Residual Hypermethylation in Early Stage Non-Small Cell Lung Cancer (NSCLC) As Part of Adjuvant Therapy and Preventive Strategy

Adjuvant chemotherapy has become an essential part for the treatment of stage IB-IIIA NSCLC
patients based on 4 randomized clinical trials showing survival advantage for patients who
received adjuvant CT, oncologists initially started to adapt this modality of treatment for
pathologically stage IB-IIIA NSCLC patients [4]. Herein, NSCLC patients will be treated in
the adjuvant setting with conventional doublet platinum-based chemotherapy followed by
5-azacitidine, a demethylating agent. The planned dosages to be used in this trial have been
extensively studied in previously conducted clinical trials involving all therapeutic
agents.

All patients will undergo thorough surgical resection of the primary lung tumor, and
evidence of promoter hypermethylation of gene(s) must be present in the tumor specimen
and/or blood sample (plasma or WBC). The collection of tumor specimen will be considered a
priority to correlate the tumor "methylation pattern/profile" with the serum "methylation
pattern/profile". If tumor specimen is not available for any reason, but the patient has at
least one of the 9 targeted genes hypermethylated in the serum, the patient is eligible to
be enrolled into the trial.

Treatment will consist of 2 parts:

Part A. Adjuvant chemotherapy.

Following surgical resection of NSCLC and test positive for promoter hypermethylation in at
least one (1) of the targeted TSGs described, patient will start adjuvant conventional
chemotherapy (doublet platinum-based chemotherapy) for stage IB-IIIA NSCLC. In the case a
patient with stage IB refuses adjuvant chemotherapy, the patient still can be enrolled into
Part B of the study if he/she understands the concept of the trial and sign an informed
consent.

Conventional chemotherapy will be selected at discretion of the treating physicians except
on those cases in which pathologic diagnosis indicates non squamous NSCLC. Those patients
will be treated with pemetrexed. Patients will receive a total of 4 cycles of adjuvant
chemotherapy (each cycle given every 21 days). Pegfilgrastim, a granulocyte-colony
stimulating factor (G-CSF), will be allowed on day 2 at discretion of the clinician.

All patients should be premedicated prior to paclitaxel or docetaxel administration in
order to prevent severe hypersensitivity reactions (HSR). Such premedication may consist of:
dexamethasone 20 mg orally administered approximately 12 and 6 hours before paclitaxel or
docetaxel; diphenhydramine 50 mg intravenously and cimetidine (300 mg) or ranitidine (50 mg)
intravenously 30 minutes before paclitaxel. In case of pemetrexed, patient will received an
injection of vitamin B-12 1,000 mcg IM a week prior to start therapy as well as folic acid 1
mg po daily a week before pemetrexed infusion and during the entire course of treatment with
this antifolate agent.

The dosages and toxicities of these chemotherapy agents are very well described in the
clinical setting, and patients will be managed as any other patient receiving standard
chemotherapy.

Part B. Targeted therapy using 5-azacitidine.

If the patient decides not to receive adjuvant chemotherapy, the patient still can be
enrolled into the study, if the patient understands the concept of chemoprevention and
consents to blood sampling during 5-azacitidine administration and follow-up (informed
consent signed). This part of the study consists of 6 cycles of 5-azacitidine (Vidaza®,
Celgene, Summit, NJ, USA).

Four weeks after completion of adjuvant chemotherapy (day 28 from day 1 of last cycle of
chemotherapy), the patient will continue his/her treatment plan with 6 cycles of
5-azacitidine. To be eligible for this part of the study, patient must have no evidence of
disease (NED). Thus, patient will be assessed by CT scan chest/abdomen/pelvis with and
without intravenous contrast prior to initiating Part B. In case of iodine
allergy/anaphylaxis history, patient will be assessed with MRI. Progression of disease will
be defined by RECIST criteria.

Patient will receive 5-azacitidine at a dose of 75 mg/m2 intravenously daily on day 1-5
every 28 days for 6 cycles. Biological correlatives will be performed during this period.
Toxicity management is described in section 6.0. The use of growth factor support (either
erythropoietin stimulating agents or G-CSF) will be also allowed at discretion of the
treating physician.

Anatomical Studies:

All patients will have surgical resection of the primary tumor. Pieces of the resected tumor
and non-tumor lung tissues that are not required for pathological evaluation will be frozen
in optimum temperature compound (OTC) and saved for molecular analysis.

As part of the enrollment process, patient will be assessed through CT scan of the
chest/abdomen/pelvis with and without intravenous contrast or with MRI in case of
contraindication for iodine contrast, prior to initiate Part B of the treatment (confirming
clinically and radiological "no evidence of disease-NED"). Another assessment will be
performed 4 weeks after the last dose of 5-azacitidine (to confirm NED status). Patient will
be clinically followed up every 3 months until completion of 2 years post-treatment. Imaging
tests will be performed every 3 months with CT scan as part of the standard follow-up until
completion of 2 years post-surgery. After 2 years of post-treatment completion, patient will
be followed up every 6 months in the clinic and also by radiographic studies until
completion of 5 years post-treatment. In the case of clinical suspicion for progression,
patient will undergo a thorough and complete work-up to rule out this possibility. This may
include imaging diagnostic tests, biopsy, or other tests and procedures.

Biological Correlatives:

The premise of the study is that certain loci will be hypermethylated in the lung cancer
specimen of the patients. For some loci (which we shall call Type I) the tumor will show
significantly elevated methylation compared to adjacent histologically normal tissue, thus
providing a cancer-specific methylation signal. Other loci (which we will refer to as Type
II) may be methylated in the tumor as well as in the adjacent "histologically" normal tissue
and/or WBCs. These loci will not be cancer-specific markers, though they may indicate
precancerous changes or they may be indicative of "cancer risk". Their hypermethylation may
be age-associated or environmentally induced, yielding "field effects" or "field defects".
Field defects would consist of molecular alterations (in this case hypermethylation) that
are not yet visible as histological changes.

Independent of whether a hypermethylated locus is specific for overt cancer (Type I) or not
(Type II), it would be of interest for the study. These Type II changes would be "hypothesis
generating". Since it has been well documented in the literature that DNA from cells can be
shed into the blood stream (more so in the case of cancer patients), it would be expected
that at least for some of the loci that are hypermethylated (Type I and II), this
methylation would be detectable in the plasma. Tumor tissue methylation will be correlated
with serum obtained at the time of tumor resection.

It might be expected that complete resection would lead to a reduction or complete
disappearance in the methylation signal for Type I loci in the plasma, since the source of
the signal has been removed. Type II also shows methylation in the tissue adjacent to the
tumor, these loci might continue to provide signal in the plasma (though it may be somewhat
diminished).

Type I loci would therefore be of great use to verify complete resection and to monitor
recurrence (one would anticipate the signal to increase again as the cancer grows back),
while Type II loci would be a great tool to monitor the efficacy of demethylating drugs. It
must be acknowledged that the spectrum of Type I and Type II methylation may not be sharply
delineated.

Inclusion Criteria:

- Patients must have a pathologic diagnosis of NSCLC.

- Patients must have surgical resection for NSCLC (stage I-IIIA) and tumor specimen
(ideally) and/or blood sample available for biological correlative studies.

- Patient's tumor specimen and/or blood sample must show hypermethylation in at least
one (1) of the following genes: DAPK, RASSF1A, CDKN2A (p16INK4a), GATA-4, GATA-5,
SPARC, MGMT, APC, and hMLH1.

- Patient's age must be 18 years or greater.

- Patients must have adequate organ and marrow function prior to be enrolled into the
trial, as defined below:

- Absolute neutrophil count (ANC) > 1500/mm3

- Platelets > 100,000/mm3

- Hemoglobin > 8.0 g/dL (with packed red blood cell transfusion or use of
erythropoietin stimulating agents allowed)

- Serum creatinine < 2.0 mg/dl.

- Total bilirubin < 2.0 mg/dl.

- AST/ALT < 2 x the upper limits of institutional normal.

- ECOG performance status of 0, 1, or 2.

- Estimated survival of > 12 months.

- Patients must be able to understand and agree to sign an IRB-approved informed
consent form, including permission to draw blood sample for correlative studies
during active treatment and follow-up.

- Women of child-bearing potential and men must agree to use adequate contraceptive
method (hormonal or barrier method of birth control) prior to study entry for the
duration of study.

- Women of childbearing potential must have a negative serum pregnancy test prior to
start targeted therapy with 5-azacitidine.

- Patients with HIV infection (but not AIDS) are eligible for this trial. Therefore, no
HIV testing will be required.

Exclusion Criteria:

- Patients who are not candidates for surgical resection.

- Patients who have received radiation therapy.

- No prior malignancy is allowed except for adequately treated basal cell or squamous
cell skin cancer, in situ cervical cancer, breast DCIS adequately treated, or other
cancer for which the subject has been disease-free ≥ 5 years.

- Subjects should not have a significant history of cardiac disease (e.g., unstable
angina, congestive heart failure, or uncontrolled arrhythmias).

- Subjects must not have an uncontrolled seizure disorder, or active neurological
disease.

- Patients who have significant systemic infections including AIDS.

- Pregnant and/or lactating women.

- Known or suspected hypersensitivity to azacitidine or mannitol.

- Prisoners or subjects who are compulsorily detained (involuntarily incarcerated) for
treatment of either a psychiatric or physical illnesses.