Genetic Mutational Panel Analyses of Extramedullary Relapses in Multiple Myeloma; No Gain of RAS Mutations

Soft tissue extramedullary (EM) disease relapse in Multiple Myeloma (MM) is considered to be a late and aggressive form of the disease with a very poor prognosis. The molecular mechanisms underlying EM disease are unknown but RAS mutations have been implied. To gain further insight in RAS and other mutations in EM relapse, we retrospectively selected MM patients from the hospital database with a relapse EM biopsy from 2000-2015. EM relapse was defined as having a previous diagnosis of MM and a soft tissue EM relapse, with or without bone marrow (BM) involvement. De study was approved by the Scientific Advisory Board Biobanking of the UMC Utrecht.

In total, 13 EM samples were retrieved and in 11 of them a BM biopsy was also available at diagnosis (BM-d) and/or at relapse (BM-r). DNA was retrieved from the biopsy material and used in a targeted panel of 50 tumor suppressor and oncogenes using next generation sequencing (NGS). NGS was performed on the IonTorrent PGM using AmpliSeq Cancer Hotspot V2 Panel. This panel primarily contains amplicons to detect currently known, actionable, mutations and amplifications in solid tumors in the following genes ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL. In addition, immunohistochemistry (IHC) for p53 protein expression was performed on EM biopsies. The EM biopsies were taken from the lymph node (2), pleura (2), skin (7), orbita (1), palate (1) and pancreas (1). The NGS results are presented in Table 1. In total 9 out of 15 BM biopsies yielded results and 10 out of 14 EM biopsies. In 6 patients paired analysis could be performed on both the BM and the EM relapse (EM-r). The most frequent detected mutations were in NRAS (Q61R/K/H) and KRAS (Q61H/L and G13C). These mutations were detected in 5 patients in their diagnostic BM biopsy and in 6 patients in a relapse biopsy. The RAS mutations were mutual exclusive. In total 9 out 13 patients (69%) had a RAS mutation in the diagnostic BM and/or the EM relapse sample. Frequency of RAS mutations in this cohort is higher than previously reported frequencies of 23-44% in newly diagnosed and relapsed MM patients. This suggests an over-representation of these mutations in MM patients with EM relapse, but also the small cohort size or other diagnostic techniques may explain the difference. Remarkable is the lack of difference in frequency of RAS mutations found at time of diagnosis and at time of EM relapse, contradicting the notion that the mutation is acquired during the disease progression from intramedullary to EM disease. TP53 mutations or frameshifts were found in 3 patients (nr 1,18,19). These patients all showed diffuse and strong nuclear expression of the p53 protein on IHC, also indicative for a TP53 mutation. Patient 9 and 10 had p53 protein overexpression in the EM relapse whereas their BM samples had normal and overexpression of TP53, respectively. This is consistent with the general understanding that TP53 mutations are rarely present at time of diagnosis but are more frequent in advanced disease and EM disease.

In conclusion, we demonstrate the feasibility of performing NGS on formalin and decalcified BM biopsy material of MM patients. Patients with an EM relapse have a high frequency of 69% of RAS mutations, in most of them already present at diagnosis. The frequency of TP53 mutations is less and mostly detected in relapsed samples. No clear mutations were associated with the progression of intramedullary to EM disease.