. We were unable to endorse any other commercial probes due to lack of experience, but any commercial probes giving consistent strong signals are likely to be acceptable. The difference between the Abbott/Vysis dual fusion t(11;14) and dual fusion TX t(11;14) probes was not considered significant. Laboratories using home grown probes are urged to ensure that they cover a large enough area on the donor chromosom : t(4;14) detection in particular is prone to underestimation if the area on 4 encompasses only FGFR3 and not MMSET due to the frequent loss of the derived chromosome 14. Laboratories may employ a hierarchical approach, attempting t(11;14) FISH first and only performing t(4;14) FISH if that is negative (but see requirement for ploidy estimation for “normal” 13q results above).

Abnormal results for the t(11;14) and t(4;14) should state the number of fusion signals seen.

It should be noted that the presence of a different IgH translocation cannot be reliably extrapolated from the CCND1/IgH or FGFR3/IgH results as loss of one or other derived chromosome is common. This would result in an apparently normal pattern of 2 IgH signals, despite a translocation being present. The presence of 3 IgH signals but no fusion with the t(11;14) and t(4;14) probes probably indicates an alternative IgH translocation rather than trisomy 14. This may sometimes be inferred from different sizes of the IgH signals, but no lab had collected data on this.

p53 The majority of labs are using the Abbott/Vysis p53 probe. We had insufficient evidence to know whether any other commercial or home-grown probe would give different results.

Other abnormalities Some labs are testing for other things as well, the most common of which are t(6;14)(p21;q32), t(14;16) and t(14;20). The use of the Abbott/Vysis 5, 9, 15 probe set is encouraged to see whether hyperdiploidy assessed from these probes in combination with the other recommended abnormalities is good enough to be used as a prognostic marker.

14. Reporting results There was strong feeling that it is important to know the proportion of plasma cells with the abnormality (particularly for deletion 13). However, there is insufficient evidence yet for what the level should be set to determine prognostic significance. It is therefore recommended that for the present reported results state the percentage of plasma cells with the abnormality.

The workshop did not endorse the use of ISCN 1995 for reporting results as it was felt that clinicians find this confusing. If laboratories are required to use this for internal reasons they must ensure that there is a very simple clear interpretation given.

Thus results reported to the clinician should be expressed as clearly as possible and must state the percentage of plasma cells involved and what method was used for plasma cell identification. A hypothetical sample report follows: . 13q14 deleted (90%) 4p16 t(4;14) single fusion (96%) 11q13 normal 17p13 (p53) normal

FISH on purified plasma cells identified a t(4;14) in 96% of plasma cells, but only one fusion was seen using a dual fusion probe, suggesting loss of one half of the translocation. From published results this is most likely to be loss of the derived 14 carrying the IgH/FGFR3 fusion. There was also a deletion of chromosome 13 seen in 90% of the plasma cells, but no abnormality of CCND1 or p53 was detected. Although the t(4;14) and deletion 13 have been associated with a poor prognosis in MM the data is not yet good enough to use these results for treatment decisions except in the context of a clinical trial.

In conclusion, FISH for myeloma is still an investigational modality. A number of guidelines and reviews mention FISH but do not definitively address its place in myeloma diagnosis and management.

Fonseca R, Barlogie B, Bataille R, Bastard C, Bergsagel PL, Chesi M, Davies FE, Drach J, Greipp PR, Kirsch IR, Kuehl WM, Hernandez JM, Minvielle S, Pilarski LM, Shaughnessy JD, Jr., Stewart AK, Avet-Loiseau H. Genetics and cytogenetics of multiple myeloma: a workshop report. Cancer Res. 2004;64:1546-1558.

NCCN.ORG, Multiple Myeloma

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 Feb;132(4):410-51. [292 references]