Study Objectives
To validate a novel test using
Dried blood spot (DBS) based RNA transcript detection against conventional
standard methods for diagnosis of leukemia in children.
Rationale:
Childhood cancers (0-14 years of
age) comprise 4% of reported cancers in India.
Leukaemia is the most common
cancer in Children in India, according to ICMR cancer registry 2022. It is also
one of the top ten cancers in adult population
Outcome in these patients is
entirely dependent on RISK STRATIFICATION as per WHO classification. Treatment
dependent on risk category and type of leukaemia.
Outcomes in West for leukaemia is
over 90%, however in India about 50-60%. Leukemia diagnostics rely on
sophisticated molecular tests not available at all centres-especially the
remote and tier two cities.
Gaps: The access and expertise in
diagnostics is limited to less than 20 centers all over India-for a population
of about 200 billion people. Specialized molecular diagnostics and prognostic
tests for leukemia are available at few tertiary centers. This restricted
accessibility results in delayed patient diagnosis, sub-optimum risk
stratification, increase in economic burden of therapy, deficiencies in the
comprehensive supportive care, burden on existing tertiary care centers and
high abandonment, relapses and poor outcomes, thus leading to an overall higher
mortality as compared to other High income countries.
Novelty:
To improve accessibility of
leukemia diagnostics and molecular subtyping, a novel protocol (Patent Applied)
based on dried blood spot (DBS) has been validated for subtyping molecular
variant of leukemia- based on WHO classification of hematolymphoid disorder.
Our background completed work-(technology TLR-5), has shown the sensitivity and
specificity of the method at par with that of conventional standard method with
advantage of implementation in less resourced areas. The transport and testing
for viable nucleic acid for leukemia detection and prognosis in tropical
temperature and humidity conditions has been completed in a pilot study. The
method is rapid, stable, sensitive, and cost effective for prognosis and
detection of leukemia in peripheral low resourced settings.
4. Study Design
Study design:
Randomized controlled trial
(double blinded, parallel assignment diagnostic trial)
Study sites: ICMR-National
Institute of pathology
•
Methodology:
Duration: 4 year,1st March 2024
to 31st May 2029
Place of sample collection:
ICMR-NIP Ground floor sample
collection room.
The diagnostic samples collected
bedside will be transported by project staff of PI from department of
Pediatrics at VMMC & SJH
A randomized diagnostic study to
validate the sensitivity and specificity and positive predictive value of this
technology versus the conventional diagnostic method to improve the leukemia
molecular subtyping and prognostication will be implemented. Patients will be
recruited as per ethical approval of VMMC & SJH, after confirmation of
flowcytometry/histopathology as leukemia patients will be randomized into one
of the Experimental Arm-1. Testing using the conventional method; Control Arm
2- using our DBS based technology for the same. So for same patients both test
will be done, treatment or management will not change for either of the arms
based on the tests, the process of the test however will not be communicated to
the clinician for blinding process. The results will then be compared for
sensitivity, specificity and positive predictive value for our technology
comparing with the conventional methodology. The results will be double
blinded.
Patient Population:
Acute leukaemia patients will be
selected as per inclusion and exclusion criteria from those who attend the
paediatric OPD of VMMC and Safdarjung Hospital will be selected for inclusion
in the study based on the following criteria:
Patient Inclusion Criteria:
1. Patients/guardians who have
given informed consent given for participation in the study
2. Untreated new diagnosed
leukaemia cases on flow cytometry.
3. Leukemia Patients with no
history of chronic disease, MDS/Chemotherapy/Immunological deficient states
4. Leukemia patients whose PB
samples have been received by ICMR-NIOP for diagnostic purposes.
Patient Exclusion Criteria:
1. Patients who are partially treated/referred
after treatment
2. Patients who are critically
ill or are in a state of shock
3. Patients who are having
biohazard exposures like HIV, Hbs Ag, HCV
4. Patients/guardians who are not
willing to give consent for participation in the study
Informed written consent will be
obtained from all patients in accordance with the Declaration of Helsinki.
Materials and Methods:
1 ml of peripheral blood samples
in acute and chronic leukemia patient samples whose TLC is more than 50,000
cells/cu mm which is left after the diagnostic reporting will be used (normally
we receive around 2 ml PB sample and only about 500Ul of it is utilized).
The approx. amount of 60ul per
DBS spot will be used. 2 DBS spots will be used per patient. The transcript
will be run on the diagnostic sample and correlated with that of the DBS
sample.
Our objective is to standardise
and derive the sensitivity & specificity versus the conventional method for
extraction of viable RNA from Dried Blood Spots (DBS) in acute and chronic
leukaemia samples for the identification of transcripts as per WHO classification
2007 and prognostication by Real-Time PCR. RNA will be extracted using
agitation-based (vortex) and with Illustra RNAspin Mini RNA Isolation kit, and
purified with Zymo RNA Concentrator kit. A spectrophotometer (Nanodrop) will be
used to quantify the extracted RNA. Then synthesis of DNA from an RNA template,
via reverse transcription, results in complementary DNA (cDNA) which will be
further used for RT-PCR diagnosis.
Patient details collected from
the patient will be the following:
Pt Age/Sex
Pt Total leucocyte count at
presentation of sample
Patient transcript status-ALL or
AML
Category of leukemia- relapsed/de
novo/adult or pediatric/treated or untreated
Chromosomal abnormality if any
Copy numbers for the specific
transcript (reported out in quantification of MRD)
Minimal residual disease status
by flowcytometry (Patient outcome indicator)-Relapsed or resistant
After testing the results will be
communicated to the treating physician based on either of the tests, in case of
doubt or discrepancy in the results the same will be confirmed by sequencing
for that patient.
Detailed Methodology:
Population:
New Diagnosed acute leukemia-Diagnosed by
flowcytometry or histopathologically as AML or ALL.
Who give consent to participation
in study. Prior treatment received or those with pancytopenia as per CTCAE ver
7.0 and those with a molecular subtyping report available will be excluded from
study.
Intervention:
The patients will be randomized into two
groups-interventional arm and comparator arm, after random allocation based on
inclusion and exclusion criteria. The patients in the interventional arm will
be tested by the novel DBS based test and the result will be provided to the
treating physician. After that the same patient will also be tested using
conventional method for transcript using PB or Bone marrow, this result however
will not be reported to the treating physician. The treatment or management
decision will not change for any intervention in the trial. Also, in case of
discrepancy between the conventional and experimental method of tests, the
confirmation will be done by NGS for that sample.
Comparator:
The patients in the comparator
arm will be tested by standard conventional subtype for leukemia based on
molecular transcript.
Primary Outcome:
Sensitivity, specificity,
positive predictive value and Negative predictive value for transcript subtype
as compared to the standard method.
a. Sample size:
A purposive sampling of
approximately 300 ALL and 150 AML samples will be tested from all participating
center for initial two years of recruitment. The patients will be randomized
based on computer generated tokens on either of the investigational arms. Only
patients willing to undergo treatment will be enrolled in the study. |