the treatment of metastatic melanoma has been revolutionized in the last three years , with the fda registration of yervoy , a monoclonal antibody blocking lymphocyte regulatory receptor cytotoxic t lymphocyte - associated antigen 4 ( ctla-4 ) , and shortly afterward , the entry to the clinic of zelboraf , a small molecule inhibitor of mutated b - raf . from being an incurable disease ,
stage iv melanoma has become an illness in which prolonged and even complete responses can be envisioned .
however , while the prospects have improved for stage iv disease , for patients with american joint committee on cancer ( ajcc ) stage iii disease no new treatment options have been developed and validated since the approval of interferon  ( ifn ) almost two decades ago [ 17 ] .
a pegylated formulation did not offer improved tolerability and treatment was discontinued due to toxicity .
both the eortc trials and the ecog pooled analysis   showed that ajcc stage iii patients with macroscopic lymph node involvement derived the smallest survival benefit , if any , from ifn. in this situation , melanoma vaccines could have been an alternative to ifn , since they could induce a tumor - specific immune response to inhibit micrometastases at a stage when the suppressive effects of an advanced tumor are not yet an obstacle . in the few controlled clinical trials reported to date , vaccinated patients did not experience a survival benefit .
this fact , together with the paucity of objective responses to active immunization in stage iv melanoma patients using different vaccination strategies , rendered a general impression of the futility of cancer vaccines [ 11 , 12 ] . the resurgence of interest in cancer vaccination was the result of several clinical trials demonstrating that a component of active immunization could improve clinical outcome of immunotherapy protocols .
examples include the addition of a peptide vaccine to the administration of high dose interleukin-2 ( il-2 )   and the use of a gmcsf - secreting tumor vaccine in combination with ctla-4 blockade for metastatic prostate cancer .
these trials led to the increasing understanding that cancer immunotherapy is a multifaceted strategy and that a single treatment modality would not suffice . noting that individuals exhibit heterogeneity of tumor antigens , the use of autologous tumor as a basis for vaccination can provide antigen authenticity .
the unique expression profile of normal and mutated proteins in the patient 's tumor cells is presented in conjunction with their own major histocompatibility complex ( mhc ) molecules , and this combination is necessary to induce antigen - specific reactive lymphocytes .
the coadministration of bacillus calmette - gurin ( bcg ) , a widely used immunological adjuvant , with the autologous vaccine , has previously been shown to enhance response to autologous vaccination protocols [ 17 , 18 ] . in this paper
we report our experience with an autologous melanoma vaccine as an adjuvant therapy for melanoma patients in the advanced categories of ajcc stage iii disease : macroscopic lymph node involvement and resectable in - transit metastases .
we demonstrate that clinical immune response ( delayed type hypersensitivity , dth ) is linked to improved survival .
furthermore , using this vaccination protocol , molecular analysis of the melanoma showed that cancer testis antigens ( ctas ) , which are generally considered targets of immune response , are predictive of survival .
interestingly , patients who had previously received the melanoma cell vaccine had improved overall survival when treated with ipilimumab immunotherapy for metastatic disease .
this prospective phase ii , single - institution , single - arm study included patients with operable ajcc stages iiib and c melanoma .
since we did not have pathological data on ulceration of the primary melanoma for all patients , ajcc stage iiib was defined for any t ( tumor ) with pathological n1b , n2b , and n2c .
the aim of the study was to document overall and disease - free survival ( primary outcome ) and to correlate skin reactivity to the autologous tumor of patients that were treated with the autologous melanoma cell vaccine with survival ( secondary outcome ) . to participate , patients had to undergo complete removal of their metastatic disease and have a normal ct scan within 30 days prior to vaccination .
additional inclusion criteria included an age of 18 years or older , normal liver and renal function tests , baseline lactate dehydrogenase ( ldh ) value below the laboratory upper limit of normal , and ability to provide informed consent .
fertile patients were requested to use adequate anticonceptive measures throughout the study period . screening procedures included baseline blood analyses ( hematological , chemical ) and computed tomography ( ct ) and/or magnetic resonance imaging ( mri ) of the entire body , including the brain , every 4 months in the first 2 years and then every 6 months for 10 years .
the study was not sponsored and was conducted following approval by the institutional ethics committee ; written informed consent was obtained from all patients .
seventy - five patients from this group were included in an immune monitoring study reported earlier .
for the preparation of the autologous vaccine , melanoma cell lines were established from resected metastases .
briefly , cells were extracted mechanically from fresh and sterile tumor specimens , frozen , and stored in liquid nitrogen in a medium containing 2.5% human albumin and 20% dmso until needed . to assure melanocytic progeny , the expressions of s100 , mart-1 , and gp100 were determined by immunostaining using polyclonal rabbit anti - s100 , monoclonal a-103 , and hmb-45 abs , respectively ( dako ) .
positive staining of more than 50% of cells with at least one of these antibodies was required .
mhc class i - related chain a ( mica ) expression was determined by flow cytometry of melanoma cell lines using anti - human mica - apc ( allophycocyanin ) , r&d systems , minneapolis , mn , usa . cell lines were routinely tested for mycoplasma contamination by ez - pcr ( biological laboratories , beth haemek , israel ) , according to the manufacturer 's instructions .
tumor cultures that were found contaminated were incubated in the presence of 10  mg / ml ciproxin 200 ( bayer ) for two weeks , with change of medium every three days .
the cells were retested after treatment and were used only after being found mycoplasma - free .
melanoma cell lines were expanded to the required number necessary for preparation of at least 8 vaccine doses of 1025  10 cells each and cryopreserved at 70c . on the day of treatment ,
one dose of cells was thawed , washed , and irradiated to a dose of 230  gy . at this stage , cells were still viable .
conjugation of melanoma cells with dnp was performed as described , leading to death of the cells ( as determined by trypan blue exclusion ) .
bcg ( statens serum institut , denmark ) was added to the vaccine prior to injection , diluted to 1  :  50 for the first three vaccine doses and up to 1  :  500 for the following doses , to avoid overreactivity at the injection site .
bcg was reported to trigger dendritic cell maturation and to aid in diverting the cd4 t cell response towards a th1 phenotype .
briefly , patients were sensitized to dnp , to enhance the response to the vaccine , by applying 0.1  ml of 2% dnp dissolved in acetone - corn oil ( sigma ) topically to the inner aspect of the arm ten days prior to injection of the first vaccine .
cyclophosphamide , 300  mg / m per dose , was given 4 days preceding the first and second vaccines .
this practice was based on the observation that cyclophosphamide prior to vaccination can reduce the proportion of t regulatory cells and enhance tumor - specific immune response [ 2225 ] .
generally , vaccines were injected in 3 adjacent sites on the upper arm or thigh , avoiding limbs with dissected lymphatic basins .
patients were evaluated periodically every 3 months and had a total body ct scan performed every 6 months , or as required according to their symptoms .
evaluation of dth to autologous melanoma cells was performed on the vaccine days 5 and 8 , by intradermal injection of 13  10 irradiated ( 170  gy ) autologous melanoma cells .
erythema diameter of 5  mm and less was arbitrarily defined as negative ; 510  mm weak ; 1015 
thirty - five melanoma cell lines that were retrieved from 35 vaccinated patients were selected for gene expression analysis based on retrospective survival data of the donors .
gene expression profiling was performed using an assay based on a collection of cellular genomic signatures that produced a pattern - matching tool and formulation - based deduction of a wider expression profile .
one thousand transcripts were identified , from which the remainder of the transcriptome could be computationally inferred .
these 1,000  landmark  transcripts were measured on luminex beads , as part of the connectivity map ( c - map ) project ( unpublished , r. narayan , broad institute of harvard university and mit , cambridge , ma ) .
cultures underwent a median of 4 passages ( range 213 ) and mrna was extracted from melanoma cell microcultures harvested at 90% to 100% confluence , produced in a synchronous way , under identical conditions of growth , in four replicates .
initial analysis was conducted with a bioconductor- ( r- ) based test ( ssat ) , which applies a cox - regression analysis followed by a second test based on rank statistics . the analysis determines the best cut - off value that separates patients into those with favorable versus worse survival time .
rna was normalized to gapdh - rna content using abi 7500 sds software , v1.2.2 ( applied biosystems inc .
positive and negative controls , as well as samples with no dna , were included in every qrt - pcr experiment .
pcr reactions were performed using abi qrt - pcr thermocycler ( 7500 real time pcr system , applied biosystems inc . , foster city , ca ) .
the qrt - pcr program was run for 45 cycles , following an initial incubation at 95c , 10  min .
each cycle consisted of 95c    15  sec and 60c   1  min . 

b - raf genotype was determined using cobas 4800 ( roche ) . during the autologous vaccine study period ,
ipilimumab ( yervoy , bms ) was administered in our institution to patients with metastatic melanoma in the framework of bms studies ca184 - 004 , 024 , and 025 . since its fda approval as a standard second - line treatment
patients from the autologous vaccine study which later developed nonresectable metastatic disease were among a larger group recruited for these protocols .
the comparison of survival curves between groups was carried out using the kaplan - meier survival analysis with the log rank test .
all tests applied were two - tailed , and a p value of 5% or less was considered statistically significant .
a total of 126 patients ( 55% male ; median age , 59 years ) with postoperative ajcc stages iiib and c ( 45% stage iiib ; 55% stage iiic ) were enrolled . for patient characteristics
twenty - four patients ( 19% ) presented with enlarged lymph nodes ( lns ) at the time of diagnosis of the primary melanoma ; 11 ( 9% ) had unknown primary lesion ; 22 ( 17% ) had metastases in transit .
nineteen patients ( 15% ) had noninvolved sentinel ln but developed macroscopic disease later , in the same lymphatic basin .
forty - two patients ( 33% ) had not undergone a sentinel ln biopsy and developed macroscopic ln involvement .
the mean number of involved lymph nodes was 2.4 , ranging from 0 ( satellites ) to 17 .
fifty - six patients ( 44% ) had undergone radiotherapy in addition to the surgical procedure , which was added when more than three nodes were involved or in cases of extracapsular invasion by melanoma cells .
no grade 3 - 4 adverse events ( ctcae v4 ) were encountered among the 126 vaccinated patients . in all patients , an erythematous nodule developed at the vaccination site and resolved in the course of 36 months leaving a depressed scar .
the os and disease - free survival ( dfs ) of participating patients were measured from the day of the first vaccine until the current analysis was performed .
a total of 126 patients were included , with a median follow - up of 44.5 months ( range 8189 months ) .
os survival data was available for all 126 patients and dfs data was available for 107 patients . os and dfs were analyzed for dth < 15  mm ( weak / positive dth ) versus dth > 15  mm ( strong positive dth ) .
overall , for the whole cohort , the 5-year os was 54% and dfs was 34% .
there was no difference between os of stage iiib and iiic patients ( p = 0.182 ) .
of 119 patients with recorded dth response , 48 patients ( 40% ) attained strong positive dth ( dth > 15  mm ) , whereas 71 ( 60% ) had a weak dth response ( < 15  mm ) .
the patients with strong dth response had a 5-year os of 75% and dfs of 47% .
in contrast , patients with weak dth had a significantly lower 5-year os of 44% ( p < 0.0001 ) and dfs of 26% ( p = 0.27 ) .
using the kaplan - meier analysis and the log rank test , the single parameter that most strongly correlated with os and dfs in a univariate analysis was the dth response ( figure 1 ) . in table 3 , os and dfs are compared between patients attaining dth responses of 10 and 15  mm and patients who did not develop such response . for patients who attained strong positive dth ( > 15  mm ) , the 5-year overall survival hazard ratio ( hr ) was 0.24 ( 95% ci 0.10.53 ; p < 0.001 ) .
the hr for 5-year disease recurrence was 0.4 ( 95% ci 0.10.83 , p = 0.015 pearson 's chi square test ) , but in a longer follow - up , the protection from recurrence decreased to a hr of 0.63 ( 95% ci 0.31.32 ; p = 0.2 ) .
age , gender , and depth of invasion of the primary melanoma had no impact on survival . in a survival analysis done for
dth cut - off of 10  mm , a similar trend was noted with a smaller p value ( 0.003 ) for improved 5-year os in patients attaining a dth response of > 10  mm ( 64% ) versus 32% in patients with dth < 10  mm .
thus , the acquisition of powerful skin reactivity against nonmodified autologous melanoma cells , which reflects the development of specific cell mediated immunity , correlates favorably with survival , supporting previous results by us and by others , for example , [ 16 , 18 , 26 ] . for
56 patients in whom more than 3 involved lymph nodes were removed , radiation therapy was added to the resected lymphatic basin .
even though the patients requiring this added treatment belonged to a grave prognostic group , radiotherapy may enhance the immune response of the patients .
five - year os of these patients was 58% compared to 33% of those who received radiotherapy and had a weak dth response ( p = 0.024 ) .
the c - map project was based on a collection of cellular genomic signatures to drugs , disease states , and cancer , in order to produce a pattern - matching tool and a formulation - based deduction of a wider expression profile .
one thousand transcripts were identified from which the remainder of the transcriptome could be computationally inferred .
these 1,000  landmark  transcripts were measured on luminex beads ( unpublished , r. narayan , broad institute of harvard university and mit , cambridge , ma ) .
thirty - five tumor samples , representing distinct subclasses of poor and good responders , were selected for c - map analysis : ( 1 ) eighteen poor responding patients with a median os of 19 months ( range 834 ) , all of whom failed to develop strong skin reactivity to their autologous tumor , and ( 2 ) seventeen good responding patients with median os of 105 months ( range 46194 ) , 12 of whom also developed strong skin reactivity ( dth data missing for one ) .  
figure 2 shows the hierarchical clustering of 50 genes expressed on patients ' melanoma cells , which yield a significantly improved or worsened hr for survival .
cancer testis antigens ctag2 ( ny - eso-2 ) , magea1 , ssx1 , and ssx4 clustered together in the hierarchical diagram ( depicted in a circle in figure 2 ) .
high expression of each of these cta genes was associated with a reduced risk of death .
as cta genes are coexpressed , we performed a principal component analysis ( pca ) of the genes to stratify the patients , using the  princomp  function in r. we found the first principal component ( pc1 ) to explain more than 80% of the variance when expression values from the 35 samples corresponding to all 51 probe sets from cta genes in the c - map array were used ( figure 3(a ) ) . to validate the predictive data extracted from c - map , we used qpcr results obtained from 21 melanoma line samples for mage - a1 , ssx1 , ssx4 , and ny - eso-1 ( ctag1b ) .
similar to the in silico data , a pca analysis based on these four genes was able to explain 75% of the variance . in both analyses , stratifying the patients into low / intermediate and high expression based on pc1 values , we show worse outcome for low cta expressors ( below quartile 1 ) compared to high expressors ( above quartile 4 ) , p = 0.02 ( figure 3 ) .
a similar analysis performed using an unrelated melanoma cohort ( gse19234 ) , which never had an autologous vaccine , did not reveal an association between cta gene expression and survival ( not shown ) , suggesting that cta gene expression is not a prognostic factor by itself but becomes one in the context of autologous vaccination .
mica ( mhc class i related chain a ) , another gene which correlated with a reduced risk of death and for which there is an antibody for flow cytometry , was used to validate the expression data ( figure 4 ) .
out of 11 samples analyzed , 10 yielded mica protein expression ( by flow cytometry ) concordant with the gene expression value . in 8 samples
the data was predictive of patient 's current status , whether alive , no evidence of disease ( ned ) , or died of disease ( dod ) .
b - raf status was determined for 32 patients of the 35 that were analyzed for gene expression .
the median survival for patients with v600e mutation was 50 months compared with 45 months for the wild type ( wt ) group ( p = 0.9 ) .
since most patients in the series who had recurrent disease died before 2012 , none of them were treated with b - raf inhibitors when they developed metastases , and consequently differences in survival could not be attributed to better treatment options .
four out of 11 patients with v600e mutations had strong dth response ( 36% ) , compared with seven out of 21 ( 33.3% ) in the wt group ( p = 0.86 ) .
thirty - five patients who received melanoma cell vaccine and developed nonresectable metastatic disease were referred to ipilimumab treatment in bms studies ca 184 - 004 , 024 , and 025 and later as a standard second - line treatment .
these patients were compared with a nonselected concurrent group of other 35 patients , who never received the vaccine ( table 2 ) .
the majority , 62 patients ( 89% ) , received ipilimumab at a dose of 3  mg / kg .
response evaluation criteria in solid tumors ( recist ) were used to define response to treatment .
complete response was achieved in six patients ( 8.6% ) , partial response in 14 ( 20% ) , and stable disease in 7 ( 10% ) .
multivariate analysis revealed that the single parameter that correlated with an improved os time was previous treatment with melanoma cell vaccine . in the group of 35 vaccinated patients , median os was 31 months with a 3-year survival of 46% , compared with a median os of 9 months and 3-year survival of only 19% in the nonvaccinated patients , ( p = 0.007 , pearson 's chi square test ) .
the results from this retrospective cohort of patients suggest that response rate and survival are improved when ipilimumab treatment succeeds autologous melanoma vaccine .
in this phase ii study we administered a vaccine composed of the autologous tumor given to melanoma patients as a postoperative adjuvant for ajcc stage iii disease following resection of macro metastases .
the study was single - armed , as it was initiated prior to the registration of ifn as standard of care for the adjuvant treatment of melanoma stages iib and iii . in an initial cohort , which included patients of worse prognoses ( stages iiib , iiic , and iv ) we observed an unexpected good survival rate with this vaccine . in view of the reduced toxicity of the vaccination regimen , we did not offer patients a high dose interferon arm after its registration .
thus , we opted to continue with the protocol in its single - arm design , to record survival rates in adjuvant stage iiib and c patients . eventually , these patients were the less likely ones to derive benefit from adjuvant ifn therapy , since treatment with the standard of care ifn-2b or peg - ifn did not yield any survival advantage for them .
the projected 5-year os for these patients was estimated at 40% and the dfs at 30% , as shown in the kaplan - meier curves generated in a meta - analysis from eortc trials 18952 and 18992 of patients with ajcc stage iii - n2 . in our group of patients with the same stage disease , the 5-year os reached 54% , with a median os of 88 months and a 5-year dfs of 34% ; these were achieved with no major adverse effects . in another series ,
the nordic study , the best median survival , 72 months , was derived from intermediate - high doses of ifn-2b for one year .
these results , worse than those we observed with the autologous vaccine , were generated in a group of patients in which 35% had earlier stage disease ( iiia ) .
notably , in our study population , patients with a strong positive skin reaction to their unmodified melanoma had much better prognosis , with a 5-year os of 75% and dfs of 44% .
the link between immune parameters that attest to the development of antitumor response and improved survival has been previously observed [ 17 , 3036 ] .
dth is a crude measurement , but an easy test to apply on all patients .
several vaccine clinical trials included a dth test as part of their clinical evaluation and reported a positive correlation between dth response and longer survival [ 37 , 38 ] .
biopsies taken from skin injection sites revealed vaccine - induced antigen - specific t cells [ 39 , 40 ] .
we previously demonstrated effective antimelanoma cd4 t cell activity associated with improved survival in a cohort included in the present study .
thus , a positive dth reaction could be indicative of the emergence of antimelanoma immunity , and the improved survival of patients attaining strong dth would attest to the vaccine 's protective effects .
since autologous tumor tissue is often not available for vaccine preparation , defined antigens , consisting of short or long peptides , are used as a substitution [ 4143 ] .
the enthusiasm for the use of defined antigens decreased when successful generation of antigen - specific t cells failed to protect against tumor progression [ 11 , 44 ] .
loss of mhc and impaired peptide presentation by the melanoma were among the reasons for vaccine failure , but another limitation is that many tumor - progeny antigens are essentially self - antigens that evoke weak responses . unlike wildtype proteins , mutations have the potential to generate neoantigens which are better targets .
the significance of mutation - derived neoantigens was illustrated when adoptively transferred tumor - infiltrating lymphocytes that destroyed melanoma in patients were surveyed and found to target mutated epitopes .
furthermore , recent data has clearly demonstrated that these mutations are , in large part , the functional targets of immune checkpoint blockade .
but until the hurdles of preparing a totally individualized vaccine are overcome , the best source of mutated antigens , as we have previously shown , is still the autologous tumor and , ideally , those tumors that express both mhc class i and class ii . another important component of melanoma cell vaccines , as reflected by our data , are a class of antigens known as cancer testis antigens .
ctas are products of several multigene families , many of which map to chromosome x , that have arisen through chromosomal duplications and were initially identified through immunologic assays . when associated with disease outcome ,
ctas sometimes confer worse prognosis , but when protective immune responses are recorded , ctas are dominant targets .
for example , rising antibody responses to cta ny - eso-1 ( ctag1b ) were recorded following shrinkage of melanoma in a patient with abscopal tumor response , and among patients treated with ipilimumab , seropositivity to ny - eso-1 with associated cd8 t cell response correlated with 77% clinical benefit . using a luminex - based method to generate a gene expression array and qpcr validation , we showed increased expression of magea1 , ctag1 , ctag2 , ssx1 , and ssx4 in patients with improved survival .
we suggest that these patients ' prolonged survival is attributed to the melanoma immunogenicity potentiated by the ctas and augmented by the vaccination procedure .
lastly , our data implies that patients who had been immunized against the autologous tumor prior to receiving ipilimumab survived longer than patients who did not receive an autologous melanoma vaccine .
ctla-4 receptor blockade prevents inhibition of activated effector lymphocytes and selective increase in the ratio between t effectors and regulatory t cells within the tumor . in patients with preexisting immune response
, the removal of inhibitory signals boosts antitumor activity , leading to the hypothesis that prior vaccination might induce in a subset of patients a population of specific antitumor cytotoxic t cells and a potent immune response following immune checkpoint blockade . in this respect , it was encouraging to note that in a cohort of vaccinated patients that had received ipilimumab , the 3-year survival was 46% , compared with 19% for the nonvaccinated patients
in this noncontrolled phase ii study we have shown that adjuvant treatment with autologous melanoma vaccine yields overall and disease - free survival rates that are not inferior to those obtained with interferon alpha . in a subgroup of patients who attained a strong positive skin response to unmodified autologous tumor ,
lastly , improved survival time following ipilimumab treatment was observed for patients who had previously been vaccinated .
thus , we suggest that autologous melanoma vaccine induces protective immunity and may offer leverage for other immunotherapies .
 
 background . 
 there is not yet an agreed adjuvant treatment for melanoma patients with american joint committee on cancer stages iii b and c. we report administration of an autologous melanoma vaccine to prevent disease recurrence . 
 patients and methods . 
 126 patients received eight doses of irradiated autologous melanoma cells conjugated to dinitrophenyl and mixed with bcg . 
 delayed type hypersensitivity ( dth ) response to unmodified melanoma cells was determined on the vaccine days 5 and 8 . 
 gene expression analysis was performed on 35 tumors from patients with good or poor survival . 
 results . 
 median overall survival was 88 months with a 5-year survival of 54% . 
 patients attaining a strong dth response had a significantly better ( p = 0.0001 ) 5-year overall survival of 75% compared with 44% in patients without a strong response . 
 gene expression array linked a 50-gene signature to prognosis , including a cluster of four cancer testis antigens : ctag2 ( ny - eso-2 ) , magea1 , ssx1 , and ssx4 . 
 thirty - five patients , who received an autologous vaccine , followed by ipilimumab for progressive disease , had a significantly improved 3-year survival of 46% compared with 19% in nonvaccinated patients treated with ipilimumab alone ( p = 0.007 ) . 
 conclusion . 
 improved survival in patients attaining a strong dth and increased response rate with subsequent ipilimumab suggests that the autologous vaccine confers protective immunity . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions