Issue 4: Multiple myeloma cytogenetic testing in the UK and Ireland: guidance vs practice
Myeloma Hub Connect is an educational resource for healthcare professionals. This edition of Myeloma Hub Connect was co-produced by Succinct Medical Communications and Takeda Oncology. Myeloma Hub Connect is edited and published by Succinct Medical Communications Ltd. on behalf of Takeda Oncology.
Dr Guy Pratt, Consultant Haematologist, University Hospitals Birmingham NHS Foundation Trust
Mr Neil Atkey, Director, Laboratory Operations, Diaceutics Ltd., UK*
Clinical outcomes in multiple myeloma (MM) are heterogeneous, with 25% of patients surviving less than 3 years and another 25% of patients surviving more than 10 years.1 While the availability of an increasing number of novel agents continues to improve clinical outcomes in these individuals,2 there remains a need for better biomarkers for prognosis and treatment selection. The search for biomarkers is, however, hindered by the extent and heterogeneity of genetic abnormalities in MM, such as chromosomal abnormalities and gene mutations. Throughout disease progression, the subclonal evolution of the cancer leads to the acquisition of new genetic abnormalities, further complicating the potential use of targeted agents. In MM, the use of fluorescence in situ hybridisation (FISH) allows us to identify several lesions—most notably the IGH translocations t(4;14), t(14;16) and t(14;20) and copy number changes 1q gain and del(17p)—that confer poor outcomes.
Due to the prognostic effects of these lesions, it has been proposed in National Guidelines, namely from the National Institute for Health and Care Excellence (NICE) and the British Society of Haematology (BSH), that these abnormalities define ‘high-risk’ disease and should be specifically sought at diagnosis in all patients.3,4
This recommendation is based on the perceived benefit of having information for patients wishing to know about prognosis but also in recognition that treatment will inevitably become tailored to particular subgroups. Currently, the data around the use of genetic information to tailor treatment are limited. There is an increasing need for more targeted therapy and better biomarkers to ensure the rational and cost-effective use of newer agents and to avoid unnecessary toxicity, particularly in an increasingly heavily-treated population.
*Prior to his position at Diaceutics Ltd. Neil had 25 years experience working in diagnostic genetic laboratories in the UK and New Zealand, predominantly in haematology and solid tumour oncology.
In MM, primary genetic events are chromosome translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14 (~40% of cases) and hyperdiploidy of odd-numbered chromosomes (~50% of cases). Frequently involved chromosomes and genes include 4p16 (FGFR3/MMSET; ~15%), 11q13 (CCND1;20%), 16q23 (MAF; 4%), 6p21 (CCND3; 4%), and 20q11 (MAFB; 1%).5 Due to the low proliferative index of MM cells, conventional karyotyping has a low sensitivity. It only detects cytogenetic abnormalities in 20-30% of cases and is therefore not routinely performed at screening. In contrast, fluorescence in situ hybridisation (FISH) is performed on interphase cells and therefore overcomes this key challenge of karyotyping, providing higher sensitivity and a quantification of cells that harbour specific genomic lesions.5 The IGH translocations t(4;14), t(14;16) and t(14;20) and copy number changes 1q gain and del(17p) confer poor outcomes. In addition, analysis of the Myeloma IX study demonstrated that the number of adverse lesions correlated with outcomes, with >1 adverse FISH lesion being associated with poor survival.6 It should be noted that unlike other lesions, deletion of chromosome 13 is not an independent marker of poor prognosis as its effects are due to its close association with abnormalities such as t(4;14).5
There is international consensus that FISH cytogenetic analysis is required at diagnosis for all MM patients.4 BSH and NICE guidelines recommend performing FISH testing on CD138-selected bone marrow plasma cells to identify the high risk abnormalities t(4;14), t(14;16), 1q gain, del(1p) and del(17p).3,4 These cytogenetic abnormalities can be combined with serum LDH, ß2 microglobulin and albumin values to determine the revised International Staging System score (R-ISS; Table 1) to provide useful prognostic information for patients wishing to discuss their outlook.7 However, the exact definition of this ‘high risk’ group is still in progress and is likely to be refined with more established gene expression profiling (GEP) and a better understanding of genetic abnormalities. Patients relapsing within 18 months of a stem cell transplant also fit into a very poor risk category, irrespective of genetic testing.8,9
While our understanding of the results of genetic testing is increasing, there are currently limited data regarding the targeted treatment of patients with high-risk cytogenetics. There is some evidence to suggest that bortezomib is more efficacious than thalidomide regimens for patients with high-risk cytogenetics10 and that thalidomide may even be detrimental as maintenance for high-risk disease, although evidence has so far only been from retrospective subgroup analyses of large trials.11 It has been shown that approximately 40% of patients with relapsed myeloma with the t(11;14) translocation respond to the BCL-2 inhibitor venetoclax†, compared to minimal responses in patients without t(11;14) translocation.12
Additionally, around 4% of patients have a BRAF mutation that may be targeted by vemurafenib† but current evidence for this approach is mainly based on case reports.13 One issue is that targeting a defect in a single gene such as BRAF, which is likely to be subclonal, will only target the proportion of tumour cells with that defect and not the entire clone. The MUK9b OPTIMUM trial (NCT03188172) focuses on treating high-risk patients (defined using FISH and GEP; see Box) using an intensive schedule of induction, consolidation and maintenance treatment based on novel combinations of drugs. This is the first time in the UK that newly diagnosed MM patients will be entered into a trial based on genetic risk and will help determine the optimal use of high-cost novel agent combinations in a high-risk subgroup of patients.14
In the context of advances in both cytogenetic testing and treatments, here we review the current landscape of FISH testing of MM in the UK and Ireland. The primary aim was to establish the national patterns of myeloma FISH testing, any variations between algorithms used in laboratories, and concordance of these algorithms with both BSH and updated NICE guidelines. We also investigate any issues regarding reimbursement, testing rates, reporting times and other technical issues which may be of importance when considering how testing is approached between different regions. We also discuss the possible changes to the genetic testing methods which may be implemented in the future.
† Vemurafenib and venetoclax are not licensed for the treatment of multiple myeloma.
This report is based on data collected by Diaceutics Ltd in 2016, following a telephone interview with the persons responsible for the MM FISH service in each laboratory. We are indebted to their willingness to share their views but their anonymity is retained in this article.Table 2 contains a full list of the survey questions. Since collecting the data, there may have been local changes to testing policies.
Table 2 contains a full list of the survey questions. Since collecting the data, there may have been local changes to testing policies.
Please note that these data were collected in 2016 and may not reflect current testing practices.
A total of 23 laboratories performing FISH testing on MM samples were identified in the UK. There were no reports of laboratories in the Republic of Ireland undertaking such testing, as it was commonplace for these to be outsourced to the UK.
Which algorithms are currently employed by laboratories?
Overall, concordance with NICE and BSH recommendations to screen with FISH for high-risk MM cytogenetic abnormalities was high. All 23 laboratories reported that they were testing for deletions of 17p using a TP53 probe with 91% (21/23) also testing for abnormalities of the long and short arms of chromosome 1.
Sequential testing for IGH gene rearrangements
Some gene fusions such as IGH-MAFB [t(14;20)] and IGH-CCND3 [t(6;14)] are observed infrequently in MM while others such as IGH-CCND1 [t(11;14)], IGH-FGFR3 [t(4;14)] and IGH-MAF [t(14;16)] are more common. If these common fusions are identified first, the need for testing for the rarer fusions is negated. As such, in order to save costs and analytical time, many laboratories use sequential testing, despite the negative impact on turnaround time (TAT).
The results of our survey showed that the use of a sequential testing algorithm for IGH rearrangements was employed by 17 laboratories (13 different algorithms in total) which consisted of a 2 or 3-step approach (Figure 1). The first step (in addition to testing for 17p deletions and 1p/q abnormalities) was to screen for an IGH rearrangement using a break-apart probe. A break-apart probe is a two-colour FISH probe with a green and red fluorescent tag flanking either side of the IGH gene; when viewed through a microscope these two signals appear overlaid. The presence of an IGH rearrangement causes the separation of the two colours, resulting in clearly distinguishable red and green signals (see Figure 2). This type of probe can be used as a screen but will not identify the gene with whichIGH has rearranged.
If an IGH rearrangement was noted, the second step in the algorithm was to employ gene-specific dual colour, dual-fusion probes with IGH to identify with which gene IGH had rearranged. A dual fusion probe is also a two-colour FISH probe, but has a green fluorescent tag labelling the IGH gene and an orange tag labelling the putative partner gene (e.g. FGFR3); this is observed as two green and two orange signals. When fusion of the two genes occurs, each gene (and the labels) are sheared in half and juxtaposed with the other; this is observed as a pair of adjacent green and orange signals in addition to single green and orange signals still present on the non-rearranged genes (Figure 3). Where no IGH rearrangement is observed, no further testing is required and a report can be issued. The extent to which laboratories went to identify the partner gene varied, but typically all attempted to identify the FGFR3 and MAF rearrangements, which are associated with poor prognosis. A number of laboratories (12/23) also tested for CCND1-IGH rearrangements. A few laboratories (3/23) used a 3-step process to test for the more common gene fusions first (CCND1, FGFR3 and MAF) followed by the rarer CCND3 and MAFB rearrangements (Figure 1). A small number of laboratories (4/23) used a sequential method with either one or both of the FGFR3-IGH and IGH-MAF dual-fusion probes as their front-line test and not an IGH break apart probe. Where a signal pattern was suggestive of a different gene fusion, IGH-MAFB rearrangement was subsequently tested for.
Parallel testing with break-apart and dual-fusion probes
A total of 6/23 laboratories reported that they did not apply a sequential approach for the identification of IGH rearrangements but instead used dual-colour, dual-fusion probes front-line (Figure 1). Of these 6 laboratories, 1 tested for the standard-risk CCND1 rearrangement and 2 for the less common MAFB partner gene. All of these laboratories tested for MAF and FGFR3 rearrangements.
Testing for other cytogenetic abnormalities
Irrespective of sequence of testing, there were just 5/23 laboratories who tested for deletions of the long-arm of chromosome 13. Of these, 3 laboratories reported that they actively tested for hyperdiploidy using probes for chromosomes 9, 11 and 15. These tended to be the larger laboratories linked to academic institutes. 13q deletions as discussed above, are shown to have a close association with the t(4;14) and consequently considered an unnecessary test by laboratories, where budgets are tight, similarly the active search for hyperdiploidy is largely the domain of large laboratories linked to academic institutions with a research interest in MM. A number of laboratories used a TP53 probe containing ATM (on 11q). Additional copies of ATM may be an indication of hyperdiploidy; indeed, some laboratories reported this observation.
A total of 3 laboratories offered testing for IGH-CCND3 fusions and 2 laboratories tested for MYC rearrangements. Only 3 laboratories offered testing for the IGH-CCND3 translocation. Only 2 laboratories currently offer testing for MYC translocations in their MM FISH panel.
CD138 enrichment and failure rates
The NICE guidelines for MM diagnosis and management recommend that FISH is performed on CD138+ cells.3 CD138 is a cell surface marker and the hallmark of plasma cells with >95% of patients with MM expressing CD138 on their plasma cells.15 Bearing in mind that one of the diagnostic criteria for MM is ≥10% plasma cells16 the accuracy of a FISH result is greatly increased by only analysing these plasma cells and not all nucleated cells in a sample. All laboratories used a method to enrich CD138+ cells. All enrichment methods were of the magnetic cell separation type, with 5/23 laboratories using an automated system. The failure rate of FISH was low (<10%) across 19/23 laboratories (Figure 4).
The TAT for FISH reporting is shown in Figure 5. A total of 9/23 laboratories achieved a reporting time of ≤14 days and 22/23 had a TAT of <21 days. There is no current clinical need for a TAT greater than 28 days. The TAT is greatly increased by the use of sequential testing to identify an IGH fusion, where parallel testing is employed a faster TAT is seen. Should there become a clinical requirement for a more rapid TAT, the use of parallel testing may have to be considered but this will, of course, not be without an increased financial burden.
Reimbursement of FISH testing
The source of reimbursement for FISH testing was found to vary between centres (Figure 6). Haematology budgets were the most common source of funding for MM FISH testing, while laboratory, local commissioning budgets, and the National Services Division Scotland provided lower but similar levels of funding. We see from these figures that 39% of laboratories claim the cost of the test direct from the referring haematologists and a further 17% have to absorb the cost of testing into their annual budget.
FISH testing rates
Both NICE and BSH guidelines recommend performing cytogenetic testing in all newly diagnosed MM patients.3,4 To determine compliance with this recommendation, we have examined the FISH testing rate per lab according to their catchment area size and expected number of new MM diagnoses per year based on published CRUK MM incidence statistics (~170 cases per 1 million persons).1 Overall, testing rates were low and varied according to the local reimbursement mechanism. Testing rates ranged from only 9% up to 144% of the minimum rate expected based on guidelines recommendations, with 18/23 labs testing <100 samples per 1 million catchment. These results suggest that many MM patients are not receiving baseline cytogenetic testing (Figure 7). Three laboratories did have testing rates in excess of their expected population. These were mainly large academic units with a specific interest in MM that test follow-up patients, patients on trials, and occasionally patients with monoclonal gammopathy of unknown significance (MGUS).
To our knowledge, this is the first survey to perform a detailed assessment of the MM cytogenetic testing landscape of laboratories in the UK and Ireland. We have also investigated issues around implementation of NICE and BSH guideline recommendations, reimbursement, testing rates, reporting times and technical issues of importance.
BSH and NICE guidelines recommend to test MM patients for t(4;14), t(14;16), 1q gain, del(1p) and del(17p).3,4 Although laboratories used a wide variety of sequential and parallel testing methodologies, we found that 100% (23/23) of laboratories routinely screened for del(17p), t(4;14) and t(14;16) and 91% (21/23) test for chromosome 1 abnormalities. This survey also revealed that only 3 laboratories offered IGH-CCND3 testing, largely because of the low incidence of this translocation in MM, and because it is not cost effective to stock the probe. Additionally, this translocation is associated with standard risk disease and is therefore not in the NICE guidance.3 The incidence of MYC translocations is reported to be high in MM17,18 and the presence of a rearrangement is shown to be associated with a poor prognosis. While only 2 laboratories offered MYC translocation testing, there is likely to be an increased demand for it in the future, if it can be used as a therapeutic target.
An interesting finding was that all laboratories used a method to enrich CD138+ cells. All enrichment methods were of the magnetic cell separation type, with 5/23 laboratories using an automated system. As this is the first survey of its kind there is no benchmark set against the previous uptake of CD138+ enrichment but the feeling was that many of the smaller laboratories had started to enrich in the last few years, some with incentives from the pharmaceutical industry. Whilst the use of enrichment is undoubtedly beneficial and indeed recommended by NICE, it does have implications on TAT as it is more cost effective to batch the use of the separation columns than to use a column for a single sample. Some laboratories reported that once cells had been enriched they were stored in fixative for 5 days prior to processing. This is reported to result in much cleaner preparations which are easier to analyse19 but does of course limit the speed in which a result can be issued.
Following CD138+ enrichment, samples are usually assessed for cell numbers and if insufficient numbers are remaining, FISH is not performed. The laboratories assessed in this survey were unable to provide data on the number of samples which were not processed for FISH. This could perhaps be seen as artificially elevating the success rate of the test, and whilst this may be the case, the reality is that unnecessary testing is not performed and the haematologists are given advance notice that a repeat bone marrow biopsy is required for genetic testing.
TATs in laboratories surveyed were high for a number of reasons. Firstly, FISH analysis is labour-intensive and requires highly skilled staff to analyse and interpret the results. NICE recommends that the same sample is used for all diagnostic and prognostic tests, meaning that when a sample arrives in a laboratory the clock starts ticking; once the cells have been enriched, the laboratory awaits instruction to perform FISH based on whether or not the sample has a plasma cell count diagnostic of MM (≥10%). Consequently, FISH testing may not be initiated until 4–5 days after a sample has been taken. The factors involved in making a diagnosis were not explored as part of this study but could include discussion by the multidisciplinary team in addition to local policies regarding automatic triggering of a FISH request.
Another factor that can increase TAT is the use of sequential testing. In larger laboratories with a high-throughput this is unlikely to be a burden, but in laboratories which may struggle to get a single step assay analysed in 14 days the introduction of sequential testing has the potential to put a two-step testing algorithm beyond the reach of a 21 day TAT, which is recommended as the maximum for non-urgent referrals.20 It should be noted that the laboratories performing
a three-step algorithm were high-throughput departments; in these cases the three-step approach did not impinge upon TAT.
There were, unsurprisingly, mixed views on reimbursement from the laboratories that were surveyed. One laboratory representative noted that “Although the NICE guidelines produced in 2016 recommend testing for key prognostic markers by FISH, no funding has been made available to do this in our region and testing levels are very low as a result”. Another was more positive, stating “Testing is paid for by haematologists as it has been incorporated into the haematology budget”. The correlation of the testing rate to the method of reimbursement is likely limited due to the small number of laboratories performing FISH testing.
- This survey showed that genetic testing in the UK is variable with regards to the methodologies employed
- This variability is reflective of a lack of central funding, which is a challenge common to many centres across the UK
- NICE and BSH guidance to test for the high-risk abnormalities del(17p), t(4;14) and t(14;16) are being followed but a significant proportion of patients are not screened for cytogenetic abnormalities at diagnosis
UK/NP/1806/0035ah – July 2018
1. Cancer research UK. http://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/myeloma/survival#heading-Zero
2. Kumar SK, Dispenzieri A, Lacy MQ, et al. Continued improvement in survival in multiple myeloma: changes in early mortality and outcomes in older patients. Leukemia 2014; 28: 1122–1128
3. NICE guideline NG35, 10 Feb 2016. https://www.nice.org.uk/guidance/ng35
4. Pratt G, Jenner M, Owen R, et al. Updates to the guidelines for the diagnosis and management of multiple myeloma. Brit J Haem 2014; 167, 127–146
5. Sonneveld P, Avet-Loiseau H, Lonial S, et al. Treatment of multiple myeloma with high-risk cytogenetics: a consensus of the International Myeloma Working Group. Blood 2016;127(24):2955-62
6. Boyd KD, Ross FM, Chiecchio L et al. A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial. Leukemia 2012; 26(2):349-55
7. Palumbo A, Avet-Loiseau H, Oliva S, et al. Revised International Staging System for Multiple Myeloma: A Report From International Myeloma Working Group. J Clin Oncol 2015; 33: 2863–2869
8. Jimenez-Zepeda VH, Reece DE, Trudel S, et al. Early relapse after single auto-SCT for multiple myeloma is a major predictor of survival in the era of novel agents. Bone Marrow Transplantation (2015) 50, 204–208
9. Kumar S, Mahmood ST, Lacy MQ, et al. Impact of early relapse after auto-SCT for multiple myeloma. Bone Marrow Transplantation 2008; 42, 413–420
10. Dispenzieri A. Myeloma: management of the newly diagnosed high-risk patient. Hematology Am Soc Hematol Educ Program. 2016: 485–494
11. Morgan GJ, Davies FE, Gregory WM, et al. Long-term follow-up of MRC Myeloma IX trial: Survival outcomes with bisphosphonate and thalidomide treatment. Clin Cancer Res 2013; 19: 6030–6038
12. Kumar S, Vij R, Kaufman JL, et al. Venetoclax Monotherapy for Relapsed/Refractory Multiple Myeloma: Safety and Efficacy Results from a Phase I Study. Blood 2016; 128:488
13. Sharman JP, Chmielecki J, Morosini D, et al. Vemurafenib response in 2 patients with posttransplant refractory BRAF V600E-mutated multiple myeloma. Clin Lymphoma Myeloma Leuk 2014; 14:e161–163
14. MUK Nine b: OPTIMUM Treatment Protocol. https://clinicaltrials.gov/ct2/show/NCT03188172
15. Bayer-Garner IB, Sanderson RD, Dhodapkar MV, et al. Syndecan-1(CD138) immunoreactivity in bone marrow biopsies of multiple myeloma:shed syndecan-1 accumulates in fibrotic regions. Mod Pathol. 2001;14(10):1052-8
16. Moreau P, San Miguel J, Ludwig H, et al. Multiple myeloma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2013 Oct;24 Suppl 6:vi133-7
17. Walker BA, Wardell CP, Brioli A, et al. Translocations at 8q24 juxtapose MYC with genes that harbour superenhancers resulting in overexpression and poor prognosis in myeloma patients. Blood Cancer Journal 2014: 4; e191
18. Talley PJ, Chantry AD, and Buckle CH. Genetics in myeloma: genetic technologies and their application to screening approaches in myeloma. British Medical Bulletin 2015; 113: 15–30
19. CEQAS – https://www.ceqas.org/sites/default/files/SOCNT%20Alert_FISH_on_immunomagnetic_selected_cells%20v3.0%20final.pdf
20. Association for clinical cytogenetics: general best practice guidelines (2007) v1.04. http://www.acgs.uk.com/media/765607/acc_general_bp_mar2007_1.04.pdf