This is a Phase II, multicenter, single-arm trial designed to evaluate the efficacy and
safety of nivolumab (NIVO), ipilimumab (IPI) and temozolomide (TMZ) combination in 27
patients with MSS, MGMT-silenced mCRC with initial clinical benefit following lead-in
treatment with single-agent TMZ.
Immune checkpoint inhibitors have been shown to trigger durable antitumor effects in a subset
of patients. A high number of tumor mutations (so called 'tumor mutational burden') has
recently been found associated with increased immunogenicity (due to a high number of
neoantigens) and improved treatment efficacy across several different solid tumors. In mCRCs,
only a small fraction of tumors (<5%) display a high mutational load and are usually
associated with inactivation of mismatch repair genes such as MLH1, MSH2 and MSH6. Checkpoint
inhibitors may have increased activity in dMMR/microsatellite instability-high (MSI-H)
tumors, a hypothesis which was tested in various Phase II trials with positive results. On
the opposite, mismatch repair proficient colorectal cancer is unresponsive to immune
checkpoint inhibitors.
Previous reports indicate that acquired resistance to TMZ may emerge through the induction of
a microsatellite-instability-positive phenotype and recent data showed that inactivation of
MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational
load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered
immune surveillance in mouse models.
On all of the above grounds, the investigators hypothesize that treatment of microsatellite
stable MGMT hypermethylated CRCs with alkylating agents could reshape the tumor genetic
landscape by increasing the tumor mutational burden, leading to achieve potential
sensitization to immunotherapy.
In advanced CRC, the occurrence of chemorefractory disease poses a major therapeutic
challenge for presence of an adequate performance status to potentially receive further
treatments. Patients who progress after all approved treatments may be generally considered
suitable for new investigational drugs or strategies. Thus, in the era of personalized
medicine, tumor molecular profiling may lead to the identification of therapeutic targets or
predictive biomarkers for pharmacological intervention. The DNA repair gene
O6-methylguanine-DNA methyltransferase (MGMT) is responsible of the elimination of alkyl
groups from the O6-position of guanine. If inactive, it may be involved in early steps of
colorectal tumor genesis leading to an increase of G-to-A point mutations. Epigenetic
silencing of MGMT during colorectal tumor genesis is associated with hypermethylation of the
CpG island in its promoter. This transcriptional gene silencing is responsible for diminished
DNA-repair of O6-alkylguanine adducts, with the consequence of enhancing chemosensitivity to
alkylating agents including dacarbazine and its oral prodrug temozolomide (TMZ). Previous
phase II studies showed that TMZ induced an average objective response rate by RECIST
criteria in 10% of heavily pre-treated patients with advanced CRC carrying MGMT promoter
methylated tumors. Thus, MGMT methylation by methylation-specific PCR (MSP) used for patients
screening seemed to be a necessary but not sufficient condition to identify response to TMZ.
Digital PCR quantification of MGMT methylation refined patients' selection, with benefit
restricted to those with highly hyper-methylated tumors. Further analysis showed that MGMT
negative/low expression by immunohistochemistry (IHC) is found in about one third of
MSP-methylated samples and is associated with increased response rate. However, even in
responding mCRC patients, acquired resistance to single agent TMZ emerges rapidly and almost
invariably within 6 months from treatment initiation.
Immune checkpoint inhibitors have been shown to trigger durable antitumor effects in a subset
of patients. A high number of tumor mutations (so called 'tumor mutational burden') has
recently been found associated with increased immunogenicity (due to a high number of
neoantigens) and improved treatment efficacy across several different solid tumors. Early
clinical testing indicated that only 1 of 33 CRC patients had a response to anti PD-1
treatment, in contrast to substantial fractions of patients with melanomas, renal-cell
cancers, and lung tumors who showed benefit from PD-1 blockade. Similarly, anti CTLA-4
treatment up today brought to unsatisfactory results in unselected mCRC patients. The
probability of response has been ascribed to a high mutational burden (that is an elevated
number of somatic mutations), which translates in increased number of neo-antigens. In mCRCs,
only a small fraction of tumors (<5%) display a high mutational load and are usually
associated with inactivation of mismatch repair genes such as MLH1, MSH2 and MSH6. Molecular
alterations in these genes occur as an initial step in colon tumor genesis leading to the
microsatellite instability (MSI) phenotype. Indeed, mismatch repair-deficient (dMMR)
colorectal cancers have 10 to 100 times as many somatic mutations as mismatch
repair-proficient colorectal cancers. Moreover, mismatch repair-deficient cancers contain
prominent lymphocyte infiltrates, a finding consistent with an immune response. Thus,
checkpoint inhibitors may have increased activity in dMMR/microsatellite instability-high
(MSI-H) tumors, a hypothesis which was tested in various Phase II trials with positive
results. On the opposite, mismatch repair proficient colorectal cancer is unresponsive to
immune checkpoint inhibitors.
Previous reports indicate that acquired resistance to TMZ may emerge through the induction of
a microsatellite-instability-positive phenotype. On the other hand, TMZ by itself has been
shown to induce an increase of mutational load in other MGMT deficient solid tumors such as
melanoma or glioblastoma. In parallel, other studies have demonstrated that alkylating
agents' side effects can influence the immune cell compartment by selectively depleting the
immuno-suppressive T regulator lymphocytes (Tregs), and activating the immuno-active T
cytotoxic lymphocytes (Tc) and natural killers (NK). The investigators recently showed that
inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide,
increased mutational load, promoted continuous renewal of neoantigens in human colorectal
cancers and triggered immune surveillance in mouse models.
On all of the above grounds, the investigators hypothesize that treatment of microsatellite
stable MGMT hypermethylated CRCs with alkylating agents could reshape the tumor genetic
landscape by increasing the tumor mutational burden either directly (by inducing G>A
mutations) or/and indirectly (by inactivating DNA repair genes such as MLH1, MSH2 or MSH6,
which in turn could lead to hypermutated phenotype) therefore enhancing formation of cancer
neoantigens and immunogenicity. TMZ treatment can also modulate the repertoire of immune
cells (Tregs, Tc, NK) favoring T cell activation. To achieve potential sensitization to
immunotherapy by means of TMZ-induced MSI-like status, treatment with TMZ should be active
(i.e. inducing a SD/PR/CR).
Inclusion Criteria:
1. Have provided written informed consent prior to any study specific procedures
2. Willing and able to comply with the protocol
3. ≥18 years of age
4. ECOG status 0 - 1
5. At least 12 weeks of life expectancy at time of entry into the study
6. Histologically confirmed metastatic or inoperable adenocarcinoma of the colon and/or
rectum, with centrally confirmed mismatch repair proficiency (microsatellite stable
[MSS]) by multiplex polymerase chain reaction (PCR), MGMT promoter methylation by
methylation-specific PCR (MSP) and MGMT low expression by IHC
7. Patients with progressive disease or that are not candidate for oxaliplatin irinotecan
fluoropirimidin based chemotherapy and anti EGFR mAbs (in RAS/BRAF wild type tumors)
in the metastatic setting
8. Patients with documented disease relapsed within 6 months from the completion of
adjuvant oxaliplatin-based chemotherapy are considered eligible
9. Measureable, unresectable disease according to RECIST 1.1. Subjects with lesions in a
previously irradiated field as the sole site of measurable disease will be permitted
to enroll provided the lesion(s) have demonstrated clear progression and can be
measured accurately.
10. Is willing and able to provide an adequate archival tumor sample (FFPE) available for
tissue screening for central tissue screening. If the tumour block is not available, a
minimum of twenty 3-micron unstained sections on charged slides of tumor will be
required.
Exclusion Criteria:
1. Requirement for treatment with any medicinal product that contraindicates the use of
any of the study medications, may interfere with the planned treatment, affects
patient compliance or puts the patient at high risk for treatment-related
complications
2. Inability to swallow pills
3. Refractory nausea and vomiting, malabsorption, external biliary shunt or significant
bowel resection that would preclude adequate absorption
4. Inadequate haematological function indicated by all of the following:
- White Blood Cell (WBC) count < 2 x 109/L
- Absolute neutrophil count (ANC) < 1.5 x 109/L
- Platelet count < 100 x 109/L
- Haemoglobin < 9 g/dL (patients may have transfusions and/or growth factors to
attain adequate Hb)
5. Inadequate liver function indicated by all of the following:
- Total bilirubin ≥ 1.5 x upper limit of normal (ULN)
- Aspartate transaminase (AST) and alanine aminotransferase (ALT) ≥ 3 x ULN (≥ 5 x
ULN in patients with known liver metastases)
- Alkaline phosphatase (ALP) ≥ 2 x ULN (≥ 5 x ULN in patients with known liver
metastases)
6. Inadequate renal function indicated by all of the following:
- Serum creatinine > 1.5 x ULN or calculated creatinine clearance < 40 ml/min
7. INR > 1.5 and aPTT > 1.5 x ULN within 7 days prior to the start of study treatment for
patients not receiving anti-coagulation
a. NOTE: The use of full-dose oral or parenteral anticoagulants is permitted as long
as the INR or aPTT is within therapeutic limits (according to the medical standard of
the enrolling institution) and the patient has been on a stable dose of anticoagulants
for at least two weeks prior to the start of study treatment
8. Active infection requiring intravenous antibiotics at the start of study treatment
9. Previous or concurrent malignancy, except for adequately treated basal or squamous
cell skin cancer, superficial bladder cancer, or carcinoma in situ of the prostate,
cervix, or breast, or other cancer for which the patient has been disease-free for
three years prior to study entry
10. Evidence of any other disease, neurologic or metabolic dysfunction, physical
examination finding or laboratory finding giving reasonable suspicion of a disease or
condition that contraindicates the use of any of the study medications, puts the
patient at higher risk for treatment-related complications or may affect the
interpretation of study results
11. Clinically significant (i.e. active) cardiovascular disease, for example
cerebrovascular accidents ≤ 6 months prior to start of study treatment, myocardial
infarction ≤ 6 months prior to study enrolment, unstable angina, New York Heart
Association (NYHA) Functional Classification Grade II or greater congestive heart
failure, or serious cardiac arrhythmia uncontrolled by medication or potentially
interfering with protocol treatment
12. History or evidence upon physical or neurological examination of central nervous
system (CNS) disease (e.g. seizures) unrelated to cancer unless adequately treated
with standard medical therapy
13. Active brain metastases or leptomeningeal metastases. Subjects with brain metastases
are eligible if these have been treated and there is no magnetic resonance imaging
(MRI except where contraindicated in which CT scan is acceptable) evidence of
progression for at least 8 weeks after treatment is complete and within 28 days prior
to first dose of study drug administration. Cases should be discussed with the medical
monitor. There must also be no requirement for immunosuppressive doses of systemic
corticosteroids (>10mg/day prednisone equivalents) for at least 2 weeks prior to study
drug administration.
14. Surgical procedure (including open biopsy, surgical resection, wound revision, or any
other major surgery involving entry into a body cavity) or significant traumatic
injury within 28 days prior to start of study treatment, or anticipation of need for
major surgical procedure during the course of the study.
15. Treatment with any chemotherapy, curative intent radiation therapy, biologics for
cancer, or investigational therapy within 28 days of first administration of study
treatment (subjects with prior cytotoxic or investigational products < 4 weeks prior
to treatment might be eligible after discussion between investigator and sponsor, if
toxicities from the prior treatment have been resolved to Grade 1 (NCI CTCAE version
4). Prior focal palliative radiotherapy must have been completed at least 2 weeks
before study drug administration.
16. All toxicities attributed to prior anti-cancer therapy other than alopecia and fatigue
must have resolved to Grade 1 (NCI CTCAE version 4) or baseline before administration
of study drug. Subjects with toxicities attributed to prior anti-cancer therapy which
are not expected to resolve and result in long lasting sequelae, such as neuropathy
after platinum based therapy, are permitted to enroll.
17. Known hypersensitivity to any of the study medications or Known hypersensitivity or
allergy to Chinese hamster ovary cell products or any component of the NIVO
formulation
18. History of severe allergic, anaphylactic, or other hypersensitivity reactions to
chimeric or humanized antibodies or fusion proteins
19. History of autoimmune disease including but not limited to myasthenia gravis,
myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis,
inflammatory bowel disease, vascular thrombosis associated with antiphospholipid
syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barré syndrome,
multiple sclerosis, vasculitis, or glomerulonephritis (see Appendix IV for a more
comprehensive list of autoimmune diseases)
a. Note: history of autoimmune-related hypothyroidism on a stable dose of thyroid
replacement hormone may be eligible. Subjects with controlled type I diabetes mellitus
on a stable insulin regimen, vitiligo or psoriasis not requiring systemic treatment
may be eligible.
20. Prior allogeneic bone marrow transplantation or prior solid organ transplantation
21. History of idiopathic pulmonary fibrosis (including pneumonitis), drug-induced
pneumonitis, organizing pneumonia (i.e., bronchiolitis obliterans, cryptogenic
organizing pneumonia), or evidence of active pneumonitis on Screening chest CT scan
22. Treatment with systemic immunostimulatory agents (including but not limited to
interferons or interleukin-2) within 4 weeks or five half-lives of the drug, whichever
is shorter, prior to start of study treatment
23. Treatment with systemic corticosteroids (>10 mg daily prednisone equivalents) or other
systemic immunosuppressive medications (including but not limited to prednisone,
dexamethasone, cyclophosphamide, azathioprine, methotrexate, thalidomide, and
anti-tumour necrosis factor [TNF] agents) within 2 weeks prior to start of study
treatment, or requirement for systemic immunosuppressive medications during the trial.
The use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone) is
allowed.
a. Note: Patients who have received acute, low-dose, systemic immunosuppressant
medications (e.g., a one-time dose of dexamethasone for nausea) may be enrolled in the
study after discussion with and approval by the Sponsor.
24. Positive test for human immunodeficiency virus (HIV)
25. Active hepatitis B (defined as having a positive hepatitis B surface antigen [HBsAg]
test prior to randomization) or hepatitis C
1. Note: Patients with past hepatitis B virus (HBV) infection or resolved HBV
infection (defined as having a negative HBsAg test and a positive antibody to
hepatitis B core antigen antibody test) are eligible. Patients with detectable
HBV-DNA are not eligible.
2. Note: Patients positive for hepatitis C virus (HCV) antibody are eligible only if
polymerase chain reaction testing is negative for HCV ribonucleic acid (RNA).
26. Active tuberculosis
27. Administration of a live, attenuated vaccine within 4 weeks prior to start of study
treatment or anticipation that such a live attenuated vaccine will be required during
the study
28. Prior treatment with CD137 agonists, anti-CTLA4, anti-PD-1, or anti-PD-L1 therapeutic
antibody or drug specifically targeting T-cell co-stimulation or immune checkpoint
pathways, including prior therapy with anti-tumor vaccines.
29. Pregnancy or lactation. A serum pregnancy test is required within 7 days prior to
start of study treatment, or within 14 days with a confirmatory urine pregnancy test
within 7 days prior start of study treatment
30. For women who are not post-menopausal (< 12 months of non-therapy-induced amenorrhea)
or surgically sterile (absence of ovaries and/or uterus): refusal to use a highly
effective contraceptive method (i.e. with a failure rate of < 1% per year such as
sexual abstinence, hormonal implants, combined oral contraceptives, vasectomised
partner), during the study drug administration and for at least 6 months after the
last dose of study medication. Periodic abstinence [e.g., calendar, ovulation,
symptothermal, postovulation methods] and withdrawal are not acceptable methods of
contraception. A combination of male condom with cap, diaphragm or sponge with
spermicide (double barrier methods) is not considered highly effective, birth control
methods. Acceptable methods of contraception may include total abstinence in cases
where the lifestyle of the patient ensures compliance. A Vasectomised partner is a
highly effective birth control method provided that partner is the sole sexual partner
of the trial participant and that the vasectomised partner has received medical
assessment of the surgical success.
31. For men: refusal to use a highly effective contraceptive method (i.e. with a failure
rate of < 1 % per year such as vasectomy, sexual abstinence or female partner use of
hormonal implants or combined oral contraceptives) during the study drug
administration and for a period of at least 6 months after the last dose of study
medication. Periodic abstinence [e.g., calendar, ovulation, symptothermal, post
ovulation methods] and withdrawal are not acceptable methods of contraception. A
combination of male condom with either, cap, diaphragm or sponge with spermicide
(double barrier methods) is not considered highly effective, birth control methods.
Acceptable methods of contraception may include total abstinence in cases where the
lifestyle of the patient ensures compliance. A vasectomised trial participant is a
highly effective birth control method provided that the trial participant has received
medical assessment of the surgical success.
