Antiviral therapy (cidofovir, an acyclic nucleoside phosphate) in combination with radiotherapy in HPV-positive tumours of the oropharynx: a phase 1 study.

1.1 Background Disease Information
Head and neck squamous cell carcinoma (HNSCC) represent about 6,5% of all new
cases of cancer. Abuse of tobacco and alcohol are well-established risk factors
for development of HNSCC. Despite new diagnostic tools and therapies, survival
rates remain low. Therefore it is important to identify new molecular markers
for improvement of therapeutic strategies for HNSCC. Human Papilloma Virus
(HPV) is associated with oropharyngeal carcinoma and with increasing incidence
in particular with tonsillar carcinoma (1). As the DNA of HPV integrates in the
human cellular genome, oncoproteins E6 and E7 are upregulated and disable tumor
suppressor genes p53 and pRb, which is a crucial step in carcinogenesis (2, 3).
Depending on tumor location and HPV-type, numbers of HPV-positive HNSCC are
varying (2-76%). The DNA type HPV-16 is found in 21 % of the HNSCC (50% of
oropharyngeal carcinomas) (3).
The standard treatment of oropharyngeal carcinomas in the Netherlands is
dependent on tumor location and TNM stage (UICC). Locoregional radiotherapy is
mostly indicated in T1 and T2 oropharynx carcinoma. In T3 and T4, the treatment
is surgery eventually followed by postoperative radiotherapy. In inoperable
(functional) T3-T4 tumors and if surgery is refused a combined chemo radiation
therapy is performed (cisplatinum).
Currently, promising results concerning the efficacy of antiviral agents for
treatment in patients with HPV-positive (HPV-16 and -18) cervical carcinomas
are found (4). Therefore it is proposed that antiviral therapeutic strategies
in addition to radiation therapy might improve tumor response and outcome
radiation in HPV-positive HNSCC.
In vitro research shows that acyclic nucleoside phosphates, like cidofovir
(HPMPC) and adefovir (PMEA), inhibit tumor cell differentiation and
angiogenesis (adefovir) and induce apoptosis (cidofovir) (5). In vivo, adefovir
acts against choriocarcinoma in rats and cidofovir in hemangioma in rats.
Cidofovir is already widely accepted in locoregional therapies for
papillomatosis in humans.
Phase 1 trials regarding cidofovir in humans already have been performed for
cytomegalovirus retinitis in patients with AIDS (6) with a dose of 5mg/kg
intravenously every 2 weeks. A following phase 2-3 analysis administered 5mg/kg
of body weight once weekly for two weeks, followed by 3 mg/kg of body weight
(low-dose) and 5mg/kg of body weight (high-dose) once every two weeks (7, 8).
Snoeck et al. (9) showed that local therapy with cidofovir gel 1% in cervix
carcinomas (stage CIN III) results in partial or complete regression of
cervical dysplasia. Tristram et al. (10) studied the effect of cidofovir in
high-grade non-cervical anogenital intraepithelial neoplasias. A complete
regression after local treatment with cidofovir was found. The research of
Sirianni et al. (11) showed that HPV-positive cells in vitro could be
radiosensitized by adding cidofovir to the radiotherapy. This was also shown in
vivo as well as in nude mouse xenografts by Abdulkarim et al. (12).
We hypothesize that combining cidofovir with radiotherapy will improve the
efficacy of radiotherapy in HPV-positive head and neck cancer. Therefore we
propose a phase 1 trial to investigate the maximum tolerated dose of cidofovir
to be combined with radiotherapy during a six-week period.

1.2 Background Therapeutic Information
Cidofovir, (S)-1-(3-Hydroxy-2-phosphonyl-methoxypropyl) cytosine, is
characterised by a stable phosphonate linkage between the acyclic nucleoside
and the phosphate moiety.
In a phase I/II clinical trial with intravenous cidofovir in patients with
human immunodeficiency virus (HIV) infection and asymptomatic CMV infection,
prolonged and dose-dependent antiviral effects were observed at doses of 3,0
and 10,0 mg/kg of bodyweight (7, 8). The dose-limiting toxicity was
nephrotoxicity, which was dose dependent and less frequent with concomitant
oral probenecid and hydration. This toxicity was characterized by proximal
tubular dysfunction and was consistent with observations in animal toxicity
studies (7, 8). Pharmacokinetic parameters for cidofovir in these patients were
dose-dependent; the mean total clearance of cidofovir from serum was
approximately 150 ml/h/kg at both dose levels. This value was significant
higher than the baseline creatinine clearance determined in the same patients,
suggesting that active tubular secretion contributed to the renal clearance of
cidofovir. There was evidence in two patients of a prolonged phase in the
elimination of cidofovir. However, in the majority of patients the drug
displayed a terminal elimination half-life of 3 to 4h. There were no
significant changes in the pharmacokinetics of cidofovir over four infusions,
suggesting that the drug did not accumulate.
The pharmacokinetics of cidofovir were examined at five dose levels in three
phase I/II studies in a total of 42 HIV-infected patients (13). Levels of
cidofovir in serum following intravenous infusion were dose proportional over
the dose range of 1,0 to 10,0 mg/kg of body weight and declined biexponentially
with an overall mean +/- standard deviation terminal half-life of 2,6 +/- 1,2h
(n=25). Approximately 90% of the intravenous dose was recovered unchanged in
the urine in 24h. The overall mean +/- standard deviation total clearance of
the drug from serum (148 +/- 25 ml//h/kg; n=25) approximated renal clearance
(129 +/- 42 ml/h/kg; n=25), which is significant higher than the baseline
creatinine clearance in the same patients (83 +/- 21ml/h/kg; n=12). Active
tubular secretion plays a significant role in the clearance of cidofovir. The
steady state volume of distribution of cidofovir was approximately 500 ml/kg,
suggesting that the drug was being distributed in total body water. Repeated
dosing with cidofovir at 3,0 and 10,0 mg/kg/week did not alter the
pharmacokinetics of the drug. Concomitant administration of intravenous
cidofovir and oral probenecid to hydrated patients had no significant effect on
the pharmacokinetics of cidofovir at a 3,0 mg/kg dose. At high doses of
cidofovir, probenecid appeared to block tubular secretion of cidofovir and
reduce its renal clearance to a level approaching glomular filtration.
The pharmacokinetics of cidofovir administered with probenecid were evaluated
in 12 HIV-infected patients with or without asymptomatic CMV infection and 10
patients with relapsing CMV retinitis. Dose-independent pharmacokinetics were
observed for cidofovir, administered with probenecid, after one hr infusions of
3.0 (n = 12), 5.0 (n = 6), and 7.5 (n = 4) mg/kg. Approximately 70 to 85% of
the cidofovir dose administered with concomitant probenecid was excreted as
unchanged drug within 24 hr. When cidofovir was administered with probenecid,
the renal clearance of cidofovir was reduced to a level consistent with
creatinine clearance, suggesting that probenecid blocks active renal tubular
secretion of cidofovir.
1.3 Hypothesis
We hypothesize that combining cidofovir with radiotherapy will improve the
efficacy of radiotherapy in HPVpositive head and neck cancer. Therefore we
propose a phase 1 trial to investigate the maximum tolerated dose of cidofovir
to be combined with radiotherapy and/or surgery during a six-week period.

Primary objective
·To define the maximum tolerated dose (MTD) of cidofovir in combination with
standard dose of radiotherapy in Human Papilloma Virus-positive head and neck
carcinomas.
Secondary Objectives
·To explore p53-related gene activity in patients before and during treatment
with Cidofovir.
·To evaluate PET/CT findings oropharyngeal carcinomas before and after
cidofovir administration.
End-points
·Incidence of locale and severe complications
·Grade IV or V: dermatology/skin
·Grade IV or V: musculoskeletal/soft tissue
·Grade III: renal/genitourinary
·Incidence of other acute toxicity, assessed according to CTC version 3.0

Phase 1 trial
Eligible patients will be registered in the trial and treated with a
combination of radiotherapy and escalating dose of cidofovir according to the
in this section described dose escalation scheme.
A biopsy of the HNSCC is taken at most two weeks prior to the combined
treatment. This biopsy will be analyzed for HPV-status and p53-related gene
activity. Ninety-six hours after the first dose of each dose level in each
patient a second biopsy is taken in case of a first HPV-positive sample. Both
samples are analyzed for p53-related gene activity. Improvement of p53-activity
is expected in at least 90% of the HPV-positive HNSCC.
Changes in p53-related gene activity (Δgene exp) will be correlated with
imaging of tumor at each dose level in patients with histological confirmed
HPV-positive HNSCC (See also Chapter 8).
A PET-CT scan is routinely performed prior to treatment and three months after
the last dose of radiotherapy. An explorative description analysis will be
performed.
Patients will be followed up until four weeks after the combined treatment.
Adverse events will be graded using the NCI Common Terminology Criteria for
Adverse Events Version 3.0 (CTCAE) (see Appendix A) at any time of presence
during and follow-up after treatment when reported by patients or encountered
during therapy.
After the defined period of the trial, patients will be routinely controlled at
the ENT- and radiation oncology department.

Dose Escalation Schedule for Cidofovir
The dose of cidofovir will be escalated in fixed increments according to the
dose escalation scheme outlined below.

Dose Dose of cidofovir given intravenously, once a week combined with
RT starting one week prior to RT (6 doses)
level Minimum number of patients
-2 1,50 mg/kg of body weight --
-1 2,50 mg/ kg of body weight --
1(SD) 4 mg/ kg of body weight 3
2 7 mg/ kg of body weight 3

Starting Dose (SD) of Cidofovir
Dose level: dose of cidofovir given intravenously, once a week during
radiotherapy starting one week prior to radiotherapy (6 doses).
The starting dose of cidofovir will be 4 mg/kg of body weight weekly during
radiotherapy. This dose is based on previously performed pharmacokinetic, phase
1, 2 and 3 trials. The rational for this starting dose is the recommended dose
given in previous studies minus 1.

Cidofovir will be solved in 100ml NaCL 0,9% and administrated during one hour.
Prior to and after administration of cidofovir the patient will receive one
litre of NaCl 0,9% during one hour.
Three hours prior to cidofovir the patient will take 2 grams of probenicid
orally and again 1gram 3 and 8 hours after administration of cidofovir.

Radiotherapy
Standard treatment of radiotherapy (3D conformal radiotherapy or MIRT) is
applied according to clinical guidelines defined by Maastricht Radiation
Oncology Clinic (appendix C).


Methods and Endpoints
Dose limited toxicity (DLT)
The rate of subject entry and escalation to the next dose level will depend
upon assessment of the safety profile of patients entered at the previous dose
level. Toxicity will be evaluated according to the NCI Common Terminology
Criteria for Adverse Events (CTCAE), Version 3.0 (see Appendix A).

3+3 design
A minimum of three patients will be entered on each dose level. All three will
be followed for one completed cycle of therapy (one dose/ week during 6 week)
and subsequent enrolment of new cohorts will be based on the toxicity
assessment in that first cycle and the documentation of any dose limiting
toxicities.

Defining Maximum Administered Dose (MAD)
·If 0/3 patients exhibit dose limiting toxicity at this dose level
Dose escalation to the next dose level may begin in a new cohort of patients 4
weeks after the last radiation.

·If 1/3 patients exhibit dose limiting toxicity at this dose level:
Expand dose level to a total of 6 patients.
If no further DLT events seen, dose escalation to the next dose level may begin
in a new cohort of patients.
If 1 or more further DLT events are seen (i.e. 2 or more of 6 patients), this
dose level will be considered the maximum administered dose (MAD).

·If 2/3 patients exhibit dose limiting toxicity:
This dose level will be considered the maximum administered dose (MAD).

·Before opening the next higher dose level all toxic effects at the preceding
dose level will be reviewed and expansion or escalation will be undertaken as
appropriate.

Dose Limiting Toxicity (DLT)
·Toxicity will be graded using CTCAE version 3.0 (see Appendix A). Any dose
limiting toxicity must be a toxicity that is considered related to study drug.
Dose limiting toxicity is defined by disorder of at least one of the following
criteria:

Hematological
1 Absolute granulocyte count (AGC) < 0.5 x 109/L
2 Febrile Neutropenia (ANC < 1.0 x 109/L, fever > 38.5oC)
3 Platelets < 25 x 109/L
4 Bleeding felt to be due to thrombocytopenia
5 Total WBC <1,0 x 109/L

Non-Hematological:
1 Diarrhea > Grade 3 despite optimal loperamide use
2 Rash > Grade 3 or grade 2 is medically concerning or unacceptable to the
patient
3 Mucositis, burns > Grade 4
4 Kidney > Grade 3 despite optimal hydration and probenecid use
5 Mucosa > Grade 4
6 Bone > Grade 4
7 Other grade 3 effects thought to be treatment related.
8 Missing > 2 doses of treatment for toxicity reasons.

Recommended Phase II Dose (RP2D)
·As described above, the MTD is that dose in which 2/3 or 2/6 patients
experience dose-limiting toxicity.
·The RP2D is the MTD dose level *-1*. If the MTD is seen at the starting dose
level, then dose level *-1* will be the recommended dose.
·At least 6 patients will be treated at the recommended dose to assure
information on the safety profile at that dose is complete.
·To assure information on the safety profile at the RP2D in HPV-positive
oropharyngeal carcinoma is complete, the RP2D will be given to an additional 6
patients with histological proven HPV-positive oropharyngeal carcinoma.
·If less then two DLT*s are observed in this cohort, the trial will be ended.

Patient Replacement
·Three patients within a dose level must be observed until 4 weeks after the
last dose of radiotherapy, before accrual to the next higher dose level may
begin.
·If a patient is withdrawn from the study for reasons other than DLT, an
additional patient may be added to that dose level. Patients one or more doses
due to toxicity will not be replaced since these patients will be considered to
have experienced DLT.
·Patients may also discontinue protocol therapy in the following instances:
Intercurrent illness which would in the judgment of the investigator affect
patient safety, the ability to deliver treatment or the primary study endpoints
Request by patient

One week before and during the five weeks of radiotherapy, patients are treated
once weekly with intravenous cidofovir (6-administrations). One administration
takesthree hours and includes observation and infusion of 1 liter of saline
during one hour, infusion of cidofovir in a 100ml saline solution during one
hour, and again infusion of 1 liter of saline during one hour. Before starting
the weekly infusion a bloodsample is taken to control haematological
parameters. This is also conducted before entering the trial and after
finishing the trial (see study design). The first biopsy and the PET/CTscan are
standardly performed according the ongoing guidelines. the second biopsy is
optional and will be taken after agrement of the patient in case of a first
HPV-positive sample, 9 hours (+/-24 hours) under local anaesthesia. Further
treatment and diagnosing is performed according ongoing national guidelines.