Description

Background: Allogeneic Hematopoietic stem cell transplant (HSCT) is often the only curative treatment option for patients with end stage hematological malignancies. Haploidentical HSCT is increasingly being adopted worldwide due to ease of access to donors. Post transplant cyclophosphamide (PTCy) has revolutionized the way the world does T cell replete haploidentical transplant. However, the success of PTCy comes at the cost of delayed engraftment and increased organ toxicities. Reduced dose of PTCy may be equally effective in GvHD prevention and translate into better engraftment kinetics and immune reconstitution Objectives: To evaluate the GvHD prophylactic effect of reduced dose PTCy in combination with Calcineurin inhibitors and MMF for haploidentical HSCT.

Novelty: The proposed study is a prospective phase 2 trial of reduced dose PTCy as GvHD prophylaxis in haplo HSCT. By demonstration of equal GvHD prevention efficacy of reduced dose PTCy this study would provide the base for phase 3 randomised trial and provide practice changing answers.

Study treatment:

Post-transplant Immunosuppression:

● GvHD prophylaxis regimen will consist of two doses of cyclophosphamide on D+3 and D+4 at 25mg/kg/day (that is half of the usual standard dose) with calcineurin inhibitor and mycophenolate mofetil / mycophenolate sodium. Mesna will be given with cyclophosphamide (this is standard of care). The use of calcineurin inhibitors and mycophenolate mofetil / mycophenolate sodium will be as per standard practice.

Administration of study treatments

• Hydration and MESNA
• Adequate intravenous hydration with normal saline will be started at least 4 hours prior to cyclophosphamide, and will be continued till 24 hours post completion of 2nd dose of cyclophosphamide as per standard institutional practice.
• Mesna will be administered as a continuous infusion starting from Day+3 till 24 hours after the second dose of PTCy.
• Cyclophosphamide
• Cyclophosphamide would be administered on D+3 and D+4 \[first dose starting between 60 - 72 hrs after stem cell infusion\] at a dose of 25 mg/kg/day intravenously as an intravenous infusion over 2 hrs. The timing of cyclophosphamide and the infusion time of 2 hours are standard of care. The actual administered dose can be rounded off to the nearest 100 mg (Eg if the dose is 1760 mg, the actual administered dose can be 1800 mg)
• Study treatment: Cyclophosphamide.
• Dosage: 25 mg/kg/day.
• Dosage schedule: Day+3 and Day+4 of transplant.
• Route/Mode of administration: Intravenous infusion over 2 hrs with Mesna. Assessment Plan (All the below assessments are according to standard institutional protocols and policies. These are not specific for this study)
• The day of neutrophil engraftment and platelet engraftment will be captured.
• Donor chimerism will be assessed by FISH\[XX/XY\] in case of sex mismatched transplants and with VNTR chimerism studies on day +14, day +28 and monthly thereafter for 1 year. Additional chimerism studies can be done if clinically indicated
• aGvHD grading will be determined by the investigator as per Modified Glucksberg criteria on a weekly basis for the first 100 days and every 28 days thereafter, as well as at scheduled study visits on Days 100, 180, and 365 and at the end of treatment. Additional assessments will be done if clinically indicated. Assessment of acute GVHD is primary clinical with additional investigations if deemed necessary. These time points are same as that followed in clinical practice and no extra study-specific assessments are needed.
• Chronic GVHD will be diagnosed and graded based on NIH and Seattle consensus criteria. The time to onset of limited and extensive chronic GVHD will also be recorded.
• Adverse events will be assessed according to National Cancer Institute Common Terminology Criteria for Adverse Events v 5.0 from the time of cyclophosphamide till day +30.
• Immune Reconstitution will be assessed at Day+30, Day+60, Day+90, Day+180 and Day +365. This assessment will be done by lymphocyte subset analysis and is being done for all transplant patients at the mentioned time point at our institute. These are not study specific but the same as what is followed in standard institutional practice.
• Cytokine analysis will be done at baseline, Day+1, Day+3, Day +5 and +7. These too are done as a standard practice for all haplo-identical transplants, and hence are not specific to the study.
• The days above assessments (not being study specific, but as usual in standard practice), can be adjusted according to patient's OPD follow up days to coincide with their routine blood collections. It may be noted that all BMT patients are followed up at least twice a week till 100 days (3 months) post BMT.

Characterization of immune cell profile using 16-color flow cytometry Peripheral blood samples will be studied for various immune cell subsets using the bulk-lyse and stain method. Antibody staining will be performed using a 16-color antibody panel and cells will be acquired on a BD LSR Fortessa flow cytometer. A minimum of 100000 cells will be studied in each panel. This panel will provide more than 100 immune cell subsets. Flow cytometry data will be analyzed using Kaluza software version 2.0 and tSNE software. The different immune cell subsets will be categorized. Absolute counts of these subsets will be calculated in PB samples with WBC counts using the dual-platform method.

Cytokine profile analysis Cytokine profile analysis for cytokines such as IL-1β, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α, IP-10, GM-CSF, etc., will be performed using the patient's peripheral blood plasma samples (within 4 h of collection) using a bead-based immunoassay (CBA, BD Biosciences, and BioLegend's LEGENDplex™) according to the manufacturer's instructions. In brief, 50 µL of prepared capture bead mixture will be mixed with 50 µL of the diluted sample and incubated at room temperature for 1.5 h. Then, 50 µL of the detection reagent will be added to this mixture and incubated for 1.5 h. The detection reagent is a mixture of analyte-specific antibodies conjugated to phycoerythrin. Incubation of the samples with capture beads and then the detection reagent leads to the formation of a "sandwich complex" of the three. These complexes will be acquired on LSRFortessa (BD Biosciences), and data will be analyzed using FCAP Array software (BD Biosciences) and LEGENDplex version 8.0 software (BioLegend).

Sample size Based on our historical data, the expected incidence of grades 3 and 4 aGvHD is estimated to be 30%. The study will enroll a total of 20 patients using a Simon 2-stage design. If in the initial 10 patients enrolled, 4 or more develop acute grade III or IV GVHD, then we will stop the study. If 3 or less patients develop acute grade III or IV GVHD, then we will complete the enrollment of the planned 20 patients. We expect to complete the enrollment in 4 years.

Statistical Analysis All continuous data will be expressed as median and IQR. Categorical data will be expressed as frequency and percentage. The incidence of acute and chronic GvHD, relapse, and NRM will be estimated based on cumulative incidence methods accounting for competing event (TRM or death due to any cause). Overall Survival and Relapse Free Survival probabilities will be estimated by the Kaplan-Meier method.

The platelet and neutrophil engraftment will be expressed using median and IQR. Statistical analysis will be performed by appropriate statistical tests using IBM SPSS v25 (statistical package for social sciences) software.

Study Timeline and Oversight

Duration: 4 years (estimated)

Site: Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)

Sample size: 20 patients

Provision of ancillary care for unrelated illness during the duration of the study:

Allogeneic stem cell transplant is associated with development of several kinds of post-transplant complications. All such complications will be managed as per standard of care by the transplant team and as mentioned previously, the patient will bear the cost of such management.

Plan for Missing Data Predefined rules will be applied to classify missing, uninterpretable, or inconsistent data.

Privacy, confidentiality and anticipated consequences of breach of confidentiality:

All the study data will remain in a password protected document which will be accessible only to the study investigators and the regulatory authorities. In any presentation originating from the study or any publication, no reference will be made to individual patients. Any data presentation will always be de-identified.

The fact that the patient has undergone an allogeneic HSCT would be available / made known to several clinicians and other medical / paramedical staff who would be involved in patient care. Since there are no additional study specific assessments or follow ups, any breach of information about your trial participation is unlikely to have any consequences.

Compensation and reimbursement:

Transplant related complications (which may lead to fatal outcomes) are inherent complications of half-matched transplants. These could occur irrespective of trial participation. Therefore, the study does not have a provision for compensation (since these complications can occur irrespective of study participation). Also, because of the same reason, there is no reimbursement and the cost of management of complications will be borne by the patient.

All trial-related data will be collected in accordance with institutional standard operating procedures (SOPs).