Randomized phase II study evaluating veliparib (ABT-888) with temozolomide in patients with metastatic melanoma.

BACKGROUND: Veliparib (ABT-888) is a potent, orally bioavailable, small-molecule inhibitor of the DNA repair enzymes poly ADP-ribose polymerase-1 and -2. Veliparib enhances the efficacy of temozolomide (TMZ) and other cytotoxic agents in preclinical tumor models. PATIENTS AND METHODS: In this multicenter, double-blind trial, adults with unresectable stage III or IV metastatic melanoma were randomized 1:1:1 to TMZ plus veliparib 20 or 40 mg, or placebo twice daily. Efficacy end points included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: Patients (N = 346) were randomized between February 2009 and January 2010. Median [95% confidence interval (CI)] PFS was 3.7 (3.0-5.5), 3.6 (1.9-4.1), and 2 (1.9-3.7) months in the 20-mg, 40-mg, and placebo arms, respectively. Median (95% CI) OS was 10.8 (9.0-13.1), 13.6 (11.4-15.9), and 12.9 (9.8-14.3) months, respectively; ORR was 10.3%, 8.7%, and 7.0%. Exploratory analyses showed patients with low ERCC1 expression had longer PFS when TMZ was combined with veliparib. Toxicities were as expected for TMZ. The frequencies of thrombocytopenia, neutropenia, and leukopenia were significantly increased in the veliparib groups. Grade 3 or 4 adverse events, mainly hematologic toxicities, were seen in 55%, 63%, and 41% of patients in the 20-mg, 40-mg, and placebo arms, respectively. CONCLUSIONS: Median PFS with 20 and 40 mg veliparib almost doubled numerically compared with placebo, but the improvements did not reach statistical significance. OS was not increased with veliparib. Toxicities were similar to TMZ monotherapy, but with increased frequency.

Initial experience using robot- assisted transaxillary thyroidectomy for Graves' disease.

INTRODUCTION: Graves' disease is the most common form of hyperthyroidism and surgery to remove the thyroid gland is a common treatment option for many of these patients. Interestingly, due to the enlarged gland size, their high vascularity, and the difficulty to control bleeding, many authors feel that Graves' disease remains a contraindication to current endoscopic techniques. We hypothesize that performing robotic subtotal thyroidectomy in Graves' disease settings could overcome the limitations of conventional endoscopic surgeries in the surgical management of this challenging thyroid disease. METHODS: Prospective study in an academic hospital. RESULTS: Sixty-seven patients had robotic transaxillary thyroidectomy within a year. Of these, five cases (7%) were done for Graves' disease. There were three females and two males (mean age, 36 years). There were no conversions to laparoscopic or open surgery. The mean (SD) thyroid volume was 16.6 (3.2) ml. The mean (SD) operative time was 159 (17.8)min and docking time was 81 (20)min. Mean blood loss was 18 mL. All patients were discharged home in 24h. There were no perioperative or postoperative complications. There was no evidence of postoperative vocal cord palsy or paresis. CONCLUSIONS: We showed that robotic transaxillary thyroidectomy is feasible and can be safe and effective in patients with Graves' disease. To our knowledge, this is the first article describing this approach for Graves' disease. These findings, however, warrant additional investigation within future prospective clinical trials.

V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors.

The Raf family includes three members, of which B-Raf is frequently mutated in melanoma and other tumors. We show that Raf-1 and A-Raf require Hsp90 for stability, whereas B-Raf does not. In contrast, mutated, activated B-Raf binds to an Hsp90-cdc37 complex, which is required for its stability and function. Exposure of melanoma cells and tumors to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in the degradation of mutant B-Raf, inhibition of mitogen-activated protein kinase activation and cell proliferation, induction of apoptosis, and antitumor activity. These data suggest that activated mutated B-Raf proteins are incompetent for folding in the absence of Hsp90, thus suggesting that the chaperone is required for the clonal evolution of melanomas and other tumors that depend on this mutation. Hsp90 inhibition represents a therapeutic strategy for the treatment of melanoma.

Genomic instability in head and neck cancer patients.

BACKGROUND: In the normal host, there are a variety of cellular systems that ensure the accurate replication and repair of DNA. Recent evidence suggests that there are individual variations in the ability to preserve the genome. Certain individuals have defects in these checkpoints and have an inherent genomic instability. They are susceptible to the accumulation of DNA damage and are prone to carcinogenesis. This article examines the role of genomic instability in the development of head and neck cancer. RESULTS: Patients with either the chromosomal instability syndromes or the Li-Fraumeni syndrome have marked defects in either DNA repair or apoptosis. These patients are prone to have head and neck neoplasms develop. Head and neck cancer patients also have a diminished ability to repair DNA damage compared with the "normal" population. Abnormalities have been identified in mutagen sensitivity, the expression of DNA mismatch repair enzymes, the expression of p53, and telomerase activity when head and neck cancer patients are compared with controls. CONCLUSION: Subpopulations exist who have increased genomic instability. These individuals are at an increased risk for the accumulation of DNA mutations and the development of head and neck cancer. More research is needed to identify specific mechanisms of genomic instability and to further define the importance of this phenomenon.

Prevention of airway complications in thyroplasty patients requiring endotracheal intubation.

Patients who have undergone silicone vocal cord medialization and require additional surgery are at risk for airway complications. There is a narrowed glottic aperture that may be prone to develop postoperative laryngeal edema and prosthesis extrusion. This study was designed to assess the management of this difficult airway and to determine the frequency of postintubation complications. We identified 82 patients who had undergone vocal cord medialization with silicone implants between 1991 and 1995. Seventeen of these patients underwent additional surgical procedures requiring general anesthesia. A retrospective review of these patients' charts was performed to determine the management of the airway and the incidence of postintubation complications. There were no postintubation complications in the 17 patients who were studied. The duration of surgery ranged from 40 minutes to 4 hours 15 minutes. Two patients were ventilated via bronchoscope, and 15 patients were intubated orally. The endotracheal tubes ranged from size 6 to size 9 (median size 8). None of the patients required perioperative steroids. All patients were successfully extubated in the recovery room. No patients required intubation or tracheotomy, and there were no implant extrusions. In this study, the incidence of postintubation airway complications in patients who had undergone previous thyroplasty was minimal. Nevertheless, the potential for airway compromise exists. We recommend preoperative discussion with the anesthesiologist, atraumatic intubation with a small endotracheal tube, and diligent observation for airway compromise.

Critical care for the severely ill head and neck patient.

OBJECTIVE: To delineate the frequency and causes of admission to a critical care environment for patients undergoing head and neck surgery at Memorial Sloan-Kettering Cancer Center. DESIGN: Retrospective clinical investigation. SETTING: Adult intensive care unit of a tertiary referral cancer center. PATIENTS: All head and neck surgery patients admitted to the special care unit (SCU) of Memorial Sloan-Kettering Cancer Center between January 1, 1994 and December 31, 1995 were included in this study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The data collected included demographic, operative procedures, clinical, laboratory, and physiologic variables at time of SCU admission, at 24 hrs, as well as vital status at the time of discharge from the SCU and hospital. Other data collected were the need for mechanical ventilation and inotropic agents. During the period of January 1, 1994 through December 31, 1995, 37 (1.5%) of 2,346 patients undergoing head and neck surgical procedures required admission to the SCU. During the same period, six patients receiving medical treatment only for head and neck malignant disease were transferred to the SCU. These 43 admissions served as the basis for the study. The causes of admission to the SCU were pulmonary (15/43), cardiac (14/43), wound related (8/43), and other (15/43). The median length of stay in the SCU was 2 days, and the median hospitalization for patients requiring critical care services was 22 days. Seventy-four percent of patients requiring critical care services were eventually discharged to home. CONCLUSIONS: Current preoperative evaluation, operative and anesthetic techniques, and perioperative care result in a low frequency of utilization of critical care services by patients undergoing head and neck surgery. There is no single identifiable cause of complications for patients after head and neck surgery leading to utilization of critical care services.

Squamous cell carcinoma of the tongue in young patients: a matched-pair analysis.

BACKGROUND: Tongue cancer is seen with increasing frequency in young individuals. There is controversy concerning the clinical course and outcome for oral tongue cancer in young patients. METHODS: A retrospective review of 36 patients under 40 years of age with squamous cell carcinoma of the tongue was performed. These patients were matched to an older population. The 5-year disease-free survival; rates of local, regional, and distant failure; and rate of second primary tumor were determined for both populations. RESULTS: The 5-year disease-free survival for the young patients was 62% versus 69% in the older population (p = .30). Ten of 36 (28%) of younger patients recurred locally versus five of 36 (14%) of the older patients (p = .11). Nine of 36 (25%) younger patients recurred regionally in the younger group versus six of 36 (17%) patients in the older group (p = .25). Sixteen of 36 (44%) of the younger patients had locoregional failure versus eight of 36 (22%) of the older patients (p < .05). The rates of metastatic disease and second primary lesions were similar in both populations. CONCLUSIONS: In this series, younger patients with squamous cell carcinoma of the oral tongue had a higher rate of locoregional recurrence rate than did older patients. This did not translate into a survival difference.

Differential DNA replication origin activities in human normal skin fibroblast and HeLa cell lines.

A modification of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.

Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein.

In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters.

Regulation of mutant p53 temperature-sensitive DNA binding.

We have examined in detail the DNA binding properties of several immunopurified tumor-derived mutant p53 proteins (Val-143 --> Ala, Arg-175 --> His, Arg-248 --> Trp, Arg-249 --> Ser, and Arg-273 --> His). While all mutants were defective for binding to DNA at 37 ;C, each bound specifically to several cognate p53 binding sites at sub-physiological temperatures (25-33 ;C), and several mutants activated transcription from a p53-responsive promoter at 26 degrees C in transfected H1299 cells. Heating mutant p53 proteins at 37 degrees C irreversibly destroyed their ability to subsequently bind at 25 degrees C. However, several different monoclonal antibodies that each share the ability to recognize an epitope encompassing amino acids 46-55 markedly stabilized binding by mutant p53 proteins at 37 degrees C. Both intact antibody and FAb fragments allowed mutant p53 to bind to DNA. By contrast, antibodies that recognize epitopes located elsewhere within p53 stabilized mutant p53 binding significantly less effectively. Our data show that the major hot-spot p53 mutants have the intrinsic ability to bind to DNA and that a unique region within the N terminus of p53 may be critical for rescuing them from loss of binding at physiological temperatures. This suggests the possibility of developing small molecules that can stabilize mutant p53 proteins under physiological conditions.

A mutant p53 that discriminates between p53-responsive genes cannot induce apoptosis.

Human wild-type (wt) p53 can induce apoptosis in transiently transfected H1299 cells maintained at 37 degrees C, whereas tumor-derived mutant forms of p53 (with the mutation Ala-143, His-175, or Trp-248) fail to do so. At 37 degrees C, p53 with a mutation to Ala at amino acid 143 (p53Ala143) was transcriptionally inactive. However, at 32 degrees C, p53Ala143 strongly activated transcription from several physiologically relevant p53-responsive promoters, to extents similar or greater than that of wt p53. Unexpectedly, p53Ala143 was defective in inducing apoptosis in H1299 cells at 32 degrees C. Concomitantly with the loss of apoptotic activity, p53Ala143 was found to be deficient in its ability to activate transcription from the p53-responsive portions of the Bax and insulin-like growth factor-binding protein 3 gene promoters. It is proposed that there may exist distinct classes of p53-responsive promoters, whose ability to be activated by p53 can be regulated differentially. Such differential regulation may have functional consequences for the effects of p53 on cell fate.

cDNA clones contain autonomous replication activity.

We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication. Subpopulations of transcripts represented in a cDNA library of human embryo lung fibroblasts (IMR90) were examined for their ability to support autonomous replication after transfection into human cells (HeLa). Two of three cDNA clones (343, 363) containing 'O'-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replication. One of these cDNA clones, 343, is enriched by selection for poly(A)+ RNA. In contrast, none of five Alu-containing transcripts was capable of autonomous replication in human cells. However, six out of ten cDNA clones contained neither 'O'-family or Alu homologous sequences and were as efficient as the cDNA clones containing 'O'-family sequences in replicating autonomously in human cells. cDNA clones, from an oligo-d(T)-primed library of human poly(A)+ enriched RNA, contain a significant proportion of independent clones that can also support autonomous replication of bacterial plasmids in human cells. cDNA clone 343 was observed to contain in a 448 bp EcoRI-HincII fragment, yeast ARS consensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and structural characteristics often associated with many prokaryotic, viral and eukaryotic origins. Sequence analysis of seven other cDNA clones (from non-'O'-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences. Two NOA clones (NOA4 and -5) did not contain any representations of ARS and SAR consensus sequences, suggesting that these two features may not be essential for autonomous replication activity in mammalian cells.

Reduction of sound levels with antinoise in MR imaging.

A combination of active and passive techniques was used to reduce the sound levels in magnetic resonance imagers. These techniques were integrated into an existing audio system. Measurements of sound reduction varied with the protocol being used and averaged 9.9 dB with coaxial cabling and 14.2 dB with fiberoptic conduction of the feedback signal to a controller. Patient comfort and communication were improved.

Effects of soft contacts of differing thickness on corneal wound healing in rabbits.

This study was undertaken to determine the effects of thin (60 microns) and thick (240 microns) soft contact lenses of equal water content (70%) and power on nonlesioned and lesioned rabbit corneas. In one group of animals, corneas were not lesioned. Thin lenses were placed on left corneas and thick lenses on right corneas. In a second group, lesions were made in both corneas. Left corneas were covered with thin lenses and right corneas with thick lenses. Post-treatment times were 8 hr and 24 hr. At sacrifice, one-half of the cornea was fixed in 4% buffered glutaraldehyde for SEM study. The other half was cut into segments, fixed in a buffered glutaraldehyde-ruthenium red (RR) solution post-osmicated in osmium containing RR and prepared for TEM. At both 8 hr and 24 hr SEM showed cell migration in lesioned corneas covered with thin lenses but not in lesioned corneas covered with thick lenses. At 8 hr, TEM of nonlesioned and lesioned corneas showed no changes in the thickness of the corneal epithelium or the RR staining of the surface. At 24 hr, in nonlesioned corneas covered with thick lenses, the RR staining of microvilli and the height of the corneal epithelium were less than in nonlesioned corneas covered with thin lenses. In lesioned corneas covered with thick lenses, the thickness of the cornea was markedly reduced, the RR staining of microvilli was less and basal cells were more compressed than in lesioned corneas covered with thin lenses. The results of this study indicate that the thickness of a soft contact lens is important in treating corneal trauma.

Effects of manno-1-deoxynojirimycin and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine on N-linked oligosaccharide processing in intestinal epithelial cells.

The effects of manno-1-deoxynojirimycin (ManDJN) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) were compared in IEC-6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N-linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H-sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7-9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7-9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP-treated cells were non-glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus-infected MDCK cells [(1984) J. Biol. Chem. 259, 12409-12413].

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture.

The lipid-linked oligosaccharides synthesized in the presence of the alpha-glucosidase inhibitors, 1-deoxynojirimycin (DJN) and N-methyl-1-deoxynojirimycin (MDJN), were compared in IEC-6 intestinal epithelial cells in culture. HPLC analysis of the oligosaccharides obtained before and after exhaustive jack bean alpha-mannosidase digestion indicates that control and MDJN-treated cells synthesize similar amounts of Glc3Man9GlcNAc2-PP-dolichol. In contrast, the formation of this compound is greatly reduced in DJN-treated cells, the major lipid-linked oligosaccharide found being Man9GlcNAc2-PP-dolichol. The decreased availability of the preferred donor for protein glycosylation may account for the impaired glycosylation and secretion of certain glycoproteins in the presence of DJN.