Trial Information

A Open Label, Phase II, Non Randomized, Clinical Trial of Chemotherapy Treatment With 5-Azacytidine Plus Valproic Acid and Eventually Atra for Patients Diagnosed With Intermediate II and High Risk Myelodysplastic Syndrome (MDS). EudraCT Number 2005-004811-31. GIMEMA Protocol MDS0205

Myelodisplastic Syndromes (MDS) are a heterogeneous group of diseases characterized by
ineffective hematopoiesis (as a result of increased apoptosis of precursor cells),
progressive peripheral cytopenia, with a tendency to evolve to acute leukemia.

The term "syndrome" represents the wide clinical spectrum of this group of diseases, ranging
from mild and stable cytopenia, with a low risk of leukemic conversion and a life expectancy
of several years, to true pre-leukemia.

The International MDS Risk Analysis Workshop recently developed a consensus risk-based
International Prognostic Score System (IPSS) for primary MDS, which has markedly improved
prognostic stratification of MDS patients. Following IPSS,it is now possible to identify
patients (i.e. Hig-Risk and Intermediate-2-Risk patients) with a bad prognosis (i.e. a
life-expectancy < 1 year) due to a high risk of leukemic evolution.

Currently, allogeneic stem cell transplantation represents the only curative therapy for
this subgroup of high-risk patients. However, this therapeutic option is often precluded for
several reasons (old age, comorbidity, lack of suitable donor).

Among the experimental treatments hitherto tested, 5-Azacitidine (5-Aza) has recently shown
promising results. Moreover, the biological experimental data suggest that the association
of 5-Aza with histone deacetylase inhibitors, such as Valproic Acid, and with
differentiating agents, such as retinoic acid, might be synergistic.

Azacitidine (Aza C), a pyrimidine nucleoside analog, was developed as an antitumor agent. In
addition to cytotoxic effects, it induces differentiation of malignant cells in vitro. Aza C
inhibits DNA methyltransferase, the enzyme in mammalian cells responsible for methylating
newly synthesized DNA, resulting in synthesis of hypomethylated DNA and changes in gene
transcription and expression. The mechanism by which Aza C produces its effects is most
likely multifactorial. Aza C can produce significant myelosuppression, particularly at
higher doses. Aza C could also be acting as a biologic response modifier. The response of
hematopoietic progenitors to cytokines is impaired in patients with MDS. This may be
attributable in part to abnormalities of the signal transduction pathway downstream from the
cytokine receptors. In vitro data suggest that Aza-C can modulate the cytokine signal
transduction pathway, rendering sensitive unresponsive cells to the effects of cytokines,
partially restoring normal hematopoietic regulation. Incorporation of Aza-C into DNA
inhibits DNA methyltransferase and induces DNA hypomethylation. 5-Azacytidine and
5-Aza-2_-deoxycytidine (decitabine), 2 potent inhibitors of cytosine methylation, have shown
strong antileukemic activity in acute myeloid leukemia (AML). In addition, both induce
trilineage responses in myelodysplastic syndromes (MDS) at dose levels allowing for
outpatient management, with moderate myelotoxicity and no significant nonhematologic
toxicity.

Azacytidine is the only drug approved by FDA for the treatment of MDS. The median survival
for patients with MDS and a Intermediate/high IPSS score, treated with conventional care
regimens, is assumed to be 11 months. Patients treated with Azacytidine in the CALGB 9221
trial had a median survival of 18 months. Responses occurred in 60% of patients on the Aza C
arm (7% complete response, 16% partial response, 37% improved) compared with 5% (improved)
receiving supportive care (P <.001). Median time to leukemic transformation or death was 21
months for Aza C versus 13 months for supportive care (P =.007). Transformation to acute
myelogenous leukemia occurred as the first event in 15% of patients on the Aza C arm and in
38% receiving supportive care (P =.001). Furthermore, Aza-C improved quality of life,
compared to supportive care.

The opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs)
allow gene expression to be exquisitely regulated through chromatin remodelling. Aberrant
transcription due to altered expression or mutation of genes that encode HATs, HDACs or
their binding partners, is a key event in the onset and progression of cancer. HDAC
inhibitors can reactivate gene expression and inhibit the growth and survival of tumour
cells. The remarkable tumour specificity of these compounds, and their potency in vitro and
in vivo, underscore the potential of HDAC inhibitors as exciting new agents for the
treatment of cancer. Histone deacetylase (HDAC) inhibitors have been shown to be potent
inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells
in vitro and in vivo. One class of HDAC inhibitors, hydroxamic acid-based hybrid polar
compounds (HPCs), induce differentiation at micromolar or lower concentrations. Studies
(x-ray crystallographic) showed that the catalytic site of HDAC has a tubular structure with
a zinc atom at its base and that these HDAC inhibitors, such as suberoylanilide hydroxamic
acid (SAHA) and trichostatin A, fit into this structure with the hydroxamic moiety of the
inhibitor binding to the zinc. HDAC inhibitors cause acetylated histones to accumulate in
both tumor and normal tissues, and this accumulation can be used as a marker of the biologic
activity of the HDAC inhibitors. Hydroxamic acid-based HPCs act selectively to inhibit tumor
cell growth at levels that have little or no toxicity for normal cells. These compounds also
act selectively on gene expression, altering the expression of only about 2% of the genes
expressed in cultured tumor cells. In general, chromatin fractions enriched in actively
transcribed genes are also enriched in highly acetylated core histones, whereas silent genes
are associated with nucleosomes with a low level of acetylation. However, HDACs can also
acetylate proteins other than histones in nucleosomes. The role that these other targets
play in the induction of cell growth arrest, differentiation, and/or apoptotic cell death
has not been determined. Our working hypothesis is that inhibition of HDAC activity leads to
the modulation of expression of a specific set of genes, which control has been disrupted by
ectopic expression of EVI1 or similar oncogene, that, in turn, result in growth arrest,
differentiation, and/or apoptotic cell death. The hydroxamic acid-based HPCs are potentially
effective agents for cancer therapy and, possibly, cancer chemoprevention.

Valproic acid Valproic acid (VPA, 2-propylpentanoic acid) is an established drug in the
long-term therapy of epilepsy. During the past years, it has become evident that VPA is also
associated with anti-cancer activity. VPA not only suppresses tumor growth and metastasis,
but also induces tumor differentiation in vitro and in vivo. Several modes of action might
be relevant for the biological activity of VPA: (1) VPA increases the DNA binding of
activating protein-1 (AP-1) transcription factor, and the expression of genes regulated by
the extracellular-regulated kinase (ERK)-AP-1 pathway; (2) VPA downregulates protein kinase
C (PKC) activity; (3) VPA inhibits glycogen synthase kinase-3beta (GSK-3beta), a negative
regulator of the Wnt signaling pathway; (4) VPA activates the peroxisome
proliferator-activated receptors PPARgamma and dagger (Rif. 25); (5) VPA blocks HDAC
(histone deacetylase), causing hyperacetylation. The findings elucidate an important role of
VPA for cancer therapy. VPA might also be useful as low toxicity agent given over long time
periods for chemo prevention and/or for control of residual minimal disease. Authors show
that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor.
Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation
of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro,
most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid
induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and
leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis
formation are significantly reduced in animal experiments. Therefore, valproic acid might
serve as an effective drug for cancer therapy. These findings constitute a "proof of
concept" to use valproic acid for the therapy of acute leukemia and myelodisplastic
syndrome.

ATRA

Retinoids have been shown to have a major role in the treatment of APL. There is also in
vitro evidence that primary blast cells from FAB groups other than M3 can demonstrate
phenotypic evidence of maturation when exposed to retinoid with or without other agents.
Additional relevant information on AML/MDS comes from a number of sources:

(I) The addition of retinoid increases the sensitivity of AML blasts to Ara C in vitro.

(II) BCL 2 protein is over-expressed in a proportion of AML cases. There is some evidence
that this correlates with treatment response and survival and, since chemotherapy results in
apoptotic cell death, this may constitute a mechanism of chemo-resistance. Retinoids down
regulate BCL 2 expression AML blasts in vitro. The increased sensitivity of AML blasts to
chemotherapy mentioned above, mediated by retinoid, is due to shortening of the BCL 2
half-life.

(III) In a pilot study by Tallman et al, 39 high risk patients with a median age of 65
years, most of whom (n=25) had failed to respond to chemotherapy, were treated with ATRA
(150 mg/m2) and LD Ara C. Forty-two percent of patients achieved a CR (25%) or PR (17%) and
a further 17% had stable disease.

(IV) In a recent study from M.D. Anderson Cancer Center, Houston, 170 patients with a
median age of 66 years with AML or high risk MDS were randomized to receive FAI
(fludarabine, Ara C and idarubicin) alone, or FAI combined with either G CSF or ATRA or
both. The overall 6 month survival was 49%. In a comparison of ATRA-treated versus
ATRA-non-treated patients, there was a more favourable outcome in recipients of retinoids,
with CR rates of 56% vs 42% respectively (p=0.06) and superior 6 month survival and event
free survival (both p=0.02) but no long-term benefit was demonstrable. In this study the
exposure to retinoid was relatively short, namely from day -2 to day +8 of chemotherapy
(Rif. 30).

(V) In a recent study from Dusseldorf, 18 patients with MDS and AML secondary to MDS were
treated with Valproic Acid (VPA) alone, and 5 patients received a combination of VPA and
ATRA. A favourable response was observed in 44% of the patients treated with VPA alone, with
a median response duration of 4 (3-9) months. Four of the five patients who subsequently
relapsed were treated with VPA + ATRA, two of them responding again. The Authors hypotize
that pre-treatment with VPA might be necessary for a synergistic effect of both drugs.

The above evidence suggests that retinoids may be a useful biological response modifier
(when combined with chemotherapy and/or VPA) with little or no additional toxicity. Their
role will therefore be examined in this trial, in association with 5-Azacytidine and
Valproic Acid.