O-036 Safe and successful ovarian transplantations post leukemia after combined various MRD excluding tests
Authors: Dereh Haim Tzfanya S , Raanani H , Amariglio N , Dick- Necula D , Shapira M , Meirow D 
Published: June 2022

Abstract

Study question
Leukemias are considered high risk for minimal residual disease (MRD) contamination in cryopreserved ovarian tissue. How improving the safety of ovarian transplantation in leukemia patients?

Summary answer
Combination of various methods; immunohistochemistry, molecular biology and long period xenotransplantation to immune-deficient mice is obligatory for safety of ovarian transplantation (OT) in high-risk patients

What is known already
With high percentage of cancer survivors and high treatment induced ovarian failure in leukemia patients, more cured patients request OT to restore fertility. Studies using different methods have identified MRD contamination in cryopreserved ovarian tissue, which could potentially result in recurrence of the primary disease after reimplantation. This risk is high in leukemia patients thus many caregivers avoid OT in these patients. Today, different methods for identifying MRD like immunohistochemistry (IHC), FISH molecular methods, xenotransplantation into immune deficient mice are used to evaluate MRD in ovary before OT

Study design, size, duration
Between 2005 and 2022 ovarian tissue cryopreservation was performed in 74 leukemia patients. Twenty one leukemic patients were evaluated for MRD in the ovarian tissue. sixteen women were diagnosed with AML, 3 ALL and 2 suffered from CML. To evaluate MRD, one stored ovarian piece was thawed prior to transplantation, and different, personally determined methods, were used according with multidisciplinary team instructions. Only when all tests were negative OT was performed

Participants/materials, setting, methods
One ovarian piece was thawed and divided into 5 for evaluation. One used for H&E and immunohistochemistry (IHC) to check for follicles presence and macroscopic malignancy evidence. One was used to check molecular markers by extracting DNA/RNA and performing molecular PCR, RT-PCR and next generation sequencing (NGS). In patients without molecular markers at diagnosis, leukemia cells were evaluated using NGS to identify new markers. Pieces were xenotransplanted into 3 SCID mice for 6 months.

Main results and the role of chance
For MRD assessment, H&E and individualized IHC were performed in all ovarian grafts with no evidence of malignancy in 20 cases. However, in one AML patient IHC identified cluster of leukemic cells and further evaluation was not performed. In all patients presence of follicles was documented.
Patient specific molecular markers were checked in ovarian tissue of 13/15 AML patients. One patient presented positive marker t(15;17) via RT-PCR while other methods were negative. Ten patients underwent ovarian tissue xenotransplantation in mice. In one patient, all 3 mice developed macroscopic leukemic masses after 5 months while other methods were negative. Eight OT were performed in 4 AML patients presenting with negative MRD tests resulting in 7 pregnancies and 4 live births. Long term follow up of up to 7 years showed no evidence of disease recurrence.
Ovaries of 3 ALL patients were evaluated. In one patient, RT-PCR identified positive marker (IGH rearrangement). No evidence of malignancy was identified in all xenotransplanted mice. One patient underwent OT and achieved a twin pregnancy.
Ovaries of 2 CML patients were checked. 1st patient showed BCR-ABL gene in ovary via RT-PCR. The ovary of second patient was free of disease, underwent OT. In this patient, no pregnancy achieved.

Limitations, reasons for caution
Safe transplantation requires checking molecular markers of diagnosis in ovary. The tumor cells aren't always available. In addition, not all leukemia patients carry molecular markers. Furthermore, since MRD is checked in one piece, malignant cells might be missed. Additionally, this piece doesn't represent the other pieces which would be transplanted

Wider implications of the findings
OT can restore fertility. High risk of ovary contamination in leukemia patients prevents centers to OT, leaving survivors infertile. Here we show safe and successful OT following meticulous MRD assessment by multiple methods. furthermore, MRD detection protocol might be practiced on testicular frozen tissue to restore fertility in young boys

Trial registration number
7222-09-SMC