November 27, 2004
2′,5′-Oligoadenylate Synthetase Activity Analysis and Human Papilloma Virus Typing As Prognostic Factors in Patients With Recurrent Respiratory Papillomatosis
Objective: Determination of early prognostic factors in patients with recurrent respiratory papillomatosis is extremely important, so the major goal of our prospective, multicentre study was to evaluate (1) the feasibility of various factors to determine prognosis of the clinical course, as well as (2) the response to interferon-alpha therapy in recurrent respiratory papillomatosis.
Methods: Forty-two patients with recurrent respiratory papillomatosis were treated with interferon-alpha (3 MU/m^sup 2^ three times per week; mean therapy duration was 2.7 1.8 years) in 1983-1994 and followed-up until 2003. Human papilloma virus (HPV) type, recurrent respiratory papillomatosis severity and 2',5'- oligoadenylate synthetase activity were determined by standard methods and analysed for correlation with the results of long-term clinical outcome.
Results and conclusion: Patients with HPV type 11, a severity score >4, a high number of surgical procedures prior to interferon- alpha therapy and a high basal 2',5'-oligoadenylate synthetase activity should be considered at high risk of an aggressive clinical course, often with spread to lower airway passages, malignant transformation and death. Human papilloma virus type, score for recurrent respiratory papillomatosis severity, number of surgical procedures and 2',5'-oligoadenylate synthetase activity showed significant association with response to interferon-alpha therapy and the long-term clinical course, so these factors have value in predicting prognosis in recurrent respiratory papillomatosis.
Keywords: Papilloma; Larynx; Papilloma Virus, Human; Interferon- alpha; 2',5'-Oligoadenylate Synthetase
Recurrent respiratory papillomatosis is associated with persistent benign tumors (papillomas) localized mainly in the larynx. The disease has a highly unpredictable course, varying from fast progression to spontaneous resolution. Growth of papillomas results from the abnormal proliferation of keratinocytes infected with human papilloma virus (HPV) types 6 and 11.1,2
Recent publications have supported previously reported differences in recurrent respiratory papillomatosis course in patients with HPV types 6 and 11.2-4 The association between HPV 11 detection and recurrent respiratory papillomatosis spread to trachea, bronchi and lungs has been shown by several investigators.2,5 Recurrent respiratory papillomatosis spread to lower airway passages tends to cause malignant transformation and frequently leads to death.6-8 Clinicians lack definite prognostic factors which may help to predict recurrent respiratory papillomatosis course.
To date, the most common treatment for recurrent respiratory papillomatosis is removal of lesions by CO2 laser or surgical debulking with adjuvant alfa-interferon (interferon-alpha) application (less frequent treatments include cis-retinoic acid, acyclovir, haematoporphyrins, ribavirin, indole-3-carbinol and cidofovir).9 Despite the presence of previously published reports on possible association between HPV type and recurrent respiratory papillomatosis course, the prognostic role of HPV typing and its influence on the choice of therapy modalities need to be defined.
Several studies have been made to assess correlation between the level of 2',5'-oligoadenylate synthetase activity and response to interferon-alpha therapy in patients with hepatitis, multiple myeloma, B-cell chronic lymphocytic leukaemia, genital HPV infection and other diseases.10-13 Measurement of 2',5'-oligoadenylate synthetase activity may have certain value for the prognosis of clinical response and results of long-term interferon-alpha therapy in recurrent respiratory papillomatosis patients.
Determination of early prognostic factors is extremely important when choosing the most suitable treatment modalities in children and young patients with primary, diagnosed recurrent respiratory papillomatosis. So, the major goal of our study was to evaluate the feasibility of using various factors, including 2',5'- oligoadenylate synthetase activity in peripheral blood mononuclear cells (PBMC), in patients with recurrent respiratory papillomatosis to determine the likely clinical prognosis and response to interferon-alpha therapy.
Materials and methods
Patients and treatment
Forty-two patients with recurrent respiratory papillomatosis were accepted into a prospective, multicentre study from 21 June 1983 to 12 August 1990. They were treated with interferon-alpha (mean therapy duration was 2.7 1.8 years) and followedup until 1 March 2003. The patients were eligible for the study if they had active, histologically confirmed squamous papillomas in the airway passages. Human leukocyte-derived interferon-alpha was supplied by the New York Blood Center or by Intron A (alfa-2binterferon-alpha) produced by Essex Company (Munich, Germany). Interferon-alpha-based treatment of 42 patients was performed by intramuscular injections in doses of 3 MU/m^sup 2^ three times per week. In patients with insufficient response to interferon-alpha after one year of therapy, doses were increased to 5 MU/m^sup 2^. The effect of interferonalpha administration in patients with recurrent respiratory papillomatosis was monitored by the analysis of 2',5'-oligoadenylate synthetase activity in the PBMC.
Assessment of recurrent respiratory papillomatosis severity and response to therapy
The recurrent respiratory papillomatosis severity for the 42 patients was classified according to a special score, designed similarly to the KL score described by Leventhal and Kashima14. The applied score estimated the severity of recurrent respiratory papillomatosis according to the localization of papillomas and the presence of trachal canula (Table I). The maximum possible score is 33; recurrent respiratory papillomatosis in patients with a score of one to four was defined as mild disease, while a score of five to 33 characterized severe disease. Responders to the interferon-alpha therapy were considered to be patients who achieved a complete macroscopic remission (i.e. absence of papillomas) at the end of interferon-alpha therapy.
2',5'-Oligoadenylate synthetase activity in human PBMC
2',5'-Oligoadenylate synthetase activity was determined as described earlier15. Briefly, lymphocytes from 2-4 ml of venous blood were isolated by the Ficoll-Hypaque method (Ficoll- Trennlosung, Biochrom KG, Berlin, Germany) and lysed for 10 min at O0C in buffer (20 mM HEPES (Sigma, Deisenhofen, Germany), pH 7.5, containing 5 mM MgCl2 120 mM KCl, 1 mM DTE (Sigma, Deisenhofen, Germany), 0.02 per cent sodium azide, 10 per cent glycerol and 0.5 per cent Nonidet P40) at a concentration of 2.5 106 cells/ml. Aliquots of the extracts were stored at -80C. Poly(I) poly(C) agarose (30 mkl, P-L Biochemicals, Freiburg, Germany), equilibrated with a buffer containing 10 mM HEPES (pH 7.5), 5 mM MgCl^sub 2^,50 mM KCl, 7 mM DTE, 0.02 per cent sodium azide and 20 per cent glycerol, incubated for 15 min with 20 mkl of lymphocyte extract in Eppendorf microtest tubes, centrifuged and washed twice with 200 mkl of buffer.
After addition of 20 mkl of a reaction mixture containing 2.5 mM adenosine triphosphate (ATP) and 1 mkCi [α-^sup 32^P] ATP (NEN, Dreieich, Germany) dissolved in buffer, the agarose beads were incubated for 20 hr at 37C. Then 10 mkl of alkaline phosphatase (Boehringer Mannheim GmbH, Mannheim, Germany; 400 U/ml in 140 mM Trisbase) was added. After a further incubation for 1 hr at 37C, 0.5 ml of 1 M glycine-HCl was added to each tube. The complete reaction mixtures were applied to small columns of alumina (0.5 4 cm; type WA-I, Sigma Chemie GmbH, Munchen, Germany) pretreated with glycine- HCl. Each of the columns was washed with 2.5 ml of the same solution.
The radioactivity of 1-ml aliquots of the effluents was measured after dilution into 5 ml of a dioxanebased scintillation cocktail. The results of the determinations, each performed at least twice, are given below relative to the enzyme activity measured in lymphocyte extracts of an untreated donor used as a laboratory standard (relative 2',5'-oligoadenylate synthetase activity). Adenosine triphosphate incorporation by reference lymphocyte extracts was about 0.5 nmol per 5 10^sup 4^ cells under the experimental conditions described above.
SCORING SYSTEM FOR RECURRENT RESPIRATORY PAPILLOMATOSIS AGGRESSIVENESS
Blood samples for 2',5'-oligoadenylate synthetase activity analysis were obtained at 0,4,8,12,24,48,72 and 96 hr after interferon-alpha induction. Relative 2',5'-oligoadenylate synthetase activity at 0 hr was considered to be the base level; the ratio between maximal 2',5'-oligoadenylate synthetase activity during interferon-alpha induction and basal 2',5'oligoadenylate synthetase activity was considered to be the increase rate of 2',5'- oligoadenylate synthetase activity during interferon-alpha induction.
Human papilloma virus typing
High-molecular-wcight DNA was isolated from the patient's lesions and analysed by restriction enzyme digestion and Southern blot hybridization as described previously.'*" Briefly, cellular DNA was digested with restriction enzymes Pstl and BamHI and electrophoresed in a 1 per cent agarose gel. After Southern transfer to a ni\trocellulose filter, the blot was hybridized with a ^sup 32^P- labelled HPV probe at 37C. The filter was washed three times at 65C and then exposed to X-ray film for 7 days with an intensifying screen at -85C.
The results are shown as means standard deviation (SD) or as percentages. Statistical analysis was conducted using Student's t- test, Fisher's exact test and Kaplan-Mcyer survival analysis. The results were considered significant when p
Aggressiveness of recurrent respiratory papillomatosis
Before the start of interferon-alpha therapy all the patients were rated, with the help of the described scoring system, for recurrent respiratory papillomatosis aggressiveness. The results obtained during long-term observation were analysed for statistical correlations with score data. The 38 followup patients (mean score 4.6 3.4) were split into two groups with cut-point 4: 22 patients with mild disease (score ≤ 4) and 16 patients with severe disease (score >4, indicating extralaryngeal spread of recurrent respiratory papillomatosis and/or need for tracheotomy).
Statistically significant differences were observed in patients grouped by HPV type (HPV 11 is associated with more aggressive recurrent respiratory papillomatosis course, p = 0.03), initial response (initial responders had less aggressive recurrent respiratory papillomatosis, p = 0.04), mortality during follow-up (all six patients who died during follow-up had severe disease, p = 0.003) and incidence of malignant transformation (all five patients who underwent malignant transformation had severe disease, p = 0.009).
Response to interferon-alpha during long-term therapy
During the first year of interferon-alpha therapy, 14/38 patients (37 per cent) achieved a macroscopic remission. Twenty-five of 37 patients (63 per cent) achieved a macroscopic remission and were considered to be responders to long-term interferonalpha therapy (32.4 21.6 months). The data available for 38 patients (initial responders at the end of interferon-alpha therapy and long-term responders on 1 August 2003) are shown in Table II. These patients were classified by sex, age at recurrent respiratory papillomatosis onset (≤4 and >4 years; age of 4 years was a median in the studied patients), age at study entry (≤16 and >16 years), number of surgical procedures received before interferon-alpha therapy commencement (≤10 and >10 procedures), frequency of surgical procedures prior to interferon-aipha therapy commencement (≤4 and >4 procedures per year), incidence of tracheotomy (patients with tracheal canula, patients with a clinical indication for canula but without one, patients with neither canula nor clinical indication), score for recurrent respiratory papillomatosis aggressiveness (≤4 and >4; score ≤4 characterized non- aggressive recurrent respiratory papillomatosis localized predominantly in the larynx) and HPV type (HPV 6, HPV 11, failed to identify HPV).
The long-term patient follow-up (172 36.8 months after the end of interferon-alpha therapy) revealed that 16/38 patients (42 per cent) remained in complete remission (i.e. relapse-free). Event- free survival evaluated by Kaplan-Meyer analysis was 42.8 per cent. During the period of follow-up, 6/38 patients died and the overall survival evaluated by Kaplan-Meyer analysis was 82.6 per cent. One of six patients died of acute bleeding after laser resection of scars during papilloma-free remission, 1/6 died of toxoplasmosis with congenital hypereosinophilic syndrome, 1/6 died of superinfection with sepsis by immunosuppression after cornea transplantation, 1/6 died of squamous cell carcinoma in the nasopharynx with lymph node metastasis, and squamous cell lung carcinoma was the cause of death for 2/6 patients; thus 3/6 patients died of malignant transformation.
2',5'-Oligoadenylate synthetase activity in PBMC before and during interferon-alpha therapy
In 31 recurrent respiratory papillomatosis patients available for 2',5'-oligoadcnylatc synthetase sampling, the basal level of relative 2',5'-oligoadenylate synthetase activity before interferon- alpha therapy was 0.6 0.4, ranging from 0.1 to 1.6. Table III shows the basal 2',5'-oligoadenylate synthetase activities in patients grouped by basic parameters of recurrent respiratory papillomatosis data. Statistically significant differences were observed only in patients grouped by their score for recurrent respiratory papillomatosis severity (p = 0.04).
CLINICOPATHOLOGICAL DATA FOR RESPONDERS AND NON-RESPONDERS
Induction of 2',5'-oligoadenylate synthetase by interferon-alpha in therapeutic doses led to a significant increase in relative 2',5'- oligoadenylate synthetase activity (p 10 surgical procedures per year (p = 0.03); patients with scores ≤4 for this parameter tended to have higher increase rates of 2',5'-oligoadenylate synthetase activity (p = 0.08).
CORRELATION BETWEEN CLINICOPATHOLOGICAL DATA AND 2',5'- OLIGOADENYLATE SYNTHETASE PARAMETERS
After the first year of interferon-alpha therapy, 2',5'- oligoadenylate synthetase activity was significantly increased (0.6 0.4 before interferonalpha therapy and 1.3 0.7 after interferon- alpha therapy, p
The increase in 2',5'-oligoadenylate synthetase activity after one year of interferon-alpha therapy showed statistical association with patients' sex, severity score, frequency of surgical procedures, response to interferon-alpha therapy and HPV type. Patients with a severity score >4 did not achieve a significant increase in 2',5'-oligoadenylate synthetase activity after one year of interferon-alpha therapy, in contrast to patients with scores 0.05) in 2',5'oligoadenylate synthetase activity was observed in men, patients with more than 10 surgical procedures per year, non- responders to interferon-alpha therapy and patients with HPV 11.
Human papilloma virus type
Thirty-one patients were successfully HPV-typed. The correlations between clinicopathological data and HPV types 6 and 11 in recurrent respiratory papillomatosis patients are shown in Table IV. Fisher's exact test showed that there was a significant difference in the number of initial and long-term responders among patients with HPV 6 and 11 (p = 0.001 and p = 0.006, respectively). Student's Mest revealed that patients with HPV 11 had significantly higher scores for recurrent respiratory papillomatosis aggressiveness (p = 0.03). Patients with HPV 11 also had a significantly higher incidence of tracheotomy (p = 0.04), malignant transformation (p = 0.01) and mortality (p = 0.03) during follow-up.
The results obtained showed that the recurrent respiratory papillomatosis aggressiveness score (which indicated spread of disease before the start of interferon-alpha therapy) correlated with initial response to treatment. Therefore, this scoring system may be used to evaluate recurrent respiratory papillomatosis severity and to predict response to interferon-alpha therapy. Total number of surgical procedures prior to interferon-alpha therapy commencement (which may also be considered an indirect indication of recurrent respiratory papillomatosis severity)17 correlates with long-term prognosis in recurrent respiratory papillomatosis. So we may conclude that patients with less aggressive recurrent respiratory papillomatosis will have a better response to interferon- alpha therapy and a better long-term prognosis. However, a previously reported association between younger age at disease onset and aggressive recurrent respiratory papillomatosis was not supported by our results.18
PATIENT DATA GROUPED BY HPV TYPE
The major etiological cause of recurrent respiratory papillomatosis is infection with HPV type 6 and/or 11, which are both considered to confer a low risk of malignant transformation but which have differing potentials to cause severe recurrent respiratory papillomatosis. A more aggressive course of recurrent respiratory papillomatosis (resulting in earlier airway obstruction and higher frequency of surgical procedures, higher incidence of tracheotomy, higher incidence of malignant transformation, etc.) is associated with HPV 11, as shown by several authors.2,3 Rabah et al.19 reported the decrease of p53 immune staining in lesions containing HPV 11, suggesting that HPV 11 E6 may be more efficient than HPV 6 E6 in degradation of p53; however, the genetic differences between HPV 11 and HPV 6 that could explain the more aggressive disease course in HPV 11 patients are unknown.
The long-term results obtained in our study support the hypothesis of a more aggressive course in recurrent respiratory papillomatosis patients with HPV \11 in comparison with HPV 6. Patients with HPV 11 had significantly higher scores for recurrent respiratory papillomatosis severity and higher incidences of tracheotomy, malignant transformation and death during follow-up. However, correlations between incidence of HPV type 11 and higher frequency and total number of surgical procedures, and younger age at disease onset were not statistically significant.
Recent publications showed that age at recurrent respiratory papillomatosis onset may influence prognosis (in one study patients with disease onset
One of the most serious disadvantages of interferon-alpha therapy for infectious diseases and neoplasms is a high percentage of non- responders. Interferon-alpha exerts its antiviral and immunomodulating properties via various interferon-stimulated genes, including 2,5oligoadenylate synthetase. It has been hypothesized that a deficiency of the interferon-dependent 2,5oligoadenylate synthetase system, which could be genetically determined, may be responsible for the failure to achieve an efficient response to interferonalpha therapy. Measurement of 2,5-oligoadenylate synthetase activity may give an indication of the basal interferon- alpha level in the patient. Thus, 2,5oligoadenylate synthetase activity is also a convenient biological marker in monitoring response to interferon-alpha therapy.21
There are few data on 2,5-oligoadenylate synthetase activity levels in HPV-associated diseases. Rusconi et al.13 revealed significantly higher basal serum 2,5-oligoadenylate synthetase activity in women with genital HPV infection than in controls. Responders had lower basal 2,5-oligoadenylate synthetase activity than did non-responders, while the peak 2,5-oligoadenylate synthetase activity during interferon-alpha therapy (day 6) was significantly higher in responders.
The results obtained in our study revealed that patients with recurrent respiratory papillomatosis had mean basal 2,5- oligoadenylate synthetase activity within the normal range. We found a significant elevation in 2,5-oligoadenylate synthetase activity during interferon-alpha therapy (p
Correlation between score for recurrent respiratory papillomatosis agressiveness and basal 2,5-oligoadenylate synthetase activity as well as correlation between number of surgical procedures and short-term capability to induce 2,5oligoadenylate synthetase allow us to conclude that a low basal level of 2,5- oligoadenylate synthetase activity and a high level of 2,5- oligoadenylate synthetase induction by interferon-alpha are prognostic markers for a mild recurrent respiratory papillomatosis course.
After one year of interferon-alpha therapy, patients with more than 10 surgical procedures per year, non-responders to interferon- alpha therapy and patients with HPV 11 had a non-significant increase in 2,5-oligoadenylate synthetase activity. These results may indicate that long-term interferon-alpha therapy causes less augmentation of 2,5oligoadenylate synthetase activity in patients with more aggressive recurrent respiratory papillomatosis (higher frequency of surgery, higher severity score, HPV 11). Non- responders did not have significant up-regulation of 2',5'- oligoadenylate synthetase (p = 0.08), in comparison with responders (p = 0.01). Inability to achieve a significant elevation of 2,5oligoadenylate synthetase activity after one year of interferon- alpha therapy indicates that nonresponsiveness to long-term interferon-alpha therapy might be connected with disruption or deficiency of 2,5-oligoadenylate synthetase induction by exogenous interferon-alpha. Overlap in 2,5oligoadenylate synthetase levels makes 2,5oligoadenylate synthetase activity less valuable for prognosis in any single patient, however, monitoring of 2,5- oligoadenylate synthetase activity during interferon-alpha therapy together with HPV-typing and evaluation of recurrent respiratory papillomatosis aggressiveness score may be useful for predicting recurrent respiratory papillomatosis course and response to long- term interferon-alpha therapy.
We conclude that several independent factors may contribute to the prognosis in recurrent respiratory papillomatosis. Patients with HPV 6, a severity score 4, a high number of surgical procedures prior to interferonalpha therapy and a high basal 2,5-oligoadenylate synthetase activity should be considered at high risk of aggressive recurrent respiratory papillomatosis, often with spread to lower airway passages, malignant transformation and death.
The results of long-term observation in the presented study (adjuvant interferon-alpha therapy) may be extrapolated to the recurrent respiratory papillomatosis course in patients treated with various other therapy options, including injections of intralesional cidofovir (application of which had evident benefit, but results lacked long-term followup). Therefore, HPV typing by polymerase chain reaction, evaluation of recurrent respiratory papillomatosis aggressiveness by the scoring system presented, and determination of 2,5-oligoadenylate synthetase activity as described may be recommended as most valuable prognostic factors in recurrent respiratory papillomatosis patients.
The authors thank Mrs B. Boctcher for excellent technical assistance and Prof. Dr M. Shurin for critically reading the manuscript. This work was financially supported by a donation from the Hilfe fr Krebskranke Kinder, Frankfurt e.V. foundation.
* This is a series of 42 patients with recurrent respiratory papillomatosis who were treated with Interferon-alpha for a mean period in excess of two years
* The response was correlated with human papilloma virus type and the severity of the disease and was also compared with the activity of an enzyme 2',5'-oligoadenylate synthetase) that has been shown to correlate with the clinical response to Interferon-alpha
* The conclusions suggest that patients with HPV type 11, a severity score of greater than 4, a high number of surgical interventions and a high basal 2',5'-oligoadenylate synthetase level are at risk of an aggressive clinical course with spread to the lower airway, malignant transformation and a lethal outcome
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V. GEREIN*[dagger], PH.D., M.D., E. RASTORGUEV*, M.SC., J. GEREIN[dagger], E. LODEMANN[double dagger], PH.D., D.SC., H. PFISTER**, PH.D., D.SC, W. DRAF[dagger][dagger], PH.D., M.D., C. DESLOOVERE[double dagger][double dagger], PH.D., M.D.
From the *Department of Paediatric Pathology, Institute of Pathology, University of Mainz, Germany, the [dagger]Clinic of Paediatric Haematology and Oncology, University of Frankfurt/Main, Germany, the [double dagger]Center for Biological Chemistry, University of Frankfurt/Main, Germany, the **Institute of Virology, University of Cologne, Cologne, Germany, the [double dagger][double dagger]Department of Otorhinolaryngology, Hospital of Fulda, Germany, and the [double dagger][double dagger]Department of Otorhinolaryngology, University of Leuven, Belgium.
Accepted for publication: 10 July 2004.
Address for correspondence:
Valentin Gerein, M.D., Ph.D.,
Department of Paediatric Pathology,
Johannes Gutenberg University of Mainz,
55101 Main/, Germany.
E-mail: [email protected]
Dr V. Gcrcin takes responsibility for the integrity of the content of the paper.
Competing interests: None declared
Copyright Royal Society of Medicine Press Ltd. Oct 2004