multiple myeloma ( mm ) is a malignant disorder characterized by multifocal proliferation of clonal , long - lived plasma cells within the bone marrow ( bm ) .
mm presents clinically with a broad range of manifestations , including skeletal destruction , immune suppression , and end - organ sequelae .
it is estimated that there will be 22 350 newly diagnosed mm cases and 10 710 mm deaths in the united states in 2013 . over the past decade
, the outcome of mm patients has been dramatically improved after the development of novel agents and the introduction of autologous stem cell transplantation .
however , mm remains an incurable disease and significant efforts aim to unravel its biology .
mm is preceded by a premalignant condition termed monoclonal gammopathy of undetermined significance ( mgus ) .
mgus is present in more than 3% of the population above the age of 50 years
. its annual progression rate to mm is of 0.5% to 3% . in some patients , an intermediate but more advanced premalignant stage ,
to date , the biological alterations that lead from mgus to smm and finally to symptomatic disease have not been elucidated .
mm evolution is characterized by an avascular stage of slow tumor progression followed by  angiogenic switch  when the balance between pro- and anti - angiogenic factors in the tumor microenvironment is deranged to promote neovascularization and disease progression .
angiogenesis is a multistep and tightly regulated process of the formation of new blood vessels , which occurs physiologically during embryonic growth and wound healing , but also in tumor growth and metastasis .
our previous study elucidated the role of angiogenic cytokines in mm pathogenesis , and our results were consistence with those of other groups [ 1115 ] .
these cytokines mainly include the vascular endothelial growth factor ( vegf ) , the angiogenin , the angiopoietins , and the basic fibroblast growth factor ( bfgf ) .
angiopoietin-1 ( ang-1 ) and its natural antagonist , angiopoietin-2 ( ang-2 ) , are involved in the biology of mm .
we have shown that the ang-1 to ang-2 ratio is an independent prognostic factor for survival in newly diagnosed mm patients . however , to our knowledge , no previous studies have evaluated the above angiogenic cytokines in a large population of each of the mm stages .
the aims of this study were to : i ) evaluate the circulating levels of important angiogenesis inducers  vegf - a , angiogenin , ang-1 , and ang-2  as representative angiogenesis inhibitors in patients with mgus , smm , and symptomatic mm ; and ii ) to evaluate the gene expression of vegf - a and determine if any variability of this predominant angiogenic cytokine is present during the evolutionary process of precursor stages to symptomatic mm , contributing to better understanding the underlying mechanisms of this transition .
we studied 109 consecutive patients , of whom 55 were newly diagnosed and symptomatic with mm ( n=55 ) , and 54 asymptomatic mm patients ( n=54 ) .
we also evaluated 27 patients with mgus at diagnosis and , as an internal control group , 22 healthy controls of similar age and gender ( 12m/10f ; median age : 67 years , range : 3983 years ) .
the study was conducted after approval from the institutional ethical committee and under the guidelines of the declaration of helsinki .
the medical history of all subgroups was reviewed to ensure that there was no disease ( e.g. , cardiovascular disorder , inflammatory disease , renal impairment , or infection ) or drug administration that could alter angiogenesis at the time of sampling .
serum samples were collected at the time of diagnosis from all patients , before the initiation of any antimyeloma treatment , including supportive treatment ( e.g. , bisphosphonate administration ) and stored at 80c until the day of measurement .
patient and control group characteristics are depicted in table 1 . peripheral blood mononuclear cells ( pbmcs ) and plasma cells from bone marrow aspirates used in gene expression study
briefly , pbmcs were isolated by using ficoll - plaque density sedimentation from freshly drawn peripheral blood as previously described .
mm plasma cells from bone marrow aspirates were purified by positive selection with anti - cd138 magnetic activated cell - sorting ( macs ) separation microbeads , as described by the manufacturer ( macs , miltenyi biotec , auburn , ca ) .
purity , as confirmed by flow cytometry cd38 and cd45 staining ( facscalibur cytometer ) , was above 95% in all mgus and mm cases , and above 90% in all mm patients .
the gene expression of vegf - a in pbmcs and in mm plasma cells was estimated in a total of 10 patients with mgus , 10 patients with smm , and 10 patients with symptomatic mm .
the gene expression of vegf - a in pbmcs was also studied in 10 healthy volunteers .
circulating levels of ang-1 , ang-2 , vegf , and angiogenin were evaluated in all patients , using an elisa methodology ( r&d systems , minneapolis , mn , ) , according to the manufacturer s instructions .
total rna was extracted with trizol according to the manufacturer s instructions ( invitrogen ) and purified from salts and residual dna using the rnease mini kit ( qiagen ) .
quantity of rna in each sample was measured by spectroscopy and integrity was determined by gel electrophoresis . only rnas with clear 18s and 28s peaks were used .
vegf - a expression was studied using semi - quantitative rt - pcr as described previously .
briefly , rt - pcr was performed as a multiplex using the titan one tube rt - pcr system according to the manufacturer s instructions ( roche diagnostics gmbh , mannheim germany ) . following rt - pcr ,
the samples were treated with exosap - it to remove excess primer and deoxyribonucleotides ( dntps ; usb ) , as described by the manufacturer .
a portion of the rt - pcr product was used in the fast start dna master sybr green i kit .
this reaction was run on the light - cycler ( roche diagnostics gmbh ) until a sample reached plateau stage .
pcr products were run on 1% agarose gel , and relative quantification was performed by spot densitometry comparisons with gapdh ( glyceraldehyde-3-phosphate dehydrogenase ) bands .
primer sequences were as follows : for gapdh , forward 5-accacagtccatgccatcac-3 and reverse 5-tccaccaccctgttgcttgta-3 ; for vegf - a , forward 5-gcccactgaggagtccaacatc-3 and reverse 5-tttttgcaggaacatttacacg-3. melting curve analysis of the pcr products was performed to confirm the presence of a single specific amplicon .
differences between patients and controls , as well as between different patient subsets , were evaluated using the mann - whitney u test .
we studied 109 consecutive patients , of whom 55 were newly diagnosed and symptomatic with mm ( n=55 ) , and 54 asymptomatic mm patients ( n=54 ) .
we also evaluated 27 patients with mgus at diagnosis and , as an internal control group , 22 healthy controls of similar age and gender ( 12m/10f ; median age : 67 years , range : 3983 years ) .
the study was conducted after approval from the institutional ethical committee and under the guidelines of the declaration of helsinki .
the medical history of all subgroups was reviewed to ensure that there was no disease ( e.g. , cardiovascular disorder , inflammatory disease , renal impairment , or infection ) or drug administration that could alter angiogenesis at the time of sampling .
serum samples were collected at the time of diagnosis from all patients , before the initiation of any antimyeloma treatment , including supportive treatment ( e.g. , bisphosphonate administration ) and stored at 80c until the day of measurement .
patient and control group characteristics are depicted in table 1 . peripheral blood mononuclear cells ( pbmcs ) and plasma cells from bone marrow aspirates used in gene expression study
briefly , pbmcs were isolated by using ficoll - plaque density sedimentation from freshly drawn peripheral blood as previously described .
mm plasma cells from bone marrow aspirates were purified by positive selection with anti - cd138 magnetic activated cell - sorting ( macs ) separation microbeads , as described by the manufacturer ( macs , miltenyi biotec , auburn , ca ) .
purity , as confirmed by flow cytometry cd38 and cd45 staining ( facscalibur cytometer ) , was above 95% in all mgus and mm cases , and above 90% in all mm patients .
the gene expression of vegf - a in pbmcs and in mm plasma cells was estimated in a total of 10 patients with mgus , 10 patients with smm , and 10 patients with symptomatic mm .
the gene expression of vegf - a in pbmcs was also studied in 10 healthy volunteers .
circulating levels of ang-1 , ang-2 , vegf , and angiogenin were evaluated in all patients , using an elisa methodology ( r&d systems , minneapolis , mn , ) , according to the manufacturer s instructions .
total rna was extracted with trizol according to the manufacturer s instructions ( invitrogen ) and purified from salts and residual dna using the rnease mini kit ( qiagen ) .
quantity of rna in each sample was measured by spectroscopy and integrity was determined by gel electrophoresis . only rnas with clear 18s and 28s peaks were used .
vegf - a expression was studied using semi - quantitative rt - pcr as described previously .
briefly , rt - pcr was performed as a multiplex using the titan one tube rt - pcr system according to the manufacturer s instructions ( roche diagnostics gmbh , mannheim germany ) . following rt - pcr ,
the samples were treated with exosap - it to remove excess primer and deoxyribonucleotides ( dntps ; usb ) , as described by the manufacturer .
a portion of the rt - pcr product was used in the fast start dna master sybr green i kit .
this reaction was run on the light - cycler ( roche diagnostics gmbh ) until a sample reached plateau stage .
pcr products were run on 1% agarose gel , and relative quantification was performed by spot densitometry comparisons with gapdh ( glyceraldehyde-3-phosphate dehydrogenase ) bands .
primer sequences were as follows : for gapdh , forward 5-accacagtccatgccatcac-3 and reverse 5-tccaccaccctgttgcttgta-3 ; for vegf - a , forward 5-gcccactgaggagtccaacatc-3 and reverse 5-tttttgcaggaacatttacacg-3. melting curve analysis of the pcr products was performed to confirm the presence of a single specific amplicon .
differences between patients and controls , as well as between different patient subsets , were evaluated using the mann - whitney u test .
symptomatic mm patients at diagnosis showed increased levels of serum ang-2 ( mean value sd : 52713370 pg / ml ) compared with smm patients ( 20431881pg / ml ; p<0.001 ) , mgus patients ( 18511516pg / ml ; p<0.001 ) , and healthy controls ( 21491756 pg / ml , p<0.001 ) .
there was no statistically significant difference between asymptomatic patients , mgus patients , and controls ( figure 1a ) .
there was no difference in circulating levels of ang-1 between patients with mm , smm , mgus , and healthy controls ( table 2 ) .
consequently , the ang-1/ang-2 ratio was reduced in symptomatic mm patients ( mean value sd : 8.07.3 ) compared to smm patients ( 19.812.3 ; p<0.001 ) , mgus patients ( 22.215.3 ; p<0.001 ) , and healthy controls ( 18.016.6 ; p<0.001 ) , but there was no significant difference between asymptomatic patients , mgus patients , and controls ( figure 1b ) .
serum levels of vegf were increased in patients with mgus ( 399188 pg / ml ; p=0.02 ) , smm ( 403273 pg / ml ; p=0.04 ) , and mm ( 613408 pg / ml ; p=0.001 ) compared to healthy controls ( 286267 pg / ml ) .
patients with mm had increased vegf serum levels compared to mgus and smm patients ( p=0.039 and p=0.009 , respectively ( figure 2a ) .
patients with mgus , smm , and symptomatic mm had increased levels of serum angiogenin ( 379.9155.6 ng / ml , 377.1137.8 ng / ml and 255.6137.2 pg / ml , respectively ) compared with the healthy controls ( 165.134.3 ng / ml ; p<0.001 for all comparisons ) . patients with mm had decreased angiogenin serum levels compared to mgus and smm patients ( p<0.001 ) for both comparisons .
there was no statistically significant difference between smm and mgus patients ( figure 2b ) .
a trend was observed in the correlation of angiogenin serum levels with ang-2 ( r=0.250 , p=0.008 ) and ang-1/ang-2 ratio ( r=0.217 , p=0.022 ) .
we found that vegf - a is expressed in pbmcs and mm plasma cells from all patients in all groups analyzed . in pbmcs
, we observed no significant difference in the levels of vegf - a mrna expression between healthy volunteers , mgus , smm , and mm patients , with median sd vegf - a / gapdh ratios of 1.0190.081 , 1.0870.093 , 1.1160.091 and 1.1190.109 , respectively .
similarly , no difference in the levels of vegf - a expression was observed in mm plasma cells among the 3 patients groups , with median sd vegf - a / gapdh ratios of 0.9330.1322 , 1.0280.1279 , and 0.9730.1406 , respectively ( figure 3 ) .
symptomatic mm patients at diagnosis showed increased levels of serum ang-2 ( mean value sd : 52713370 pg / ml ) compared with smm patients ( 20431881pg / ml ; p<0.001 ) , mgus patients ( 18511516pg / ml ; p<0.001 ) , and healthy controls ( 21491756 pg / ml , p<0.001 ) .
there was no statistically significant difference between asymptomatic patients , mgus patients , and controls ( figure 1a ) .
there was no difference in circulating levels of ang-1 between patients with mm , smm , mgus , and healthy controls ( table 2 ) .
consequently , the ang-1/ang-2 ratio was reduced in symptomatic mm patients ( mean value sd : 8.07.3 ) compared to smm patients ( 19.812.3 ; p<0.001 ) , mgus patients ( 22.215.3 ; p<0.001 ) , and healthy controls ( 18.016.6 ; p<0.001 ) , but there was no significant difference between asymptomatic patients , mgus patients , and controls ( figure 1b ) .
serum levels of vegf were increased in patients with mgus ( 399188 pg / ml ; p=0.02 ) , smm ( 403273 pg / ml ; p=0.04 ) , and mm ( 613408 pg / ml ; p=0.001 ) compared to healthy controls ( 286267 pg / ml ) .
patients with mm had increased vegf serum levels compared to mgus and smm patients ( p=0.039 and p=0.009 , respectively ( figure 2a ) .
patients with mgus , smm , and symptomatic mm had increased levels of serum angiogenin ( 379.9155.6 ng / ml , 377.1137.8 ng / ml and 255.6137.2 pg / ml , respectively ) compared with the healthy controls ( 165.134.3 ng / ml ; p<0.001 for all comparisons ) . patients with mm had decreased angiogenin serum levels compared to mgus and smm patients ( p<0.001 ) for both comparisons .
there was no statistically significant difference between smm and mgus patients ( figure 2b ) .
a trend was observed in the correlation of angiogenin serum levels with ang-2 ( r=0.250 , p=0.008 ) and ang-1/ang-2 ratio ( r=0.217 , p=0.022 ) .
we found that vegf - a is expressed in pbmcs and mm plasma cells from all patients in all groups analyzed . in pbmcs
, we observed no significant difference in the levels of vegf - a mrna expression between healthy volunteers , mgus , smm , and mm patients , with median sd vegf - a / gapdh ratios of 1.0190.081 , 1.0870.093 , 1.1160.091 and 1.1190.109 , respectively .
similarly , no difference in the levels of vegf - a expression was observed in mm plasma cells among the 3 patients groups , with median sd vegf - a / gapdh ratios of 0.9330.1322 , 1.0280.1279 , and 0.9730.1406 , respectively ( figure 3 ) .
several cytokines are implicated in the angiogenesis process . the cross - talk among angiogenic and inflammatory cytokines plays an important role in the modulation of blood vessel growth in several pathological conditions , including cancer . in the abnormal tumor microenvironment ,
pro - angiogenic cytokines and inflammatory mediators prevail and promote structurally and functionally aberrant vessel formation . during tumor angiogenesis ,
quiescent endothelial cells become sensitive to vegf , then proliferate and migrate to form new vessels .
bone marrow - derived endothelial cell progenitors are found in the peripheral blood and are recruited by angiogenin at sites of angiogenesis [ 2023 ] .
mm was the first hematologic malignancy in which angiogenesis were defined as a prognostic factor .
first demonstrated that tumor growth is angiogenesis - dependent , by reporting a high correlation between the extent of bm angiogenesis ( microvessel area ) and the proliferating fraction of bm plasma cells ( labeling index ) in mgus and mm patients .
angiogenesis itself was significantly associated with active , as opposed to non - active , mm and mgus .
angiogenesis is possibly important in the progression of mgus to mm and in the progression of early - stage myeloma to advanced , refractory disease .
previous reports have shown that bone marrow angiogenesis progressively increases along the spectrum of plasma cell disorders , from the more benign stage of mgus to advanced myeloma , indicating that angiogenesis may be related to disease progression .
a proposed mechanism is that angiogenesis contributes to myeloma pathogenesis and progression by ensuring an adequate tumor oxygen and nutrient supply and by paracrine stimulation of tumor cell migration and proliferation .
however , the precise mechanism underlying the observed alterations of angiogenic cytokines in the evolution of mm has not been fully investigated ; our study aimed to answer this critical question .
vegf was increased in the serum of patients with mm as well as in patients with mgus and smm compared with healthy control samples , indicating that myeloma genesis , the transition process to symptomatic disease , occurs via mechanisms that are less vegf - dependent .
nevertheless , patients with symptomatic disease had higher vegf serum levels than other patients , perhaps due to greater tumor burden .
however , post - transcriptional modifications such as stabilization of mrna , induction of translation and stabilization of final protein could be responsible , since we found no significant difference in the levels of vegf - a mrna expression from plasma cells of mgus , smm , and mm patients . reduced angiopoietin-1/angiopoietin-2 ratio has been previously correlated with advanced disease features , including international staging system ( iss)-3 stage , renal impairment , 2-microglobulin levels , and extensive bone disease , as well as with inferior survival in newly diagnosed patients with mm who received therapy with novel antimyeloma agents . in the present study ,
symptomatic mm patients , but not mgus or smm patients , had increased serum levels of ang-2 and decreased ang-1/ang-2 ratio compared with healthy controls .
interestingly , the patients with symptomatic mm exhibit decreased serum levels of angiogenin compared to mgus and smm patients , but had increased levels compared to healthy controls .
furthermore , it is well established that there is a widespread expression pattern of angiogenin , suggesting a physiological function that is not restricted to the neovascularization process .
further investigation is needed to unravel the complex network of angiogenic cytokines and their precise role in the biology of mm .
patients with smm have similar levels of circulating angiopoietins , angiogenin , and vegf compared with mgus patients , but these levels are altered compared to symptomatic mm patients .
ang-1/ang-2 and angiogenin is reduced in mm patients with symptomatic disease compared to mgus or smm .
these results suggest that in the progression of mgus to smm , circulating angiogenic molecules appear to be the same .
in contrast , in symptomatic disease the alterations of angiopoietins pathway along with vegf contribute to myeloma cell growth . because there is great need for novel drugs active against myeloma cells , our data support that the ang-1/ang-2/tie-2 axis may be an effective target for the development of novel antimyeloma agents .
 backgroundsymptomatic multiple myeloma ( mm ) evolves from an asymptomatic precursor state termed monoclonal gammopathy of undetermined significance ( mgus ) and smoldering myeloma ( smm ) . 
 angiogenesis plays a key role in the pathogenesis of mm but there are very limited data for angiogenesis in smm.material/methodswe measured the circulating levels of angiopoietin-1 ( ang-1 ) , angiopoietin-2 ( ang-2 ) , vascular endothelial growth factor ( vegf ) , and angiogenin in 54 patients with smm . 
 the results were compared with those of 27 mgus patients , 55 mm patients , and 22 healthy controls . 
 the expression of vegf - a gene was also evaluated in 10 patients with smm , 10 with symptomatic mm , and 10 with mgus.resultsthe ratio of circulating ang-1/ang-2 was reduced in mm patients with symptomatic disease due to a dramatic increase of ang-2 ( p<0.001 ) , but not in patients with smm or mgus , in whom it did not differ compared to controls . vegf and angiogenin were increased in all patients compared to controls . however , circulating vegf was higher in symptomatic mm compared to smm and mgus , while angiogenin was reduced . 
 there were no differences in the expression of vegf - a among the 3 patients categories.conclusionssmm has a circulating angiogenic cytokine profile similar to that of mgus , but has altered profile compared to symptomatic mm . 
 thus , in the progression of mgus to smm , circulating angiogenic cytokines seem to be the same . on the contrary , in symptomatic myeloma , the alterations of angiopoietins along with vegf contribute to myeloma cell growth , supporting the target of these molecules for the development of novel anti - myeloma agents . 
Background
Material and Methods
Patients and samples processing
ELISA assay and reagents
Gene expression  PCR assay
Statistical analysis
Results
Measurement of serum levels of angiogenic cytokines
VEGF-A expression by semi-quantitative RT-PCR
Discussion
Conclusions