Rakesh K. Goyal

TERC and TERT gene mutations in patients with bone marrow failure and the significance of telomere length measurements

Dyskeratosis congenita (DC) is a rare inherited form of bone marrow failure (BMF) caused by mutations in telomere maintaining genes including TERC and TERT. Here we studied the prevalence of TERC and TERT gene mutations and of telomere shortening in an unselected population of patients with BMF at our medical center and in a selected group of patients referred from outside institutions. Less than 5% of patients with BMF had pathogenic mutations in TERC or TERT. In patients with BMF, pathogenic TERC or TERT gene mutations were invariably associated with marked telomere shortening (<< 1st percentile) in peripheral blood mononuclear cells (PBMCs). In asymptomatic family members, however, telomere length was not a reliable predictor for the presence or absence of a TERC or TERT gene mutation. Telomere shortening was not pathognomonic of DC, as approximately 30% of patients with BMF due to other causes had PBMC telomere lengths at the 1st percentile or lower. We conclude that in the setting of BMF, measurement of telomere length is a sensitive but nonspecific screening method for DC. In the absence of BMF, telomere length measurements should be interpreted with caution.

Ajuba, a Novel LIM Protein, Interacts with Grb2, Augments Mitogen-Activated Protein Kinase Activity in Fibroblasts, and Promotes Meiotic Maturation of Xenopus Oocytes in a Grb2- and Ras-Dependent Manner

ABSTRACT LIM domain-containing proteins contribute to cell fate determination, the regulation of cell proliferation and differentiation, and remodeling of the cell cytoskeleton. These proteins can be found in the cell nucleus, cytoplasm, or both. Whether and how cytoplasmic LIM proteins contribute to the cellular response to extracellular stimuli is an area of active investigation. We have identified and characterized a new LIM protein, Ajuba. Although predominantly a cytosolic protein, in contrast to other like proteins, it did not localize to sites of cellular adhesion to extracellular matrix or interact with the actin cytoskeleton. Removal of the pre-LIM domain of Ajuba, including a putative nuclear export signal, led to an accumulation of the LIM domains in the cell nucleus. The pre-LIM domain contains two putative proline-rich SH3 recognition motifs. Ajuba specifically associated with Grb2 in vitro and in vivo. The interaction between these proteins was mediated by either SH3 domain of Grb2 and the N-terminal proline-rich pre-LIM domain of Ajuba. In fibroblasts expressing Ajuba mitogen-activated protein kinase activity persisted despite serum starvation and upon serum stimulation generated levels fivefold higher than that seen in control cells. Finally, when Ajuba was expressed in fully developed Xenopus oocytes, it promoted meiotic maturation in a Grb2- and Ras-dependent manner.

Reduced-intensity allogeneic transplantation in pediatric patients ineligible for myeloablative therapy: results of the Pediatric Blood and Marrow Transplant Consortium Study ONC0313

The role of reduced-intensity conditioning (RIC) regimens in pediatric cancer treatment is unclear. To define the efficacy of a busulfan/fludarabine/antithymocyte globulin RIC regimen in pediatric patients ineligible for myeloablative transplantation, we completed a trial at 23 institutions in the Pediatric Blood and Marrow Transplant Consortium. Forty-seven patients with hematologic malignancies were enrolled. Sustained engraftment occurred in 98%, 89%, and 90%, and full donor chimerism was achieved in 88%, 76%, and 78% of evaluable related bone marrow/peripheral blood stem cells (BM/PBSCs), unrelated BM/PBSCs, and unrelated cord blood recipients. With a median follow-up of 24 months (range, 11-53 months), 2-year event-free survival, overall survival (OS), transplantation-related mortality, and relapse were 40%, 45%, 11%, and 43%, respectively. Univariate analysis revealed an inferior outcome when patients had undergone previous total body irradiation (TBI)-containing myeloablative transplantation (2-year OS, 23% vs 63% vs 52%, previous TBI transplantation vs no TBI transplantation vs no transplantation, P = .02) and when patients not previously treated with TBI had detectable disease at the time of the RIC procedure (2-year OS, 0% vs 63%, detectable vs nondetectable disease, P = .01). Favorable outcomes can be achieved with RIC approaches in pediatric patients in remission who are ineligible for myeloablative transplantation. This study was registered at www.clinicaltrials.gov as #NCT00795132.

The genomic landscape of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 or CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and could therefore be candidates for experimental therapies. In addition, few molecular pathways aside from the RAS-MAPK pathway have been identified that could serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia to expand knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, splicing, Polycomb repressive complex 2 (PRC2) and transcription. Notably, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.

Lung Function, Pulmonary Complications, and Mortality after Allogeneic Blood and Marrow Transplantation in Children

Pulmonary complications (PC) remain a significant barrier to the success of allogeneic blood and marrow transplantation (BMT). Pretransplant pulmonary function tests (PFTs) have been correlated with risk of early respiratory failure and mortality in adult BMT recipients. There is limited data on their relationship to posttransplant outcomes in pediatric patients. We sought, in pediatric allo-BMT recipients (1) to analyze the spectrum of infectious and noninfectious PCs, (2) to evaluate the prevalence and course of PFT abnormalities before and after transplant, and (3) to correlate pretransplant PFT findings with patient outcomes, specifically risk of PC, respiratory failure, and death. We conducted a retrospective review of PC in all patients who underwent allo-BMT at Childrens Hospital of Pittsburgh during 1996 to 2006. PFTs were performed in children 6 years and older pretransplant, 3, 6, 12, and 24 months after transplant. PCs occurring within 100 days of BMT were considered early. One hundred ten consecutive children who underwent allo-BMT were included (median age = 9.7 years; 67 males, 43 females). Seventy-five of 110 patients had 370 PFT studies performed; 62 of 73 patients >6 years of age (85%) underwent PFT studies pre-BMT. There was a significantly higher risk of early respiratory failure in patients with reduced pretransplant forced expiratory volume in 1 second (FEV(1)) (P = .0001, odds ratio [OR] 5.1) or forced vital capacity (FVC) (P = .0001, OR 8.5). Forty-three of 110 (39%) patients required mechanical ventilation, and in 30 episodes (70%), patients remained ventilator-dependent until time of death. Posttransplant, we observed statistically significant reductions in FEV(1), FVC, total lung capacity (TLC), and diffusing capacity of the lungs (DLco) at 3 months post-BMT and similar reductions at 6 months post-BMT except for DLco (not significant). Between 12 and 24 months, FEV(1), FVC, TLC, and DLco improved significantly from earlier declines post-BMT; however FEV(1) and FVC remained significantly below pretransplant values. At a median follow-up of 5.5 (1.6-11.6) years, 58 of 110 (53%) patients were surviving. The majority of the patients who died from transplant-related complications suffered from 1 or more PCs (31/32, 97%). Early PC was associated with over 4-fold reduction in probability of survival at 10 years (8/44, 18% with early PC versus 50/66, 76% without early PC). On multivariate analysis, risk of death was significantly associated with high-risk disease status (P = .015; hazard ratio [HR] = 2.5), unrelated donor (P = .03; HR = 2.1), early PC (P = .0001; HR = 7.7) and pathogen identification (P = .02; HR = 2.7). These results suggest that, in children undergoing allo-BMT (1) compromised pretransplant lung function is significantly correlated with risk of early respiratory failure but not of overall survival (OS), (2) reductions in lung volumes and diffusion capacity are common 3- to 6-month posttransplant with partial recovery by 12 to 24 months, (3) there is high mortality following mechanical ventilation, and (4) early PCs are associated with significantly worse OS.

Focal nodular hyperplasia of the liver: a sequela of tumor therapy

Focal nodular hyperplasia (FNH) of the liver occurs with increased frequency in oncology patients after completion of tumor therapy. Its development may be related to the vascular damage induced by such therapy. We present three children who developed FNH after undergoing antineoplastic therapy for non-hepatic primary tumors. Recognition of this association in the appropriate patient population might obviate the need for biopsy.

The addition of sirolimus to tacrolimus/methotrexate GVHD prophylaxis in children with ALL: A phase 3 children's oncology group/pediatric blood and marrow transplant consortium trial

Sirolimus has activity against acute lymphoblastic leukemia (ALL) in xenograft models and efficacy in preventing acute graft-versus-host disease (aGVHD). We tested whether addition of sirolimus to GVHD prophylaxis of children with ALL would decrease aGVHD and relapse. Patients were randomized to tacrolimus/methotrexate (standard) or tacrolimus/methotrexate/sirolimus (experimental). The study met futility rules for survival after enrolling 146 of 259 patients. Rate of Grade 2-4 aGVHD was 31% vs 18% (standard vs experimental, P = .04), however, grade 3-4 aGVHD was not different (13% vs 10%, P = .28). Rates of veno-occlusive disease (VOD) and thrombotic microangiopathy (TMA) were lower in the nonsirolimus arm (9% vs 21% VOD, P = .05; 1% vs 10% TMA, P = .06). At 2 years, event free survival (EFS) and overall survival (OS) were 56% vs 46%, and 65% vs 55% (standard vs experimental), respectively (P = .28 and .23). Multivariate analysis showed increased relapse risk in children with ≥0.1% minimal residual disease (MRD) pretransplant, and decreased risk in patients with grades 1-3 aGVHD (P = .04). Grades 1-3 aGVHD were associated with improved EFS (P = .02), whereas grade 4 aGVHD and extramedullary disease at diagnosis led to inferior OS. Although addition of sirolimus decreased aGVHD, survival was not improved. This study is registered with ClinicalTrials.gov as #NCT00382109.

The amino terminus of the glial glutamate transporter GLT-1 interacts with the LIM protein Ajuba

We have identified a cytoplasmic LIM protein, Ajuba, which interacts with the amino terminus of GLT-1, the most abundant plasma membrane glutamate transporter in the brain. Ajuba has a cytoplasmic location when expressed alone in COS cells, but translocates to colocalize with GLT-1 at the plasma membrane when GLT-1 is coexpressed. Ajuba is expressed in cerebellum, cortex, hippocampus, and retina and also in organs outside the CNS. Ajuba is found with GLT-1 in astrocytes, cerebellar Bergmann glia and retinal neurons, and antibodies to Ajuba coimmunoprecipitate GLT-1 from brain. For GLT-1 expressed in COS cells, coexpression of Ajuba did not affect the transporters K(m) or V(max) for glutamate. Since Ajuba is known to activate MAP kinase enzymes, and its homologue Zyxin binds to cytoskeletal proteins, we propose that Ajuba is a scaffolding protein allowing GLT-1 to regulate intracellular signaling or interact with the cytoskeleton.

The Ajuba LIM Domain Protein Is a Corepressor for SNAG Domain–Mediated Repression and Participates in Nucleocytoplasmic Shuttling

The SNAG repression domain is comprised of a highly conserved 21-amino acid sequence, is named for its presence in the Snail/growth factor independence-1 class of zinc finger transcription factors, and is present in a variety of proto-oncogenic transcription factors and developmental regulators. The prototype SNAG domain containing oncogene, growth factor independence-1, is responsible for the development of T cell thymomas. The SNAIL proteins also encode the SNAG domain and play key roles in epithelial mesenchymal differentiation events during development and metastasis. Significantly, these oncogenic functions require a functional SNAG domain. The molecular mechanisms of SNAG domain-mediated transcriptional repression are largely unknown. Using a yeast two-hybrid strategy, we identified Ajuba, a multiple LIM domain protein that can function as a corepressor for the SNAG domain. Ajuba interacts with the SNAG domain in vitro and in vivo, colocalizes with it, and enhances SNAG-mediated transcriptional repression. Ajuba shuttles between the cytoplasm and the nucleus and may form a novel intracellular signaling system. Using an integrated reporter gene combined with chromatin immunoprecipitation, we observed rapid, SNAG-dependent assembly of a multiprotein complex that included Ajuba, SNAG, and histone modifications consistent with the repressed state. Thus, SNAG domain proteins may bind Ajuba, trapping it in the nucleus where it functions as an adapter or molecular scaffold for the assembly of macromolecular repression complexes at target promoters.

Unrelated Donor Bone Marrow Transplantation for Children With Acute Myeloid Leukemia Beyond First Remission or Refractory to Chemotherapy

PURPOSE Identify prognostic factors that influence outcome after unrelated donor bone marrow transplantation in children with acute myeloid leukemia (AML). PATIENTS AND METHODS Included are 268 patients (age <or= 18 years) with AML in second complete remission (n = 142), relapse (n = 90), or primary induction failure (n = 36) at transplantation. All patients received bone marrow grafts from an unrelated donor and a myeloablative conditioning regimen. Cox regression models were constructed to identify risk factors that influence outcome after transplantation. RESULTS In this analysis, the only risk factor that predicted leukemia recurrence and overall and leukemia-free survival was disease status at transplantation. The 5-year probabilities of leukemia-free survival were 45%, 20%, and 12% for patients who underwent transplantation at second complete remission, relapse, and primary induction failure, respectively. As expected, risk of acute but not chronic graft-versus-host disease (GVHD) was lower with T-cell-depleted bone marrow grafts; T-cell-depleted grafts were not associated with higher risks of leukemia recurrence. We observed similar risks of leukemia relapse in patients with and without acute and chronic GVHD. CONCLUSION Children who underwent transplantation in remission had a superior outcome compared with children who underwent transplantation during relapse or persistent disease. Nevertheless, 20% of children who underwent transplantation in relapse are long-term survivors, suggesting that unrelated donor bone marrow transplantation is an effective therapy in a significant proportion of children with recurrent or primary refractory AML.