Study Of Pediatric Acute Promyelocytic Leukemia And IPS Cells From A Pediatric Leukemia Patient

Part1Retrospective Analysis of119Cases of Pediatric Acute Promyelocytic Leukemia:Comparisons of four RegimesObjective:Clinical trials have demonstrated that pediatric Acute promyelocytic leukemia (APL) is highly curable. Small-scale studies have been reported on treatment of APL on one or two treatment regimes. Here we report a multiple center-based study of119cases of pediatric APL treated with four regimes based on all trans-retinoic acid (ATRA). Procedures:We retrospectively analyzed the clinical and laboratorial characteristics and treatment outcome of the pediatric APL patients. Regime1used a protocol developed in house, regime2was modified from the PETHEMA LPA99protocol, regime3was modified from the European-APL93protocol, and regime4used a protocol suggested by the British Committee for Standards. Results:the overall complete remission rates for the four regimes were88.9%,87.5%,97.1%, and87.5%, respectively, which have no statistic difference. However, better results were observed with regimes2and3than regimes1and4, in terms of estimated3.5-year disease-free survivals, relapse rates, drug toxicity (including hepatotoxicity, cardiac arrhythmia, and differentiation syndrome), and sepsis. Conclusion:The overall outcomes are better with regimes2and3than with regimes1and4, which may benefit from the specific compositions of regimes2and3. Part2Case study of pediatric acute promyelocytic leukemia:why did some patients abandon treatment?Background:Treatment refusal and abandonment are major causes for death of pediatric acute promyelocytic leukemia (APL) patients, especially in developing countries, which is often associated with the socioeconomic status of patient's parents. Here we report the reasons identified for refusal and abandonment of pediatric APL treatment in six hospitals in central and southern China. Methods:we retrospectively analyzed the medical records of pediatric APL patients admitted from September1997to December2009, and interviewed the families of those who refused or abandoned the treatment to identify reasons associated to the dropout. Results:Of158admitted patients in total,39(23.7%) refused or abandoned treatment. Interviews were successfully conducted through telephone or mail with the parents or guardians of37of the dropout patients. The reasons for the dropout, although varying among these cases, are associated with financial hardships, misbelief that APL is incurable, the female gender, age younger than5years, and parents'educational levels. Conclusion:Effective health insurance systems, sufficient and adequate communications between health care providers and patients/parents, legal protection from discrimination against female and young patients, educational programs for patients/parents, and psychosocial support from communities are essential to reduce the treatment refusal and abandonment, and increase the survival and quality life of pediatric APL patients. Part3Generation and hematopoietic differentiation of induced pluripotent stem cells from a pediatric leukemia patientObjective:the purpose of this study is to derive induced pluripotent stem (iPS) cells from the skin fibroblasts of a leukemia patient, to differentiate the iPS cells into hematopoietic stem/progenitor cells, and to reconstitute the hematopoietic system and model leukemogenesis in sublethally irradiated nude mice. Methods:we used the skin fibroblasts from a one-year-old, acute myeloid leukemia, female patient with Rob(13;22) as parental cells named TC. Transduction of TC with lentiviral vectors expressing OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28was performed to derive iPS cells. An established iPS cell line from this patient was named TZ4. Fluoroimmunoassay for expression of the pluripotency markers SSEA3and SSEA4, and teratomas formation assay were used to confirm the pluripotency of TZ4. Karyotyping of the TC fibroblasts and TZ4iPSCs was conducted. The TZ4and a wild-type iPSC line TK5were induced to differentiate into hemangioblasts (HBs), which were subjected to flow cytometry analysis for CD34+and CD45+cell ratios. The HB cells differentiated from TZ4or TK5iPS cells were injected intravenously through the tail vein into sublethally irradiated NOG-SCID (immunocompromised) mice to observe hematopoietic reconstitution and leukemogenesis. Results:An iPS cell line TZ4was successfully derived from the skin fibroblasts of the patient. The pluripotency of TZ4was confirmed for expression of the pluripotency markers SSEA3and SSEA4, and the capability of teratoma formation. The karyotyping showed that the dominant karyotypes of TC and TZ4were mainly contained Rob(13;22), which TZ4tested as46, XX,+5, Rob(13;22)(q10;q10), respectively. Both TZ4and TK5iPS cells could differentiate into hemangiobloasts (HBs), progenitors for both hematopoietic and angiogenic cells. The HBs differentiated from TZ4and TK5contained similar ratio of CD34+and CD45+cells based on flow cytometry analysis. However, following transplantation of the HBs into sublethally irraidated NOG-SCID mice, no human cell engraftment was observed in the mice. Conclusions:Fibroblasts with Rob(13;22) karyotype from a pediatric AML patient could be reprogrammed to generate iPS cells, which sustained the Rob(13;22) karyotype with some additional chromosomal abnormalities. Like wild-type iPS cells, the patient-derived iPS cells could also differentiate into HB cells. However, in vivo experiments on sublethally irraidated NOG-SCID mice did not show engraftment of the human iPS cell-derived HB cells. Further studies are necessary to further characterize the HB cells and optimize the methodologies for blood reconstitution in mice with the human HB cells.