Molecular Characterisation of TP53 Aberrations in 1,777 Myeloma Trial Patients

INTRODUCTION

Aberrations of TP53 remain the most important adverse genetic prognostic factor in multiple myeloma (MM), even in the context of novel biologic combination therapies. However, there is still no consensus approach on how to assess this molecular marker in daily practice and within clinical trials. Main controversies exist in A) methods used to assess TP53 aberrations, B) definition of the optimal cut-off for calling TP53 deletions, C) whether heterozygous alterations have independent prognostic impact. Here, we report on comprehensive TP53 profiling of 1,777 myeloma patients from the UK NCRI Myeloma XI trial to inform the discussion towards a consensus regarding these questions.

PATIENTS AND METHODS

In the phase III multicenter Myeloma XI trial newly diagnosed patients with MM were randomised between thalidomide (CTD) or lenalidomide (CRD) based induction, a response-dependent bortezomib (CVD) intensification and lenalidomide +/- vorinostat maintenance or no maintenance. Transplant eligible patients received high dose melphalan+ ASCT after induction. The data cut-off for this analysis was July 2016 with a median follow-up of 36 months.

DNA from CD138-purified (>95%) bone marrow MM cells from 1,777 Myeloma XI patients was used for copy-number aberration (CNA) profiling with the MLPA P425 probemix (MRC Holland), which assays 3 representative exons of TP53 (Shah V, et al, Leukemia 2017). 1,178 patient tumors were further analysed with probemix X073, covering all exons of TP53 . Previously published exome sequencing data was available for 422 patients (Walker B, et al, JCO 2015). Survival analyses were performed in R 3.4.1, C-statistics were used for evaluating adequacy of risk prediction.

RESULTS

We systematically tested different clonal fraction cut-off thresholds for calling TP53 deletions and confirmed an optimal cut-off of <0.75 by MLPA, equivalent to a 20% cut-off by iFISH (Boyle EM, et al, GenChromCanc 2015), identifying 9% of patients with a hazard ratio (HR) for OS of 2.4 (CI: 1.9-3.1; P=1.1x10^-12; C-statistics 0.55). Increasing stringency of the cut-off resulted in a loss of sensitivity, e.g. a cut-off of <0.55 by MLPA (equivalent to 60% by iFISH) identified 3.4% of patients with a HR for OS of 2.3 (CI: 1.6-3.5; P=3.3x10^-6; C-statistics 0.52). Accordingly, a less stringent cut-off of <0.85 by MLPA (10% clonal fraction) decreased specificity with a HR for OS of only 1.6 (CI: 1.3-2.0; P=1.29x10^-6; C-statistics of 0.54). Results were consistent for progression free survival (PFS).

We next examined the impact of homozygous and heterozygous alterations of TP53 on outcome. Seven of 1,178 MM tumors (0.6%) carried homozygous deletions of at least one exon of TP53 . Homozygous deletions were associated with very short median OS of 22.4 months and a HR for OS of 4.0 (CI: 1.7-9.7; P=0.002). Heterozygous deletions were detected in 8.1% of cases and were also clearly associated with shorter survival (median OS 33.5 vs. 60.3 months without deletion) and a HR for OS of 2.2 (CI: 1.7-3.0; P=8.4x10^-8) (Figure 1). The association with outcome for homo- and heterozygous deletions was independent of ISS, gain of 1q and adverse translocations by multivariable analysis.Results were consistent in the sub-group of patients receiving intensive therapy.

In the 422 patients assessed for TP53 deletions and mutations, bi-allelic loss (e.g. deletion and mutation) was found in 1.9% (n=8) and mono-allelic loss in 8.3% (n=35) of tumors. Again, both bi- and mono-allelic TP53 loss were associated with inferior survival, with median OS of 12.2 months (HR 5.2; CI: 2.4-11.5; P=2.6x10^-5) for bi-allelic loss vs. 31.0 months for mono-allelic loss (HR 2.1; CI: 1.3-3.3; P=0.002) vs. 60.3 months in the group with normal TP53 status (Figure 1).

CONCLUSION

With this detailed analysis of TP53 aberrations in 1,777 trial patients from a single trial, we confirm the independent prognostic significance of mono-allelic TP53 alterations. Our data establish MLPA with a cut-off at <0.75, equivalent to 20% by iFISH, as a suitable molecular method for TP53 CNA detection that can be readily applied in standard diagnostic laboratories. Harmonisation of TP53 assessment is highly warranted for consistent outcome reporting and to facilitate meta-analyses, inside and outside of clinical trials.

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