Marc S. Raab

Baseline characteristics, chromosomal alterations, and treatment affecting prognosis of deletion 17p in newly diagnosed myeloma.

Deletion 17p13, del(17p), is associated with poor outcome in myeloma but some patients show long‐term survival. With the current study we intended to identify factors impacting outcome of such high risk patients. We analyzed 110 newly diagnosed, symptomatic patients with del(17p) detected by fluorescence in situ hybridization (FISH) in CD138‐purified myeloma cells to identify prognostic factors for survival. Age >65 years, ISS III, and elevated LDH negatively impacted survival. Patients with subclonal (10–60% of plasma cells) del(17p) had longer progression‐free survival (PFS) than patients with del(17p) in >60% of plasma cells (26 vs. 19 months, P = 0.03). Additional gain of 1q21 was associated with shorter PFS (17 vs. 25 months, P = 0.01). Hyperdiploidy did not ameliorate impact of del(17p), but gain 19q13 predicted longer PFS (30 vs. 18 months, P = 0.01) and overall survival (50 vs. 29 months, P = 0.01). Multivariate analysis in transplant eligible patients (≤65 years) revealed better survival for patients treated with upfront autologous transplantation (hazard ratio, [95% confidence interval]: 0.15 [0.04, 0.58], P = 0.006). Application of maintenance therapy was associated with better survival in transplant‐eligible patients (0.30 [0.09, 0.99], P = 0.05). We demonstrate heterogeneous outcome of patients with del(17p) according to baseline characteristics and treatment. 19q13 should be included in routine FISH panel, since gains were associated with better survival. Am. J. Hematol. 91:E473–E477, 2016.

Longitudinal fluorescence in situ hybridization reveals cytogenetic evolution in myeloma relapsing after autologous transplantation

To investigate cytogenetic evolution after upfront autologous stem cell transplantation for newly diagnosed myeloma we retrospectively analyzed fluorescence in situ hybridization results of 128 patients with paired bone marrow samples from the time of primary diagnosis and at relapse. High-risk cytogenetic abnormalities (deletion 17p and/or gain 1q21) occurred more frequently after relapse (odds ratio: 6.33; 95% confidence interval: 1.86–33.42; P<0.001). No significant changes were observed for defined IGH translocations [t(4;14); t(11;14); t(14;16)] or hyperdiploid karyotypes between primary diagnosis and relapse. IGH translocations with unknown partners occurred more frequently at relapse. New deletion 17p and/or gain 1q21 were associated with cytogenetic heterogeneity, since some de novo lesions with different copy numbers were present only in subclones. No distinct baseline characteristics were associated with the occurrence of new high-risk cytogenetic abnormalities after progression. Patients who relapsed after novel agent-based induction therapy had an increased risk of developing high-risk aberrations (odds ratio 10.82; 95% confidence interval: 1.65–127.66; P=0.03) compared to those who were treated with conventional chemotherapy. Survival analysis revealed dismal outcomes regardless of whether high-risk aberrations were present at baseline (hazard ratio, 3.53; 95% confidence interval: 1.53–8.14; P=0.003) or developed at relapse only (hazard ratio, 3.06; 95% confidence interval: 1.09–8.59; P=0.03). Our results demonstrate cytogenetic evolution towards high-risk disease after autologous transplantation and underline the importance of repeated genetic testing in relapsed myeloma (EudraCT number of the HD4 trial: 2004-000944-26).

A systematic classification of death causes in multiple myeloma

The introduction of high-dose therapy followed by autologous blood stem cell transplantation (HDT/ ABSCT) and the novel agents led to continuously improving survival in multiple myeloma (MM). However, the majority of MM patients ultimately relapses or progresses, and deceases of disease related conditions such as severe infections, renal failure or toxicity. Nonetheless, the increasing life expectancy of the MM population might translate into an increase in causes of death (COD) unrelated to MM. The aim of this study was to develop and apply a reliable systematic classification for COD in MM, combining the assessment of a specific COD and a causal link between the COD and MM, MM therapy or unrelated conditions, and to assess the impact of known MM prognostic factors on COD. A number of 818 MM patients, who had received an upfront HDT/ABSCT between June 1992 and October 2013 at the University Hospital Heidelberg, Heidelberg, Germany were included in this single-center, retrospective analysis (Supplemental Table 1). Until April 2014, 483 of the eligible patients were deceased. The median overall survival of the cohort was 5.9 years (Supplemental Figure 1). This analysis was approved by the ethics committee of the University of Heidelberg (Number S-337/2009). All patients gave written informed consent. To construct a systematic COD classification, the process of qualitative content analysis was applied using MAXQDA (VERBI GmbH, Berlin, Germany) (Supplemental Figure 2). A preliminary classification was developed based on the results of a PubMed literature search on COD in MM and existing COD classifications. Next, the preliminary COD classification (Supplemental Figure 3) was examined on the collective to evaluate feasibility and practicality. Based on this initial evaluation, a final, systematic COD classification for MM (Fig. 1) and a corresponding rule-based allocation algorithm (Supplemental Figure 4) were built and applied to our cohort. The final COD classification is hierarchically and systematically structured (Fig. 1). A superordinate system of categories determines the causal link between the COD and MM or side effects of MM therapy and distinguishes the following categories: (1) MM-dependent, (2) MMindependent, (3) not attributable to (1)/(2), and (4) unknown (Fig. 1). In addition, MM-dependent COD (1) are subdivided into (1A) MM progression-related, (1B) therapy-related, and (1C) not attributable to (1A)/(1B). The subordinate system defines COD at four levels of different specificity applying the MedDRA terminology: the System Organ Class (SOC) has the lowest and the Preferred Term (PT) the highest specificity (Fig. 1). According to the developed algorithm, COD were allocated to the different categories by consulting the available medical documentation within 90 days before death (Supplemental Figure 4). Examples for each category can be found in the Supplemental Methods. Additionally, a validity system was developed and applied to the cohort to evaluate the reliability of the consulted medical documentation (Supplemental Methods). Statistical analyses were conducted using R version 3.2.218 and survival package (Supplemental Methods).

Cytogenetic abnormalities in monoclonal gammopathy of undetermined significance

Multiple myeloma (MM) is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS), and patients with MGUS have a 1% per year risk of progression into MM [1–3]. Recently, colleagues from the Mayo Clinic in Rochester analyzed for the first time the prognostic significance of cytogenetic abnormalities detected by interphase fluorescence in situ hybridization (iFISH) in MGUS [1]. They showed that most cytogenetic abnormalities occur less frequently in MGUS compared to MM and identified t(4;14) as well as del(17p) as risk factors for progression from MGUS to MM. Authors concluded that cytogenetics provide a powerful tool for risk stratification in MGUS, and underlined that their results need to be validated in an independent cohort of patients. Therefore, we searched our iFISH database for patients, who were diagnosed with MGUS between 2002 and 2017 at the University Hospital Heidelberg, Germany. FISH analysis was performed on CD138-purified plasma cells as described previously using probes for: 1q21, 4p16, 5p15, 5q35, 8p21, 9q34, 11q23, 13q14.3, 15q22, 17p13, and 19q13 [4]. We also investigated immunoglobulin H (IgH) translocations t (11;14), t(4;14), and t(14;16). Hyperdiploidy (HD) was assessed by the score of Wuilleme et al. (gains of at least two of the chromosomes 5, 9, and 15) [5]. Statistical analyses were carried out according to the manuscript by Lakshman et al., with time to progression (TTP) being defined by the period from iFISH testing to progression into smoldering MM (SMM) and MM [3]. The study was approved by the local ethics committee review board (numbers S-337/2009 and S-096/20), and carried out in accordance with the Declaration of Helsinki. We identified 155 patients with MGUS and cytogenetic abnormalities detected by iFISH. Median age was 62 years compared to 64 years in the Mayo Clinic cohort and 51% were male (Mayo Clinic 56%). The most frequently detected cytogenetic abnormality in our and the Mayo Clinic cohort was del(13q) (Heidelberg 23.9% and Mayo Clinic 28.0%). We identified del(17p) in 1.3% (Mayo Clinic 1.8%) and t(4;14) in 5.2% (Mayo Clinic 2.9%). Although we investigated different odd numbered chromosomes than the colleagues from Mayo Clinic, rates of HD were comparable between both groups (Heidelberg 18.4% and Mayo Clinic 22.5%). The most significant discrepancies between the groups were the occurrence of t(11;14) (Heidelberg 5.8% and Mayo Clinic 22.5%) and proportion of patients with normal cytogenetics. While after the exclusion of patients with insufficient plasma cells 26.2% of patients in the Mayo Cohort harbored a normal karyotype, only in 11 patients (7.1%) from our cohort cytogenetic abnormalities were detected in less than 10% of purified cells. This difference might be explained by the fact that in Heidelberg iFISH is performed on purified plasma cells isolated by magnetic-activated cell sorting with anti-CD138 microbeads [4]. Afterwards purity is confirmed by CD38/CD138 phenotypes by using flow cytometry resulting in a high purity of plasma cells even in MGUS or SMM [6]. Table 1 summarizes frequencies of cytogenetic abnormalities detected by iFISH in the Heidelberg and Mayo Clinic cohorts. During the median follow-up of 3.9 years (95% confidence interval [CI] 2.8–4.6 years), ten patients progressed * Maximilian Merz maximilian.merz@med.uni-heidelberg.de

Spectrum and functional validation of PSMB5 mutations in multiple myeloma

Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.