Between December 1996 and September 1998, 13 patients with advanced recurrent malignant brain tumors (9 with glioblastoma multiforme, 1 with gliosarcoma, and 3 with anaplastic astrocytoma) were treated with a single intratumoral injection of 2 x 10(9), 2 x 10(10), 2 x 10(11), or 2 x 10(12) vector particles (VP) of a replication-defective adenoviral vector bearing the herpes simplex virus thymidine kinase gene driven by the Rous sarcoma virus promoter (Adv.RSVtk), followed by ganciclovir (GCV) treatment. The VP to infectious unit ratio was 20:1. Our primary objective was to determine the safety of this treatment. Injection of Adv.RSVtk in doses <==2 x 10(11) VP, followed by GCV, was safely tolerated. Patients treated with the highest dose, 2 x 10(12) VP, exhibited central nervous system toxicity with confusion, hyponatremia, and seizures. One patient is living and stable 29.2 months after treatment. Two patients survived >25 months before succumbing to tumor progression. Ten patients died within 10 months of treatment, 9 from tumor progression and 1 with sepsis and endocarditis. Neuropathologic examination of postmortem tissue demonstrated cavitation at the injection site, intratumoral foci of coagulative necrosis, and variable infiltration of the residual tumor with macrophages and lymphocytes.

For patients with local recurrence of prostate cancer after definitive irradiation therapy there is no treatment widely considered safe and effective. After extensive preclinical testing of prodrug gene therapy in vitro and in vivo, we conducted a phase I dose escalation clinical trial of intraprostatic injection of a replication-deficient adenovirus (ADV) containing the herpes simplex virus thymidine kinase gene (HSV-tk) injected directly into the prostate, followed by intravenous administration of the prodrug ganciclovir (GCV). Our goal was to determine safe dose levels of the vector for future trials of efficacy. Patients with a rising serum prostate-specific antigen (PSA) level and biopsy confirmation of local recurrence of prostate cancer without evidence of metastases one or more years after definitive irradiation therapy were eligible for the trial. After giving informed consent, patients received injections of increasing concentrations of ADV/HSA-tk in 1 ml into the prostate under ultrasound guidance. Ganciclovir was then given intravenously for 14 days (5 mg/kg every 12 hr). Patients were monitored closely for evidence of toxicity and for response to therapy. Eighteen patients were treated at 4 escalating doses: group 1 (n = 4) received 1 x 10(8) infectious units (IU); group 2 (n = 5) received 1 x 10(9) IU; group 3 (n = 4) received 1 x 10(10) IU; group 4 (n = 5) received 1 x 10(11) IU. Vector was detected by PCR of urine samples after treatment, increasing in frequency and duration (up to 32 days) as the dose increased. All cultures of blood and urine specimens were negative for growth of adenovirus. Minimal toxicity (grade 1-2) was encountered in four patients. One patient at the highest dose level developed spontaneously reversible grade 4 thrombocytopenia and grade 3 hepatotoxicity. Three patients achieved an objective response, one each at the three highest dose levels, documented by a fall in serum PSA levels by 50% or more, sustained for 6 weeks to 1 year. This study is the first to demonstrate the safety of ADV/HSV-tk plus GCV gene therapy in human prostate cancer and the first to demonstrate anticancer activity of gene therapy in patients with prostate cancer. Further trials are underway to identify the optimal distribution of vector within the prostate and to explore the safety of repeat courses of gene therapy.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10−14 for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.