Summary
The gene expression profiles (GEPs) of 96 selected genes were analysed by real‐time quantitative polymerase chain reaction (qPCR) with a TaqMan low‐density array card in isolated tumour ...plasma cells (PCs) from 157 newly diagnosed multiple myeloma (MM) patients. This qPCR‐based GEP correctly classified cases following the Translocation‐cyclin D classification. Classic prognostic parameters and qPCR‐based GEP predicted MM patient outcome and, although multivariate analyses revealed that cytogenetic risk (standard vs. high risk) was the variable that most strongly predicted prognosis, GEP added significant information for risk stratification. Considering only the standard risk cytogenetic patients, multivariate analyses revealed that high β2‐microglobulin, low CDKN1A and high SLC19A1 gene expression levels independently predicted a short time‐to‐progression (TTP), while high International Staging System stage, low CDKN2B and high TBRG4 gene expression predicted poor overall survival (OS). A gene expression risk score enabled the division of standard risk patients into two groups with different TTPs (83% vs. 38% at 3 years, P < 0·0001) and OS rates (88% vs. 61% at 5 years; P = 0·003). This study demonstrates that quantitative PCR is a robust, accurate and feasible technique for implementing in the daily routine as a surrogate for GEP‐arrays.
Introduction: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 del(5q). The clinical efficacy of ...lenalidomide in MDS patients has been extensively reviewed and although the mechanisms of action in del(5q) clone have been previously described, in vivo sequential studies of modulatory effect on T lymphocytes are lacking. Our study was conducted in patients included in the Sintra-REV Clinical Trial: Lenalidomide (Revlimid) phase III, multicenter, randomized, double-blind study versus placebo in patients with low-risk MDS (low and intermediate IPSS-1) with del(5q), with anemia (HB≤12gr/dl) and without transfusion needs.
Aim: The aim of this study was to explore the effect of lenalidomide in T-lymphocytes in MDS patients with del(5q) and without transfusion dependence.
Materials and Methods: Sequential study was carried out in 26 samples from 13 paired MDS patients with del (5q). Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated-patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR < 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite.
Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; 199 of them were over-expressed after the administration of this drug (Fig 1a). Of note, none of them were observed in non-treated patients after one month. The ORA revealed significant differences in the gene expression profile of sixteen cytokines and enrichment of genes of the cell cycle pathway (35 genes). The most relevant up-regulated cytokines were: IL10, TNFSF10, IFNGand IL6. These data explain lenalidomide-induced activation of an antileukemic immune response and secretion of anti-inflammatory cytokines. Although lenalidomide has been reported to reduce the expression of IL6 secreted by myeloid cell derived from MDS clon, we have observed upregulation of this gene in T-lymphocytes. Moreover, our study showed a downregulation of MBP6 that may help to correct the anemia and also attenuate inflammation signaling in MDS patients with del(5q) (Fig 1b). In addition, the most represented up-regulated genes related to cell cycle pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C), BUB1 mitotic checkpoint genes (BUB1, BUB1B), and genes involved in cell division (CDC6, CDC7,CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells (Fig 1b).
Conclusions: The immunomodulatory properties of lenalidomide can be summarized in two: a) regulation of antileukemic and anti-inflammatory cytokines production, b) activation of cell cycle and proliferation in T cells. To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action.
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Sanz:Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Fenaux:Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Diez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.
Lenalidomide was administered in anemic but not transfusion-dependence patients with low-risk MDS and del(5q)
•Low dose of irradiation on human MSCs improves their hematopoietic-supporting ability.•Low-dose irradiation decreases the adipogenic differentiation of human MSCs.•Osteogenesis is increased after ...low-dose irradiation on human MSCs.
Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.
The incidence of EOCRC (age < 50 years at diagnosis) with unknown causes is rising worldwide, necessitating the mechanistical analysis of its molecular basis. The NOMO1 gene is deleted in a high ...number of EOCRC tumors compared to LOCRC. In this work, we aimed to test the NOMO1 gene mutational profile in EOCRC tumors and to characterize the effect of NOMO1 loss in different CRISPR/cas9-edited cell lines, as well as in murine models. Here, we show that the NOMO1 gene can be inactivated not only by deletion but also by pathogenic mutations in EOCRC. Our results indicate that NOMO1 loss could be a passenger mutation in the development of EOCRC, although it contributes significantly to colon cancer cell migration. The incidence of early-onset colorectal cancer (EOCRC; age younger than 50 years) has been progressively increasing over the last decades globally, with causes unexplained. A distinct molecular feature of EOCRC is that compared with cases of late-onset colorectal cancer, in EOCRC cases, there is a higher incidence of Nodal Modulator 1 (NOMO1) somatic deletions. However, the mechanisms of NOMO1 in early-onset colorectal carcinogenesis are currently unknown. In this study, we show that in 30% of EOCRCs with heterozygous deletion of NOMO1, there were pathogenic mutations in this gene, suggesting that NOMO1 can be inactivated by deletion or mutation in EOCRC. To study the role of NOMO1 in EOCRC, CRISPR/cas9 technology was employed to generate NOMO1 knockout HCT-116 (EOCRC) and HS-5 (bone marrow) cell lines. NOMO1 loss in these cell lines did not perturb Nodal pathway signaling nor cell proliferation. Expression microarrays, RNA sequencing, and protein expression analysis by LC–IMS/MS showed that NOMO1 inactivation deregulates other signaling pathways independent of the Nodal pathway, such as epithelial–mesenchymal transition and cell migration. Significantly, NOMO1 loss increased the migration capacity of CRC cells. Additionally, a gut-specific conditional NOMO1 KO mouse model revealed no subsequent tumor development in mice. Overall, these findings suggest that NOMO1 could play a secondary role in early-onset colorectal carcinogenesis because its loss increases the migration capacity of CRC cells. Therefore, further study is warranted to explore other signalling pathways deregulated by NOMO1 loss that may play a significant role in the pathogenesis of the disease.
Introduction: Filanesib (ARRY-520) is a novel inhibitor of the "kinesin spindle protein" (KSP), which has demonstrated efficacy in heavily pretreated patients with refractory MM, (Lonial et al, ASH ...2013). Our preliminary studies demonstrated synergy with standard anti-MM agents, especially with pomalidomide and dexamethasone. This set the stage for a recently activated trial being run by the Spanish MM group investigating FPD in relapsed MM patients. In this abstract we investigate the mechanisms underlying the synergy of the combination.
Methods: In vitro action of FPD was evaluated in MM cell lines by MTT assay, bioluminescence, Annexin V staining, cell cycle profile analysis and TMRE staining by flow cytometry. Synergy was quantified with the Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice (The Jackson Laboratory, Bar Harbor, ME, USA). The mechanism of action was analyzed by Western blot, flow cytometry, genomic techniques, immunohistochemistry and immunofluorescence techniques.
Results: The triple combination of FPD resulted in clear synergy in multiple myeloma cell lines (MM1S, OPM2, and RPMI8226) with combination indices between 0.4-0.7, and abrogated the effect of the soluble cytokines IL-6 and IGF-I and the protective effect of the adhesion of plasma cells to BMSCs, HS-5 and TERT cells.
FPD caused cell cycle arrest in G2/M and specific apoptosis of cells arrested in these proliferative phases (with apoptosis percentage of 5, 23, 58 and 88 for control, poma+dexa, filanesib and FPD, respectively) demonstrated by flow cytometry with DRAQ5 and Annexin-V. Thus, FPD and filanesib in monotherapy treatments induced a similar effect on the cell cycle profile (arrest in G2/M) with a concordant increase of cyclin B1 and phosphorylated Histone H3. Although a secondary increase of KSP protein levels would be expected, pomalidomide and dexamethasone induced a decrease of the levels of this protein, which was still present in the triple combination (FPD). This fact could be contributing to the potentiation observed with the combination. Attending to apoptosis mechanism, proapoptotic stimulus from the extrinsic and intrinsic apoptotic pathways were promoted by pomalidomide and dexamethasone and filanesib, and converged in the triple combination. In this regard, a decrease of MCL-1 (antiapoptotic protein) and a significant increase of the proapoptotic BCL2 family members of the intrinsic pathway like NOXA and BIMEL BIML, BIMS(this last one being the most potent proapoptotic isoform), tBID (extrinsic pathway) and Bax protein were observed. We confirmed that all these proteins were translocated into the mitochondria, resulting in a decrease of the mitochondrial membrane potential by TMRE, increase of permeability and a release of cytochrome C and AIF.
These results were confirmed in vivo in a model of subcutaneous plasmacytoma in small (70 mm3) and large (2000 mm3) tumors. In this model we observed a significant reduction of tumor growth, which was correlated with a statistically significant improvement in survival. Changes induced by FPD in the gene expression profile were concordant with the in vitro results as several overexpressed genes belonging to the previous pathways were identified, such as spindle assembly checkpoint (CENP-E and CENP-F) and apoptosis (BCL2L11, gene that codifies BIM protein). Furthermore, IHC of tumors treated with FPD showed more apoptosis by TUNEL and a significant increase of monopolar spindles (2, 0, 53 and 140 per 10 high-power fields, for control, poma+dexa, filanesib and FPD, respectively).
Conclusions: The synergy observed with filanesib in combination with pomalidomide and dexamethasone is the result of several coincidental mechanisms: a potentiation of the KSP inhibition with a subsequent increase in monopolar spindle formation and a simultaneous activation of the intrinsic and extrinsic pathways of apoptosis. In this regard, NOXA, BIM, BAX and tBID are probably the central players that, through different mechanisms, inhibit antiapoptotic proteins (MCL-1, BCL2 and BCL-XL) and promote mitochondrial outer membrane permeabilization and the release of apoptogenic factors such us cytochrome C and AIF.
This work was funded in part by the company Array BioPharma.
Tunquist:Array BioPharma: Employment. Mateos:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ocio:Jassen: Honoraria; Celgene: Honoraria, Research Funding; Pharmamar: Consultancy, Research Funding; MSD: Research Funding; Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding.
The Brigham and Women's Hospital and the Tübingen cutaneous squamous cell carcinoma (SCC) stratification systems propose different criteria from the American Joint Committee on Cancer, eighth ...edition. Our group identified prognostic subgroups within T3 stage according to the American Joint Committee on Cancer eighth edition's classification, the most common classification for high-risk cutaneous SCCs.
To compare the performance and prognostic accuracy of these staging systems in a subset of high-risk cutaneous SCCs.
Homogeneity, monotonicity, and McNemar tests for pairwise comparisons were assessed. Distinctiveness and relative risk of poor outcome were calculated by stage. Prognostic accuracy was compared with respect to quality (Akaike and Bayesian information criteria), concordance (Harrell C-index and Gönen and Heller concordance probability estimate), and predictive accuracy (sensitivity, specificity, negative predictive value, positive predictive value, and global accuracy).
The Brigham and Women's Hospital and Salamanca systems were more distinctive, homogeneous, and monotonic than the Tübingen system. The Tübingen system was the most specific, whereas the Salamanca and Brigham and Women's Hospital systems were more sensitive. Negative predictive value was high in all 3 systems, but positive predictive value and accuracy were low overall.
Alternative staging systems may partially overcome the heterogeneity and low prognostic accuracy of the American Joint Committee on Cancer, eighth edition and enable high-risk cutaneous SCCs to be stratified more reliably, but their prognostic accuracy is still low. Considering the accumulation of risk factors may improve high-risk cutaneous SCC risk stratification.
Introduction: While dexamethasone (Dex) has been commonly used in the treatment of multiple myeloma (MM), its immunosuppressive effect is becoming a matter of concern with the advent of immune-based ...therapies. One example is the combination of lenalidomide and Dex (LenDex) because it has been reported that Dex abrogates the immunomodulatory effects of Len; however, most of the studies have been performed in vitro, using high-doses of Dex, and in small series of relapsed patients previously exposed to other drugs.
Methods: Because the potentially antagonist effect of Dex may represent a dilemma in the design of clinical trials, here we aim to shed light into the question about whether or not low-dose Dex abrogates the immunomodulatory effect of Len by studying the phenotypic profile of T-lymphocytes, NK-cells and dendritic cells (DCs) of 31 previously untreated high-risk smoldering MM (SMM) patients enrolled in the Quiredex trial at baseline, after 3 and 9 cycles of LenDex, and during maintenance with Len as single-agent.
Results: Patients with high-risk SMM showed at baseline normal numbers of CD4 and CD8 T-lymphocytes as well as CD56dim and CD56bright NK-cells compared to age-matched healthy individuals. By contrast, they displayed an increment of TCRγδ positive T-lymphocytes (P =.02) and Tregs (P =.04), as well as an altered distribution of BDCA-1 positive myeloid DCs (P =.02) and tissue macrophages (P =.06). Moreover, the expression levels of activation markers (CD25, CD28 and CD54) as well as Th1-related markers (CD195, IFN-γ, TNF-α, or IL-2) were significantly inferior in T-lymphocytes from high-risk SMM patients. A significantdown-regulation of proliferation-related markers (CD119 and CD120b) was also noted. To assess the combined effect of LenDex in T-lymphocytes and NK-cells, we compared the immune status of the 31 high-risk SMM patients at baseline vs. after 3 and 9 cycles of LenDex. Interestingly, TCRγδ positive T-lymphocytes as well as Tregs were further increased with LenDex; conversely, CD4 T-lymphocytes were significantly decreased at the end of induction. There was a marked shift on the distribution of antigen-related maturation subsets induced by LenDex, and reflected by a significant increase of central memory CD4 (P<.001) and effector memory CD8 (P<.001) T-lymphocytes. Accordingly, CD4 and/or CD8 T-lymphocytes showed an increased expression of activation markers (CD69, CD25, CD28, and CD54), together with an up-regulation of the Th1 related chemokine CCR5 (CD195) and increased cytokine production of IFNγ, TNFα, and IL-2. NK-cells showed an up-regulation of the activation marker HLA-DR (P<.001), the ADCC associated receptor CD16 (P≤0.005), and the adhesion molecules CD11a (P≤0.001) and CD11b (P≤0.005) after 3 and 9 courses of LenDex. The percentage of cells in S-phase progressively increased from baseline vs. 3 and 9 cycles of LenDex for CD4 (P<0.001) and CD8 (P<0.001) T-lymphocytes as well as NK-cells (P<0.001). Most interestingly, high-risk SMM patients treated with LenDex and without disease progression showed higher numbers of functionally active T-lymphocytes as compared to those progressing to MM. To address the question whether Dex antagonizes Len, we compared the immune profile of 13 patients with PB samples collected at cycle 9 of induction vs. during maintenance (single-agent Len at least 3 months after Dex discontinuation). No significant differences were observed for the absolute numbers of all cell populations analyzed. From the total 63 phenotypic parameters analyzed, only 7 were found to be differently expressed. Namely, the expression of CD94, CD154 and CD212 positive T-lymphocytes as well as CD11a in T-lymphocytes and NK-cells were down-regulated during maintenance.
Conclusions: Our results, obtained from a carefully selected population of patients without previous exposure to anti-MM therapy and with available longitudinal samples after consecutive cycles of LenDex, shed new light on the synergy between lenalidomide and dexamethasone which, at low doses, does not abrogate the immune modulatory effects of lenalidomide here analyzed. Accordingly, high-risk SMM patients have an impaired immune system that could be re-activated with LenDex, and support the value of therapeutic immunomodulation to delay the progression to MM.
Paiva:Millenium: Consultancy; BD Bioscience: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Sanofi: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen-Cilag: Honoraria; Sanofi-Aventis: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Onyx: Honoraria.
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