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Cidofovir as an Useful Adjunct in Treating Recurrent Respiratory Papillomatosis
By American Society of Pediatric Otolaryngology
May 30, 2005 - 8:57:00 PM

Recurrent Respiratory Papillomatosis (RRP) is a chronic disease evidenced by wart-like lesions in the larynx, particularly on the vocal folds. Debulking or removing a major part of the lesions is generally the treatment of choice in order to preserve as much laryngeal function as possible and limit the morbidity associated with surgical treatment of this generally benign disorder.

The papillomas cause hoarseness, and if left untreated can grow large enough to obstruct the airway and cause death. Some patients (6-17 percent) develop spread of the papillomas into the trachea, bronchi, or even the pulmonary parenchyma. On rare occasions the papilloma may undergo malignant transformation.

A majority of the patients afflicted with this disease are children, and the papillomas often demonstrate a more aggressive behavior in children. Recurrence is the norm, with the average child undergoing 10 surgeries in their lifetime, and seven percent undergoing over 100 surgeries. There are great financial costs of treating this chronic disease with estimates of direct lifetime medical costs of treating a single person with RRP reaching $200,000.

The human papilloma virus ( HPV ) 6 and 11 causes the disease is usually caused by. Human papillomaviruses ( HPVs ) are small, double-stranded DNA viruses that infect strictly squamous epithelial tissues and induce hyperproliferative lesions.

Cidofivir is an acyclic nucleoside phosphonate HPMPC [( S )-1-( 3-hydroxy-2-phosphonylmethoxypropyl )cytosine]. It has proved to be effective in vitro and in vivo against a wide variety of DNA virus and RNA retrovirus infections. Cidofovir is activated by multi-step phosphorylation.

The long-lasting antiviral effect of cidofovir allows infrequent dosing of the drug ( i.e., only once a week or every other week ), which clearly distinguishes cidofovir from other antiviral drugs ( acyclovir, penciclovir, and ganciclovir ), which have to be administered several times daily to sustain an antiviral response.

Complete remissions of papillomatous lesions have been achieved following either topical gel application or direct intralesional injections of cidofovir. The potential of cidofovir to treat laryngeal papillomatosis has also been demonstrated by studies in which patients with severe recurrent laryngeal papillomatosis, treated with intralesional injections of cidofovir resulted in a complete regression of the papillomatous lesions in the majority of the patients.

Although response to cidofovir is not universal, long-term follow-up of patients treated with cidofovir have demonstrated its usefulness as an adjunct in treating RRP.

Serial analysis of gene expressions ( SAGE ) was developed in 1995 as an open system technique for probing the expressed genome. As such, it identifies cellular transcripts without relying on known genes for detection. SAGE allows detailed qualitative and quantitative analysis of large numbers of cellular transcripts. A new study uses SAGE to study gene expression of papilloma cells before and after the administration of the anti-viral adjuvant cidofovir. The researchers hypothesize that there is a differential gene expression in the papilloma after treatment with cidofovir. This differential expression may help better understand the intracellular effects of cidofovir, and more importantly, potential genetic alterations that occur with antiviral therapy in general.

Methodology:

Papilloma samples were obtained from one patient during regularly schedule RRP d�bridements. During the first tissue harvest, samples were taken, after which cidofovir was injected into the base of the lesions. Two months later, the patients were taken back to the OR and samples were again harvested.

SAGE library was constructed using the I-SAGE kit from Invitrogen; total RNA was isolated from a patient sample using Trizol reagent. RNA integrity was tested prior to use by gel electrophoresis. NlaIII ( restriction site 5'-CATG-3' ) was used as the anchoring enzyme and BsmFI ( Type IIS restriction site ) as the tagging enzyme.

Sequence files were first analyzed with PHRED base-calling software to determine the accuracy of the sequencing run. A minimum PHRED score of 20 was used to filter out tags with a higher probability of sequencing error. This data was imported into eSAGE which also excludes tags of unexpected larger or smaller size to ensure the analysis of valid tags. The program then filters out duplicate dimers ( ditags with the same two tags ) as this suggests non-random ligation and a non-quantitative library.

Results:

Over 19,000 tags were found in each of the libraries, with 7,993 unique transcripts identified in the pre-treatment and 6,749 identified in the post-cidofovir library. A total of 997 transcripts were found in both libraries. Following cidofovir treatment, the greatest up-regulation was in gene families associated with cell proliferation, metabolism, transport and response to biotic stimuli. Post-treatment up-regulation was seen in numerous specific genes, such as Interferon Regulatory Factor, which has been associated with virus-host interactions, passive viral induction of host immune response, and response to DNA damage stimulus. Down-regulation was demonstrated in gene families associated with transcription, regulation of nucleic acid metabolism, and signal transduction.

Conclusions:

The researchers demonstrated a long list of genes that are expressed in RRP using the SAGE technique. More importantly, they have demonstrated how the expression of various gene families changes when exposed to the anti-viral drug cidofovir. Additionally, identification of more than 1400 unique transcripts was achieved that may help with new gene discovery and better understanding of the response to cidofovir and other anti-viral drugs on a genetic and molecular level.

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