Aaron D. Bossler

Oligodeoxynucleotide CpG 7909 delivered as intravenous infusion demonstrates immunologic modulation in patients with previously treated non-Hodgkin lymphoma.

Oligodeoxynucleotides containing CpG motifs (CpG ODN) can alter various immune cell subsets important in antibody therapy of malignancy. We undertook a phase I trial of CPG 7909 (also known as PF-3512676) in patients with previously treated lymphoma with the primary objective of evaluating safety across a range of doses, and secondary objectives of evaluating immunomodulatory effects and clinical effects. Twenty-three patients with previously treated non-Hodgkin lymphoma received up to 3 weekly 2-hour intravenous (IV) infusions of CPG ODN 7909 at dose levels 0.01 to 0.64 mg/kg. Evaluation of immunologic parameters and clinical endpoints occurred for 6 weeks. Infusion-related toxicity included grade 1 nausea, hypotension, and IV catheter discomfort. Serious adverse hematologic events observed more than once included anemia (2=Gr3, 2=Gr4), thrombocytopenia (4=Gr3), and neutropenia (2=Gr3), and were largely judged owing to progressive disease. Immunologic observations included: (1) The mean ratio of NK-cell concentrations compared with pretreatment at day 2 was 1.44 (95% CI=0.94-1.94) and at day 42 was 1.53 (95% CI=1.14-1.91); (2) NK activity generally increased in subjects; and (3) Antibody-dependent cellular cytotoxicity activity increased in select cohorts. No clinical responses were documented radiographically at day 42. Two subjects demonstrated late response. We conclude CpG 7909 can be safely given as a 2-hour IV infusion to patients with previously treated non-Hodgkin lymphoma at doses that have immunomodulatory effects.

The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth.

ABSTRACT The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Rasv12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cultures from Cystic Fibrosis Patients

ABSTRACT The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting Gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).

Impaired PTPN13 phosphatase activity in spontaneous or HPV-induced squamous cell carcinomas potentiates oncogene signaling through the MAP kinase pathway

Human papillomaviruses (HPVs) are a causative factor in over 90% of cervical and 25% of head and neck squamous cell carcinomas (HNSCCs). The C terminus of the high-risk HPV 16 E6 oncoprotein physically associates with and degrades a non-receptor protein tyrosine phosphatase (PTPN13), and PTPN13 loss synergizes with H-RasV12 or ErbB2 for invasive growth in vivo. Oral keratinocytes that have lost PTPN13 and express H-RasV12 or ErbB2 show enhanced Ras/RAF/MEK/Erk signaling. In co-transfection studies, wild-type PTPN13 inhibited Ras/RAF/MEK/Erk signaling in HEK 293 cells that overexpress ErbB2, EGFR or H-RasV12, whereas an enzymatically inactive PTPN13 did not. Twenty percent of HPV-negative HNSCCs had PTPN13 phosphatase mutations that did not inhibit Ras/RAF/MEK/Erk signaling. Inhibition of Ras/RAF/MEK/Erk signaling using MEK inhibitor U0126 blocked anchorage-independent growth in cells lacking PTPN13. These findings show that PTPN13 phosphatase activity has a physiologically significant role in regulating MAP kinase signaling.

Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells

Human papillomavirus 16 (HPV16) has been associated with head and neck squamous cell carcinoma (HNSCC) in up to 60% of sampled specimens.

Genomic Sequencing Procedure Microcosting Analysis and Health Economic Cost-Impact Analysis: A Report of the Association for Molecular Pathology

The increasing use of advanced nucleic acid sequencing technologies for clinical diagnostics and therapeutics has made vital understanding the costs of performing these procedures and their value to patients, providers, and payers. The Association for Molecular Pathology invested in a cost and value analysis of specific genomic sequencing procedures (GSPs) newly coded by the American Medical Association Current Procedural Terminology Editorial Panel. Cost data and work effort, including the development and use of data analysis pipelines, were gathered from representative laboratories currently performing these GSPs. Results were aggregated to generate representative cost ranges given the complexity and variability of performing the tests. Cost-impact models for three clinical scenarios were generated with assistance from key opinion leaders: impact of using a targeted gene panel in optimizing care for patients with advanced non–small-cell lung cancer, use of a targeted gene panel in the diagnosis and management of patients with sensorineural hearing loss, and exome sequencing in the diagnosis and management of children with neurodevelopmental disorders of unknown genetic etiology. Each model demonstrated value by either reducing health care costs or identifying appropriate care pathways. The templates generated will aid laboratories in assessing their individual costs, considering the value structure in their own patient populations, and contributing their data to the ongoing dialogue regarding the impact of GSPs on improving patient care.

Exophiala oligosperma Causing Olecranon Bursitis

ABSTRACT A 62-year-old male with a history of Wegeners granulomatosis and immunosuppressive therapy presented with chronic olecranon bursitis. A black velvety mould with brown septate hyphae and tapered annellides was isolated from a left elbow bursa aspirate and was identified as an Exophiala species. Internal transcribed sequence rRNA sequencing showed the isolate to be identical to Exophiala oligosperma. The patient was successfully treated with aspiration and intrabursal amphotericin B.

Human Papillomavirus (HPV) Type 18 Induces Extended Growth in Primary Human Cervical, Tonsillar, or Foreskin Keratinocytes More Effectively than Other High-Risk Mucosal HPVs

ABSTRACT Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in ∼25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of full-length mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or “crisis,” and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.

Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1

Abstract Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-β synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-β as a potential boost in infection. Treatment with low levels of human IFN-β induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1 −/− , IRF-2 −/− and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.

Performance of the COBAS ® AmpliPrep/COBAS TaqMan ® automated system for hepatitis C virus (HCV) quantification in a multi-center comparison

BACKGROUND Quantitative HCV RNA testing is considered standard of care for monitoring during treatment of patients infected with HCV. The COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test fully automates specimen processing and reaction assembly for HCV viral load testing using reverse transcription and real-time PCR amplification. OBJECTIVES The performance of the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test was evaluated in a multi-center study. STUDY DESIGN Typical plasma based specimens were tested for accuracy, analytic range of measurement, reproducibility and genotype specific quantitation. RESULTS Linear regression analysis of the quantitative results demonstrated a linear range of detection from 50 to 5 million (1.7-6.7 log(10))IU/mL and a coefficient of determination (R(2)) of 0.9948. The precision of the assay was highly reproducible within and between runs and among laboratories with coefficients of variance (CV) ranging from 6.7% to 40.0% across the seven laboratories. A representative sample for each of the six major HCV genotypes demonstrated reproducible quantitation between the seven laboratories. CONCLUSIONS The COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test is a reliable and sensitive assay for HCV RNA quantitation.