Review Article
Innovative Exploratory Clinical Approaches for Relapsed and/or Refractory Metastatic Ewing’s Sarcoma
Ghisoli M1,2,3, Manning L4, Senzer N1,4,5 and Nemunaitis J1,2,3,4,5*
1Mary Crowley Cancer Research Centers, USA
2Medical Oncology and Hematology, Texas Oncology, USA
3Medical City Dallas Hospital, USA
4Gradalis, Inc., USA
5Strike Bio, USA
*Corresponding author: John Nemunaitis, Medical Oncology and Hematology, Mary Crowley Cancer Research Centers, 12222 Merit Drive Suite 1500, Dallas, Texas 75251, USA
Published: 08 Sep, 2016
Cite this article as: Ghisoli M, Manning L, Senzer N,
Nemunaitis J. Innovative Exploratory
Clinical Approaches for Relapsed
and/or Refractory Metastatic Ewing’s
Sarcoma. Clin Oncol. 2016; 1: 1079.
Abstract
Relapsed and/or refractory Ewing’s Sarcoma is a devastating pediatric disease with rapid progression and oftentimes severe side effects related to generally ineffective high dose multi-agent chemotherapy. Eighty five percent of diagnosed Ewing’s Sarcoma is characterized by EWS/FLI1 fusion gene expression that provides a unique opportunity for targeted therapeutics development. The EWS/FLI1 gene is a “driver gene” with transformative potential and integral to Ewing’s cancer progression. Although encoding a transcription factor, which is pharmacologically “undruggable”, it connects with potentially targetable molecular signals and, in addition, as a fusion gene along with accompanying tumor specific mutations provides unique neoantigens some of which process into immunogenic epitope. Very few cutting edge advances for the management and control of Ewing’s Sarcoma have been made in the last 20 years due, in part, to low incidence (one case per million people), a narrow therapeutic window, and a limited availability of tissue suitable for biomarker studies. However, recent advances in DNA/RNA manipulation [CRISPR and RNA interference (siRNA)] as well as in molecular and immune technologies have transformed both the understanding of signaling pathways and molecular mechanisms of actions and, consequently, the approach to target identification. We review the innovative exploratory approaches to five unique therapies (a EWS-FLI1 co-activator, the EWS-FLI1 fusion gene itself, a signaling receptor, a DNA damage repair component, and the antigenic matrix) currently undergoing clinical assessment in Ewing’s Sarcoma for which preliminary preclinical and clinical results suggest therapeutic benefit.
Background
Ewing’s Family Sarcoma is a highly aggressive and malignant bone tumor that metastasizes
frequently. The median age of diagnosis is 14-15 years [1,2] and the incidence rate is 1 case per
million people in the United States but as high as 9-10 cases per million in the 10 to 19 year old age
range [3].
Eighty five percent of Ewing’s Sarcoma patients show a balanced translocation of the EWS gene
at chromosome 22q12 with the FLI1 gene at chromosome 11q24 [4]. The EWS-FLI1 translocation
can occur at one of several different gene fusion breakpoint sites. Most frequently seen are Type
1 (accounting for 60%) and Type 2 (25%). In Type 1 EWS-FLI1, exons 1-7 of EWS are fused with
exons 6-9 of the FLI1 gene [5]. Some of the early clinical studies suggested a relationship of fusion
type to rapid progression of disease [6,7] that more recent studies have not confirmed [8]. The Type
2fusion comprised of EWS exons 1-7 juxtaposed to exons 5-9 of FLI1 [9], is associated with a higher
Ewing’s Sarcoma proliferation rate that may or may not have clinical significance. Ten to 15 percent
of patients with Ewing’s Sarcoma show other translocations; the EWS-ERG gene fusion (t(22;21)
(q22;q12)) [10-12], and the less frequent EWS-ETS fusion group (EWS-ETV1 t(7:22), EWS-ETV4
t(17;22), EWS-FEV t(2;22)) [13-16]. Methodologies used to categorize the EWS-FLI1 translocations
include real-time polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH),
and next generation sequencing (NGS) [17,18].
At diagnosis, less than 25% of patients present with metastatic disease, however up to 90%
of Ewing’s adolescents eventually experience either disease progression or relapse after frontline
treatment [3,19]. The most important prognostic factor for survival following failure of first-line
treatment is relapse-time ≤2years, which is associated with 5-year
event free survival (EFS) of only 5% [2,20]. After second-line treatment,
only 9-13% of patients will achieve second-line remission and most
of these relapse rapidly on completion [20-22]. In a retrospective
analysis of 195 advanced, ≥second-line therapy, metastatic Ewing’s
Sarcoma patients 86% did not achieve second remission and of those
(n=26) who did so, the majority either re-relapsed or died within the
year [20].There are no standard of care (SOC) NCI recommendations
for second-line treatment with advanced Ewing’s Sarcoma, although
multi-agent regimens including irinotecan, temozolomide, topotecan,
and cyclophosphamide are commonly utilized today. In addition to
relative ineffectiveness, the use of intensive chemotherapy in both
frontline and second-line treatments of Ewing’s Sarcoma is associated
with a severe toxicity and morbidity.
Given the limitation of cumulative toxicity associated with
chemotherapy and the emergence of resistance, it is not surprising
that third-line management is even more challenging. Single or
combined chemotherapy regimens only show limited response (in
both rate and durability). Some regimens include high dose ifosfamide
or gemcitabine/docetaxel [23-25]. Unfortunately, to date there has
been no significant survival advantage to any ≥third-line therapy for
patients with relapsed or refractory, disease Ewing’s Sarcoma [26].
Bottom line: there is a need for both innovative treatment
approaches and a greater array of therapeutic options in second- and
third-line management of Ewing’s Sarcoma. In the following we focus
on experimental therapies currently in clinical trial for ≥ third-line
treatment of metastatic refractory and/or relapsed Ewing’s Sarcoma
(Table 1).
Table 1
Table 1
Treatment options for patients with advanced, refractory or recurrent Ewing's Sarcoma. Comparison of objective response rate (ORR) and progression free
survival (PFS).
YK-4-279
YK-4-279 [27] is a small molecule that interacts with RNA Helicase A (RHA, encoded by the DHX9 gene) thereby affecting EWS/ FLI1 signaling activity. The EWS/FLI1 fusion protein binds RHA in a unique region not targeted by other transcriptional proteins and thereby inhibits helicase activity in a dose dependent manner [28]. YK-4-279 binds to RHA adjacent to its helicase domain and to an as yet not completely specified region on the EWS/FLI1 fusion protein to disinhibit helicase activity but without affecting ATPase activity. Erkizan and colleagues have shown that YK-4-279 may significantly shift the RNA binding profiles of both EWS/FLI1 and RHA. RHA is a transcriptional co-activator regulating both transcription and mRNA splicing and plays a role in both oncogenesis and tumor maintenance. Whatever the dominant mechanism, YK-4-279 results in inhibition of oncogenic activity and activation of caspase-3-induced apoptosis in vitro in a range of Ewing’s Sarcoma cell lines in vitro (TC32, A4573, TC71, and ES925 in Figure 1) and in vivo [29-31]. Preclinical data suggest a chimeric structure of the small molecule with (S) and (R)-enantiomers with a (S)-YK-4-279 enantiomer-specific effect in EWS/FLI1 cells [29,30], (Figure 1). Disruption of protein-protein interactions, such as the transcription complex in Ewing’s Sarcoma cells, comprising RNA polymerase II, CREB-binding protein (CBP), and RNA Helicase a (RHA) [32], thus seems to be a reasonable goal for therapeutic effectiveness. Interestingly, the same small molecule also demonstrates activity in other tumors with ETS family translocations such as ETV1 fusion-positive prostate cancer. The preclinical data demonstrates YK-4-279inhibition of tumor growth as well as decreased motility and invasion of prostate cancer xenografts [33]. While EWS/FLI1 Types 1 and 2 are the two most common translocations in Ewing’s Sarcoma and a further 10% of the patients reveal a EWS/ERG gene fusion, there is a less frequent population that presents with EWS/ETS-like fusions [13]. A similarly modified small molecular inhibitor, TK216, is listed on clinical trials.gov as a Phase I trial opportunity in patients with advanced Ewing’s Sarcoma.
Figure 1
Figure 1
YK-4-279 reverses the inhibitory effect of EWS-FLI1 on RHAin
an enantiomer-specific manner. Immobilized fulllengthRHA on a CM5
chip and purified EWS-FLI1 were used in RHA assays. Figure shows
recombinant RHA activity and ssRNA product in presence of either single
recombinant RHA, or recominant RHA / recombinant EWS-FLI1/YK-4-279,
or recombinant RHA/recombinant EWS-FLIW1/S-YK-4-279, orrecombinant
RHA/recombinant EWS-FLI1, or recombinant RHA/recombinant EWS-FLI1/
R-YK-4-279. Results were plotted over time; x-axis represents time (sec)
and y-axis is percent (product in helicase assay). Bothracemic YK-4-279 and
(S)-YK-4-279 disinhibited the helicasereaction, showing restoration of 80%
helicase activity, while (R)-YK-4-279 does not restore RHA activity(Erkizan,
Schneider et al. 2015).
Figure 2
Figure 2
Tumor Growth Inhibition and survival (a, b, c) demonstrated in
3 separate studies (studies RE-PTL-003, RE-PTL-004, RE-PTL-146).
Correlation (d) with EWS/FLI1 knockdown and downstream CD99
knockdown was demonstrated for study RE-PTL-147 (enclosed) harvested
tumor xenograft samples. Study RE-PTL-146 utilized GMP constructed
EWS/FLI1 LPX (same lot as utilized for pig toxicology). Characterized in
SK-N-MC Xenograft Model with bi-shRNA EWS/FLI1 Type 1 LPX. *indicates
GMP product(Rao, Jay et al. 2016).
Bi-sh (Bifunctional Short Hairpin) RNA EWS-FLI1 Type 1 Lipoplex (LPX)
The dual stem-loop bi-shRNA EWS-FLI1 (Type 1) incorporated into the pUMVC3 plasmid construct transcribes both siRNA and miRNA-like effectors that target the identical junction region of the EWS-FLI1 fusion gene encoded mRNA [34-36]. The plasmid is systemically delivered in a DOTAP (cationic lipid dioleoyl trimethyl ammonium propane)/cholesterol delivery vehicle [37] as a lipoplex (LPX). This RNAi technology obviates the inherent difficulty of targeting the undruggable EWS-FLI1 protein and by targeting the Type 1 breakpoint to down regulates the expression of the EWS/FLI1 encoded mRNA and protein. Preclinical testing in vitro and in vivo demonstrated 85-92% type-specific knockdown of target protein [38]. Bi-shRNA simultaneously induces RISC (RNA induced silencing complex)-cleavage-dependent mRNA degradation and RISC-cleavage-independent degradation of same nucleotide sequence. Bi-sh RNA affects targeted protein down regulation at a 5-log lower dose in comparison to si-RNA targeting the same strand sequence [34]. Furthermore, the bi-sh RNA EWS/FLI1 Type 1 dual effect or target sequence-specific activity limits the potential for offtarget effects against “non” Type 1 Ewing’s Sarcoma fusion constructs [38]. A significant tumor growth delay and survival advantage in vivo was demonstrated in human Type 1 EWS/FLI1 cells treated with the bi-sh RNA EWS/FLI1 Type 1 LPX (Figure 2). As hypothesized, the specificity of fusion gene encoded protein knockdown as compared with wild type FLI1 protein which was not knocked down was confirmed in HEK 293 cells that contained both the wild type EWS and wild type FLI1. Figure 3 shows the predicted specificity by comparing the response of SK-N-MC cells containing the EWS/ FLI1 Type 1 fusion gene to the response of HEK-293 cells with wild types EWS and FLI1 genes. Importantly, GMP (good manufacturing practice) safety testing in large animals revealed excellent tolerability [38] at active human equivalent dose ranges. Clinical investigation has been initiated in refractory/relapsed Ewing’s Sarcoma patients with the Type 1 EWS/FLI1 gene fusion (ClinicalTrials.gov).
IGF-1R Inhibitors
The EWS/FLI1 translocation is associated with dysregulation of
the insulin growth factor receptor (IGF-1R) pathway. An oncogenic
role for co-activation of IGF-1R signaling has been suggested based
on preclinical assessment [39]. In Ewing’s Sarcoma there is evidence
for autocrine activation of the IGF-1R pathway as well as EWS-FLI1
induced over expression of the caveolin-1 membrane transport protein
by way of which IGF-1R internalizes [40]. In addition, the fusion
product represses IGFBP-3 that binds IGF-1in the plasma thereby
up regulating ligand-receptor induced signaling. Enhanced IGF-1R
mediated activity can stream through two parallel pathways: 1) the
PI3k/AKT pathway inhibiting apoptosis, increasing protein synthesis
and promoting glucose metabolism and 2) the Ras/MAPK pathway
promoting cancer cell proliferation [41]. Preclinical studies in cancer
have demonstrated the relationship of the intrinsic tyrosine kinase
activity of the IGF-1R with tumor proliferative and anti-apoptotic
activity. Therefore, based on rationale and preclinical support, trials
of therapeutic anti-tumor targeting of IGF-1R have been initiated
[42-44]. In fact, IGF-1R inhibitor therapy has demonstrated benefit in
subsets of patients with Ewing’s Sarcoma [45,46]. However, activity
is limited with response rates between 6% to 14% in Phase I and II
clinical trials of patients with advanced Ewing’s Sarcoma who are
generally undergoing second or third-line treatment (Table 1) [47-
50]. Lack of effectiveness can be ascribed to multiple mechanisms
including, but not limited to, lower than expected surface membrane
receptor density, release from negative feedback inhibition pathways,
and alternative signaling via enhanced insulin receptor (IR)-A
homodimer formation.
Tap et al. [50] enrolled 38 patients including 22 with Ewing’s
Sarcoma and 16 with desmoplastic small round cell tumor (DSRCT)
into a Phase II study to test the monoclonal antibody (MAb) IGF-1R
inhibitor ganitumab (AMG479). They observed an objective response
rate of 6%. Forty-nine percent of the patients had stable disease (SD),
only four of whom achieved SD for ≥24 weeks. In another trial 115
patients with refractory or recurrent Ewing’s Sarcoma were treated
with a different MAbIGF-1R inhibitor, R1507, achieving an overall
response of 10% with a median OS of 7.6 months (95% CI, 6 to 9.7
months) [49].
A third MAb IGF-1R inhibitor figitumumab was tested in a 29
patient study (16 of whom had Ewing’s Sarcoma). Two patients
(12.5%), both with Ewing’s Sarcoma, had partial responses and
37.5% (6/16) stable disease [47]. A Phase I/II study was subsequently
conducted to investigate the safety and effectiveness of figitumumab
in patients with Ewing’s Sarcoma [48]. However, only 1 of 31 patients
achieved partial response (PR). Despite limited efficacy a Phase II
study was performed involving 106 heavily pre-treated (≥1 – 4 prior
lines of chemotherapy) patients with refractory or recurrent Ewing’s
Sarcoma. Fourteen percent(15/106) of these patients achieved PR and
23% (25/106) stable disease(SD); the median progression free survival
was 1.9 months, and median overall survival 8.9 months (95% CI,
7.2 to 11.1) [48]. Although treatment was generally well tolerated,
three cases of leukemia were observed; one attributed to figitumumab
(after one cycle in a patient previously treated with doxorubicin and
etoposide), another to concurrent rapamycin, and a third to prior
etoposide. Figitumumab associated leukemia has not been reported
in studies of other cancer types with IGR-1R inhibitors. Even though
therapy related myelodysplasia and acute myeloid leukemia are
known adverse events in Ewing’s Sarcoma patients treated with
chemotherapy, particularly in association with ifosfamide [51-53],
the possibility of figitumumab associated leukemia cannot be entirely
excluded.
These trials suggest mild to moderate clinical activity in Ewing’s
Sarcoma and establish a reasonable safety profile. Few of the studies
incorporated predictive biomarkers (e.g., increased expression of
IRS2 (insulin receptor substrate), IR, growth hormone (GH) and
decreased expression of IGF-binding protein-5) to help identify
those patients with a higher likelihood of response. Based on pathway
analysis, rationale based clinical trials involving IGF-1R inhibitors are
being explored; the concurrent administration of IGF-1R and mTOR
inhibitors to attenuate negative feedback inhibition and dual IGF-1R/
IR kinase inhibitors (e.g., Linsitinib) to block compensatory increased
expression of IR [54], (ClinicalTrials.gov ID: NCT02546544).
Figure 3a
Figure 3a
(a) Quantitative comparison of EWS-FLI1 fusion protein knockdown
by Western immunoblot. In Vitro EWS-FLI1 Fusion Gene Protein Knockdown
of SK-N-MC, EWS/FLI1 Type 1 cells with pGBI-140 (bi-shRNA EWS-FLI1
Type 1) Plasmid. (b) Growth inhibition of SK-N-MC EWS/FLI1 Type 1 cells
in correlation with EWS/FLI1 Type 1 protein knockdown. (c) Specificity of
bi-shRNA EWS/FLI1 Type 1 plasmid was further verified in demonstration of
no adverse effect on HEK-293 (non-malignant cell line not containing EWS/
FLI1 Type 1 fusion gene) cell growth. (d) The knockdown specificity was
further demonstrated on wild type FLI1 protein expression in HEK-293 cells.
FLI-1 expression in HEK293 cells is not affected by pGBI-140 treatment.
HEK-293 cells were transfected with equal amount of total plasmid DNA
containing either 9:1, 1:1 or 1:9 ratio of pGBI-140 + pIDO1-myc (transfection
control). Two days after the transfection, cells were harvested for western
immunoblot. Western immunoblot was sequentially probed with FLI-1, c-myc
or GAPDH specific antibodies on the same PVDF membrane transfer. V=
empty vector control. Quantification is normalized to GAPDH load control and
relative to empty vector transfected samples.
PARP Inhibitors
Poly-ADP-ribose-polymerases (PARP1 and PARP2) are
comprised of enzymes that transfer ADP-ribose onto target proteins
(PARylation), thereby modifying a wide range of cellular processes
including genome maintenance, transcriptional regulation, cell cycle
control, proliferation, differentiation, necrosis and apoptosis [55,56].
PARP1, activated by DNA damage, binds to DNA single-strand
breaks (SSB) and double strand breaks (DSB), then catalyzes and
promotes multiple DNA repair processes [56]. Patients with cancer
related mutations inBRCA1 or BRCA2, suppressor proteins involved
in DSB repair, demonstrate enhanced sensitivity to PARP1-inhibitors
with a consequent increase in apoptosis [57]. The anti-tumor activity
of PARP-inhibitors has been confirmed in BRCA-mutant breast,
ovary and prostate cancers [58-60]. Garnett et al. [61] was able to show
Ewing’s Sarcoma cell line sensitivity to PARP1-inhibitors by way of
decreased viability of EWS/FLI1 cancer cells (Figure 4). Brenner and
colleagues [62] hypothesized a reciprocal positive feedback loop in
Ewing’s Sarcoma; EWS-FLI1 protein driving PARP1 expression,
the latter then facilitating EWS-FLI1 transcriptional activation. In
addition, 7% of Ewing’s Sarcoma patients have been shown to have
BRCA2 mutations [63].
Choy et al. [64] conducted a two-part Phase II clinical trial
of olaparib enrolling 12 patients with advanced Ewing’s Sarcoma
progressing following chemotherapy. None of the patients had
an objective response (RECIST 1.1 criteria PR/CR). Four of the
12 patient’s sustained SD for10.9 to17.9 weeks. Median time to
progression was 5.7 weeks [64]. Based on the results of Part 1,
enrollment to Part 2 was put on hold. However, reanalysis and future
assessment of PARP1 inhibitor effectiveness as well as protocol
design need to take into account the mechanistic differences between
two recently described classes of PARP1 inhibitors; 1) those that
effect catalytic inhibition of PARP enzyme activity and 2) those that
result in formation of PARP-traps that function as cytotoxic PARPDNA
complexes [65]. On the basis of catalytic inhibitory activity, the
effectiveness of the three clinical PARP inhibitors ranks as follows:
olaparib>veliparib>niraparib. Based on active cytotoxic PARP-DNA
formation the ranking is niraparib>olaparib>veliparib.
Drug-sensitivity testing of PARP inhibition in combination with
various S-phases DNA damaging agents in Ewing’s Sarcoma cell
lines [66] showed enhanced activity with the combination of olaparib
and temozolomide. Engert et al. [67] demonstrated that combined
olaparib and temozolomide up-regulated the pro-apoptotic proteins
BAX and BAK and caspase activation. Synergistic activity was also
demonstrated with the combination of niraparib and temozolomide
[68]. These results are presumably due to interaction with the
normally sublethal effects of temozolomide induced lesions insofar
as Ewing’s Sarcoma cell lines are MGMT (O-6-methylguanine-DNA
methyltransferase) expressers and relatively resistant to the single
chemotherapeutic agent [66,69] (Figure 5). Clinical trials are ongoing
in advanced Ewing’s Sarcoma for safety and efficacy.
Figure 4
Figure 4
Sensitivity to olaparib of EWS-FLI1 (and FUS-CHOP) transformed
mouse mesenchymal cells compared to the SK-N-MC cell line (which harbors
the EWS-FLI1 fusion (Garnett, Edelman et al. 2012).
Figure 5
Figure 5
Temozolomide enhanced PARP-trapping in Ewing Sarcoma
cell lines. Fold-induction of caspase 3/7 activation in EWS-cells following
treatment with vehicle, olaparib single, temozolomide singe, olaparib/
temozolomide combination (Gill, Travers et al. 2015).
Vigil®
Vigil vaccine is a DNA engineered autologous whole tumor cell
immunotherapy which activates the afferent arm of the immune
response arc by i) using autologous tumor cells as a source of the
full matrix of tumor antigens, ii) recruiting, enhancing function and
stimulating maturation of antigen-presenting cell (APC) populations
via DNA encoded GMCSF expression and iii) dampening the escape
of immune tolerance via knockdown of immunosuppressive TGFβ
isoforms.
Vigil contains a plasmid comprised of both a DNA segment
encoding for GMCSF protein expression and a bi-functional
shRNAFurin DNA segment encoding for knockdown of Furin protein
expression(a proprotein convertase which activates TGFβ1 and 2
isoforms) and consequent knockdown of both TGFβ1 and 2 (Figure
6).
In established cancers, TGFβ is an immune-suppressive cytokine,
released by T-regulatory cells and cancer cells. Interestingly, it
has paradoxical and context-dependent effects functioning as
a tumor suppressor early in tumorigenesis and as an immune
suppressive protein in the immune escape process and in established
malignancies. In the latter context, TGFβ promotes cancer
progression and proliferation, enhances activation of T-regulatory
cells that contribute to apoptosis in APCs, and significantly decreases
IFN-γ, granzymes A and B, and perforin release by cytotoxic T-cells
[70]. By blocking these pathways, TGFβ suppresses the immune
response and promotes immune-tolerance in cancer cells [71-74].
The immunogenic activity of plasmid-encoded, cell-secreted GMCSF
has been extensively studied in a variety of GVAX trials and [genemodified]
oncolytic viral products [38,75-79].
The effective functionality of the Vigil encoded vectors is
confirmed by product release criteria which require ≥30pg of GMCSF
secreted protein/106 cells and TGFβ1,2 knockdown of ≥30% [78,80].
The gene modified autologous cells are irradiated to prevent tumor
replication, placed in pharmaceutical standard vials containing
1x107 cells and then cryopreserved. The treatment protocol specifies
intradermal administration of 1 mL vaccine every 4 weeks for ≥4-
12 doses depending on the quantity of vaccine available, Immune
effectiveness is assessed by serial INFγ-ELISPOT analysis of PMBC
response to autologous non-processed tumor cells as antigen source.
Phase I testing of Vigil in patients with advanced solid tumor types
(heavily pre-treated and burdened with tumor volume) with evidence
of progression following SOC therapies and/or FDA approved
phase I/II clinical trials demonstrates safety and suggests efficacy
of Vigil (1x107 cells or [in earlier trials] 2.5x107 cells per injection)
with a correlation of survival to ELISPOT response (>10 spots/106
cells and 2x baseline) [78,80]. A long-term update of survival status
of all Phase I treated patients [81] revealed a cohort of advanced
Ewing’s Sarcoma patients, predominantly third-line or greater, with
suggestive evidence of survival benefit; i.e., >75% survival at 1 year
compared to less than 25% survival based on historical experience.
Longer term follow up of these patients also confirmed product safety
with no evidence of Vigil related Grade ≥3 toxic effect.
More recently [82], a long term follow up of an expanded subset of
advanced, late stage metastatic Ewing’s Sarcoma patients treated with
Vigil (n=16) was performed. The results of treatment in these patients
were compared to the outcome of a non-randomized, concurrently
treated group of Ewing’s Sarcoma patients who underwent similar
surgical procedures to harvest tissue for vaccine construction but
who did not receive Vigil (n=14) for a variety of personal and/or
physician determined reasons [82]. The median OS of Vigil treated
patients (n=16) was 24 months vs. 6.8 months (Kaplan-Meier)
in the control-group (no-Vigil treatment); a 17.2 month survival
improvement (Figure 7) [82].The update also showed a 75% 1-year
survival of patients that received Vigil vs. 23% no-Vigil. Based on
these findings, a randomized Phase IIb clinical trial in patients with
advanced relapsed or refractory Ewing’s Sarcoma who have not
received ≥third-line therapy is active and ongoing.
Figure 6
Figure 6
Vigil™ is a plasmid of a bi-functional shRNA-furin DNA sequence
which prevents cleavage of pro-TGFβ precursor into functional TGFβ1
& TGFβ2, as well as a GMCSF DNA sequence which stimulates MHCI
expression, antigen presentation and adaptive immune response. Autologous
tumor cells are transfected with the plasmid via electroporation providing
full tumor antigen profile. Prior studies demonstrated in Phase I, II testing
induction of circulating cytotoxic T lymphocytes with increased immune
response to tumor cells, measured by IFNy ELISPOT response. Locally
injected transfected autologous tumor cells increasingly express antigens to
attracted dendritic cells, while immunosuppressive cytokines (such as TGFβ)
are blocking tumor-induced immune tolerance and escape. Antigen-loaded
dendritic cells activate naïve CD8+ T cells in primary lymphatic tissue and
enhance activation of cytotoxic CD8+ effector T cells that are then enabled
to circulate to target lesions for cancer-antigen specific immune response.
(Ghisoli, Barve et al. 2016).
Figure 7
Figure 7
Kaplan-Meier Survival Curve. Comparison of Vigil vs. no Vigil in
pilot trial of patients with advanced, relapsed or refractory Ewing's Sarcoma.
X-axis is time (months since tissue procurement) and y-axis is cumulative
survival. The blue curve represents patients that received Vigil, the red
curve is for the representative control group. The survival comparison shows
approx. 17.2 month survival increase of patients with advances, relapsed
Ewing’s cancer, that received Vigil compared to patients that had similar
patient demographics but were not eligible for Vigil treatment or chose other
treatment options. (Ghisoli, Barve et al. 2016).
Conclusion
Given historical lack of demonstrable effectiveness as well as a narrow therapeutic window, there are no FDA indicated treatment options for second- and third-line therapy of Ewing’s Sarcoma patients who frequently have cumulative chemotherapy related toxic thereby limiting experimental treatment eligibility opportunity. As a result of advances in “-omics” analysis and molecular immunology as well as their directed application to Ewing’s Sarcoma patients, a long needed window of opportunity has opened for exploration of innovative therapeutic options. Some evidence of activity has been suggested with single agent IGF-R1 and PARP inhibitors but, more importantly, even with failures data has been accrued and next generation studies have been implemented. Elements including biomarker identification, “-omic” analysis, pharmacokinetics and pharmacodynamics are now in place to help identify agent-specific sensitive subsets of Ewing’s Sarcoma patients and guide protocol construction. Preclinical results with YK-4-279 and bi-shRNA EWS/ FLI1 Type 1 LPX are encouraging, but on the basis of clinical results to date remain preliminary. Results from the phase I/II Vigil studies have matured and, based on analysis of outcomes, Vigil immunotherapy is currently undergoing randomized testing to determine qualification for FDA registration opportunity.
Acknowledgement
We gratefully acknowledge the generous support of the Alan B. Slifka Foundation, the Carson Sarcoma Foundation, the Chemo Warrior Foundation, Don and Linda Carter, The Crystal Charity Ball, the Jasper L. and Jack Denton Wilson Foundation, the Helen L. Kay Charitable Trust, Hyundai Hopes On Wheels, The Marilyn Augur Family Foundation, Michele Ashby, the Rutledge Foundation, the family and friends of Sam Day, the Speedway Children’s Charities, Summerfield G. Roberts Foundation, Triumph Over Kid Cancer, Wipe Out Kids’ Cancer and Young Texans Against Cancer. The authors would like to acknowledge Michelle Watkins and Brenda Marr for their competent and knowledgeable assistance in the preparation of the manuscript.
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