M. F. Ozkaynak

Telomerase activity and telomere length in pediatric patients with malignancies undergoing chemotherapy

Telomerase activity and telomere length in mononuclear cells (MNCs) and granulocytes from peripheral blood (PB) and bone marrow (BM) specimens were studied in pediatric acute leukemia (ALL, n = 15; AML, n = 1) and pediatric solid tumor (ST) patients (n = 9) at diagnosis, during and after chemotherapy. In four ST patients, tumor tissue was also available. For comparative analysis, MNCs from healthy donors (n = 53) were analyzed. Telomerase was evaluated using a modified telomeric repeat amplification protocol (TRAP) assay, and telomere length by terminal restriction fragment (TRF) analysis. At diagnosis, high telomerase activity was detected in MNCs from all leukemia patients, which was similar to the activity from ST biopsy specimens. This exceeded by 10- to 20-fold the activity in PB MNCs from ST patients and healthy donors (P < 0.05). granulocyte fractions lacked telomerase activity in all groups. bm mncs in leukemia patients revealed a four-fold higher telomerase activity than pb (P = 0.005). After induction chemotherapy and response to treatment, telomerase activity decreased to borderline or undetectable levels in PB MNCs in leukemia (P < 0.01). average telomeres in pb mncs from pediatric patients were significantly longer (n = 25; 10.9 kbp) than telomeres in PB and BM MNCs from adult healthy donors (7.45 kbp) (P < 0.0001). at diagnosis, telomeres were shorter from bm compared to pb specimens in leukemia (P < 0.05), and two peak trfs were observed corresponding to the malignant and normal cell clones. with the attainment of remission, the lower trf peak, reflecting the leukemic population, was lost. in leukemia patients, mean trfs increased on average 2.2 kbp after induction chemotherapy, but decreased thereafter on consolidation and maintenance chemotherapy (1 kbp). this was comparable to an average telomere loss of 1.2 kbp in pb specimens from st patients after chemotherapy. in all patients, telomere loss in granulocytes as compared to mncs was more pronounced with 1.8 vs 1 kbp, respectively (P = 0.014). Our results demonstrate that at diagnosis, telomerase was consistently and highly upregulated in BM and PB specimens in leukemia, decreased after induction therapy, and correlated with remission. BM specimens in leukemia had higher telomerase activity, probably due to the greater leukemic burden than in PB. Telomeres were significantly longer in children than in adults, but shortened as a consequence of chemotherapy with repeated cycles of hematopoietic regeneration. In acute leukemia, with the loss of the leukemic burden after induction chemotherapy, longer mean TRFs were found, a reflection of the repopulation with normal cells. Our findings suggest that telomerase activity may be useful in the management of childhood malignancies. The significance of telomere length shortening in pediatric patients undergoing chemotherapy and possible telomere regeneration after myelosuppressive treatment remain to be determined.

Telomere dynamics in childhood leukemia and solid tumors: a follow-up study

Telomeres of hematopoietic cells shorten with age, possibly contributing to the aging-associated hematopoietic pathology (immunosenescence, malignant transformation). Accelerated telomere shortening is seen with replicative stress, such as during administration of serial chemotherapy cycles for the treatment of childhood cancer. To define the long-term consequences of pediatric cancer treatment on hematopoietic cell telomere length, we undertook a prospective 4-year follow-up study of a 61-patient cohort of pediatric malignancies in a community-based setting. We found that mononuclear cells (MNC) and granulocytes of children with standard-risk acute lymphoblastic leukemia (ALL) suffered minimal telomere shortening throughout therapy (less than 1 kbp; average follow-up, 20 months), while those of children with solid tumors showed greater and more heterogenous telomere attrition (0.5–2.8 kbp, average follow-up, 9 months). In addition, we evaluated the role of telomerase, the enzyme commonly up-regulated in pediatric leukemic and solid tumor cells for telomere length maintenance, as a disease marker. Serial determinations of telomerase in MNC were useful to confirm disease remission in leukemia, but play no role in the follow-up of children with solid tumors.

Real-time quantitative PCR: Standardized detection of minimal residual disease in pediatric acute lymphoblastic leukemia

Purpose To develop a standardized real-time polymerase chain reaction (PCR) method of quantifying minimal residual disease (MRD) in patients with pre-B acute lymphoblastic leukemia (ALL). Patients and Methods In a series of 24 follow-up bone marrow (BM) samples in 11 patients (14 clonal markers), we performed real-time PCR assays using one consensus and one clone-specific primer for each marker. The markers analyzed included immunoglobulin heavy chain (IgH), T-cell receptor (TCR) and TEL-AML1 rearrangements. Results We achieved a detection limit of 3.3 × 10−5 ± 1.2 × 10−5 and an accurate quantitation (r = −0.99) limit of 2.0 × 10−4 ± 8.8 × 10−5 blasts. Both inter- and intra-assay reproducibility were exceptional. Additionally, we found comparable results to those of a “gold standard” limiting-dilution PCR assay (r = 0.62). Conclusions The IgH, TCR and TEL-AML1 markers can be used as targets by real-time PCR under the same cycling profile, allowing quantitation of MRD in more 95% of patients with pre-B ALL. This standardized, real-time PCR technique should simplify monitoring MRD in clinical trials.

Rituximab therapy to prevent relapse in chronic relapsing thrombotic thrombocytopenic purpura (TTP) in a child.

Our patient first developed thrombotic thrombocytopenic purpura (TTP) at age 10 years with an initial platelet count of 10,000/μL. She achieved remission with plasmapheresis (PE), but suffered 2 relapses in the next 2 years, each approximately 1 year from PE, with ADAMTS13 levels of <5%. Early in her third remission, with vincristine (weekly × 4 doses) and prednisone (for 2 weeks) her ADAMTS13 increased to 99% in 24 weeks, but decreased to <4% in the next 38 weeks. After 4 weekly doses of rituximab (375 mg/m2), her ADAMTS13 level reached 101% in 9 weeks and has remained consistently above 97% on bimonthly monitoring for more than a year. She remains in continuous clinical and hematologic remission with an ADAMTS13 level of 108% at 60 weeks from rituximab therapy and 124 weeks from her second relapse. This case report suggests that monitoring ADAMTS13 level at regular intervals in recurrent TTP may help us identify patients at risk for further relapse; and such a relapse may be prevented, or at least delayed with timely rituximab therapy, thus reducing morbidity from relapsed TTP and its treatment.

Recurrent immune thrombocytopenic purpura in children.

Recurrent immune thrombocytopenic purpura (ITP) is defined as the recurrence of ITP after at least 3 months of remission sustained without treatment. Among 340 children with ITP, 14 had recurrent ITP (4.1%). Ten were females. The initial course was acute in 8 patients and chronic in 6. The median time to recurrence was 33 months (range 4–120). Only 1 patient had a second recurrence. Twelve (86%) achieved complete (n = 10) or partial (n = 2) remission, two of them after splenectomy. One patient continued to require treatment at 10 months from recurrence. One child died of intracranial hemorrhage despite aggressive treatment including splenectomy and craniotomy.

Favorable Outcome of Primary Cns Anaplastic Large Cell Lymphoma in an Immunocompetent Patient

A 9-year-old immunocompetent male patient with primary central nervous system anaplastic large cell lymphoma was treated with 5 cycles of intensive chemotherapy including high-dose Ara-C, high-dose methotrexate, etoposide, and carmustine along with intraventricular chemotherapy followed by high-dose thiotepa and carboplatin with autologous peripheral blood hematopoietic stem cell transplantation. He received radiotherapy as the final therapy and has remained in remission for 26 months off therapy.

T-cell acute lymphoblastic leukemia after renal transplantation in childhood

Purpose: Lymphoproliferative disorders in solid organ recipients are usually of B-cell type and have rarely been described in childhood. This study describes the development of T-cell acute lymphoblastic leukemia (ALL) in a child occurring 6 years after renal transplantation. Patient: An 11 -year-old boy had received a renal allograft from his father at 5 years of age. He was receiving imuran, prednisone, and cyclosporin A prophylaxis for graft rejection after transplant until T-cell ALL was diagnosed. Although an acute Epstein-Barr virus (EBV) infection was noted at the time of diagnosis, the EBV genome was not detected by Southern blot analysis and polymerase chain reaction (PCR) in the leukemic cells. Results: A large mediastinal mass and malignant pleural effusion were noted at diagnosis. Leukemic cells of his bone marrow and pleural fluid expressed T-cell antigens with unique cytogenetic features, including add(1)(p36.1), del(11)(q14), and monosomy 7. EBV serology was consistent with a recent infection but EBV genome was not detected by Southern blot and PCR analysis in his leukemic cells. Human T-lymphotropic virus-1 (HTLV-I) antibody titer was negative. He has been on chemotherapy for 9 months, maintaining his first remission. Conclusions: Malignancies developing after renal transplantations are usually lymphoproliferative disorders and of B-cell origin. In the majority of these patients, EBV plays an etiologic role. Although adult T-cell leukemia developing during immunosup-pressive treatment in renal transplant recipients has been reported, T-cell leukemia after transplant in pediatric patients has not been reported to date. This case is unique in terms of the patients age, the T-cell immunophenotype, the cytogenetic features, and the absence of an EBV genome within the leukemic cells despite an acute EBV infection before diagnosis.

HLA-Bw4-I-80 isoform differentially influences clinical outcome as compared to HLA-Bw4-T-80 and HLA-A-Bw4 isoforms in rituximab or dinutuximab-based cancer immunotherapy

Killer-cell immunoglobulin-like receptors (KIRs) are a family of glycoproteins expressed primarily on natural killer cells that can regulate their function. Inhibitory KIRs recognize MHC class I molecules (KIR-ligands) as ligands. We have reported associations of KIRs and KIR-ligands for patients in two monoclonal antibody (mAb)-based trials: (1) A Children’s Oncology Group (COG) trial for children with high-risk neuroblastoma randomized to immunotherapy treatment with dinutuximab (anti-GD2 mAb) + GM-CSF + IL-2 + isotretinion or to treatment with isotretinoin alone and (2) An Eastern Cooperative Oncology Group (ECOG) trial for adults with low-tumor burden follicular lymphoma responding to an induction course of rituximab (anti-CD20 mAb) and randomized to treatment with maintenance rituximab or no-maintenance rituximab. In each trial, certain KIR/KIR-ligand genotypes were associated with clinical benefit for patients randomized to immunotherapy treatment (immunotherapy in COG; maintenance rituximab in ECOG) as compared to patients that did not receive the immunotherapy [isotretinoin alone (COG); no-maintenance (ECOG)]. Namely, patients with both KIR3DL1 and its HLA-Bw4 ligand (KIR3DL1+/HLA-Bw4+ genotype) had improved clinical outcomes if randomized to immunotherapy regimens, as compared to patients with the KIR3DL1+/HLA-Bw4+ genotype randomized to the non-immunotherapy regimen. Conversely, patients that did not have the KIR3DL1+/HLA-Bw4+ genotype showed no evidence of a difference in outcome if receiving the immunotherapy vs. no-immunotherapy. For each trial, HLA-Bw4 status was determined by assessing the genotypes of three separate isoforms of HLA-Bw4: (1) HLA-B-Bw4 with threonine at amino acid 80 (B-Bw4-T80); (2) HLA-B-Bw4 with isoleucine at amino acid 80 (HLA-B-Bw4-I80); and (3) HLA-A with a Bw4 epitope (HLA-A-Bw4). Here, we report on associations with clinical outcome for patients with KIR3DL1 and these separate isoforms of HLA-Bw4. Patients randomized to immunotherapy with KIR3DL1+/A-Bw4+ or with KIR3DL1+/B-Bw4-T80+ had better outcome vs. those randomized to no-immunotherapy, whereas for those with KIR3DL1+/B-Bw4-I80+ there was no evidence of a difference based on immunotherapy vs. no-immunotherapy. Additionally, we observed differences within treatment types (either within immunotherapy or no-immunotherapy) that were associated with the genotype status for the different KIR3DL1/HLA-Bw4-isoforms. These studies suggest that specific HLA-Bw4 isoforms may differentially influence response to these mAb-based immunotherapy, further confirming the involvement of KIR-bearing cells in tumor-reactive mAb-based cancer immunotherapy.

Osteosarcoma Following Growth Hormone Therapy in Recurrent Acute Lymphoblastic Leukemia and Rapadilino Syndrome

A 16-year old Caucasian girl was evaluated for scoliosis 9 years after bone marrow transplant (BMT) and 13 years after her initial ALL diagnosis. A chest radiograph showed a lesion of the right third rib. Histopathology was consistent with a high-grade OS. She died 5 months after OS diagnosis. There is no family history of cancer. Prior therapy comprised BFM standard risk for de novo ALL, St. Jude Children’s Research Hospital regimen and cranial-spinal radiation for central nervous system (CNS) recurrence, Children’s Cancer Group (CCG) 1941, and matched unrelated allo-transplant (conditioned with TBI and thiotepa, cyclophosphamide, and ATG) for bone marrow recurrence. Two years after transplant GH therapy commenced. A 15-year-old Hispanic girl with RAPADILINO syndrome experienced left lower extremity pain. Magnetic resonance imaging (MRI) showed a destructive lesion and biopsy was consistent with OS. At 11 years of age short stature was diagnosed. Growth hormone therapy was started and discontinued once OS was diagnosed. There is no family history of cancer. The Childhood Cancer Survivor Study reported the 25-year follow-up among survivors of childhood ALL. The cumulative incidence of SMN of 5.2% (6.2% for those receiving radiation therapy) at 25 years from diagnosis [2]. St. Jude Children’s Research Hospital (SJCRH) investigators reported the incidence of SMN as a first event in 2169 patients treated between 1962 and 1998 [3]. The cumulative incidence of SMN was 4.17% at 15 years and 10.85% at 30 years. Two cases of OS constituting 1.2% of SMN were observed.

Laparoscopic Surgery for Acute Appendicitis in Children With Cancer.

Introduction: Abdominal pain during cancer chemotherapy may be caused by medical or surgical conditions. A retrospective review of 5 children with cancer who had appendicitis while receiving chemotherapy was performed. Case Descriptions: Three had acute lymphoblastic leukemia, and 1 each had T-cell lymphoblastic lymphoma and rhabdomyosarcoma. Two of the patients had a Pediatric Appendectomy Score of 6, and 1 each had a score of 7, 5, and 2. All had evidence of appendicitis on computed tomography. Laparoscopic appendectomy was performed without any perioperative complication. Discussion: Appendicitis is an important diagnosis in children with cancer, and laparoscopic appendectomy is safe and the procedure of choice.