A diagnostic randomized controlled trial to study to validate novel DBS based technology for prognosis and screening for leukemia transcripts

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

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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.