Molecular Long-Term Analysis of the GMMG-HD4 Trial in Multiple Myeloma-Patterns of Association of Chromosomal Aberrations with Response and Proliferation Determining Survival in Selecting Treatments in View of Limited Resources in Low- and Middle-Income Countries.

Based on the lack of differences in progression-free and overall survival after a median follow-up of 93 months in our HOVON-65/GMMG-HD4 trial (German part; n = 395) randomizing VAD induction (vincristin/adriamycin/dexamthasone)/tandem-transplantation/thalidomide-maintenance vs. PAD induction (bortezomib/adriamycin/dexamethasone)/tandem transplantation/bortezomib maintenance, we discern how chromosomal aberrations determine long-term prognosis by different patterns of association with proliferation and treatment-dependent response, whether responses achieved by different regimens are equal regarding prognosis, and whether subpopulations of patients could be defined as treatable without upfront "novel agents" in cases of limited resources, e.g., in low- or middle-income countries. Serum parameters and risk factors were assessed in 395 patients. CD138-purified plasma cells were subjected to fluorescence in situ hybridization (n = 354) and gene expression profiling (n = 204). We found chromosomal aberrations to be associated in four patterns with survival, proliferation, and response: deletion (del) del17p13, del8p21, del13q14, (gain) 1q21+, and translocation t(4;14) (all adverse) associate with higher proliferation. Of these, del17p is associated with an adverse response (pattern 1), and 1q21+, t(4;14), and del13q14 with a treatment-dependent better response (pattern 2). Hyperdiploidy associates with lower proliferation without impacting response or survival (pattern 3). Translocation t(11;14) has no association with survival but a treatment-dependent adverse response (pattern 4). Significantly fewer patients reach a near-complete response or better with "conventional" (VAD) vs. bortezomib-based treatment after induction or high-dose melphalan. These patients, however, show significantly better median progression-free and overall survival. Molecularly, patients responding to the two regimens differ in gene expression, indicating distinct biological properties of the responding myeloma cells. Patients with normal renal function (89.4%), low cytogenetic risk (72.5%), or low proliferation rate (37.9%) neither benefit in progression-free nor overall survival from bortezomib-based upfront treatment. We conclude that response level, the treatment by which it is achieved, and molecular background determine long-term prognosis. Chromosomal aberrations are associated in four patterns with proliferation and treatment-dependent responses. Associations with faster and deeper responses can be deceptive in the case of prognostically adverse aberrations 1q21+ and t(4;14). Far from advocating a return to "outdated" treatments, if resources do not permit state-of-the-art-treatment, normal renal function and/or molecular profiling identifies patient subpopulations doing well without upfront "novel agents".

Daratumumab for systemic AL amyloidosis: prognostic factors and adverse outcome with nephrotic-range albuminuria.

Daratumumab has shown promising first results in systemic amyloid light-chain (AL) amyloidosis. We analyzed a consecutive series of 168 patients with advanced AL receiving either daratumumab/dexamethasone (DD, n = 106) or daratumumab/bortezomib/dexamethasone (DVD, n = 62). DD achieved a remission rate (RR) of 64% and a very good hematologic remission (VGHR) rate of 48% after 3 months. Median hematologic event-free survival (hemEFS) was 11.8 months and median overall survival (OS) was 25.6 months. DVD achieved a 66% RR and a 55% VGHR rate. Median hemEFS was 19.1 months and median OS had not been reached. Cardiac organ responses were noted in 22% with DD and 26% with DVD after 6 months. Infectious complications were common (Common Terminology Criteria [CTC] grade 3/4: DD 16%, DVD 18%) and likely related to a high rate of lymphocytopenia (CTC grade 3/4: DD 20%, DVD 17%). On univariable analysis, hyperdiploidy and gain 1q21 conferred an adverse factor for OS and hemEFS with DD, whereas translocation t(11;14) was associated with a better hemEFS. N-terminal prohormone of brain natriuretic peptide >8500 ng/L could not be overcome for survival with each regimen. Multivariable Cox regression analysis revealed plasma cell dyscrasia (difference between serum free light chains [dFLC]) >180 mg/L as an overall strong negative prognostic factor. Additionally, nephrotic-range albuminuria with an albumin-to-creatinine-ratio (ACR) >220 mg/mmol was a significantly adverse factor for hemEFS (hazard ratio, 2.1 and 3.1) with DD and DVD. Daratumumab salvage therapy produced good results and remission rates challenging any therapy in advanced AL. Outcome is adversely influenced by the activity of the underlying plasma cell dyscrasia (dFLC) and nephrotic-range albuminuria (ACR).

Resemblance-Ranking Peptide Library to Screen for Binders to Antibodies on a Peptidomic Scale.

A novel resemblance-ranking peptide library with 160,000 10-meric peptides was designed to search for selective binders to antibodies. The resemblance-ranking principle enabled the selection of sequences that are most similar to the human peptidome. The library was synthesized with ultra-high-density peptide arrays. As proof of principle, screens for selective binders were performed for the therapeutic anti-CD20 antibody rituximab. Several features in the amino acid composition of antibody-binding peptides were identified. The selective affinity of rituximab increased with an increase in the number of hydrophobic amino acids in a peptide, mainly tryptophan and phenylalanine, while a total charge of the peptide remained relatively small. Peptides with a higher affinity exhibited a lower sum helix propensity. For the 30 strongest peptide binders, a substitutional analysis was performed to determine dissociation constants and the invariant amino acids for binding to rituximab. The strongest selective peptides had a dissociation constant in the hundreds of the nano-molar range. The substitutional analysis revealed a specific hydrophobic epitope for rituximab. To show that conformational binders can, in principle, be detected in array format, cyclic peptide substitutions that are similar to the target of rituximab were investigated. Since the specific binders selected via the resemblance-ranking peptide library were based on the hydrophobic interactions that are widespread in the world of biomolecules, the library can be used to screen for potential linear epitopes that may provide information about the cross-reactivity of antibodies.

CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is still incurable and characterized by clonal expansion of plasma cells in the bone marrow (BM). Therefore, effective therapeutic interventions must target both myeloma cells and the BM niche. METHODS: Cell proliferation, drug resistance, and chromosomal instability (CIN) induced by CHEK1 were confirmed by Giemsa staining, exon sequencing, immunofluorescence and xenograft model in vivo. Bone lesion was evaluated by Tartrate-resistant acid phosphatase (TRAP) staining. The existence of circCHEK1_246aa was evaluated by qPCR, Sanger sequencing and Mass Spectrometer. RESULTS: We demonstrated that CHEK1 expression was significantly increased in human MM samples relative to normal plasma cells, and that in MM patients, high CHEK1 expression was associated with poor outcomes. Increased CHEK1 expression induced MM cellular proliferation and evoked drug-resistance in vitro and in vivo. CHEK1-mediated increases in cell proliferation and drug resistance were due in part to CHEK1-induced CIN. CHEK1 activated CIN, partly by phosphorylating CEP170. Interestingly, CHEK1 promoted osteoclast differentiation by upregulating NFATc1 expression. Intriguingly, we discovered that MM cells expressed circCHEK1_246aa, a circular CHEK1 RNA, which encoded and was translated to the CHEK1 kinase catalytic center. Transfection of circCHEK1_246aa increased MM CIN and osteoclast differentiation similarly to CHEK1 overexpression, suggesting that MM cells could secrete circCHEK1_246aa in the BM niche to increase the invasive potential of MM cells and promote osteoclast differentiation. CONCLUSIONS: Our findings suggest that targeting the enzymatic catalytic center encoded by CHEK1 mRNA and circCHEK1_246aa is a promising therapeutic modality to target both MM cells and BM niche.

Lenalidomide and dexamethasone in relapsed/refractory immunoglobulin light chain (AL) amyloidosis: results from a large cohort of patients with long follow-up.

Lenalidomide and dexamethasone (RD) is a standard treatment in relapsed/refractory immunoglobulin light chain (AL) amyloidosis (RRAL). We retrospectively investigated toxicity, efficacy and prognostic markers in 260 patients with RRAL. Patients received a median of two prior treatment lines (68% had been bortezomib-refractory; 33% had received high-dose melphalan). The median treatment duration was four cycles. The 3-month haematological response rate was 31% [very good haematological response (VGHR) in 18%]. The median follow-up was 56·5 months and the median overall survival (OS) and haematological event-free survival (haemEFS) were 32 and 9 months. The 2-year dialysis rate was 15%. VGHR resulted in better OS (62 vs. 26 months, P < 0·001). Cardiac progression predicted worse survival (22 vs. 40 months, P = 0·027), although N-terminal prohormone of brain natriuretic peptide (NT-proBNP) increase was frequently observed. Multivariable analysis identified these prognostic factors: NT-proBNP for OS [hazard ratio (HR) 1·71; P < 0·001]; gain 1q21 for haemEFS (HR 1·68, P = 0·014), with a trend for OS (HR 1·47, P = 0·084); difference between involved and uninvolved free light chains (dFLC) and light chain isotype for OS (HR 2·22, P < 0·001; HR 1·62, P = 0·016) and haemEFS (HR 1·88, P < 0·001; HR 1·59, P = 0·008). Estimated glomerular filtration rate (HR 0·71, P = 0·004) and 24-h proteinuria (HR 1·10, P = 0·004) were prognostic for renal survival. In conclusion, clonal and organ biomarkers at baseline identify patients with favourable outcome, while VGHR and cardiac progression define prognosis during RD treatment.

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells.

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.

Identification of patients with smouldering multiple myeloma at ultra-high risk of progression using serum parameters: the Czech Myeloma Group model.

Smouldering multiple myeloma (SMM) presents without MM defining symptoms. We aimed to identify patients with SMM with an 80% risk of progression within 2 years using only serum parameters. In total, 527 patients with SMM were included and divided into a training group (287 patients from the Czech Myeloma Group [CMG]) and an independent validation group (240 patients from Heidelberg). The median follow-up was 2·4 and 2·5 years, respectively. Progression to MM occurred in 51·9% of the CMG and 38·8% of the Heidelberg patients, respectively. The median risk of progression was 11·0% (CMG) and 9·7% (Heidelberg) per year, during the 5 years after diagnosis. A serum involved/uninvolved free light-chain ratio of >30, immunoparesis, and serum monoclonal (M) protein of ≥2·3 g/dl emerged as powerful predictors of 2-year progression rate with a hazard ratio (HR) of 2·49 (95% confidence interval [CI] 1·49-4·17), HR of 2·01 (95% CI 1·36-2·96) and HR of 2·00 (95% CI 1·44-2·79) (P < 0·001) in univariate Cox regression analysis, respectively. Based on this, the CMG model identified patients with SMM with a 2-year risk of progression of 78·7% (95% CI 53·1-95·7; HR 6·8; P < 0·001, CMG) and 81·3% (95% CI 47·1-98·8; HR 38·63; P < 0·001, Heidelberg). Serum parameters in the CMG model allow identification of patients with SMM with an 80% risk of progression to symptomatic MM within 2 years.

Kinome expression profiling to target new therapeutic avenues in multiple myeloma.

Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma.

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.

DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells.

BACKGROUND: Multiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy. METHODS: Since HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment. RESULTS: We report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines. CONCLUSION: In conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

Prognostic impact of cytogenetic aberrations in AL amyloidosis patients after high-dose melphalan: a long-term follow-up study.

Cytogenetic aberrations detected by interphase fluorescence in situ hybridization (iFISH) of plasma cells are routinely evaluated as prognostic markers in multiple myeloma. This long-term follow-up study aimed to assess the prognosis of systemic light chain amyloidosis (AL) patients treated with high-dose melphalan (HDM) chemotherapy and autologous stem cell transplantation, depending on iFISH results. Therefore, we analyzed a consecutive cohort of 123 AL patients recruited from 2003 to 2014. HDM was safe, with only 1 of 123 patients dying as a result of treatment-related mortality, and effective, with a complete remission (CR) rate of 34%. Translocation t(11;14) as the most prevalent aberration (59%) led to an improved CR rate after high-dose therapy (41.2% vs 20.0%; P = .02), translating into a prolonged hematologic event-free survival (hemEFS; median, 46.1 vs 28.1 months; P = .05) and a trend for better overall survival (median, not reached vs 93.7 months; P = .07). In multivariate analysis, t(11;14) was confirmed as a favorable prognostic factor regarding hemEFS along with lower values for the difference between involved and uninvolved free light chains. Conversely, deletion 13q14, gain of 1q21, and hyperdiploidy had no significant prognostic impact. The high-risk cytogenetic aberrations t(4;14), t(14;16), and del(17p13) conferred an unfavorable prognosis, although statistical significance was reached only for univariate CR analysis in this small group of 9 patients. Thus, t(11;14) positivity in HDM-treated AL patients conferred superior CR rates and hemEFS. In view of the reduced response of t(11;14) to bortezomib, this highlights the impact of therapy on the prognostic role of cytogenetic aberrations.

A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model.

BACKGROUND: In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. METHODS: Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. RESULTS: We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. CONCLUSION: This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.

Novel recurrent chromosomal aberrations detected in clonal plasma cells of light chain amyloidosis patients show potential adverse prognostic effect: first results from a genome-wide copy number array analysis.

Immunoglobulin light chain (AL) amyloidosis is a rare plasma cell dyscrasia characterized by the deposition of abnormal amyloid fibrils in multiple organs, thus impairing their function. In the largest cohort studied up to now of 118 CD138-purified plasma cell samples from previously untreated immunoglobulin light chain amyloidosis patients, we assessed in parallel copy number alterations using high-density copy number arrays and interphase fluorescence in situ hybridization (iFISH). We used fluorescence in situ hybridization probes for the IgH translocations t(11;14), t(4;14), and t(14;16) or any other IgH rearrangement as well as numerical aberrations of the chromosome loci 1q21, 8p21, 5p15/5q35, 11q22.3 or 11q23, 13q14, 15q22, 17p13, and 19q13. Recurrent gains included chromosomes 1q (36%), 9 (24%), 11q (24%), as well as 19 (15%). Recurrent losses affected chromosome 13 (29% monosomy) and partial losses of 14q (19%), 16q (14%) and 13q (12%), respectively. In 88% of patients with translocation t(11;14), the hallmark chromosomal aberration in AL amyloidosis, a concomitant gain of 11q22.3/11q23 detected by iFISH was part of the unbalanced translocation der(14)t(11;14)(q13;q32) with the breakpoint in the CCND1/MYEOV gene region. Partial loss of chromosome regions 14q and 16q were significantly associated to gain 1q. Gain 1q21 detected by iFISH almost always resulted from a gain of the long arm of chromosome 1 and not from trisomy 1, whereas deletions on chromosome 1p were rarely found. Overall and event-free survival analysis found a potential adverse prognostic effect of concomitant gain 1q and deletion 14q as well as of deletion 1p. In conclusion, in the first whole genome report of clonal plasma cells in AL amyloidosis, novel aberrations and hitherto unknown potential adverse prognostic effects were uncovered.

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).

Chetomin, targeting HIF-1α/p300 complex, exhibits antitumour activity in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. The constitutive expression of HIF-1α in MM suggests that inhibition of HIF-1α-mediated transcription represents an interesting target in MM. METHODS: As p300 is a crucial co-activator of hypoxia-inducible transcription, disrupting the complex HIF-1α/p300 to target HIF activity appears to be an attractive strategy. RESULTS: We reported that chetomin, an inhibitor of HIF-1α/p300 interaction, exhibits antitumour activity in human myeloma cell lines and primary MM cells from patients. CONCLUSIONS: Our data suggest that chetomin may be of clinical value in MM and especially for patients characterised by a high EP300/HIF-1α expression and a poor prognosis.

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. © 2016 Wiley Periodicals, Inc.

Tumor cells in multiple myeloma patients inhibit myeloma-reactive T cells through carcinoembryonic antigen-related cell adhesion molecule-6.

Although functionally competent cytotoxic, T cells are frequently observed in malignant diseases, they possess little ability to react against tumor cells. This phenomenon is particularly apparent in multiple myeloma. We here demonstrate that cytotoxic T cells reacted against myeloma antigens when presented by autologous dendritic cells, but not by myeloma cells. We further show by gene expression profiling and flow cytometry that, similar to many other malignant tumors, freshly isolated myeloma cells expressed several carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) at varying proportions. Binding and crosslinking of CEACAM-6 by cytotoxic T cells inhibited their activation and resulted in T-cell unresponsiveness. Blocking of CEACAM-6 on the surface of myeloma cells by specific monoclonal antibodies or CEACAM-6 gene knock down by short interfering RNA restored T-cell reactivity against malignant plasma cells. These findings suggest that CEACAM-6 plays an important role in the regulation of CD8+ T-cell responses against multiple myeloma; therefore, therapeutic targeting of CEACAM-6 may be a promising strategy to improve myeloma immunotherapy.

Subcutaneous versus intravenous bortezomib in two different induction therapies for newly diagnosed multiple myeloma: an interim analysis from the prospective GMMG-MM5 trial.

We investigated the impact of subcutaneous versus intravenous bortezomib in the MM5 trial of the German-Speaking Myeloma Multicenter Group which compared bortezomib, doxorubicin, and dexamethasone with bortezomib, cyclophosphamide, and dexamethasone induction therapy in newly diagnosed multiple myeloma. Based on data from relapsed myeloma, the route of administration for bortezomib was changed from intravenous to subcutaneous after 314 of 604 patients had been enrolled. We analyzed 598 patients who received at least one dose of trial medication. Adverse events were reported more frequently in patients treated with intravenous bortezomib (intravenous=65%; subcutaneous=56%, P=0.02). Rates of grade 2 or more peripheral neuropathy were higher in patients treated with intravenous bortezomib during the third cycle (intravenous=8%; subcutaneous=2%, P=0.001). Overall response rates were similar in patients treated intravenously or subcutaneously. The presence of International Staging System stage III disease, renal impairment or adverse cytogenetic abnormalities did not have a negative impact on overall response rates in either group. To our knowledge this is the largest study to present data comparing subcutaneous with intravenous bortezomib in newly diagnosed myeloma. We show better tolerance and similar overall response rates for subcutaneous compared to intravenous bortezomib. The clinical trial is registered at eudract.ema.europa.eu as n. 2010-019173-16.

Fetal calf sera can distort cell-based luminescent proteasome assays through heat-resistant chymotrypsin-like activity.

Luminescence-based proteasome activity assays use specific substrates that are supposed to be cleaved by cellular proteasome activity leading to luciferase substrates. Usually, control wells containing cell culture medium supplemented with antibiotics and fetal calf serum are used as background. Using the Proteasome-Glo chymotrypsin-like cell-based assay from Promega, we show here that fetal calf sera from different manufacturers contain heat-resistant, bortezomib-inhibitable, chymotrypsin-like activities that can interfere with proteasome activity assays. These data strongly recommend the use of pure phosphate-buffered saline (PBS) or serum-free medium during proteasome activity assays to diminish background luminescence and, thus, to obtain reliable results.

Regulation of integrin and growth factor signaling in biomaterials for osteodifferentiation.

Stem cells respond to the microenvironment (niche) they are located in. Under natural conditions, the extracellular matrix (ECM) is the essential component the in stem cell niche, in which both integrin ligands and growth factors are important regulators to directly or indirectly modulate the cell behavior. In this review, we summarize the current knowledge about the potential of integrin ligands and growth factors to induce osteogenic differentiation of stem cells, and discuss the signaling pathways that are initiated by both individual and cooperative parameters. The joint effect of integrin ligands and growth factors is highlighted as the multivalent interactions for bone therapy.