[Pathogenesis of multiple myeloma]. / Pathogenese des Multiplen Myeloms.

BACKGROUND: Multiple myeloma (MM) is a hematologic malignancy characterized by monoclonal plasma cells infiltrating the bone marrow thereby causing anemia and lytic bone lesions. Despite significant improvement in overall survival, most MM patients inevitably, yet unpredictably, develop refractory disease and MM remains largely incurable. OBJECTIVE: This article describes the stages of progression and presents current insights into the pathogenesis of MM. MATERIAL AND METHODS: Discussion of basic conceptional works and most recent scientific publications. RESULTS: Genetic predisposition, inflammation and abnormal immune response are responsible for the pathogenesis of MM. The initiating genomic event occurs during B cell maturation and clonal plasma cells are disseminated within the bone marrow (BM). This early stage is called monoclonal gammopathy of undetermined significance. The next stage of asymptomatic myeloma, shows a BM infiltration >10%. End organ damage defines symptomatic MM requiring treatment. According to most recent studies MM is characterized by spatial clonal heterogeneity, with aggressive clones frequently being restricted to focal lesions and therefore not being detectable at the iliac crest. Aggressive clones often present with complete inactivation of tumor suppressor genes, such as TP53 and are selected during treatment, which explains the difficulties in treating relapsed MM. CONCLUSION: The tumor biology determines the progression of MM and underlies the heterogeneous response to treatment, which can be observed despite intensive treatment.

Correction: Role of growth arrest-specific gene 6-mer axis in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Meeting report of the 7th Heidelberg Myeloma Workshop: today and tomorrow.

PURPOSE: The 7th Heidelberg Myeloma Workshop was held on April 5th and 6th, 2019 at the University Hospital Heidelberg. METHODS AND RESULTS: Main topics of the meeting were (1) diagnostics and prognostic factors, (2) role of immunotherapy in multiple myeloma (MM), (3) current therapy of MM, (4) biology and genomics of MM as well as (5) novel treatment concepts. A debate on the status of minimal residual disease (MRD) driven therapy was held. CONCLUSION: Diagnostics and treatment of newly diagnosed and relapsed MM are continuously evolving. While advances in the field of (single cell) genetic analysis now allow for characterization of the disease at an unprecedented resolution, immunotherapeutic approaches and MRD testing are at the forefront of the current clinical trial landscape.

Combination of flow cytometry and functional imaging for monitoring of residual disease in myeloma.

The iliac crest is the sampling site for minimal residual disease (MRD) monitoring in multiple myeloma (MM). However, the disease distribution is often heterogeneous, and imaging can be used to complement MRD detection at a single site. We have investigated patients in complete remission (CR) during first-line or salvage therapy for whom MRD flow cytometry and the two imaging modalities positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) were performed at the onset of CR. Residual focal lesions (FLs), detectable in 24% of first-line patients, were associated with short progression-free survival (PFS), with DW-MRI detecting disease in more patients. In some patients, FLs were only PET positive, indicating that the two approaches are complementary. Combining MRD and imaging improved prediction of outcome, with double-negative and double-positive features defining groups with excellent and dismal PFS, respectively. FLs were a rare event (12%) in first-line MRD-negative CR patients. In contrast, patients achieving an MRD-negative CR during salvage therapy frequently had FLs (50%). Multi-region sequencing and imaging in an MRD-negative patient showed persistence of spatially separated clones. In conclusion, we show that DW-MRI is a promising tool for monitoring residual disease that complements PET and should be combined with MRD.

Carfilzomib resistance due to ABCB1/MDR1 overexpression is overcome by nelfinavir and lopinavir in multiple myeloma.

Proteasome inhibitor (PI) carfilzomib (CFZ) has activity superior to bortezomib (BTZ) and is increasingly incorporated in multiple myeloma (MM) frontline therapy and relapsed settings. Most MM patients ultimately experience PI-refractory disease, an unmet medical need with poorly understood biology and dismal outcome. Pharmacologic targeting of ABCB1 improved patient outcomes, including MM, but suffered from adverse drug effects and insufficient plasma concentrations. Proteomics analysis identified ABCB1 overexpression as the most significant change in CFZ-resistant MM cells. We addressed the functional role of ABCB1 overexpression in MM and observed significantly upregulated ABCB1 in peripheral blood malignant plasma cells (PCs) vs untreated patients' bone marrow PC. ABCB1 overexpression reduces the proteasome-inhibiting activity of CFZ due to drug efflux, in contrast to BTZ. Likewise, the cytotoxicity of established anti-MM drugs was significantly reduced in ABCB1-expressing MM cells. In search for potential drugs targeting ABCB1 in clinical trials, we identified the HIV protease inhibitors nelfinavir (NFV) and lopinavir (LPV) as potent functional modulators of ABCB1-mediated drug export, most likely via modulation of mitochondria permeability transition pore. NFV and LPV restored CFZ activity at therapeutically relevant drug levels and thus represent ready-to-use drugs to be tested in clinical trials to target ABCB1 and to re-sensitize PC to established myeloma drugs, in particular CFZ.

Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of "fitter" clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.In multiple myeloma, malignant cells expand within bone marrow. Here, the authors use multi-region sequencing in patient samples to analyse spatial clonal architecture and heterogeneity, providing novel insight into multiple myeloma progression and evolution.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Genome-wide association study of immunoglobulin light chain amyloidosis in three patient cohorts: comparison with myeloma.

Immunoglobulin light chain (AL) amyloidosis is characterized by tissue deposition of amyloid fibers derived from immunoglobulin light chain. AL amyloidosis and multiple myeloma (MM) originate from monoclonal gammopathy of undetermined significance. We wanted to characterize germline susceptibility to AL amyloidosis using a genome-wide association study (GWAS) on 1229 AL amyloidosis patients from Germany, UK and Italy, and 7526 healthy local controls. For comparison with MM, recent GWAS data on 3790 cases were used. For AL amyloidosis, single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at P<10-5 with homogeneity of results from the 3 sample sets; some of these were previously documented to influence MM risk, including the SNP at the IRF4 binding site. In AL amyloidosis, rs9344 at the splice site of cyclin D1, promoting translocation (11;14), reached the highest significance, P=7.80 × 10-11; the SNP was only marginally significant in MM. SNP rs79419269 close to gene SMARCD3 involved in chromatin remodeling was also significant (P=5.2 × 10-8). These data provide evidence for common genetic susceptibility to AL amyloidosis and MM. Cyclin D1 is a more prominent driver in AL amyloidosis than in MM, but the links to aggregation of light chains need to be demonstrated.

Clinical value of molecular subtyping multiple myeloma using gene expression profiling.

Using a data set of 1217 patients with multiple myeloma enrolled in Total Therapies, we have examined the impact of novel therapies on molecular and risk subgroups and the clinical value of molecular classification. Bortezomib significantly improved the progression-free survival (PFS) and overall survival (OS) of the MMSET (MS) subgroup. Thalidomide and bortezomib positively impacted the PFS of low-risk (LoR) cases defined by the GEP70 signature, whereas high-risk (HiR) cases showed no significant changes in outcome. We show that molecular classification is important if response rates are to be used to predict outcomes. The t(11;14)-containing CD-1 and CD-2 subgroups showed clear differences in time to response and cumulative response rates but similar PFS and OS. Furthermore, complete remission was not significantly associated with the outcome of the MAF/MAFB (MF) subgroup or HiR cases. HiR cases were enriched in the MF, MS and proliferation subgroups, but the poor outcome of these groups was not linked to subgroup-specific characteristics such as MAF overexpression per se. It is especially important to define risk status if HiR cases are to be managed appropriately because of their aggressive clinical course, high rates of early relapse and the need to maintain therapeutic pressure on the clone.

Genetic factors influencing the risk of multiple myeloma bone disease.

A major complication of multiple myeloma (MM) is the development of osteolytic lesions, fractures and bone pain. To identify genetic variants influencing the development of MM bone disease (MBD), we analyzed MM patients of European ancestry (totaling 3774), which had been radiologically surveyed for MBD. Each patient had been genotyped for ~6 00 000 single-nucleotide polymorphisms with genotypes for six million common variants imputed using 1000 Genomes Project and UK10K as reference. We identified a locus at 8q24.12 for MBD (rs4407910, OPG/TNFRSF11B, odds ratio=1.38, P=4.09 × 10(-9)) and a promising association at 19q13.43 (rs74676832, odds ratio=1.97, P=9.33 × 10(-7)). Our findings demonstrate that germline variation influences MBD and highlights the importance of RANK/RANKL/OPG pathway in MBD development. These findings will contribute to the development of future strategies for prevention of MBD in the early precancerous phases of MM.

Role of Growth arrest-specific gene 6-Mer axis in multiple myeloma.

Multiple myeloma is a mostly incurable malignancy characterized by the expansion of a malignant plasma cell (PC) clone in the human bone marrow (BM). Myeloma cells closely interact with the BM stroma, which secretes soluble factors that foster myeloma progression and therapy resistance. Growth arrest-specific gene 6 (Gas6) is produced by BM-derived stroma cells and can promote malignancy. However, the role of Gas6 and its receptors Axl, Tyro3 and Mer (TAM receptors) in myeloma is unknown. We therefore investigated their expression in myeloma cell lines and in the BM of myeloma patients and healthy donors. Gas6 showed increased expression in sorted BMPCs of myeloma patients compared with healthy controls. The fraction of Mer(+) BMPCs was increased in myeloma patients in comparison with healthy controls whereas Axl and Tyro3 were not expressed by BMPCs in the majority of patients. Downregulation of Gas6 and Mer inhibited the proliferation of different myeloma cell lines, whereas knocking down Axl or Tyro3 had no effect. Inhibition of the Gas6 receptor Mer or therapeutic targeting of Gas6 by warfarin reduced myeloma burden and improved survival in a systemic model of myeloma. Thus, the Gas6-Mer axis represents a novel candidate for therapeutic intervention in this incurable malignancy.

[In vitro evaluation of MR thermometry in the implementation of laser-induced thermotherapy]. / In-vitro-Evaluierung der MR-Thermometrie zum Einsatz der laserinduzierten Thermotherapie.

PURPOSE: To optimize the MR sequences parameter for monitoring hyperthermic effects in the tissue during laser induced thermotherapy (LITT). MATERIAL AND METHODS: Experimental studies were performed for the evaluation of MR-thermometry using a contrast-agent-water solution and a pig-liver. A T1-weighted TurboFLASH sequence and a FLASH-2D sequence were used. The TurboFLASH sequence was used with various T1 settings (between 100 and 1250 ms). MR findings were correlated with temperature measurements using a fluoride optical temperature measuring system in a distance of 1, 2, and 5 cm from the laser applicator. RESULTS: Using the contrast-agent-water solution demonstrated the temperature sensitivity of both sequences. In vitro evaluations using pig liver demonstrated a near linear increase of signal versus increasing tissue temperatures in a distance of 1 cm to the tip of the laser applicator. Optimal visualization of the temperature effects was obtained using a T1 between 100 ms and 400 ms. Using the FLASH-2D sequence a signal loss was documented at a TR of 110 ms. CONCLUSION: MR-thermometry using sequentially TurboFLASH and FLASH-2D sequences allowed a non-invasive monitoring of the laser induced temperature changes.

[Verification of MR thermometry by means of an in vivo intralesional, fluoroptic temperature measurement for laser-induced thermotherapy ov liver metastases]. / Verifizierung der MR-Thermometrie mittels in vivo intraläsionaler, fluoroptischer Temperaturmessung für die laserinduzierte Thermotherapie von Lebermetastasen.

PURPOSE: To evaluate the correlation of MR-measured changes of signal intensity and invasive fluoroptic temperature measurements during MR-guided LITT of liver metastases. MATERIALS AND METHODS: In 15 patients with proven liver metastases of colorectal carcinoma, MR-guided LITT was performed with a percutaneous approach in a multiapplicator technique. Two temperature sensitive T1-weighted sequences (FLASH-2D- and TurboFLASH-sequences) were used to map the spatial and temporal distribution of Nd:YAG laser effects. Parallel fluoroptic temperature measurements were carried out by means of an inserted probe in a distance of 5-26 mm (mean: 14 mm) from the laser applicator. RESULTS: In both sequences a gradually increasing signal loss could be documented during laser application which proved to be reversible after cessation of energy deposition. the percentage of decrease in signal intensity correlated directly with the measured increase of temperature. Invasive fluoroptical evaluation of temperature distribution after 10 min exposure time showed at 5 mm distance from the applicator an increase of temperature of 35 degrees C, in 10 mm distance a mean increase of 9 degrees C +/- 1.7, in 15 mm a mean increase of 7 degrees C +/- 1.6 and in 20 mm a mean increase of 3 degrees C +/- 0.5. This is evidence of thermal tissue damage up to 3 cm in diameter with laser monoapplication. The qualitative evaluation revealed a reproducible correlation of the extent of signal loss around the applicator and the finally induced degree of necrosis. CONCLUSION: Invasive fluoroptical temperature measurements prove the diagnostic reliability of MR thermometry for the online monitoring of LITT of liver metastases.

[Early clinical experiences with MR-guided laser-induced thermotherapy (LITT) of liver metastases in preoperative care]. / Erste klinische Erfahrungen zur MR-gesteuerten laserinduzierten Thermotherapie (LITT) von Lebermetastasen im präoperativen Einsatz.

PURPOSE: To evaluate the LITT-induced changes with the aid of MRT and correlate these with histopathological findings. MATERIAL AND METHODS: Five patients with solitary colorectal liver metastases were treated by means of MR-guided LITT before liver resection. Application time and energy of the Nd:YAG laser (1064 nm) was 10-20 minutes and 4.5-8.8 W. MRT monitoring during the LITT was carried out with temperature-sensitive T1 weighted sequences (FLASH-2-D, turbo FLASH). The extent of the induced necrosis as seen on MR was compared with the unfixed specimen and with the histopathological findings. RESULTS: The extent of necrosis visible by MRT correlated with the histopathological findings with an accuracy of 95.3% +/- 4.2%. Following single treatments (three cases) the metastases suffered a reduction of 24%-55% of their original volume. In two patients a second application produced laser-induced necrosis of 78% and 98% of volume. In these two patients a temperature sound was used for measuring regional heating and showed an exact correlation with MR thermometry. CONCLUSION: The results of pre-operative MR-guided LITT indicates the potential of this form of treatment for obtaining reproducible tumor necrosis of liver metastases.

Inherited genetic susceptibility to multiple myeloma.

Although the familial clustering of multiple myeloma (MM) supports the role of inherited susceptibility, only recently has direct evidence for genetic predisposition been demonstrated. A meta-analysis of two genome-wide association (GWA) studies has identified single-nucleotide polymorphisms (SNPs) localising to a number of genomic regions that are robustly associated with MM risk. In this review, we provide an overview of the evidence supporting a genetic contribution to the predisposition to MM and MGUS (monoclonal gammopathy of unknown significance), and the insight this gives into the biological basis of disease aetiology. We also highlight the promise of future approaches to identify further specific risk factors and their potential clinical utility.

Cost-effective multiplexing before capture allows screening of 25 000 clinically relevant SNPs in childhood acute lymphoblastic leukemia.

Genetic variants, including single-nucleotide polymorphisms (SNPs), are key determiners of interindividual differences in treatment efficacy and toxicity in childhood acute lymphoblastic leukemia (ALL). Although up to 13 chemotherapeutic agents are used in the treatment of this cancer, it remains a model disease for exploring the impact of genetic variation due to well-characterized cytogenetics, drug response pathways and precise monitoring of minimal residual disease. Here, we have selected clinically relevant genes and SNPs through literature screening, and on the basis of associations with key pathways, protein-protein interactions or downstream partners that have a role in drug disposition and treatment efficacy in childhood ALL. This allows exploration of pathways, where one of several genetic variants may lead to similar clinical phenotypes through related molecular mechanisms. We have designed a cost-effective, high-throughput capture assay of ∼25,000 clinically relevant SNPs, and demonstrated that multiple samples can be tagged and pooled before genome capture in targeted enrichment with a sufficient sequencing depth for genotyping. This multiplexed, targeted sequencing method allows exploration of the impact of pharmacogenetics on efficacy and toxicity in childhood ALL treatment, which will be of importance for personalized chemotherapy.