Validation of digital-PCR analysis through programmed imatinib interruption in PCR negative chronic myeloid leukemia patients.

Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) represents 7-20% of all leukemia cases, with a
worldwide incidence projected at one to two per 100,000 people. CML is caused
by the unregulated activity of the tyrosine kinase BCR-ABL, which is formed by
the fusion of exons belonging to the BCR and ABL genes, located on chromosomes
22 and 9 respectively, that generates the Philadelphia (Ph) chromosome.
Untreated CML commonly progresses through three disease phases: chronic phase
(CP), accelerated phase (AP) and blast phase (BP), each corresponding to
increasing leukemic blast counts and clinical severity. The chronic phase (CP),
that usually lasted 2-3 years in the pre-imatinib era, is characterized by an
abnormal expansion of the clonal hematopoiesis retaining an apparent normal
differentiation; the AP's median duration is 3-9 months, while BP*s median
survival is 3-6 months. The last two phases are marked by the development of a
differentiation block typical of acute leukemia which fatally closes the course
of the disease.

Imatinib Therapy

The main goal of CML therapy is the suppression of Ph+ clone in the chronic
phase (CP). Since BCR-ABL translocation represents the molecular cause of CML,
the targeting of its enzymatic activity represents a truly *targeted* attempt
to cancer therapy. In fact, over the last two decades, the therapy evolved from
the use of non-specific cytotoxic agents (i.e. hydroxurea, busulfan) to
interferon-α (IFN-α) or allogeneic stem cell transplantation (allo-SCT) and
more recently to imatinib, a competitive inhibitor of the BCR-ABL kinase that,
with a 5-year survival rate greater than 90%, is now recognized as the
first-line treatment of CML and could allow a normal life expectancy.
Imatinib induces complete cytological response (CCyR) in up to 80% of patients
and major molecular response (MMR) in 33-90% of the patients, according to
treatment duration. Moreover, approximately 1/3 of long term treated patients
who are in CCyR show complete molecular response (CMR, i.e. undetectable
BCR-ABL transcripts) and absence of residual sign of leukemia. Anyway,
undetectable BCR-ABL may not equate to eradication of minimal residual disease
(MRD) because the sensitivity of the standard diagnostic method, the Q-RT-PCR,
is limited and significant numbers of residual leukemic cells can remain in a
patient.

The digital-PCR assay

The development of a more sensitive Q-RT-PCR to monitor MRD was recently
included in the ILTE study, aimed to present a global picture of imatinib long
term effects (serious adverse events, toxicities not qualifying as serious
adverse events, loss of CCyR, survival). A pilot study was performed on blood
samples obtained from patients who remained negative for at least one year,
using a new diagnostic method, the digital-PCR (dPCR), developed by
Gambacorti-Passerini, Saglio and Kim, able to detect until 1 BCR-ABL+ cell out
of 10000000 cells, which corresponds to a 100 times increased sensitivity as
compared to conventional Q-RT-PCR. The results showed that, among 30 patients
negative by conventional Q-RT-PCR for at least one year, only 10 patients (33%)
were negative by dPCR. Interestingly, these numbers are compatible with those
observed in a French study in which about 40% of patients were relapse-free
within 12 months from imatinib suspension. Therefore, it is possible that dPCR,
assessing with more sensitivity the presence of MRD, could better identify the
patients where CML is truly eradicated.

Primary Objective

To assess the capability of the dPCR technique to predict the absence of
disease relapses after imatinib discontinuation in CML patients with negative
Q-RT-PCR results for longer than 18 months.

Secondary Objectives

• To estimate relapse rate after imatinib discontinuation.
• To characterize the performances of the dPCR technique.
• To quantify the maintenance of the molecular remission, after imatinib
discontinuation, in CML patients with negative Q-RT-PCR for longer than 18
months.
• To evaluate the impact of imatinib treatment (discontinuation and resumption)
on quality of life.
• To assess the timing of recurrence.
• To quantify the occurrence of progression/resistance in patients who relapse
after imatinib resumption.
• To evaluate whether survival of patients dPCR negative and relapse free at 36
months is comparable to the one of normal population.
• To evaluate whether survival of relapsing patients is comparable to the
survival of the non relapsing ones.

This is a multi-center, multi-national trial, sponsored by a non commercial
public institution, that will involve approximately 15 investigational centers
in EU (Italy, Spain, Germany and The Netherlands) and in non EU countries
(Israel and Canada) and will recruit approximately 100 CML patients under
imatinib therapy in complete molecular remission with a history of at least 18
months of consecutive negative standard Q-RT-PCR as performed in their own
centers. After signing the informed consent form (ICF), the patients will be
tested for dPCR and will discontinue imatinib therapy. Then they will be
monitored by standard Q-RT-PCR to assess the maintenance of the molecular
remission; collection of data will be prospective as each center will collect
the data for 36 months. At the end of this period, a peripheral blood sample
for dPCR analysis will be obtained from those patients who will still have
undetectable BCR-ABL transcripts by Q-RT-PCR to verify CML eradication. The
maintenance of molecular remission by Q-RT-PCR and the survival will be
monitored every six months during an additional follow-up of 24 months.
Patients found to be positive to BCR-ABL transcripts by standard Q-RT-PCR will
repeat the test every 2 to 4 weeks until the loss of molecular remission,
defined as two consecutive BCR-ABL positive tests with at least one with
BCR-ABL/BCR value above 0.1%, or until the end of the study, whichever come
first.
The patients losing molecular remission will immediately resume imatinib
treatment at the same dosage used before interruption. The patient losing
molecular remission during the first 36 months period will be followed for 12
months or until progression of disease/resistance to imatinib treatment; the
disease status will be then monitored by Q-RT-PCR and/or cytogenetic analysis
with the patient survival every six months during a follow-up of additional 24
months.The patient losing molecular remission after the first 36 months period
will not be followed any further and will go off study.
Patient*s quality of life during imatinib discontinuation/resumption will be
evaluated trough the EORTC - C30 Quality of Life (QoL) questionnaire.
The main analysis of the study, including both primary and secondary endpoints
will be carried out upon completion of the 36 months period by all the patients
who did not relapse earlier. Interim evaluations of the main endpoint will take
place at one and two years after the completion of the enrollment,
respectively. Evaluation of survival will be repeated at the end of the
follow-up period.
The eligible patients will be entered between September 2011 and August 2012.
The end of the trial is defined as the date of the last visit of the last
patient, including follow up.

The CML patients under imatinib therapy, with at least 18 months of consecutive
negative standard Q-RT-PCR, will stop imatinib treatment.

In case of loss of molecular remission during the 36 months of the study or the
2 years follow up, the patients will resume imatinib treatment at the same
dosage used before interruption.

During the course of the study some blood samples will be taken for laboratory
analysis.

In addition to the amount of blood needed for normal diagnostic tests provided
by the hospital clinical practice, blood will be taken for dPCR test; this
examination will take place during the initial study visit, and even after 3
years from drug suspension. During the surveillance period, after imatinib
discontinuation, conventional Q-RT-PCR analysis will be performed every month
during the first 6 months and every two months for the next 30 months. In
addition, the vital signs (e.g. blood pressure, weight) and blood samples for
biochemistry and hematology assessments will be carried out after 1 month and
every 6 months for 36 months from the imatinib suspension date. The purpose of
these regular reviews is to assess the patients' health status and to restart
as soon as possible the drug therapy in case of diagnosis of CML relapse.

In the event that during the first 3 years of imatinib suspension, diagnostic
tests revealed the presence of leukemic cells, the bone marrow will be taken in
order to check the status of the disease and immediately the imatinib treatment
will be resumed at the same dosage used before interruption; subsequently, in
order to assess the disease status, any bone marrow aspirate/biopsies could be
necessary every 3 months for up to one year. In addition, the state of health
will be followed by vital signs assessment, biochemistry and hematology
assessments, conventional Q-RT-PCR, at relapse and every month for the first 3
months and then every three months for an additional 9 months from the date of
the first bone marrow aspirate.

In the final study period of 2 years the state of health and the disease
stability will be monitored every 6 months with the Q-RT-PCR and in case of
imatinib resumption, with conventional cytogenetics assessments.

At different times during the study, patients will be asked to fill out a
questioonnaire (EORTC-QLQ-C30) to evaluate the quality of life.

Blood analysis: blood sampling may bring a slight risk of pain or bruising and
infection.
Bone Marrow Aspirate/Biopsy: different individuals feel the pain caused by
injection of the local anesthetic and the remainder of the procedure to a
variable extent. There may be dull soreness for a day or two. Significant
complications are very unusual but can include bleeding, infection, and
prolonged pain.
X-Ray, CT Scan, MRI, Ultrasound analysis: they can be performed to assess the
presence of extramedullary disease (EMD) in the body. These are painless
procedures.