Robert E. Goldsby

Fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP), a rare and disabling genetic condition of congenital skeletal malformations and progressive heterotopic ossification (HO), is the most catastrophic disorder of HO in humans. Episodic disease flare-ups are precipitated by soft tissue injury, and immobility is cumulative. Recently, a recurrent mutation in activin receptor IA/activin-like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor, was reported in all sporadic and familial cases of classic FOP, making this one of the most highly specific disease-causing mutations in the human genome. The discovery of the FOP gene establishes a critical milestone in understanding FOP, reveals a highly conserved target for drug development in the transforming growth factor (TGF)-beta/BMP signalling pathway, and compels therapeutic approaches for the development of small molecule signal transduction inhibitors for ACVR1/ALK2. Present management involves early diagnosis, assiduous avoidance of iatrogenic harm, and symptomatic amelioration of painful flare-ups. Effective therapies for FOP, and possibly for other common conditions of HO, may potentially be based on future interventions that block ACVR1/ALK2 signalling.

Defective DNA polymerase-δ proofreading causes cancer susceptibility in mice

To the editor—Tumor development is a multistep process that requires an accumulation of mutations in genes regulating cell growth and metastasis. Spontaneous mutations occur rarely in normal cells, and thus it is argued that defects in pathways governing genetic stability may affect tumorigenesis. Consistent with this idea is the observation that loss of DNA mismatch repair confers both a mutator phenotype and increased cancer susceptibility. However, defects in polymerase proofreading have not been causally linked to cancer, even though proofreading and mismatch repair contribute almost equally to mutation avoidance. To determine the consequences of defective proofreading in mammals, we created mice with an inactivating point mutation in the proofreading domain of DNA polymerase-δ (pol-δ). Pol-δ is a primary replicative enzyme in eukaryotes. Its catalytic subunit contains a proofreading active site formed by the conserved motifs Exo I, Exo II and Exo III that include four invariant carboxylate residues thought to be essential for catalysis. In Saccharomyces cerevisiae, haploid and homozygous diploid mutations of these residues cause a large increase in spontaneous mutation rate (‘mutator phenotype’). A particularly strong mutator results from substitution of alanine for aspartic acid at position 407 (D407A) in the Exo II motif. This amino-acid substitution selectively disrupts exonucleolytic proofreading while preserving overall polymerase activity. To generate mice with this mutation, we cloned the mouse Pold1 gene, created a point mutation in the exonuclease-encoding domain (D400A corresponding to yeast D407A) and introduced the mutant allele into SvJ-129 mouse embryonic stem (ES) cells by homologous recombination. Reverse-transcriptase PCR showed that the mutant and wild-type alleles were expressed at equal levels in the resultant Pold1 clones. We created two chimeric males by aggregation of these recombinant ES cells with C57BL/6J morulas, and germline transmission of the mutant Pold1 allele was confirmed by Southern blot, PCR and sequence analyses. To determine the phenotype of the mutant allele, we crossed a chimeric male with C57BL/6J females, and the resultant heterozygous F1 mice were interbred to generate a cohort of F2 animals. A total of 199 F2 mice were evaluated from 11 F1 breeding pairs (mean litter size, 6.8 pups). We obtained 53 wild-type, 97 Pold1 and 49 Pold1 mice. Thus, the mutant allele did not affect embryo viability. Subsequent breeding experiments showed that both male and female Pold1 mice are fertile. All mice exhibited normal development and weight gain. Therefore, pol-δ proofreading is not required for mouse reproduction or development. Beyond two months of age, Pold1 mice began to develop cancer and die (Fig. 1a). Twenty-one of the 49 Pold1 mice and none of the Pold1 or wild-type mice have developed tumors 12 months into the study; 1 Pold1 animal had 3 different primary tumors, bringing the total count to 23 tumors (Fig. 1b). The tumors were derived from two different cell lineages. Sixteen of the 23 tumors originated from mesenchymal tissue (12 thymic lymphomas, 2 diffuse lymphomas and 2 sarcomas) and 7 originated from epithelial tissue (6 tail-skin squamous-cell carcinomas and 1 lung adenocarcinoma). There were no significant differences in cancer incidence by gender or F1 breeding pairs. Surviving Pold1 mice continue to develop early signs of tail skin lesions (cracking, small nodules) and are being monitored for further tumor progression and metastatic disease. Interestingly, tumors developed in homozygous but not heterozygous mutant mice. Western blot analyses of fibroblasts cultured from Pold1, Pold1 and Pold1 embryos showed similar levels of total pol-δ protein, indicating that the wild-type and mutant proteins are equally expressed. It is not clear why the Pold1 allele is recessive. Defects in yeast pol-δ (including the analogous D407A allele) also exhibit limited codominance, suggesting that this is a general property of pol-δ proofreading. The effects of a single mutant allele might be masked by mismatch repair, intermolecular proofreading, competition by wild-type pol-δ molecules and/or novel error-processing pathways. Our findings reveal the importance of polymerase proofreading in mammals and significantly advance the view that increased spontaneous mutation results in increased cancer susceptibility. Defects in mismatch repair also confer a mutator phenotype and increase cancer susceptibility. However, loss of proofreading and mismatch repair are different in important ways. Although both exhibit mutator phenotypes and develop early lymphomas, these mice display different epithelial tumors. Mice with defects in the mismatch-repair proteins Mlh1, Msh2 or Msh6 tend to develop gastrointestinal tumors, whereas pol-δ proofreading-deficient mice develop squamous-cell skin cancer. This varied tumor response might reflect tissue-/celltype–specific differences in replication error repair, DNA damage, mutagenic processing of pol-δ errors and/or mismatch-repair–mediated apoptosis. Proofreading and mismatch repair also Defective DNA polymerase-δ proofreading causes cancer susceptibility in mice

Temozolomide and intravenous irinotecan for treatment of advanced Ewing sarcoma

Preclinical models show sequence‐dependent synergy with the combination of temozolomide and irinotecan, and a Phase I trial has demonstrated the combination to be tolerable and active in children with relapsed solid tumors. Because responses were seen in patients with Ewing sarcoma (ES) on that trial, additional patients were treated with this combination following study completion.

Phase II Study on the Effect of Disease Sites, Age, and Prior Therapy on Response to Iodine-131-Metaiodobenzylguanidine Therapy in Refractory Neuroblastoma

PURPOSE To evaluate the effect of disease sites and prior therapy on response and toxicity after iodine-131-metaiodobenzylguanidine (131I-MIBG) treatment of patients with resistant neuroblastoma. PATIENTS AND METHODS One hundred sixty-four patients with progressive, refractory or relapsed high-risk neuroblastoma, age 2 to 30 years, were treated in a limited institution phase II study. Patients with cryopreserved hematopoietic stem cells (n = 148) were treated with 18 mCi/kg of 131I-MIBG. Those without hematopoietic stem cells (n = 16) received 12 mCi/kg. Patients were stratified according to prior myeloablative therapy and whether they had measurable soft tissue involvement or only bone and/or bone marrow disease. RESULTS Hematologic toxicity was common, with 33% of patients receiving autologous hematopoietic stem cell support. Nonhematologic grade 3 or 4 toxicity was rare, with 5% of patients experiencing hepatic, 3.6% pulmonary, 10.9% infectious toxicity, and 9.7% with febrile neutropenia. The overall complete plus partial response rate was 36%. The response rate was significantly higher for patients with disease limited either to bone and bone marrow, or to soft tissue (compared with patients with both) for patients with fewer than three prior treatment regimens and for patients older than 12 years. The event-free survival (EFS) and overall survival (OS) times were significantly longer for patients achieving response, for those older than 12 years and with fewer than three prior treatment regimens. The OS was 49% at 1 year and 29% at 2 years; EFS was 18% at 1 year. CONCLUSION The high response rate and low nonhematologic toxicity with 131I-MIBG suggest incorporation of this agent into initial multimodal therapy of neuroblastoma.

DNA polymerase ε and δ proofreading suppress discrete mutator and cancer phenotypes in mice

Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols ε and δ, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown. Here we show that mouse Pol ε and δ proofreading suppress discrete mutator and cancer phenotypes. We found that inactivation of Pol ε proofreading elevates base-substitution mutations and accelerates a unique spectrum of spontaneous cancers; the types of tumors are entirely different from those triggered by loss of Pol δ proofreading. Intercrosses of Pol ε-, Pol δ-, and mismatch repair-mutant mice show that Pol ε and δ proofreading act in parallel pathways to prevent spontaneous mutation and cancer. These findings distinguish Pol ε and δ functions in vivo and reveal tissue-specific requirements for DNA replication fidelity.

High incidence of epithelial cancers in mice deficient for DNA polymerase δ proofreading

Mutations are a hallmark of cancer. Normal cells minimize spontaneous mutations through the combined actions of polymerase base selectivity, 3′ → 5′ exonucleolytic proofreading, mismatch correction, and DNA damage repair. To determine the consequences of defective proofreading in mammals, we created mice with a point mutation (D400A) in the proofreading domain of DNA polymerase δ (polδ, encoded by the Pold1 gene). We show that this mutation inactivates the 3′ → 5′ exonuclease of polδ and causes a mutator and cancer phenotype in a recessive manner. By 18 months of age, 94% of homozygous Pold1D400A/D400A mice developed cancer and died (median survival = 10 months). In contrast, only 3–4% of Pold1+/D400A and Pold1+/+ mice developed cancer in this time frame. Of the 66 tumors arising in 49 Pold1D400A/D400A mice, 40 were epithelial in origin (carcinomas), 24 were mesenchymal (lymphomas and sarcomas), and two were composite (teratomas); one-third of these animals developed tumors in more than one tissue. Skin squamous cell carcinoma was the most common tumor type, occurring in 60% of all Pold1D400A/D400A mice and in 90% of those surviving beyond 8 months of age. These data show that polδ proofreading suppresses spontaneous tumor development and strongly suggest that unrepaired DNA polymerase errors contribute to carcinogenesis. Mice deficient in polδ proofreading provide a tractable model to study mechanisms of epithelial tumorigenesis initiated by a mutator phenotype.

Phase II Study of High-Dose [131I]Metaiodobenzylguanidine Therapy for Patients With Metastatic Pheochromocytoma and Paraganglioma

PURPOSE To evaluate the safety and efficacy of high-dose [(131)I]metaiodobenzylguanidine ([(131)I]MIBG) in the treatment of malignant pheochromocytoma (PHEO) and paraganglioma (PGL). METHODS Fifty patients with metastatic PHEO or PGL, age 10 to 64 years, were treated with [(131)I]MIBG doses ranging from 492 to 1,160 mCi (median, 12 mCi/kg). Cumulative [(131)I]MIBG administered ranged from 492 to 3,191 mCi. Autologous hematopoietic stem cells were collected and cryopreserved before treatment with [(131)I]MIBG greater than 12 mCi/kg or with a total dose greater than 500 mCi. Sixty-nine [(131)I]MIBG infusions were given, which included infusions to 35 patients treated once and infusions to 15 patients who received two or three treatments. Response was evaluated by [(123)I]MIBG scans, computed tomography/magnetic resonance imaging, urinary catecholamines/metanephrines, and chromogranin A. RESULTS The overall complete response (CR) plus partial response (PR) rate in 49 evaluable patients was 22%. Additionally, 35% of patients achieved a CR or PR in at least one measure of response without progressive disease, and 8% of patients maintained stable disease for greater than 12 months. Thirty-five percent of patients experienced progressive disease within 1 year after therapy. The estimated 5-year overall survival rate was 64%. Toxicities included grades 3 to 4 neutropenia (87%) and thrombocytopenia (83%). Grades 3 to 4 nonhematologic toxicity included acute respiratory distress syndrome (n = 2), bronchiolitis obliterans organizing pneumonia (n = 2), pulmonary embolism (n = 1), fever with neutropenia (n = 7), acute hypertension (n = 10), infection (n = 2), myelodysplastic syndrome (n = 2), and hypogonadism (n = 4). CONCLUSION Although serious toxicity may occur, the survival and response rates achieved with high-dose [(131)I]MIBG suggest its utility in the management of selected patients with metastatic PHEO and PGL.

Phase I Trial of Temozolomide and Protracted Irinotecan in Pediatric Patients with Refractory Solid Tumors

Purpose: The purpose is to estimate the maximum-tolerated dose (MTD) of temozolomide and irinotecan given on a protracted schedule in 28-day courses to pediatric patients with refractory solid tumors. Experimental Design: Twelve heavily pretreated patients received 56 courses of oral temozolomide at 100 mg/m2/day for 5 days combined with i.v. irinotecan given daily for 5 days for 2 consecutive weeks at either 10 mg/m2/day (n = 6) or 15 mg/m2/day (n = 6). We assessed toxicity, the pharmacokinetics of temozolomide and irinotecan, and the DNA repair phenotype in tumor samples. Results: Two patients experienced dose-limiting toxicity (DLT) at the higher dose level; one had grade 4 diarrhea, whereas the other had bacteremia with grade 2 neutropenia. In contrast, no patient receiving temozolomide and 10 mg/m2/day irinotecan experienced DLT. Myelosuppression was minimal and noncumulative. No pharmacokinetic interaction was observed. Drug metabolite exposures at the MTD were similar to exposures previously associated with single-agent antitumor activity. One complete response, two partial responses, and one minor response were observed in Ewing’s sarcoma and neuroblastoma patients previously treated with stem cell transplant. Responding patients had low or absent O6-methylguanine-DNA methyltransferase expression in tumor tissue. Conclusions: The MTD using this schedule was temozolomide (100 mg/m2/day) and irinotecan (10 mg/m2/day), with DLT being diarrhea and infection. Drug clearance was similar to single-agent values, and clinically relevant SN-38 lactone and MTIC exposures were achieved at the MTD. As predicted by xenograft models, this combination and schedule appears to be tolerable and active in pediatric solid tumors. Evaluation of a 21-day schedule is planned.

Her-2/neu expression in osteosarcoma increases risk of lung metastasis and can be associated with gene amplification.

The purpose of this study was to investigate whether Her-2/ neu expression at diagnosis of osteosarcoma could provide biologic and prognostic information that predicts the risk of pulmonary metastases and outcome. Human epidermal growth factor (Her-2/ neu) expression in 25 initial pretreatment osteosarcoma biopsies and 12 posttreatment pulmonary metastatic osteosarcoma resection specimens was assessed by standard immunohistochemical techniques on formalin-fixed paraffin-embedded tissue. As a screening analysis to determine if gene amplification may be a mechanism for increased Her-2/ neu expression, FISH analysis was conducted on seven Her-2/ neu immunostain-positive samples and five Her-2/ neu immunostain-negative samples. Cytoplasmic Her-2/ neu reactivity was identified in 11/25 (44%) of primary tumors and in 7/12 (58%) resection specimens from pulmonary metastases. Cytoplasmic Her-2/ neu expression was associated with shorter overall metastasis-free survival. Her-2/ neu gene amplification was identified by FISH analysis in six of the seven immunostain-positive samples but was also identified in two of the five immunostain-negative samples. Her-2/ neu expression in patients with osteosarcoma is associated with an increased risk of metastasis and may define a subset of patients with a more aggressive tumor phenotype. Her-2/ neu gene amplification may provide a mechanism for Her-2/ neu overexpression in certain cases of osteosarcoma. Whether Her-2/ neu expression influences outcome needs to be examined further in a prospective fashion. The hope is that Her-2/ neu expression will identify patients who may benefit from the addition of directed biologic therapy.

Twenty years of follow-up among survivors of childhood and young adult acute myeloid leukemia: A report from the Childhood Cancer Survivor Study

Limited data exist on the comprehensive assessment of late medical and social effects experienced by survivors of childhood and young adult acute myeloid leukemia (AML).