The Ins and Outs of Endosteal Niche Disruption in the Bone Marrow: Relevance for Myeloma Oncogenesis.

Multiple Myeloma (MM) and its preexisting stage, termed Monoclonal Gammopathy of Undetermined Significance (MGUS), have long been considered mainly as genomic diseases. However, the bone changes observed in both conditions have led to a reassessment of the role of the bone microenvironment, mainly the endosteal niche in their genesis. Here, we consider the disruption of the endosteal niche in the bone marrow, that is, the shift of the endosteal niche from an osteoblastic to an osteoclastic profile produced by bone senescence and inflammaging, as the key element. Thus, this disrupted endosteal niche is proposed to represent the permissive microenvironment necessary not only for the emergence of MM from MGUS but also for the emergence and maintenance of MGUS. Moreover, the excess of osteoclasts would favor the presentation of antigens (Ag) into the endosteal niche because osteoclasts are Ag-presenting cells. As such, they could significantly stimulate the presentation of some specific Ag and the clonal expansion of the stimulated cells as well as favor the expansion of such selected clones because osteoclasts are immunosuppressive. We also discuss this scenario in the Gaucher disease, in which the high incidence of MGUS and MM makes it a good model both at the bone level and the immunological level. Finally, we envisage that this endosteal niche disruption would increase the stochasticity (epigenetic and genetic instability) in the selected clones, according to our Tissue Disruption-induced cell Stochasticity (TiDiS) theory.

A bone paradigm challenging the standard model of myeloma oncogenesis.

The standard model of multiple myeloma (MM) oncogenesis from monoclonal gammopathy of undetermined significance (MGUS) relies on genetic instability in the normal counterparts of MM cells. However, the importance of both MGUS-associated and MM-induced bone changes has been recently re-appraised, emphasizing the bone microenvironment (BME) as a tissue of significance. In this review, we propose that early BME alterations (bone senescence and inflammation, i.e., bone inflamm'aging) at the pre-MGUS stage could be causal, and not simply permissive, and creative of phenotypic instability and genetic alterations thanks to the concept of tissue disruption-induced cell stochasticity (TiDiS). This article offers a bone scenario challenging the chromosome-and-gene-centric standard model of MM oncogenesis. The high incidence of both MGUS and MM in Gaucher disease supports such a scenario.

Multiple Myeloma as a Bone Disease? The Tissue Disruption-Induced Cell Stochasticity (TiDiS) Theory.

The standard model of multiple myeloma (MM) relies on genetic instability in the normal counterparts of MM cells. MM-induced lytic bone lesions are considered as end organ damages. However, bone is a tissue of significance in MM and bone changes could be at the origin/facilitate the emergence of MM. We propose the tissue disruption-induced cell stochasticity (TiDiS) theory for MM oncogenesis that integrates disruption of the microenvironment, differentiation, and genetic alterations. It starts with the observation that the bone marrow endosteal niche controls differentiation. As decrease in cellular stochasticity occurs thanks to cellular interactions in differentiating cells, the initiating role of bone disruption would be in the increase of cellular stochasticity. Thus, in the context of polyclonal activation of B cells, memory B cells and plasmablasts would compete for localizing in endosteal niches with the risk that some cells cannot fully differentiate if they cannot reside in the niche because of a disrupted microenvironment. Therefore, they would remain in an unstable state with residual proliferation, with the risk that subclones may transform into malignant cells. Finally, diagnostic and therapeutic perspectives are provided.

The international St-aging system as a prognostic marker in general senior population: Findings from the EPIDOS cohort study.

The International Staging System (ISS) -calculated from serum albumin and beta-2 microglobulin (ß2m)- is an established prognostic marker in multiple myeloma (MM), which has also been suggested to account for survival among general senior population. Our objective was to examine long-term survival of older women free of MM according to baseline ISS. The study included 230 community-dwelling healthy older women without known MM from the EPIDOS cohort (mean ±â€¯SD, 80.4 ±â€¯3.4 years). Serum albumin and ß2m were measured at baseline, and used to calculate the ISS a posteriori. Abnormal ISS was defined as ISS = 2 or ISS = 3, although ISS = 1 was considered normal. The vital status was sought after a mean follow-up of 17.6 ±â€¯0.2 years (range, 16.8-18.3). Age, body mass index, mean arterial pressure, diabetes mellitus, hypertension, coronary heart disease, stroke, use corticosteroids, number of drugs daily taken, smoking, physical activity, fall history, bone mineral density, and creatinine clearance were used as potential confounders. All participants died during the 17-year follow-up. Compared to women with normal ISS, those with abnormal ISS (n = 24) had shorter survival time (4.9 ±â€¯4.3 versus 8.7 ±â€¯5.2 years, P = .001) and died earlier (85.6 ±â€¯4.8 versus 89.1 ±â€¯5.6 years old, P = .003). Survival time after blood test correlated with both serum albumin (r = 0.16, P = .015) and ß2m (r = -0.27, P < .001). Cox regression revealed that abnormal ISS was associated with mortality (adjusted HR = 3.03, P < .001). Kaplan-Meier distributions showed that participants with abnormal ISS died earlier than those with normal ISS (log-rank P < .001). In conclusion, community-dwelling older women with abnormal ISS had shorter survival time than the others, suggesting that ISS could be considered as a universal prognostic "aging system" rather than a specific MM "staging system".

Multiple Myeloma Exemplifies a Model of Cancer Based on Tissue Disruption as the Initiator Event.

The standard model of multiple myeloma (MM) oncogenesis is based on the genetic instability of MM cells and presents its evolution as the emergence of clones with more and more aggressive genotypes, giving them surviving and proliferating advantage. The micro-environment has a passive role. In contrast, many works have shown that the progression of MM is also characterized by the selection of clones with extended phenotypes able to destroy bone trabeculae, suggesting a major role for early micro-environmental disruption. We present a model of MM oncogenesis in which genetic instability is the consequence of the disruption of normal interactions between plasma cells and their environment, the bone remodeling compartment. These interactions, which normally ensure the stability of the genotypes and phenotypes of normal plasma cells could be disrupted by many factors as soon as the early steps of the disease (MGUS, pre-MGUS states). Therapeutical implications of the model are presented.

Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma.

Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects.

Multiple myeloma international staging system: "staging" or simply "aging" system?

Because of the wide variation in multiple myeloma (MM) survival, numerous studies have focused over the past 40 years on the biological and cytogenetic prognostic values in MM patients. Since 2005, the MM International Staging System (ISS) has recognized the combination of beta-2 microglobulin (ß2M) with serum albumin (SA) concentrations as the most simple and potent combination to determine the prognosis in MM patients. Curiously, the reasons for the efficiency of the combination of ß2M with SA remain not clear-cut. In 2007, Fonseca and San Miguel (Prognostic factors and staging in multiple myeloma. Hematol Oncol Clin North Am 2007; 21:1115-40) underlined that cytogenetic assessment might also be useful for evaluating MM prognosis. Furthermore, new perspectives recently appeared with the genomic approach. Here, we (1) question the specific rationale for ß2M and SA as prognostic markers in MM, (2) emphasize the well-documented prognostic implications of ß2M and SA as potent biomarkers of comorbidity in older adults, and (3) conclude that the current MM-ISS is rather a staging system for age-related comorbidity burden (ie, aging system) than a specific MM staging system, and should not be used alone. Thus, we suggest that: (1) cytogenetics with the superscript MM-ISS could be the standard method; (2) some factors discovered using genomics could reflect the comorbidity burden and the intrinsic malignancy of MM clone, and thus needs more investigation; and (3) while waiting for standard genomic classification.

Cell death via DR5, but not DR4, is regulated by p53 in myeloma cells.

Myeloma cells are sensitive to TRAIL through the two death receptors DR4 and DR5. Because p53 directly modulates expression of death receptors, we investigated here whether p53 can modulate myeloma sensitivity to TRAIL. We found that p53 affects the sensitivity of myeloma cells to the DR5 agonistic human antibody lexatumumab but not the DR4 antibody mapatumumab. TP53 wild-type myeloma cells overexpressed DR5 in correlation with sensitivity to lexatumumab. Both nongenotoxic (nutlin-3a) and genotoxic (melphalan) p53-inducing stresses increased DR5 expression only in TP53 wild-type cells and synergistically increased lexatumumab efficiency yet did not increase DR4 expression, nor sensitivity to mapatumumab. Silencing of p53 strongly decreased DR5 expression and induced resistance to nutlin-3a and lexatumumab but did not modulate DR4 expression or sensitivity to mapatumumab. Increase of lexatumumab efficiency induced by nutlin-3a was related to a p53-dependent increase of DR5 expression. In primary myeloma cells, nutlin-3a increased DR5 expression and lexatumumab efficiency but did not increase mapatumumab efficiency. Taken together, our findings indicate that p53 controls the sensitivity of myeloma through DR5 but not DR4 and suggest that a subset of patients with multiple myeloma may benefit from DR5 therapy.

Critical role of the NOTCH ligand JAG2 in self-renewal of myeloma cells.

The purpose of this study was to identify the pathways associated with the ability of CD138(+) human myeloma cells to form colonies in a serum-free semi-solid human collagen-based assay. Only 26% (7 of 27) of human myeloma cell lines were able to spontaneously form colonies. This spontaneous clonogenic growth correlated with the expression of the NOTCH ligand JAG2 (p<0.001). Blocking JAG-NOTCH interactions with NOTCH-Fc chimeric molecules impaired self-colony formation, indicating a role for JAG-NOTCH pathway in colony formation. In two cell lines, silencing of JAG2 blocked both colony formation and in vivo tumor formation in immunocompromised mice. RT-PCR and flow cytometry analysis revealed that JAG2 is often expressed by CD138(+) primary cells. Our results indicate that spontaneous clonogenic growth of myeloma cells requires the expression of JAG2.

Increased singlet oxygen-induced secondary ROS production in the serum of cancer patients.

Photodynamic therapy (PDT) generates singlet oxygen ((1)O(2)) and Reactive Oxygen Species (ROS) that are counteracted by patient's defenses. As cancer treatments are among the most important PDT applications the aim of this pilot study was to determine whether the serum of cancer patients produces more or less secondary ROS or peroxides after a photoreaction as compared to healthy persons. Fifty-three volunteers and 105 cancer patients were recruited. The capacity of (1)O(2) or secondary oxidant production was found to be increased in 6 healthy donors and 36 cancer patients (23/69 women and 13/31 men p<0.007 and p<0.04) with a mean value of 1.52 as compared to 1.29 in the healthy subjects (p<0.05) when considering values higher than the normal range (norm=1±10%) or 1.1 vs. 0.85 (p<0.01) in the whole cohort. This increase correlated with a poor prognosis, TNM and SBR classification. Serum (1)O(2) deactivation capacity was impaired and secondary ROS were more produced during cancer progression. Although it is currently unclear whether this is the cause or effect of cancer, this finding may hold interest as a potential marker of cancer severity. It would also support the interest of PDT as an adjuvant for cancer treatment, even for aggressive tumors particularly when associated to surgery for bulk removal.

ABT-737 is highly effective against molecular subgroups of multiple myeloma.

Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.

A high-risk signature for patients with multiple myeloma established from the molecular classification of human myeloma cell lines.

BACKGROUND: Multiple myeloma is a plasma-cell tumor with heterogeneity in molecular abnormalities and treatment response. DESIGN AND METHODS: We have assessed whether human myeloma cell lines have kept patients' heterogeneity using Affymetrix gene expression profiling of 40 human myeloma cell lines obtained with or without IL6 addition and could provide a signature for stratification of patient risk. RESULTS: Human myeloma cell lines, especially those derived in the presence of IL6, displayed a heterogeneity that overlaps that of the patients with multiple myeloma. Human myeloma cell lines segregated into 6 groups marked by overexpression of MAF, MMSET, CCND1, FRZB with or without overexpression of cancer testis antigens (CTA). Cell lines of CTA/MAF and MAF groups have a translocation involving C-MAF or MAFB, cell lines of groups CCND1-1 and CCND1-2like have a t(11;14) and cell lines of group MMSET have a t(4;14). The CTA/FRZB group comprises cell lines that had no or no recurrent 14q32 translocation. Expression of 248 genes accounted for human myeloma cell line molecular heterogeneity. Human myeloma cell line heterogeneity genes comprise genes with prognostic value for survival of patients making it possible to build a powerful prognostic score involving a total of 13 genes. CONCLUSIONS: Human myeloma cell lines derived in the presence of IL6 recapitulate the molecular diversity of multiple myeloma that made it possible to design, using human myeloma cell line heterogeneity genes, a high-risk signature for patients at diagnosis. We propose this classification to be used when addressing the physiopathology of multiple myeloma with human myeloma cell lines.

Patients with drug-free long-term graft function display increased numbers of peripheral B cells with a memory and inhibitory phenotype.

Several transplant patients maintain stable kidney graft function in the absence of immunosuppression. Here we compared the characteristics of their peripheral B cells to that of others who had stable graft function but were under pharmacologic immunosuppression, to patients with chronic rejection and to healthy volunteers. In drug-free long-term graft function (DF) there was a significant increase in both absolute cell number and frequency of total B cells; particularly activated, memory and early memory B cells. These increased B-cell numbers were associated with a significantly enriched transcriptional B-cell profile. Costimulatory/migratory molecules (B7-2/CD80, CD40, and CD62L) were upregulated in B cells; particularly in memory CD19(+)IgD(-)CD38(+/-)CD27(+) B cells in these patients. Their purified B cells, however, responded normally to a polyclonal stimulation and did not have cytokine polarization. This phenotype was associated with the following specific characteristics which include an inhibitory signal (decreased FcgammaRIIA/FcgammaRIIB ratio); a preventive signal of hyperactive B-cell response (an increase in BANK1, which negatively modulates CD40-mediated AKT activation); an increased number of B cells expressing CD1d and CD5; an increased BAFF-R/BAFF ratio that could explain why these patients have more peripheral B cells; and a specific autoantibody profile. Thus, our findings show that patients with DF have a particular blood B-cell phenotype that may contribute to the maintenance of long-term graft function.

Serum vitamin D concentration and short-term mortality among geriatric inpatients in acute care settings.

INTRODUCTION: Vitamin D insufficiency is related to acute medical conditions known to increase the risk of short-term death in older adults. The objective of this study was to determine whether serum 25-hydroxyvitamin D (25OHD) concentrations were associated with the occurrence of in-hospital mortality in geriatric acute care settings while taking into account all characteristics likely to improve the rate of in-hospital mortality. METHODS: Three hundred ninety-nine Caucasian adults admitted between January and October 2009 to the geriatric acute care unit of Angers University Hospital, France were included in this cross-sectional study. The occurrence of all-cause in-hospital death and the measurement of serum 25OHD were assessed. Age, gender, body mass index, supine systolic blood pressure, numbers of acute diseases, chronic diseases, and hospital days, serum albumin, creatinine clearance, and season of hospital admission were used as potential confounders. RESULTS: Mean serum 25OHD was 34.88+/-1.7 nmol/L. Seventeen deaths occurred in the acute care unit. Only serum 25OHD concentration was significantly and independently associated with in-hospital death (adjusted odds ratio [OR] 0.65; 95% CI: 0.44, 0.96; P=0.029 for full adjusted logistic regression. OR 0.87; 95% CI: 0.76, 0.99; P=0.029 fo or step-wise backward model). CONCLUSION: Increased serum 25OHD concentrations were associated with a low in-hospital mortality rate in this cohort of acute care geriatric inpatients. It is not only a new orientation of research,but also an additional argument for prescribing vitamin D in deficient older adults.

Phosphorothioate-modified TLR9 ligands protect cancer cells against TRAIL-induced apoptosis.

Hypomethylated CpG oligodeoxynucleotides (CpG ODNs) target TLR9 expressed by immune cells and are currently being evaluated as adjuvants in clinical trials. However, TLR signaling can promote some tumor growth and immune evasion, such as in multiple myeloma (MM). Therefore, deciphering the effects of CpG ODNs on cancer cells will help in preventing these adverse effects and in designing future clinical trials. TLR activation induces multiple signaling pathways, notably NF-kappaB that has been involved in the resistance to TRAIL. Thus, we wondered if CpG ODNs could modulate TRAIL-induced apoptosis in different models of tumors. Here, we show that TLR9+ (NCI-H929, NAN6, KMM1) and TLR9- MM cells (MM1S) were protected by CpG ODNs against recombinant TRAIL-induced apoptosis. By using two fully human, agonist mAbs directed against TRAIL receptors DR4 and DR5 (mapatumumab and lexatumumab, respectively), we show that the protection was restricted to DR5-induced apoptosis. Similar results were observed for two colon cancer (C45 and Colo205) and two breast cancer cell lines (HCC1569 and Cal51). The protection of CpG ODNs was mediated by its nuclease-resistant phosphorothioate backbone independent of TLR9. We next demonstrated by surface plasmon resonance that phosphorothioate-modified CpG ODNs directly bound to either TRAIL or lexatumumab and then decreased their binding to DR5. Finally, NK cell lysis of a DR5-sensitive MM cell line (NCI-H929) through TRAIL was partially inhibited by phosphorothioate-modified CpG ODNs. In conclusion, our results suggest that the phosphorothioate modification of CpG ODNs could dampen the clinical efficacy of CpG ODN-based adjuvants by altering TRAIL/TRAIL receptor interaction.

The imbalance between Survivin and Bim mediates tumour growth and correlates with poor survival in patients with multiple myeloma.

Survivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock-down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3-only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult-to-treat cancer.

TLR3 ligand induces NF-{kappa}B activation and various fates of multiple myeloma cells depending on IFN-{alpha} production.

Multiple myeloma (MM) cells express TLR. It has been shown that TLR ligands induce the proliferation, survival, and immune surveillance escape of MM cells through MyD88-TLR pathways. Deciphering TLR function in MM cells will help in understanding the mechanisms of tumor cell growth. In this study, we examined the response of MM cells to the MyD88-independent/TIR-domain-containing adapter-inducing IFN-beta-dependent TLR3. Deregulation of NF-kappaB pathway is a feature of MM cells, and we wondered whether TLR3 activation could mobilize the NF-kappaB pathway. We show that five of seven human myeloma cell line (HMCL) cells expressed TLR3. In the presence of the synthetic TLR3 ligand (poly(I:C)), activation of NF-kappaB pathway was observed in three of five selected TLR3(+) HMCL, NCI-H929, RPMI 8226, and KMM1. In agreement with NF-kappaB activation, only these three HMCL responded to poly(I:C), although by either an increase (KMM1) or a decrease (NCI-H929, RPMI 8226) of proliferation. We show that KMM1 increase of proliferation was prevented by NF-kappaB inhibitor. In contrast, inhibition of proliferation in both NCI-H929 and RPMI 8226 was due to IFN-alpha-induced apoptosis. We next demonstrated that p38 MAPK pathway controlled both IFN-alpha secretion and IFN-alpha-mediated cell death. Moreover, cell death also involved activation of ERK1/2 pathway. In conclusion, our results show that TLR3 ligand induces NF-kappaB pathway activation in MM and support a switching function of type I IFN in the functional outcome of TLR3 triggering in tumor cells.

A 38-gene expression signature to predict metastasis risk in node-positive breast cancer after systemic adjuvant chemotherapy: a genomic substudy of PACS01 clinical trial.

Currently, no prognostic gene-expression signature (GES) established from node-positive breast cancer cohorts, able to predict evolution after systemic adjuvant chemotherapy, exists. Gene-expression profiles of 252 node-positive breast cancer patients (median follow-up: 7.7 years), mostly included in a randomized clinical trial (PACS01), receiving systemic adjuvant regimen, were determined by means of cDNA custom array. In the training cohort, we established a GES composed of 38 genes (38-GES) for the purpose of predicting metastasis-free survival. The 38-GES yielded unadjusted hazard ratio (HR) of 4.86 (95% confidence interval = 2.76-8.56). Even when adjusted with the best two clinicopathological prognostic indexes: Nottingham prognostic index (NPI) and Adjuvant!, 38-GES HRs were 3.30 (1.81-5.99) and 3.40 (1.85-6.24), respectively. Furthermore, 38-GES improved NPI and Adjuvant! classification. In particular, NPI intermediate-risk patients were divided into 2/3 close to low-risk group and 1/3 close to high-risk group (HR = 6.97 [2.51-19.36]). Similarly, Adjuvant! intermediate-risk patients were divided into 2/3 close to low-risk group and 1/3 close to high-risk group (HR = 4.34 [1.64-11.48]). The 38-GES was validated on gene-expression datasets from three external node-positive breast cancer subcohorts (n = 224) generated from different microarray platforms, with HR = 2.95 (1.74-5.01). Moreover, 38-GES showed prognostic performance in supplementary cohorts with different lymph-node status and endpoints (1,040 new patients). The 38-GES represents a robust tool able to type systemic adjuvant treated node-positive patients at high risk of metastatic relapse, and is especially powerful to refine NPI and Adjuvant! classification for those patients.