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Conceived and designed the experiments: CP GE FH FB JD CD SSC SFC JM PC BW CM. Performed the experiments: FH FB JD. Analyzed the data: CP GE FH FB JD CD SSC SFC JM PC BW SS JS CM DT HR. Wrote the paper: FB JD. Other: Enrolled subject and provided clinical specimens: FB. Enrolled subjects and provided clinical specimens: SSC SFC CM JM PC BW DT JS SS CD. Edited the paper: SSC SFC CM JM PC BW DT JS SS CP GE FH CD.

The authors have declared that no competing interests exist.

RRP is a devastating disease in which papillomas in the airway cause hoarseness and breathing difficulty. The disease is caused by human papillomavirus (HPV) 6 or 11 and is very variable. Patients undergo multiple surgeries to maintain a patent airway and in order to communicate vocally. Several small studies have been published in which most have noted that HPV 11 is associated with a more aggressive course.

Papilloma biopsies were taken from patients undergoing surgical treatment of RRP and were subjected to HPV typing. 118 patients with juvenile-onset RRP with at least 1 year of clinical data and infected with a single HPV type were analyzed. HPV 11 was encountered in 40% of the patients. By our definition, most of the patients in the sample (81%) had run an aggressive course. The odds of a patient with HPV 11 running an aggressive course were 3.9 times higher than that of patients with HPV 6 (Fisher's exact p = 0.017). However, clinical course was more closely associated with age of the patient (at diagnosis and at the time of the current surgery) than with HPV type. Patients with HPV 11 were diagnosed at a younger age (2.4y) than were those with HPV 6 (3.4y) (p = 0.014). Both by multiple linear regression and by multiple logistic regression HPV type was only weakly associated with metrics of disease course when simultaneously accounting for age.

The course of RRP is variable and a quarter of the variability can be accounted for by the age of the patient. HPV 11 is more closely associated with a younger age at diagnosis than it is associated with an aggressive clinical course. These data suggest that there are factors other than HPV type and age of the patient that determine disease course.

It is now well established that human papilloma virus (HPV) causes recurrent respiratory papillomatosis (HPV)

As of April 2007, we have collected fresh, laryngeal biopsies from over 130 unique patients with RRP in collaboration with the RRP Task Force. To our knowledge, this collection of specimens represents the largest of its type in the world. The current study brings the largest dataset yet developed to bear on the potential relationship between clinical course and HPV type. Equally importantly, and often neglected, we explore how much of the variability of the disease can be attributed to the type of HPV while simultaneously accounting for other variables such as age.

Between January 2003 and April 2007 fresh papilloma biopsies were preserved in TRIzol® from 135 unique patients with RRP and were subjected to HPV typing. By using both allele specific PCR and RFLP typing on all cases we were able determine whether HPV 6 versus HPV 11 was present in the papilloma. Papillomas from 8 cases were positive for both HPV 6 and HPV 11 by both methodologies. Thus data was available on 127 unique patients who either had HPV 6 or HPV 11. Eight of the cases were excluded since we had less than one year of clinical course data (HPV 6 = 7, HPV 11 = 1, median age = 6.8y). Of the remaining 119 unique cases only one had adult-onset RRP and therefore was excluded. (

Gender | male = 68 (58%), female = 50 (42%) |

Race | White = 67 (57%), Black = 41 (35%), Asian, Other or Not Recorded = 10 (8%) |

Hispanic ethnicity | Hispanic = 11 (9%), Non-Hispanic = 107 (91%) |

Age at diagnosis | median = 3.0 years, range = (0.1, 13.1) |

Duration of follow up (years) | median = 5.2 years, range = (0.9, 33) |

Total number of surgeries | median = 19, range = (2, 402) |

Max number of surgeries in a 12 month period | median = 4, range = (1, 52) |

Interval since previous surgery | median = 113 days, range = (10 days, 3 years) |

Distal Involvement | involved = 30 (25%), uninvolved = 88 (75%) |

Tracheostomy | currently or previously = 12 (10%), never = 106 (90%) |

Aggressiveness of course | aggressive = 95 (81%), indolent = 23 (19%) |

HPV type | HPV 6 = 71 (60%), HPV 11 = 47 (40%) |

Initial analysis was directed at determining whether or not HPV type and clinical course were associated or independent of each other. There was indeed an association between HPV 11 and a more aggressive course (

indolent | Aggressive | odds ratio (p-value) | |

HPV 6 | 19 | 52 (73% of all HPV 6 cases) | |

HPV 11 | 4 | 43 (91% of all HPV 11 cases) | 3.9 (0.02) |

Surgery Count | total procedures <10 | total procedures ≥10 (row percentage) | odds ratio (p-value by Fisher Test) |

HPV 6 | 25 | 46 (64% of all HPV 6 cases) | |

HPV 11 | 8 | 39 (83% of all HPV 11 cases) | 2.6 (0.04) |

procedure frequency never ≥4 per year | procedure frequency ever ≥4 per year | ||

HPV 6 | 31 | 40 (56%) | |

HPV 11 | 13 | 34 (72%) | 2.0 (0.08) |

distal involvement: never | has had distal involvement | ||

HPV 6 | 60 | 11 (15%) | |

HPV 11 | 28 | 19 (40%) | 3.7 (0.004) |

tracheostomy: never | has had tracheostomy | ||

HPV 6 | 67 | 4 (6%) | |

HPV 11 | 39 | 8 (17%) | 3.4 (0.06) |

Rather than simply looking at the data dichotomously (aggressive vs. indolent) the total number of surgeries were analyzed as a count variable. Total number of surgeries thus far in the clinical course was log transformed since the untransformed variables were highly skewed. The geometric mean number of surgeries in the cases with HPV 6 was 16 and in the cases with HPV 11 it was 30 (t-test p-value = 0.005) suggesting that subjects infected with HPV 11 tend to follow an aggressive course of the disease.

The above data not only represents yet another set supporting the association between HPV 11 and aggressive course but it is also the largest data set to ever be analyzed. At this point one may conclude the case finally settled and resolve to use HPV typing as a clinical prognosticator. Yet that would be premature since other variables, and not just HPV type may be associated with clinical course.

Gender and Race are categorical variables just as HPV type is. Yet there was no association between them and disease course. Age can be cut into a dichotomous variable but with no obvious cut point. The variable fitted a Gaussian distribution best when it was transformed by taking the square root. Thus the mean age at the time of diagnosis of the children who had experienced an aggressive course was 2.7 years, but was 4.3 years for those with an indolent course (t-test p-value = 0.01). For the 6 cases with pulmonary involvement, the mean age of diagnosis(based upon square root transformed data) was 1.3 years for those affected and 3.1 years for those with no recorded pulmonary involvement (t-test p-value = 0.002). The range of age of diagnosis for those with pulmonary involvement was 0.5 to 2.6 years. In other words, all 6 cases fell below the 42nd percentile for the sample as a whole. While age of diagnosis has been associated with aggressiveness as measured by the frequency of surgery, Reeves et al. ^{−7}). On the other hand, HPV type was not related to the interval since the last surgery (t-test p-value = 0.25). (

For those cases where the enrollment surgery was not their first surgery, the interval in days since the last surgery was recorded. Variables were transformed so that they would comply with a Gaussian distribution. The natural log of the number of days since the last surgery are depicted on the y axis. The left panel depicts a linear regression against the square root of the age at the time of enrollment. The model predicts that a 4 year old last had surgery 69 days (2.3 months) ago and a 9 year old last had surgery 113 days (3.7 months) but only 20% of the variability is accounted for by this model. The right panel depicts a boxplot of the same data but clustered by HPV type. The geometric mean of the number of days since the last surgery was 119 days (3.9 months) for those with HPV 6 and a similar 97 days (3.2 months) for those with HPV 11. The t-test p-value was 0.25.

It is quite conceivable that the duration between any two surgeries may be a function of more than just the current aggressiveness of the disease. Some surgeons schedule subsequent surgery as symptoms dictate, whereas other surgeons set a pre-determined duration based upon the agressiveness of the clinical course over the preceding year. Others have adopted a hybrid of the two methods. The variation in practice style was controlled for by coding the surgeon's response for each patient as 1 for “as required (prn)”, 2 for “hybrid” and 3 for “predetermined”. Adding the surgeon's style to the linear model accounted for an additional 7% of the variability (p-value = 0.010). Age at diagnosis was added to the model and further accounted for some of the variability in a statistically significant manner (p-value = 0.014) albeit that it only accounted for 6% of the variability. So while age at diagnosis was correlated with the duration since the preceding surgery, this correlation was very weak when contrasted against that of age at the time of enrollment in the study. Together, age at enrollment+age at diagnosis+surgeon's style of determining surgical interval accounted for 31% of the variability in the time since the preceding surgery. Adding HPV type accounted for none of the residual variability. Thus in the multiple linear regression equation where y = log (days between last and second last surgery), coefficients were calculated for 4 variables simultaneously; in other words it was not a step-wise hierarchical model where a subsequent term is allowed to explain the residual left over after a preceding term has already been entered into the model. The p-values for each of the estimated coefficients were as follows: HPV type = 0.95, square root of at age of enrollment = 6×10^{−5}, square root of age at diagnosis = 0.015 and surgeon's style = 0.013. Fourteen observations were excluded either because the surgeon's style was missing or the patient had been enrolled at the time of the first surgical intervention and thus there was no duration between the last and the second last surgery. Similarly, mother's highest educational qualification and gross household income accounted for none of the variability. Even the anatomical component of the Derkay/Coltrera score at the time of enrollment did not account for the variability in the time since the last surgery given that the age variables and the surgeon's style had already been accounted for.

Since the duration between surgery at the time of enrollment in the study and the preceding surgery utilizes information from only a small interval of the subject's clinical course it may be highly variable. Thus, a similar analysis was conducted using the annual frequency of surgeries around the time of the enrollment. The sampling period was a median of 1.8 years (range 0.9 to 2). As above, the age was transformed to the square root and the frequency of procedures in the time period surrounding enrollment was log transformed. Results were similar to that obtained above; a simple linear regression model declared that square root of the age at the time of enrollment accounted for 20% of the variability in the natural log of the annual frequency of surgeries around the time of enrollment (p = 4.6*10^{−7}). Together, age at enrollment+age at diagnosis+surgeon's style of determining surgical interval accounted for 37% of the variability in the time since the preceding surgery. However, unlike for the variability in duration since preceding surgery, annual frequency variability was very highly significantly correlated with both age variables: age at time of measuring frequency p-value = 0.00023; age at time of diagnosis p-value = 0.00020. Adding HPV type once again accounted for none of the residual variability.

We have seen above that HPV 11 is associated with a more aggressive clinical course as is young age of the patient, with clinical course being far more correlated with age than with HPV type. In fact HPV type is correlated with age of the patient and this is why one sees an association between HPV type and clinical course, if not simultaneously controlling for the age of the patient. This is most easily seen if one performs logistic regression with clinical course measured as a binomial variable as defined above. Children with HPV 11-induced disease are diagnosed at a younger age than children with HPV 6; the average age of diagnosis amongst those with HPV 6 was 3.4 years and the average age of diagnosis for those with HPV 11 was 2.4 years (t test p-value = 0.014). Logistic regression in which HPV-type was stipulated as the sole independent variable accounted for 5.6% of the deviance (p-value = 0.02) and when age of diagnosis (transformed to square root) was the sole independent variable, it accounted for 7.4% of the deviance (p-value = 0.005). When the two variables were handled simultaneously in a multiple logistic regression then 12% of the deviance was accounted for with the individual p-values being 0.057 for HPV Type and 0.017 for age of diagnosis.

RRP is a devastating disease-sometimes because of the urgency of symptoms at presentation (such as respiratory distress), but far more commonly because of the chronicity of the disease and the multitude of surgeries undertaken. Amongst the many patients with RRP there is considerable variability in the clinical course. For many years investigators have explored the relationship between the clinical course, and whether the patient was infected by HPV type 6 or type 11, but many studies suffered from small sample size. Now, thanks to a large multi-center collaboration that was founded to explore the underlying genetic susceptibility to RRP, we have access to the largest dataset ever assembled. With confidence one can note that HPV 11 is associated with a more aggressive disease but that the clinical course is more closely associated with the age of the child which in turn is associated with HPV type.

Younger children tend to have a more aggressive clinical course and so do children with HPV 11. However, HPV type is associated with both the age of the child and with the outcome, namely an aggressive clinical course. Once the age of the child is controlled for, then the relationship between HPV type and clinical course becomes one of borderline significance. We can speculate upon the association between the age of the child and frequency of surgery. Investigators have believed that the relationship is due to the relatively small airway of younger children demanding more frequent surgery to keep it clear. In our study we did not measure the diameter of the airway following papilloma removal. The closest approximation to such a value can be garnered from an article in which appropriate endotracheal tube diameter was discussed for each age

Previous studies

A possible criticism of our findings is that we have analyzed a biased sample of aggressive RRP cases based upon our acquisition process. The data described herein was acquired for the principal purpose of determining the underlying genetic susceptibility to RRP. The design is cross-sectional and acquires probands at any stage of their disease. Our enrollment mechanism was unlikely to reach cases diagnosed a long time ago who subsequently had an indolent course and went into remission before our study was initiated. On the contrary those who were diagnosed at a similar time but have endured an aggressive and protracted course would have been reached by our acquisition mechanism. Furthermore, we excluded 8 cases from analysis since the duration of their follow up had been shorter than a year. These factors combined, bias our sample to a generally more aggressive clinical course than the population of all incident cases tracked over their lifetime. Another weakness of this cross-sectional study is that data was not collected from each and every surgical intervention. Rather, physical findings were recorded only at the time of enrollment and summary data pertaining to history were recorded at enrollment and 1 year later. An ideal metric of papilloma burden would have been a documentation of the full Derkay/Coltrera Staging Score for each individual from the time of diagnosis to the time of remission. The area under such a curve would be less subject to stochastic phenomena in each individual's course.

Less than half of RRP surgeons send papillomas for HPV typing

Given the variability of the clinical course it would be useful to be able to predict when a particular child would require subsequent surgery. The current study suggests that HPV type would be of no or little utility in making this determination. Age of the patient at the time and age of the patient at diagnosis would be more accurate predictors but even with these factors taken into effect we could only account for about one third of the variability.

Malignant transformation of RRP is a devastating but rare complication that has been investigated by others. Malignancy was not specifically addressed by the incumbent study. Nevertheless, it is important for the reader to be aware that other groups have investigated the features of malignant transformation and have associated them with pulmonary involvement and with HPV 11 exclusively (that is no isolates of HPV 6 from malignant RRP biopsies)

The cohort described herein had a similar rate of co-infection compared to that which has already appeared in the literature. In the initial cohort of 135 cases (before exclusions for insufficient clinical course data) for whom we had fresh papilloma specimens, only 8 (6%) were deemed to harbor both HPV 6 and 11. Following exclusion of cases with insufficient clinical course data there were only 5 cases with co-infection; statistical analysis of such a small group would be of questionable meaningfulness. Furthermore, one would not know if both HPV types were leading to the phenotype since it is conceivable that one of the types had caused the disease and the other was merely present much in the same way that HPV 6 and 11 have been found in the mucosa of people without RRP. Our method of typing used two methods on each specimen (allele-specific PCR and RFLP). Co-infection rates between papers could be difficult to interpret since methodologies vary from study to study. (

Study | HPV 6 | HPV 11 | coinfection |

Rabah 2001 | 29 (48%) | 32 (52%) | 0 (0%) |

Wiatrak 2004 | 31 (53%) | 23 (40%) | 4 (7%) |

Gabbott 1997 | 19 (43%) | 24 (55%) | 1 (2%) |

Pou 1995 | 21 (88%) | 2 (8%) | 0 (0%) |

Draganov 2006 | 6 (26%) | 14 (61%) | 3 (13%) |

Padayachee 1993 | 5 (25%) | 15 (75%) | 0 (0%) |

Rimell 1997 | 9 (47%) | 6 (36%) | 4 (21%) |

Szeps 2005 | 12 (66%) | 6 (33%) | 0 (0%) |

Maloney 2006 | 4 (27%) | 4 (27%) | 7 (47%) |

Pou 2004 | 6 (46%) | 7 (54%) | 0 (0%) |

The RRP Task Force and the Center for Genomic Sciences, Allegheny-Singer Research Institute in Pittsburgh, PA are currently collaborating to determine the underlying genetic susceptibility in RRP. A secondary objective is to determine what the genetic factors are that account for the variability in clinical course. One of the reasons for obtaining a papilloma specimen at the time of enrollment was to determine whether the patient was infected with HPV 6 or 11. We believed that HPV type would be an important factor to control for in trying to observe an association between aggressiveness and underlying genotype. Given the data contained in this report it appears as if HPV type may not be a significant variable, or if it is, then only weakly so. While it is not known what factor(s) account for the remaining two thirds of the variability in clinical course, a reasonable hypothesis would be that host genetic factors account for the remainder of the clinical variability. A genome wide search for those factors is underway.

The RRP patients studied here were identified through an ongoing collaborative study on the genetics of RRP with the coordinating site located at Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA

Information about the clinical course of each case was supplied by their otolaryngologist at the time of enrollment and one year later updated clinical information was supplied. Metrics of clinical course included total number of airway procedures (count), frequency of procedures (count per year), ever had involvement in the trachea or distally (binomial), ever had a tracheostomy (binomial). A clinical course was defined as “aggressive” if any of the following criteria were met: total procedures ≥10, procedure frequency ever ≥4 per year, ever had distal involvement, ever had a tracheostomy tube. If none of these criteria were met the patient was considered to be experiencing an indolent course. Numeric cuttoffs can appear arbitrary but the metrics used in this study are based on precedence set by other investigator groups and importantly, were decided upon a priori. Doyle et al, working in New Orleans and Derkay, reporting a national survey of otolaryngologists, used identical or similar cut offs for classifying aggressive versus benign

For each patient the surgeon was asked about the manner in which he or she decided to perform the next surgical intervention. The exact wording on the forms was, “Some surgeons operate on patients according to pre-determined intervals. Others only return to the operating room on a prn (as required) basis. Some adopt a hybrid approach whereby only the next interval is pre-determined in response to what happened over the last one or two intervals. What approach are you closest to with respect to this patient over the past year? “The surgeon's response for each patient was coded as 1 for “as required (prn)”, 2 for “hybrid” and 3 for “pre-determined”.

During surgery to control the patient’s disease, two 1mm by 1mm papilloma biopsies were collected by the patient's attending physician and preserved in 700 µl of TRIzol® solution contained in a 1.5 ml microcentrifuge tube. The biopsy specimens were shipped to Allegheny-Singer Research Institute, Pittsburgh, PA, the specimen and data coordinating site. DNA was extracted from laryngeal biopsies according to the manufacturer's guidelines (Invitrogen, Carlsbad, California). To assess the quantity and quality of the isolated DNA, samples were tested by spectrophotometry using a ND-1000 spectrometer (NanoDrop) and by PCR using the GH20 (

DNA was analyzed for the presence of HPV sequences by both Type-Specific PCR primers (TS-PCR) and Restriction Fragment Polymorphism (RLFP) on all laryngeal specimens. These two HPV typing techniques were systematically used in our specimens to ensure reproducibility and accuracy of genenotyping results. In fact, like any other PCR-based genotyping, TS-PCR is very sensitive and can produce either false positives or false negatives, which is a potential explanation for the very high frequency of dual infection reported in some RRP studies. RLFP typing using a different set of PCR primers followed by specific endonuclease digestion was used to replicate TS-PCR results and confirm HPV genotype.

For each HPV type, two Type-Specific (TS) primers were designed in the E6 (HPV 6) and L2 (HPV 11) region. Prior to selecting the primers, HPV 6 and HPV 11 DNA sequences were downloaded from the public database at Los Alamos National Laboratory (

The first 2 lanes after the ladder (both labeled “1”) represent the wells for HPV 6 positive control clone for the TS-PCR using either primer pair 6930–6932 (designed to be specific for HPV 6) or 6931–6933 (designed to be specific for HPV 11). The same pattern applies to the remaining lane couplets. The couplet labeled “2” represents HPV 11 positive control clone. Couplets 3–8 are RRP specimens and couplet 9 is the PCR negative control. In this gel we see that that laryngeal DNA used in couplet 3 contained only HPV 11 and the remainder contained only HPV 6.

HPV6 and 11 consensus E7 and E1 primers Forward primer:

Prior to any subject enrollment the primary metric of clinical course was defined as the composite label “aggressive” or “indolent” as stated above under “clinical course”. While clinical course was considered a dependent outcome measure, the following variables were considered to be independent potential predictor variables: HPV type, age at diagnosis, age at enrollment, gender, race, educational status of mother, gross household income, surgeon's style. The dataset was initially inspected by conducting a descriptive statistical summary of all the variables in isolation (R statistical software (

The authors would like to thank the following individuals who have contributed to ascertainment of clinical materials and data.

Ms. Marilyn R. Smith and Ms. Mary O'Toole , Allegheny-Singer Research Institute, Pittsburgh PA; Dr. Anthony Mortelliti, SUNY Upstate Medical Center, Syracuse, NY; Dr. Lisa Elden, Children's Hospital of Philadelphia, PA; Dr. David Tunkel, Johns Hopkins Children's Center, Baltimore, MD; Dr. Jerome Thompson, Le Bonheur Children's Medical Center, Memphis, TN; Dr. Joseph Dohar, Children's Hospital of Pittsburgh, PA; Dr. Frederick Kozak, British Columbia's Children's Hospital, Vancouver, Canada.