Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers.

STUDY QUESTION: Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized processing/freezing for centers that do not have those capabilities? SUMMARY ANSWER: Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice. WHAT IS KNOWN ALREADY: Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient's tissue was donated to research and 75% was stored for patient's future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg-6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size. WIDER IMPLICATIONS OF THE FINDINGS: Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested. STUDY FUNDING/COMPETING INTEREST(S): Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests.

High-dose carboplatin, thiotepa, and etoposide with autologous stem-cell rescue for patients with recurrent medulloblastoma. Children's Cancer Group.

PURPOSE: Medulloblastoma is a highly lethal disease when it recurs. Very few patients survive with conventional treatment. This study evaluated the use of high-dose carboplatin, thiotepa, and etoposide with autologous stem-cell rescue (ASCR) in patients with recurrent medulloblastoma. METHODS: Chemotherapy consisted of carboplatin 500 mg/m2 (or area under the curve = 7 mg/mL x min via Calvert formula) on days -8, -7, and -6; and thiotepa 300 mg/m2 and etoposide 250 mg/m2 on days -5, -4, and -3; followed by ASCR on day 0. In addition to the study-prescribed therapy, 21 patients received other treatment: neurosurgical resection in seven, conventional chemotherapy in 17, and external-beam irradiation in 11 cases. RESULTS: Twenty-three patients with recurrent medulloblastoma, aged two to 44 years (median, 13 years) at ASCR, were treated. Three patients died of treatment-related toxicities within 21 days of ASCR; multiorgan system failure in two, and Aspergillus infection with venoocclusive disease in one. Seven of 23 patients (30%) are event-free survivors at a median of 54 months post-ASCR (range, 24 to 78 months). Kaplan-Meier estimates of event-free (EFS) and overall survival are 34% +/- 10% and 46% +/- 11%, respectively, at 36 months post-ASCR. CONCLUSION: This strategy may provide long-term survival for some patients with recurrent medulloblastoma.

Multidrug resistance gene expression in pediatric primitive neuroectodermal tumors of the central nervous system.

Pediatric primitive neuroectodermal tumor (PNET) is a malignancy of the central nervous system currently treated with surgery, radiation therapy, and chemotherapy. Despite aggressive management, tumors recur in almost one-half of all patients. Drug resistance of tumor cells may, in part, explain the poor outcome. Resistance to chemotherapeutic agents may be related to expression of the multidrug resistance gene (MDR1) and its protein product, P-glycoprotein. The role of MDR1 in 16 instances of PNET was investigated using Western blot analysis to detect the expression of P-glycoprotein, messenger ribonucleic acid (mRNA), polymerase chain reaction to detect MDR1 mRNA expression, and Southern blot analysis to assess gene amplification. Analysis of proteins extracted from 15 tumors revealed that two of the 15 patients expressed detectable levels of P-glycoprotein. Polymerase chain reaction of ribonucleic acid from 12 PNET's revealed that six of the 12 patients (four of 10 de novo tumors and both recurrent tumors) expressed MDR1 mRNA. Southern blot analysis of deoxyribonucleic acid from 16 PNET's revealed no evidence of MDR1 amplification in any tumor. This is the first report of MDR1 expression in pediatric brain tumors. These data suggest a possible role for MDR1 in de novo and acquired drug resistance in PNET's.

Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer.

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.

Results of a phase I/II trial of recombinant human granulocyte-macrophage colony-stimulating factor in very low birthweight neonates: significant induction of circulatory neutrophils, monocytes, platelets, and bone marrow neutrophils.

Neonates, especially those of very low birthweight (VLBW), have an increased risk of nosocomial infections secondary to deficiencies in development. We previously demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) production and mRNA expression from stimulated neonatal mononuclear cells are significantly less than that from adult cells. Recombinant murine GM-CSF administration to neonatal rats has resulted in neutrophilia, increased neutrophil production, and increased survival of pups during experimental Staphylococcus aureus sepsis. In the present study, we sought to determine the safety and biologic response of recombinant human (rhu) GM-CSF in VLBW neonates. Twenty VLBW neonates (500 to 1,500 g), aged < 72 hours, were randomized to receive either placebo (n = 5) or rhuGM-CSF at 5.0 micrograms/kg once per day (n = 5), 5.0 micrograms/kg twice per day (n = 5), or 10 micrograms/kg once per day (n = 5) given via 2-hour intravenous infusion for 7 days. Complete blood counts, differential, and platelet counts were obtained, and tibial bone marrow aspirate was performed on day 8. Neutrophil C3bi receptor expression was measured at 0 and 24 hours. GM-CSF levels were measured by a sandwich enzyme-linked immunosorbent assay at 2, 4, 6, 12, and 24 hours after the first dose of rhuGM-CSF. At all doses, rhuGM-CSF was well tolerated, and there was no evidence of grade III or IV toxicity. Within 48 hours of administration, there was a significant increase in the circulating absolute neutrophil count (ANC) at 5.0 micrograms/kg twice per day and 10.0 micrograms/kg once per day, which continued for at least 24 hours after discontinuation of rhuGM-CSF. When the ANC was normalized for each patient's first ANC, there was a significant increase in the ANC on days 6 and 7 at each dose level. By day 7, all tested doses of rhuGM-CSF resulted in an increase in the absolute monocyte count (AMC) compared with placebo-treated neonates. In those receiving rhuGM-CSF 5.0 micrograms/kg twice per day, there was additionally a significant increase in the day 7 and 8 platelet count. Tibial bone marrow aspirates demonstrated a significant increase in the bone marrow neutrophil storage pool (BM NSP) at 5.0 micrograms/kg twice per day and 10.0 micrograms/kg once per day. Neutrophil C3bi receptor expression was significantly increased 24 hours after the first dose of rhuGM-CSF at 5.0 micrograms/kg once per day. The elimination half-life (T1/2) of rhuGM-CSF was 1.4 +/- 0.8 to 3.9 +/- 2.8 hours.(ABSTRACT TRUNCATED AT 400 WORDS)