DCs subsets and receptors for immunovaccine (LC – Langerhans cell; pDC - plasmacytoid-derived DC; MoDC – monocyte-derived DC; DEC205 – C-type lectin receptor on DC; BDCA-2 – blood dendritic cell antigen, specific to pDC subset; DCIR - dendritic cell immunoreceptor; DC-SIGN - dendritic cell specific ICAM-3 grabbing non-integrin; MR – mannose receptor; TLR – toll-like receptor )
In this chapter the maturity of immune-therapy in cancer, with emphasis on melanoma will be discussed.
The heterogeneity of melanoma tumour regarding primary and metastatic variants will be argued. Therefore the mutational heterogeneity of this type of tumor triggers complex immune-therapy approach. Notions such as
Physiologically, the immune system can recognize cells that display an aberrant proliferation like neoplasia. The immune system is equipped with cells that can destroy cancer cells them during their early development. Years ago, when the theory of immunoediting was initiated , immunosurveillance was defined as a complex pathway that supervises and controls the elimination of transformed self cells/tissue. When the immune system cannot properly control these aberrantly proliferating cells, and the equilibrium is deregulated, tumour cells escape and form a clinically significant tumoral tissue .
In cancer immune-therapy, several approaches that aim to start and sustain the immune response and eventually elicit an immunological memory were lately tackled. The therapy armentarium that started the “bumpy road” from bench to bedside comprises cancer vaccines, adoptive T cell therapy, anti-tumor antibodies, immune checkpoint blockade and/or various immune combinations. Some of the conclusions of the last Congress of SITC (
Early diagnosed stages of melanoma are resolved mainly by surgery and large margin excision, but for advanced stages, systemic therapies, whether chemotherapy, immune-therapy or combined ones have had very low efficacy. Advanced melanoma remains a continuous clinical provocation for the physicians who use therapeutical approaches with low response rates, un-manageable toxicities, and reduced efficacy.
One of the main
Recently, a study was published on single melanoma cells and the report shows that there are 114 genes expression that could distinguish the proliferative and invasive phenotype of cells. Among these genes, regulatory networks were found along with genes that encode for pluripotency factor (e.g. POUF51); all these genes were found associated with cell’s tumorigenic potential. Authors report that among the regulatory network genes, MITF (microphthalmia-associated transcription factor) is one of the key players in the heterogeneous character of tumour cells populations. Moreover, the heterogeneity of cells depends on the 2D or 3D status of the cell cultures, thus TPBG (trophoblast glycoprotein) is expressed in a melanoma cell line, 501Mel, only in 3D cultures . In the same way, in 1205Lu melanospheres, PI3K/AKT (phosphatidylinositol 3' –kinase/protein kinase B) signaling pathway is enhanced , while DAPK1 (death-associated protein kinase 1) expression is decreased in 501Mel experimental tumors, finding that is in line with the hypermethylated gene promoter associated to melanoma . This study emphasizes that when transformed melanocytes start to organize in growing tumors, the heterogeneity of the cellular populations’ increases, the tumoral tissue having MITF-low/negative cells [4, 7]. In experimental tumour spheres, cells that grow on the exterior layers have an active proliferation, while in the interior of the tumour, due to hypoxic conditions, cells are arrested in the G1 phase . The overall mechanism is that 3D growth enhances tumour-initiating properties .
Cell’s heterogeneity is important from the immunological point of view. Tumour heterogeneous tissues have different expression of tumor antigens, thus any type of therapy that addresses only one tumour epitope is proned to have low efficacy. This is the rationale to investigate tumor antigens and evaluate the patient’s immune responses prior to any immune-therapy .
Extensive studies performed by large groups of researchers and extended networks like
Starting from 2011, metastatic melanoma beneficiated from four new approved drugs, all these drugs proving in clinical trials the improvement of patient’s survival. From this four drugs, one is a B-Raf enzyme inhibitor (vemurafenib), one is an inhibitor of the associated enzyme B-Raf (dabrafenib), and one is a MEK inhibitor (trametinib); the only one that is an actual immune-therapy, is an anti-CTLA-4 (ipilimumab) antibody . Therefore searching new efficacious immune-therapies in this disease is still an open subject of intense research.
This chapter summarizes the main achievements gathered in the last 3 years regarding immune-therapy as the ultimate approach for melanoma treatment.
2. Reducing specific immune-suppression
Until recently, the approved therapy armentarium in advanced melanoma was comprising only dacarbazine (DTIC), hydroxyurea, while the only approved immune agent was high-dose interleukin-2 (IL-2) . These drugs cannot provide satisfactory overall survival (OS) rates in advanced stages. After searching various combination of immune-therapies, from vaccination  to drugs that inhibit immune-signaling pathways , in 2011 FDA approved, ipilimumab and vemurafenib, agents that significantly increased OS and long-term improvement in advanced melanoma .
In this case, monoclonal antibodies (mAbs) as immune-therapy agents have the intrinsic role to establish an antineoplastic action through stimulation of a specific immune response. This action can be performed by inducing
The most advanced, in terms of positive research results, were the immune-related drugs that abrogated the immune –suppression such as CTLA4 or PD-1. Other mAbs were designed to aim toward stimulation of co-stimulatory receptors, molecules that are expressed by antigen presenting cells (APC); the aimed molecules were CD40 or OX40 (member 4 tumor necrosis factor receptor superfamily) or GITR (TNFRSF18), expressed on activated T lymphocytes.
Tremelimumab, also an anti CTLA4 mAb, is being evaluated in solid tumors. Nivolumab, an anti PD-1 mAb is also in the evaluation phase. This year (2014) clinical trials assessing OX40-and GITR-activating mAbs were initiated as well .
2.1. Anti-CTLA-4 antibodies
Activated T lymphocytes express transiently CTLA-4 transmembrane protein, while on T regulatory lymphocytes (Tregs) this protein is expressed constitutively. Two possible mechanisms are known accounting for CTLA-4 immune-suppressive effect. One of the mechanisms is the competitive binding to B7-1 and B7-2 in the detriment of the normal binding to CD28, delivering thus an immune-suppressive signal . Actually CTLA-4 competes with the binding of CD28 to B7, thus hindering a normal activation. The other possible mechanism is that cells expressing CTLA-4, endocytose the appropriate ligands of other cells, as such, CD28 cannot trigger activation .
Overall in cancer research, drugs that aim B7 family can enhance the therapeutic panel and thorough studies are further needed for elucidating these regulatory pathways .
The particular action of monoclonal anti-CTLA-4 antibody is to link to CTLA-4, blocking the inhibitory immune-suppressive signal, T cell can perform thus its activation pathways, proliferate while infiltrating the tumors, and in the end, set off tumour apoptosis. Designed to bind to CTLA-4, ipilimumab was approved by FDA in 2011 for advanced melanoma  and in the same year the European Commission issued a marketing authorization for ipilimumab .This type of immune-drug aims to downregulate the inhibitory activity of T-lymphocytes leading to the normal activation of T cells by allowing the binding of CD28 to B7, this costimulatory process participates to the main coupling of MHC (major histocompatibility complex) that presents the tumour antigen to TCR (T cell receptor). Re-establishing these physiological molecular interactions, T-cells can mount an efficient antitumor immune response (Figure 1) . CTLA-4 expressed on T cells is omnipresent and is not dependent upon the tumour’s particularities, thus the drug’s action should not be dependent on tumor’s characteristics.
EMA recommendation after analyzing the post-phase III results extended clinical trials [25,17] is a dose of intravenously 3 mg/kg ipilimumab over a 90 min period, this procedure needs to be repeated every 3 weeks; overall four doses should be administered . If the patients are treated in combination with DTIC it is recommended that a dose of 10 mg/kg should be used [25,17].
The actual clinical results of using this immune-therapy showed that patients with advanced melanoma increased their median OS to 10 months when compared to the 6 months OS in gp100 melanoma vaccine. When comparing OS in patients treated with ipilimumab in combination with DTIC, OS was 11 months, while DTIC monotherapy just 9 months.
As stated above ipilimumab’s action is not dependent on specific and individual tumor cell mutations, hence it can be efficient in different patients and stages [26, 27]. The beneficial clinical results regarding OS were registered as independent of various parameters such as age, gender, stage and/or previous therapy regimens.
Investigating the patients survival curves it has been shown that there are groups surviving more than 4 years , and that their clinical response was durable during the follow-up . Durability is an important differentiating criterion when recommending first-line therapy with immunomodulators in comparison to the duration obtained in kinase inhibitors treatment.
As we are focusing on an immune therapy and as
Non-cutaneous melanomas were also therapeutic targets for ipilimumab therapy. Thus, in an Australian study published in 2014 over 100 patients were followed after ipilimumab therapy. Median OS for mucosal and uveal melanoma patients was half of that registered in cutaneous melanoma patients. This report underlines the severities of adverse effects, even death-related to therapy cases, thus administration and follow-up by an experienced clinical team is extremely necessary in this type of clinical trials .
Stage IV patients presenting brain metastasis were another therapy target group as blood-brain barrier is permeable to activated T lymphocytes, cells capable of inducing a local immune response . In a large study comprising over 800 patients with brain metastasis treated with ipilimumab, up to 25% survived at least 1 year, as the un-treated median OS is only 5 months .
Adverse reactions in ipilimumab therapy are associated with hyper-immune reactions, but these can be solved by the physician using additional classic therapies. Over 10% of patients experienced gastrointestinal deregulations (e.g. diarrhea, nausea, vomiting, decreased appetite and abdominal pain), rash, pruritus, fatigue. Side-effects are manageable by the physician and, seldom, the severity can lead to treatment discontinuation (www.ema.europa.eu). These immune-related adverse effects (irAEs) are mild to moderate toxicity and are experienced by around 60% of the patients, while around 15% developed grade 3 or 4 toxicity . Endocrine system – related adverse effects were also reported in this therapy; in patients group receiving ipilimumab, 8% have experienced hypophysitis and 6% hypothyroidism/thyroiditis. Combined therapy, ipilimumab and nivolumab, induce in 22% of the patients thyroiditis or hypothyroidism and in 9% hypophysitis. Authors report hormone replacement as adjuvant therapy and immediate initiation of this therapy reverses symptoms . If the ipilimumab therapy is combined with vemurafenib, important hepatotoxicity was reported, thus caution should be taken when combining immune-therapy with this B-Raf enzyme inhibitor .
2.2. Anti-PD-1 or PD-1L antibodies
In July 2014, the first human monoclonal antibody against programmed death receptor-1 was announced as approved in Japan-
Nivolumab displayed a good tolerability profile; grade 3 or 4 adverse effects were reported in les than 18 % of patients, mainly an increased γ-glutamyl transferase .
Another group studying T lymphocyte interaction with tumour cell, interaction mediated by PD-1 receptor linking to PD-L 1, has shown that in phase I/II studies this antibody can lead to tumor regression and can enhance OS in various cancers including skin melanoma. Studying antibodies that target PD-1 or PD-1L (e.g. nivolumab, MK-3475, pidilizumab, MPDL3280A, BMS-936559, MEDI4736, MSB0010718C) the authors show that the positive clinical response goes to a maximum of 50% response rate when antibodies against PD-1 combined with anti-CTLA-4 were used. The clinical responses start early upon treatment and continue after the treatment is finished .
Another study published in 2014 searched to evaluate the survival of patients upon discontinuation of the therapy. After enrolling over 100 patients with advanced melanoma, the authors concluded that the median OS was 16.8 months .
Melanoma patients along with other cancer diagnosed patients were treated with anti-PD-1 (nivolumab). This early-phase clinical trial published in 2014 aimed to elucidate the link between PD-1, PD-L1, and PD-L2 expression, immune cell infiltration and the clinical efficacy of this therapy. The degree of PD-L1 expression depicted on tumor cells was associated with its receptor PD-1 expressed by lymphocytes. The other ligand of PD-1, PD-L2 corroborated with PD-L1 expression. The expression of PD-L1 on the tumors was correlated with the clinical efficacy of the anti-PD-1 therapy, and the found best correlation when compared to other studied factors, such as PD-1 expression and/or TIL (tumor infiltrating lymphocytes). The study concludes that, when achieving maximum efficacy with novilumab, tumor PD-L1 expression is the base of anti-PD-1 therapeutical blockade .
In 2013, results from phase I clinical trials, of nivolumab and MK-3475 (anti-PD-1 and anti-PD-L1 antibodies) were released. For MK-3475 an objective response of 38 % with only 13 % of the patients reporting grade 3/4 toxicities was shown . These results were probably the ground for further approval (see below).
Phase III clinical trails are on-going and, it seems that PD-1-PD-L1 triggers a sequence of intracellular signaling that brings important clinical benefits .
In September 2014, FDA granted accelerated approval to pembrolizumab (formerly known as MK-3475), an antibody targeting PD-1, to be used following ipilimumab therapy. A recently published report showed the efficacy and safety results of this antibody at two doses (2 mg/kg and 10 mg/kg) given every 21 days. The enrolled patients that received the therapy were refractory to ipilimumab therapy. Similar safety profiles were reported whether patients were treated with 2 mg/kg or 10 mg/kg and authors show that no drug-related deaths were registered . Another published study had similar results with the difference that the response rate between patients with or without prior ipilimumab treatment were not statistically different. Positive clinical outcome was registered with the overall median progression-free survival exceeding 7 months. Patients with advanced melanoma, prior refractory to ipilimumab, proved in this study a high rate of tumor regression .
Drug-related adverse effects were fatigue in one third of the patients and around 20% of them experienced pruritus and rash. Grade 3 fatigue was the single drug-related grade 3 to 4 adverse effect in 3% of the patients .
The positive results in a difficult to manage patients, like the ones refractory to ipilimumab, probably accelerated this drug authorization with two months ahead of its planned approval and the clinical study is still on going .
After ipilimumab approval, finding another antibody that could be used as immune-therapy for blocking an immune checkpoint like PD-1 and PD-1L gained an intense research frenzy in the last years.Therapeutical approaches that use immunomodulatory drugs have completely different mode of action in comparison to the well-known chemotherapeutical procedures. From this point of view investigating the intimate mechanisms that underlie their effect is of outmost importance because it can reveal new signaling molecules, future to be drug targets. Moreover biomarkers that can clinically predict the patient response could optimize the approach and personalize the immune-therapy .
2.3. Biomarkers for clinical benefit prediction
In the last couple of years there is a less abundant literature focusing on predictive and/or prognostic biomarkers in the immune-therapy of cutaneous melanoma. Biomarkers that were published lately range from classic serum LDH, to membrane molecules and circulating cells without any
In the last year researchers were focusing on biomarkers that can predict immune-therapy with ipilimumab outcome. Thus some studies show that ipilimumab therapy was correlated with an increase in peripheral blood absolute lymphocyte count when patients had a good clinical outcome in terms of OS. More specifically OS was 11.9 months for patients that had more than 1,000 lymphocyte count / μL peripheral blood in comparison to OS of 1.4 months in patients with lower counts [48, 49]. These results were confirmed this year when increased absolute lymphocyte count was associated with increased progression free survival (PFS) but not with OS. Any other parameters including classic serum LDH did not relate to OS or PFS .
Another cell biomarker, forkhead box P3 (FoxP3) expressed by T-regs, was correlated with positive clinical outcome in advanced melanoma patients .
Correlations were investigated regarding circulatory myeloid-derived suppressor cells (MDSC) in treated patients. Authors report that circulatory MDSC with Lin(-) CD14(+) HLA-DR(-) phenotype are increased in patients compared to normal. After surgically removing the tumour and subjecting patients’ to ipilimumab treatment, this immune parameter did not change. Then again, an interesting finding was that patients could be stratified in the ipilimumab-responders and non-responders based on the lower and respectively higher concentration of circulatory MDSC, thus pinpointing these cells as possible predictive markers of response to ipilimumab. This candidate immune-marker did not correlate with baseline serum LDH, but showed higher values in severe metastasis compared to localized metastasis to skin and/or to lymph nodes .
As to the possible efficacy biomarkers for nivolumab therapy, in a phase I clinical trial, stage III or IV patients were followed after this therapy by several biomarkers evaluation. This recent study reports that high circulatory T lymphocytes with NY-ESO-1 and MART-1-specific CD8(+) phenotype are associated with disease progression. After therapy, increased circulatory Tregs and decreased antigen-specific T cells are the two immune biomarkers that were found associated with disease progression. The expression of PD-L1 on the tumor did not correlate with the clinical response .
2.4. Animal models studying immune-therapy mechanisms
There are few studies focusing on animal models that bring new data regarding the intimate cellular mechanisms in immune-therapy. Having in mind the fact that these recent immune-therapies are limited to certain groups of patients, the published animal models searched for the resistance mechanisms that could hinder this therapy.
In 2013, the role of IDO upon an experimental anti CTLA-4 blockade was shown. Authors used IDO knockout mice and showed that, upon treatment with anti-CTLA-4 antibody, B16 melanoma was growing more slowly and that, the animals’ overall survival increased compared to normal mice expressing IDO. The mechanisms were similar when the animal model was treated with anti PD-1/ anti-PD-L1 and GITR. The authors show in this animal model that CTLA-4 and IDO inhibitors converge and that the inhibitory role of IDO can be the background mechanisms accounting for the resistance to anti-CTLA-4 therapy. Moreover, the process is T lymphocyte dependent and, if this resistance is overridden, effector T cells are found increased in tumour infiltration, the effector-to-regulatory T cell ratio increases as well . The molecular mechanisms of IDO expression are intimately related to the immune response. Several years ago it was reported that IDO expression is controlled by T activated lymphocytes through their secreted cytokines. IL-13 can repress the induction of IDO mediated by IFN-gamma . Regarding possible emerging therapies, authors report that fludarabine that hinders the up-regulation of IDO in a T lymphocytes dependent manner, can be tested as a pre-treatment drug for melanoma patients. These patients can receive afterwards immunotherapies that would have been less efficient when IDO was over-expressed .
Using animal models, new emerging therapies can be discovered, overriding the resistance to immune-therapies in certain patients groups.
3. Dendritic cells pulsed with specific antigens as inductors of specific immune-response
Treatment paradigms aim to include naturally occurring dendritic cells subsets in a single vaccine. The studies that are in the pre-clinical phases show synergistic effects between various antigen-presenting cells. We will present different types of methodologies to pulse dendritic cells, starting with mere cultivation of dendritic cells in total tumour lysates and ending with newer technologies such as electroporation mRNA-pulsed dendritic cells. Recently, the first clinical trials released their results and showed increased survival rates and broader anti-cancer immune responses. These new clinical findings will be presented.
In just a short period of time, the cancer immunotherapy field has gained new combatants through sipuleucel-T FDA approval, first DC immunovaccine for metastatic prostate cancer patients, followed closely by ipilimumab, an antibody specific to CTLA-4 as major target in metastatic melanoma . Due to its clear tumor immunogenicity, melanoma treatment could be handled from an immunotherapeutic viewpoint. However, an important issue in melanoma immunovaccine success is the highly heterogeneous composition of antigens expressed within tumor site along with different genetic patterns of melanoma patients . Deciphering this complex (intra)tumor heterogeneity of melanoma is directly linked to the possibility for clearly identifying, targeting and manageing drug-resistant cell subpopulations from the tumour site . The genetic profile of melanoma patient is one of the foremost rationales for an autologous whole cell vaccine acting more efficiently in treating the micrometastasis than would an allogeneic designed one.
Revisiting the melanoma vaccines, it was pointed out that a preponderant cytokine-driven therapy activates a robust antitumoral T-cell mediated response representing a class of individualized auto-vaccination formula, surmounting thus the melanoma intratumoral heterogeneity . Assembling a cancer vaccine aims to activate a specific anti-tumor immune response and/or to better access the tumor-associated antigens. In cutaneous melanoma, the pattern of tumor specific and tumor associated antigens is both large and heterogeneous, making melanoma immunogenicity exploitable in therapeutic approaches. Therefore, the plenty of discovered or yet hidden melanoma tumor antigens could open the way for vaccination of patients groups with the same vaccine type . Being explored with synthetic peptides, whole tumor cells, cellular lysates or autologous immunovaccine, dendritic cells (DCs) take advantages in treatment or even in prevention approaches of cancer .
Dendritic cell in melanoma immunotherapy undergo a sequential number of actions. Thus, loading DCs with a tumor antigen and a specific adjuvant will induce the maturation state which involves antigens processing by proteasomal degradation and presenting the resulted peptides to T cells
3.1. Dendritic cells subsets
In malignant skin, the principal subsets of DC responsible for Ag-specific T cell immune response comprise
|LC/dermal DC||FcR; DEC205; CD40; DCIR||Langerin||TLR-3|
|pDC||FcR; DEC205; CD40; DCIR||CD303; CD123; BDCA-2||TLR-7; TLR-9 specific to pDC|
|MoDC||FcR; DEC205; CD11c; CD40; DCIR||DC-SIGN; MR||TLR-4; 8; 3; 7.|
3.2. Exploring dendritic cells in melanoma immunovaccine
As skin is an abundant cellular immune network and hence an accessible portal for therapeutical approaches, DC cells remain an attractive target in melanoma therapy both as
3.2.1. Loading DC with tumor associated-antigens
Loading effector immune cells with antigenic peptides or a whole tumor-associated antigen (TAA) was initially designed as an immune vaccination system with T lymphocytes
Monocyte-derived DCs could be loaded in different conditions with a mixture of peptides, tumor lysates or even with a single tumor peptide such as from Mage-3A1. For 8 of 11 patients enrolled in the study it was registered an increase of Mage-3A1-specific (CD8+) T cells, with regression of a few metastases for 6 advanced melanoma patients; a lack of Mage 3A1 expression was observed in some non-regressed areas of melanoma . Even immature DCs could be exploited in vaccination, thus DCs generated from CD34+ progenitor cells were cytokines-stimulated and pulsed
Engineered DCs loaded with peptides or antigens could be delivered by lymphatic nodes or intradermally; the last type being the optimum method for generating T cell antitumor immunity .
3.2.2. DC electroporation with mRNA
Cellular electroporation is a transfection method to efficiently introduce mRNA encoding for a certain biomolecule in order to express at high level that specific antigen. A main advantage of mRNA transfection is the prolongation of the exposure and an accurate antigens processing. This approach was translated for DCs as a promising opportunity to facilitate access to tumor antigens and thus priming the T cell specific antitumor melanoma response, being applicable even in advanced stages of melanoma .
In the last few years, mRNA was proposed as an innovative vehicle for antigen delivery appropriate for cancer vaccination purposes. DCs were evaluated as the most suitable immune cells for mRNA transfection due to their professional quality in processing and presenting antigens for inducing specific immune responses by T cells. mRNA as an antigen delivery tool could generate a whole antigenic protein with all epitopes ready to be viewed by MHC molecules; last but not least, the interest mRNA molecule could be produced in large quantities with high purity.
The successful use of
The foremost parameter to be monitored for immunovaccine efficacy is the induction or the enhancement of melanoma anti-tumor immune response. Several technologies like ELISPOT technique allows quantifying the precise number of active immune cells by counting antigen-specific cells that secrete a particular anti-tumor cytokine e.g. IFN-γ . Since 2006, clinical studies with tumor mRNA transfected DCs administrated usually intradermic in patients with metastatic melanoma, detect an immunological T cell response with promising results at least for disease stabilization. In addition, advanced melanoma patients were treated intradermic with DCs simultaneously co-electroporated for 4 tumor antigens – Mage A3, Mage C2, Tyrosinase and gp100/DC-LAMP followed by TriMix mRNA single or in combination with additional therapy like IFNa-2b. In both clinical set-ups, the CD4/CD8 T cell responses were enhanced .
Recently, a small study on seven melanoma patients involved DCs from peripheral blood mononuclear cells pulsed with gp100 peptides and maturated further
4. Translating up-dated knowledge to clinics — Therapeutical future scenery
There is a recent trend that advises as first therapy a BRAF inhibitor in
In the clinical management of melanoma it is certain that immune-therapy is one of the pillars. Among this new therapeutical approaches, MEK inhibitors can overcome the resistance induced by BRAF inhibitors [79, 80]. has shown in phase 3 clinical studies, an improved .
Combination of these antibodies with the new anti-CTLA4 drug induces an astonishing 80% or more tumor regression in patients (http://www.clinicaltrials.gov/show/NCT01783938; http://trialsunited.com/studies/NCT01024231/) . Newer therapeutical combination of ipilimumab with oncolytic immunotherapy with GM-CSF-expressing engineered herpes simplex virus, have shown in phase 3 clinical studies an improved OS in advanced melanoma .
On-going studies seek to establish the clinical boundaries of NK adoptive transfer in melanoma. Last year a study was published searching to rationalize the clinical trial framework  as the prior published results of a pilot trial showed no tumor regression in spite of the increased concentration of circulating autologous NK cells persistence .
Immunotherapy is the only therapeutical “light” that can increase OS in advanced melanomas. Eliciting an efficient immune response takes a certain amount of time in order to have an efficient anti-tumoral response, but as
We strongly believe, taking into account our experience in melanoma patients follow-up [86, 87] that immune-therapy is a major therapy weapon, increasing survival in advanced melanomas. The only draw-back is that, in order to develop its full efficacy, immune-therapy takes time, and this time interval can be up to 2–4 months. Thus, an early diagnostic in the metastatic stage would make a huge difference for a positive clinical outcome .
Besides imaging-based follow-up, immune parameters should complete the panel of investigation. New data regarding circulatory MDSC can enhance this panel and prospective clinical trials should soon validate them. Resistance to immune-therapy, like IDO expression opens new therapeutical avenues aiming at immune checkpoints and combining specific antibodies with IDO inhibitors.
The immune system is still not fully explored in this type of skin cancer [89, 90], thus new insights in tumour microenvironment and the involvement of innate immunity cells could enhance the panel of new therapeutical targets.
Balancing antitumor efficacy and reconstitution of a proper functioning immune system are processes aimed by immune-therapy in cutaneous melanoma. Owing to cutaneous melanoma immunogenic outline, this disease treatment could be addressed from an immunotherapeutic viewpoint. As we are facing the great success of having the first immune-therapy approved drug in melanoma, there is an open research combat of targeting personalized/individual antigens or undifferentiating antigens-stem-like to tackle the aggressive character of this disease.
The study was financed by the following Research Projects: PN-II-PT-PCCA-2013-4-1407, PN-II-ID-PCE-2011-3-0918 and PN.09.33-01.01/2009.
Dunn GP, Bruce AT, Ikeda H, et al. Cancer Immunoediting: From Immunosurveillance To Tumor Escape. Nature Immunology 2002; 3 991–998
Dunn GP, Old LJ, Schreiber RD. The Immunobiology Of Cancer Immunosurveillance And Immunoediting. Immunity 2004; 21 137–148
Raval RR, Sharabi AB, Walker AJ, Drake CG, Padmanee Sharma P. Tumor Immunology And Cancer Immunotherapy: Summary Of The 2013 SITC Primer. Journal for Immunotherapy of Cancer 2014; 2 14
Ennen M, Keime C, Kobi D, Mengus G, Lipsker D, Thibault-Carpentier C, Davidson I. Single-Cell Gene Expression Signatures Reveal Melanoma Cell Heterogeneity. Oncogene 2014; doi: 10.1038/onc.2014.262. [Epub ahead of print]
Widmer DS, Cheng PF, Eichhoff OM, Belloni BC, Zipser MC, Schlegel NC et al. Systematic Classification Of Melanoma Cells By Phenotype-Specific Gene Expression Mapping. Pigment Cell Melanoma Research 2012; 25 343–353
Liu S, Ren S, Howell P, Fodstad O, Riker AI. Identification Of Novel Epigenetically Modified Genes In Human Melanoma Via Promoter Methylation Gene Profiling. Pigment Cell Melanoma Research 2008; 21 545–558
Thurber AE, Douglas G, Sturm EC, Zabierowski SE, Smit DJ, Ramakrishnan SN et al. Inverse Expression States Of The BRN2 And MITF Transcription Factors In Melanoma Spheres And Tumour Xenografts Regulate The NOTCH Pathway. Oncogene 2011; 30 3036–3048
Haass NK, Beaumont KA, Hill DS, Anfosso A, Mrass P, Munoz MA, Kinjyo I, Weninger W. Real-Time Cell Cycle Imaging During Melanoma Growth, Invasion, And Drug Response. Pigment Cell Melanoma Research 2014; 27(5) 764-776
Perego M, Tortoreto M, Tragni G, Mariani L, Deho P, Carbone A et al. Heterogeneous Phenotype of Human Melanoma Cells with In Vitro and In Vivo Features of Tumor-Initiating Cells. The Journal of Investigative Dermatology 2010; 130 1877–1886
Md. Selim Ahmed, Yong-Soo Bae. Dendritic Cell-Based Therapeutic Cancer Vaccines: Past, Present And Future. Clinical and Experimental Vaccine Research 2014;3 113-116
Khattak M, Fisher R, Turajlic S, Larkin J. Targeted Therapy And Immunotherapy In Advanced Melanoma: An Evolving Paradigm. Therapeutic Advances in Medical Oncology 2013; 5 105–118
Menzies AM, Long GV. New Combinations And Immunotherapies For Melanoma: Latest Evidence And Clinical Utility. Therapeutic Advances in Medical Oncology 2013; 5(5) 278–285
Saraceni MM, Khushalani NI, Jarkowski A 3rd. Immunotherapy in Melanoma: Recent Advances and Promising New Therapies. Journal of Pharmacy Practice. 2014 Mar 27. Epub ahead of print]
Garbe C, Eigentler TK, Keilholz U, Hauschild A, Kirkwood JM. Systematic Review Of Medical Treatment In Melanoma: Current Status And Future Prospects. Oncologist 2011;16(1) 5-24
Slingluff CL JrPetroni GR, Olson W, Czarkowski A, Grosh WW, et al. Helper T-Cell Responses and Clinical Activity of a Melanoma Vaccine with Multiple Peptides from MAGE and Melanocytic Differentiation Antigens. Journal of Clinical Oncology 2008; 26, 4973-4980
Bernardo-Faura M, Massen S, Falk CS, Brady NR, Eils R. Data-Derived Modeling Characterizes Plasticity of MAPK Signaling in Melanoma. PLOS Computational Biology 2014; 10(9) e1003795. doi:10.1371/journal.pcbi.1003795
Robert C, Thomas L, Bondarenko I, et al. Ipilimumab Plus Dacarbazine for Previously Untreated Metastatic Melanoma. The New England Journal of Medicine 2011;364(26) 2517-2526
Aranda F, Vacchelli E, Eggermont A, Galon J, Fridman WH, Zitvogel L, Kroemer G, Galluzzi L. Trial Watch: Immunostimulatory Monoclonal Antibodies in Cancer Therapy. Oncoimmunology 2014; 3(1) e27297
Salama AK, Hodi FS. Cytotoxic T-Lymphocyte-Associated Antigen-4. Clinical Cancer Research 2011; 17 4622–4628
Qureshi OS et al. Trans-Endocytosis Of CD80 And CD86: A Molecular Basis for the Cell-Extrinsic Function of CTLA-4. Science 2011; 332 600–603
Ceeraz S, EC, Noelle RJ. B7 family checkpoint regulators in immune regulation and disease. Trends in Immunology 2013; 34 (11) 556-563
Yervoy (ipilimumab) prescribing Information. Bristol-Myers Squibb Co. Revised December 2013. Available at: http://packageinserts.bms.com/pi/pi_yervoy.pdf. (accessed August 2014).
Hanaizi Z, van Zwieten-Boot B, Calvo G, Sancho Lopez A, van Dartel M, Camarero J, Abadie E, Pignatti F. The European Medicines Agency Review of Ipilimumab (Yervoy) for the Treatment of Advanced (Unresectable Or Metastatic) Melanoma In Adults who Have Received Prior Therapy: Summary of the scientific assessment of the Committee for Medicinal Products for Human Use, European Journal of Cancer 2012; 48(2) 237–242
Tarhini A, Lo E, Minor DR. Releasing the brake on the immune system: ipilimumab in melanoma and other tumors. Cancer Biotherapy and Radiopharmaceuticals 2010; 25(6) 601-613
Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. The New England Journal of Medicine 2010; 363(8) 711-723
Wolchok JD, Weber JS, Maio M, et al. Four-Year Survival Rates for Patients with Metastatic Melanoma who Received Ipilimumab in Phase II Clinical Trials. Annals of Oncology 2013; 24(8) 2174-2180
Eggermont AM. Can Immuno-Oncology Offer a Truly Pan-Tumour Approach to Therapy? Annals of Oncology 2012; 23(Suppl 8) viii53-57
Olszanski AJ. Current and Future Roles of Targeted Therapy and Immunotherapy in Advanced Melanoma. Journal of Managed Care Pharmacy JMCP 2014; 20(4) 346-356
Prieto PA, Yang JC, Sherry RM, et al. CTLA-4 Blockade with Ipilimumab: Long-Term Follow-Up of 177 Patients with Metastatic Melanoma. Clinical Cancer Research 2012;18(7) 2039-2047
National Comprehensive Cancer Network website. NCCN Clinical Practice Guidelines in Oncology. Melanoma v2.2014. (Available at: http://www.nccn.org/professionals/physician_gls/pdf/melanoma.pdf)
Robert C, Schadendorf D, Messina M, Hodi FS, O’Day S, for the MDX010-20 investigators. Efficacy and Safety of Retreatment with Ipilimumab in Patients with Pretreated Advanced Melanoma who Progressed After Initially Achieving Disease Control. Clinical Cancer Research 2013;19(8) 2232-2239
Alexander M, Mellor JD, McArthur G, Kee D. Ipilimumab in Pretreated Patients with Unresectable Or Metastatic Cutaneous, Uveal And Mucosal Melanoma. Medical Journal of Australia 2014; 201(1) 49-53
[Becher B, Bechmann I, Greter M. Antigen Presentation In Autoimmunity And CNS Inflammation: How T Lymphocytes Recognize The Brain. Journal of molecular medicine (Berlin, Germany) 2006; 84(7) 532-543
Eigentler TK, Figl A, Krex D, et al. on behalf of the Dermatologic Cooperative Oncology Group and the National Interdisciplinary Working Group on Melanoma. Number Of Metastases, Serum Lactate Dehydrogenase Level, And Type Of Treatment Are Prognostic Factors In Patients With Brain Metastases Of Malignant Melanoma. Cancer 2011;117(8) 1697-1703
Ryder M, Callahan M, Postow MA, Wolchok J, Fagin JA. Endocrine-Related Adverse Events Following Ipilimumab In Patients With Advanced Melanoma: A Comprehensive Retrospective Review From A Single Institution. Endocrine-related cancer 2014; 21(2) 371-381
Ribas A, Hodi FS, Callahan M, Konto C, Wolchok J. Hepatotoxicity With Combination Of Vemurafenib And Ipilimumab. The New England Journal of Medicine 2013; 368(14) 1365-1366
Nivolumab Receives Manufacturing and Marketing Approval in Japan for the Treatment of Unresectable Melanoma http://www.esmo.org/Oncology-News/Nivolumab-Receives-Manufacturing-and-Marketing-Approval-in-Japan-for-the-Treatment-of-Unresectable-Melanoma
Deeks ED. Nivolumab: A Review Of Its Use In Patients With Malignant Melanoma. Drugs 2014;74(11) 1233-1239
Lu J, Lee-Gabel L, Nadeau MC, Ferencz TM, Soefje SA. Clinical Evaluation Of Compounds Targeting PD-1/PD-L1 Pathway For Cancer Immunotherapy. Journal of oncology pharmacy practice: official publication of the International Society of Oncology Pharmacy Practitioners 2014; pii: 1078155214538087 [Epub ahead of print]
Topalian SL, Sznol M, McDermott DF, et al. Survival, Durable Tumor Remission, and Long-Term Safety In Patients With Advanced Melanoma Receiving Nivolumab. Journal of Clinical Oncology 2014; 32(10) 1020-1030. doi: 10.1200/JCO.2013.53.0105.
Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, Chen L, Pardoll DM, Topalian SL, Anders RA. Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti-PD-1 Therapy. Clinical Cancer Research 2014 Apr 8. [Epub ahead of print]
Hamid O, Sosman JA, Lawrence DP, Sullivan RJ, Ibrahim N, Kluger HM, et al. Clinical activity, safety, and biomarkers of MPDL3280A, an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM). 2013 ASCO Annual Meeting (May 31-June 4, 2013, Chicago, IL. 31(15suppl) 9010
Mamalis A, Garcha M, Jagdeo J. Targeting the PD-1 pathway: a promising future for the treatment of melanoma. Archives of dermatological research 2014; 306(6) 511-519
Robert C, Ribas A, Wolchok JD, Hodi FS, Hamid O, Kefford R, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet. 2014; 384(9948):1109-17
Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, et al.Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med. 2013;369(2):134-44.
ClinicalTrials.gov identifier: NCT01295827
Naidoo J, Page DB, Wolchok JD. Immune checkpoint blockade. Hematology/Oncology Clinics of North America 2014; 28(3) 585-600
Ku GY, Yuan J, Page DB, et al. Single-Institution Experience With Ipilimumab In Advanced Melanoma Patients In The Compassionate Use Setting: Lymphocyte Count After 2 Doses Correlates With Survival. Cancer 2010;116(7) 1767–1775
Berman DM, Wolchok J, Weber J, et al. Association Of Peripheral Blood Absolute Lymphocyte Count (ALC) And Clinical Activity In Patients (Pts) With Advanced Melanoma Treated With Ipilimumab. Journal of Clinical Oncology 2009; 27(Suppl 15) Abstr 3020
Hamid O, Chasalow SD, Tsuchihashi Z, et al. Association Of Baseline And On-Study Tumor Biopsy Markers With Clinical Activity In Patients (Pts) With Advanced Melanoma Treated With Ipilimumab. Journal of Clinical Oncology 2009; 27(Suppl 15) Abstr 9008
Meyer C, Cagnon L, Costa-Nunes CM, Baumgaertner P, Montandon N, Leyvraz L, Michielin O, Romano E, Speiser DE. Frequencies Of Circulating MDSC Correlate With Clinical Outcome Of Melanoma Patients Treated With Ipilimumab. Cancer Immunology, Immunotherapy 2014; 63(3) 247-257
Weber JS, Kudchadkar RR, Yu B, Gallenstein D, Horak CE, Inzunza HD, et al. Safety, efficacy, and biomarkers of nivolumab with vaccine in ipilimumab-refractory or -naive melanoma. Journal of Clinical Oncology 2013; 31(34) 4311-438
Holmgaard RB, Zamarin D, Munn DH, Wolchok JD, Allison JP. Indoleamine 2,3-Dioxygenase Is A Critical Resistance Mechanism In Antitumor T Cell Immunotherapy Targeting CTLA-4. The Journal of Experimental Medicine 2013; 210(7) 1389-1402
Godin-Ethier J, Pelletier S, Hanafi LA, Gannon PO, Forget MA, et al. Human activated T lymphocytes modulate IDO expression in tumors through Th1/Th2 balance. The Journal of Immunology 2009; 183 7752–7760
Hanafi L-A, Gauchat D, Godin-Ethier J, et al. Fludarabine Downregulates Indoleamine 2,3-Dioxygenase in Tumors via a Proteasome-Mediated Degradation Mechanism, PLOS ONE 2014; 9(6) e99211
Iversen TZ, Andersen MH, Svane IM. The Targeting of IDO-Mediated Immune Escape in Cancer. Basic & Clinical Pharmacology & Toxicology 2014; doi: 10.1111/bcpt.12320.
Elias EG, Sharma BK. Melanoma Vaccines, Revisited a Review, Update. G Ital Dermatol Venereol. 2014 Jul 31. [Epub ahead of print]
Seftor EA, Seftor RE, Weldon DS, Kirsammer GT, Margaryan NV, Gilgur A, Hendrix MJ. Melanoma Tumor Cell Heterogeneity: A Molecular Approach to Study Subpopulations Expressing the Embryonic Morphogen Nodal. Semininars in Oncology 2014;41(2) 259-266. doi: 10.1053/j.seminoncol.2014.02.001.
Butterfield LH, Buffo MJ. Immunologic Monitoring of Cancer Vaccine Trials Using the ELISPOT assay. Methods in Molecular Biology 2014;1102 71-82. doi: 10.1007/978-1-62703-727-3_5
Ahmed MS, Bae YS. Dendritic cell-based therapeutic cancer vaccines: past, present and future. Clin Exp Vaccine Res. 2014 Jul;3(2):113-6. doi: 10.7774/cevr.2014.3.2.113.
Shtivelman E, Davies MQ, Hwu P, Yang J, Lotem M, Oren M, Flaherty KT, Fisher DE. Pathways And Therapeutic Targets In Melanoma. Oncotarget 2014; 5(7) 1701-1752
Oosterhoff D, Sluijter BJ, Hangalapura BN, de Gruijl TD. The Dermis as A Portal For Dendritic Cell-Targeted Immunotherapy Of Cutaneous Melanoma. Current Topics in Microbiology and Immunology 2012; 351 181-220.
Benencia F, Muccioli M, Alnaeeli M. Perspectives On Reprograming Cancer-Associated Dendritic Cells For Anti-Tumor Therapies. Frontiers in Oncology 2014; 4 72. doi: 10.3389/fonc.2014.00072. eCollection 2014.
Gerlini G, Di Gennaro P, Borgognoni L. Enhancing Anti-Melanoma Immunity By Electrochemotherapy and In Vivo Dendritic-Cell Activation. Oncoimmunology 2012;1(9) 1655-1657
Palucka K, Banchereau J. Cancer Immunotherapy via Dendritic Cells. Nature Reviews Cancer 2012;12 265-277
Butterfield LH. Dendritic Cells In Cancer Immunotherapy Clinical Trials: Are We Making Progress? Frontiers in Immunology 2013;4 454.
Li H, Ma W. Intracellular Delivery Of Tumor Antigenic Peptides In Biodegradable-Polymer Adjuvant For Enhancing Cancer Immunotherapy. Current Medicinal Chemistry. 2014; 21(21) 2357-2366
Ibarrondo FJ, Yang OO, Chodon T, Avramis E, Lee Y, Sazegar H, et al. Natural Killer T Cells In Advanced Melanoma Patients Treated With Tremelimumab. PLoS One 2013; 8(10) e76829. doi: 10.1371/journal.pone.0076829. eCollection 2013
Markowicz S, Nowecki ZI, Rutkowski P, Lipkowski AW, Biernacka M, Jakubowska-Mucka A, et al. Adjuvant vaccination with melanoma antigen-pulsed dendritic cells in stage III melanoma patients. Medical Oncology 2012; 29(4) 2966-2977
Thurner B, Haendle I, Roder C, Dieckmann D, Keikavoussi P, Jonuleit H, et al. Vaccination With Mage-3A1 Peptide-Pulsed Mature, Monocyte-Derived Dendritic Cells Expands Specific Cytotoxic T Cells And Induces Regression Of Some Metastases In Advanced Stage IV Melanoma. The Journal of experimental medicine. 1999; 190(11) 1669-1678
Mackensen A, Herbst B, Chen JL, Kohler G, Noppen C, Herr W, et al. Phase I Study In Melanoma Patients Of A Vaccine With Peptide-Pulsed Dendritic Cells Generated In Vitro From CD34(+) Hematopoietic Progenitor Cells. International journal of cancer Journal international du cancer 2000; 86(3) 385-392
Lesterhuis WJ, de Vries IJ, Schreibelt G, Lambeck AJ, Aarntzen EH, Jacobs JF, Scharenborg NM, van de Rakt MW, de Boer AJ, Croockewit S, van Rossum MM, Mus R, Oyen WJ, Boerman OC, Lucas S, Adema GJ, et al. Route Of Administration Modulates The Induction Of Dendritic Cell Vaccine-Induced Antigen-Specific T Cells In Advanced Melanoma Patients. Clinical cancer research: an official journal of the American Association for Cancer Research 2011; 17(17) 5725-5735
Aarntzen EH, Schreibelt G, Bol K, Lesterhuis WJ, Croockewit AJ, de Wilt JH, van Rossum MM, Blokx WA, Jacobs JF, Duiveman-de Boer T, Schuurhuis DH, Mus R, Thielemans K, de Vries IJ, Figdor CG, Punt CJ, et al. Vaccination With Mrna-Electroporated Dendritic Cells Induces Robust Tumor Antigen-Specific CD4+ And CD8+ T Cells Responses In Stage III And IV Melanoma Patients. Clinical cancer research: an official journal of the American Association for Cancer Research 2012; 18(19) 5460-5470
Van Lint S, Heirman C, Thielemans K, Breckpot K. mRNA: From A Chemical Blueprint for Protein Production to an Off-The-Shelf Therapeutic. Human vaccines & immunotherapeutics 2013; 9(2) 265-274
Salio M, Cella M, Vermi W, Facchetti F, Palmowski MJ, Smith CL, et al. Plasmacytoid Dendritic Cells Prime IFN-Gamma-Secreting Melanoma-Specific CD8 Lymphocytes And Are Found In Primary Melanoma Lesions. European Journal of Immunology 2003; 33 1052–1062
Wilgenhof S, Van Nuffel AM, Corthals J, Heirman C, Tuyaerts S, Benteyn D, et al. Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients with advanced melanoma. Journal of Immunotherapy 2011; 34 448-456
Schuurhuis DH, Verdijk P, Schreibelt G, Aarntzen EH, Scharenborg N, de Boer A, van de Rakt MW, Kerkhoff M, Gerritsen MJ, Eijckeler F, Bonenkamp JJ, Blokx W, van Krieken JH, Boerman OC, Oyen WJ, Punt CJ, Figdor CG, Adema GJ, de Vries IJ. In Situ Expression of Tumor Antigens by Messenger RNA-Electroporated Dendritic Cells in Lymph Nodes of Melanoma Patients. Cancer Research 2009; 69(7) 2927-2934
Carreno BM, Becker-Hapak M, Huang A, Chan M, Alyasiry A, Lie WR, Aft RL, Cornelius LA, Trinkaus KM and Linette GP. IL-12p70-Producing Patient DC Vaccine Elicits Tc1-Polarized Immunity. The Journal of Clinical Investigation 2013; 123(8)3383-3394
Flaherty KT, Robert C, Hersey P, et al., for the METRIC Study Group. Improved Survival With MEK Inhibition In BRAF-Mutated Melanoma. The New England Journal of Medicine 2012; 367(2) 107-114
Falchook GS, Lewis KD, Infante JR, et al. Activity of the Oral MEK Inhibitor Trametinib in Patients with Advanced Melanoma: A Phase 1 Dose Escalation Trial. The Lancet Oncology 2012; 13(8) 782-789
Brahmer JR, Tykodi SS, Chow LQ, et al. Safety And Activity Of Anti-PD-L1 Antibody In Patients With Advanced Cancer. The New England Journal of Medicine 2012; 366(26) 2455-2465
Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab Plus Ipilimumab In Advanced Melanoma. The New England Journal of Medicine 2013; 369(2) 122-133
Kaufman HL, Ruby CE, Hughes T, Slingluff CL Jr. Current Status of Granulocyte-Macrophage Colony-Stimulating Factor in the Immunotherapy of Melanoma. Journal for ImmunoTherapy of Cancer 2014; 13(2) 11
Besser MJ1, Shoham T, Harari-Steinberg O, Zabari N, Ortenberg R, Yakirevitch A, Nagler A, Loewenthal R, Schachter J, Markel G. Development of allogeneic NK cell adoptive transfer therapy in metastatic melanoma patients: in vitro preclinical optimization studies. PLoS One 2013; 8(3) e57922
Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA. Adoptive Transfer Of Autologous Natural Killer Cells Leads To High Levels Of Circulating Natural Killer Cells But Does Not Mediate Tumor Regression. Clinical Cancer Research 2011; 17(9) 6287–6297
Neagu M, Constantin C, Manda G et al. Biomarkers Of Metastatic Melanoma. Biomarkers In Medicine 2009; 3(1) 71-89
Neagu M, Constantin C, Zurac S. Immune Parameters In Prognosis And Therapy Monitoring Of Cutaneous Melanoma Patients - Experience, Role And Limitations. BioMed Research International, special issue Molecular Biomarkers: Tools of Medicine (MBTM) 2013; 2013 Article ID 107940
Gore M. Introduction Optimal approach for melanoma. European Journal of Cancer Supplements 2013; 11(2), 80
Neagu M. The Immune System-A Hidden Treasure for Biomarker Discovery in Cutaneous Melanoma In: Makowski G.S. (ed.) Advances In Clinical Chemistry. Book Series: Advances in Clinical Chemistry; 2012. p89-140
Neagu M, Constantin C, Tanase C. Immune-Related Biomarkers For Diagnosis/Prognosis And Therapy Monitoring Of Cutaneous Melanoma. Expert Review Of Molecular Diagnostics 2011; 10(7) 897-919