Usefulness of Urine Mass Spectrometry to Identify Women Infected with HPV 16 or Diagnosed with Higher Grades of Cervical Intraepithelial Neoplasia

Research Article

A Proteomics. 2014;1(1): 9.

Usefulness of Urine Mass Spectrometry to Identify Women Infected with HPV 16 or Diagnosed with Higher Grades of Cervical Intraepithelial Neoplasia

Chandrika Piyathilake1*, Andres Azuero2, Roland Matthews3, Mi Kyung Kim4 and Senait Asmellash5

1Department of Nutrition Sciences, University of Alabama at Birmingham, USA

2School of Nursing, University of Alabama at Birmingham, USA

3Morehouse School of Medicine, USA

4Department of Cancer Epidemiology, National Cancer Center, Korea

5Department of Surgery, University of Alabama at Birmingham, USA

*Corresponding author: Chandrika Piyathilake, Department of Nutrition Sciences, University of Alabama at Birmingham (UAB), Wallace Tumor Institute 420D, 1824 6th Avenue South, Birmingham, AL 35294-3300, USA

Received: May 29, 2014; Accepted: August 08, 2014; Published: August 13, 2014


Identification of women infected with human papilloma virus 16 (HPV 16) or with higher grades of cervical intraepithelial neoplasia (CIN 2+) using cost-effective and non-invasive tests will help to eliminate the worldwide burden of cervical cancer. The aim of the study was to explore the usefulness of matrix-assisted laser desorption/ionization (MALDI) time-of- flight (TOF) mass spectrometry (MS) to identify women infected with HPV 16 or diagnosed with CIN 2+ using urine samples. An additional aim was to compare several statistical and data mining techniques used to build predictive algorithms. The study used urine samples collected from 235 women diagnosed with abnormal cervical cytology. 87 and 148 women were diagnosed with CIN 2+ (cases) and ≤CIN 1 (non-cases), respectively. 126 and 109 women were tested positive or negative for HPV16, respectively. The cross-validated accuracy for detecting CIN 2+ varied from 62-73% based on the predictive technique used suggesting the usefulness of comparing different predictive modeling techniques. The positive predictive value (PPV) for detecting CIN 2+ was higher than previous studies and varied from 70% to 79%, with highest PPV noted among HPV 16 negative and African American (AA) women. Similar to CIN 2+ predictive models, the cross validated predictive accuracy for HPV 16 infections varied based on the predictive technique used, from 53% to 75%. The best PPV (75%) for HPV 16 infections was observed for AA women and the worst PPV for Caucasian American (CA) women (62%), suggesting racial differences in the usefulness of MALDI-TOF-MS based tests. The PPVs for detecting CIN 2+ or HPV16 infections were ~ 75%, a reasonably good result given the fact that non-invasively collected samples used may allow repeat testing, especially if cost-effective ELISA tests based on the discriminatory features identified in our study can be developed in the future.


Keywords: Urine; Protein profiles; CIN


Certain types of carcinogenic or high risk Human papilloma viruses (HR-HPVs), which are sexually transmitted, represent the most important risk factors for the development of invasive cervical cancer (ICC) as well as cervical intraepithelial neoplasia (CIN), precursor lesions for ICC [1-3]. This is unique in cancer etiology because no other human cancer has yet been shown to have a necessary cause that is so clearly identified. Information on HPV prevalence worldwide, however, is inconsistent since there is no standardized method for detecting HPV. Currently in the US, several HPV test systems exist. The US Food and Drug Administration (FDA)-approved Hybrid Capture 2 assay (HC-2) targets 13 HR-HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) but does not distinguish individual HPV types. On April, 2014, the cobas HPV Test was approved by the US FDA for use as a first-line primary screening tool in women aged 25 years or older to assess risk of ICC. The test simultaneously provides pooled results for HR-HPV genotypes (HPV-31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and individual results for HPV 16 and HPV 18.

More than 90% of cervical cancers are associated with HR-HPV DNA [4]. Studies that used improved HPV testing procedures have established HPV as a causative agent for CIN as well [5]. In the New York Cervical Disease Study, Wright et al. [6] detected HR-HPV DNA in 75% and 100% of women with low grade squamous intraepithelial lesions (LSILs) and high grade squamous intraepithelial lesions (HSILs) respectively. Eighty-five percent of women diagnosed with low grade cervical lesions in the cohorts established at the University of Alabama at Birmingham (UAB) are positive for HR-HPV based on Roche diagnostics linear array HPV genotyping test [7]. Results from this cohort also showed that among women diagnosed with LSIL and HSIL where a large majority are positive for HR-HPV, only 23% and 50% respectively have biopsy-confirmed higher grades of CIN (CIN 2+). These observations demonstrate that the presence of any HR-HPV genetic material is not by itself indicative of cervical disease. Therefore, novel markers with higher specificity for the presence of CIN 2+ among HR-HPV positive are needed and this will improve cervical cancer screening and reduce the cost associated with patient care.

Infection with HPV 16 is the biggest causative agent of cervical cancer and it is the most prevalent HR-HPV in the USA. Campion et al. [8] reported that 58% of HPV 16-positive women who had mild cervical atypia progressed to CIN 3 within 2 years. The Centers for Disease Control (CDC) estimates that 20 million people in the US are infected with HPV 16 and that every year there are about 5.5 million new infections. Results from our studies [7] demonstrate that the prevalence of HPV 16 in our population can vary from 21% to 55% depending on the grade of cervical lesions.

Because there is no cure for infection with HPVs, prevention and control of these infections could be used for primary cancer prevention, possibly saving thousands of lives. Because HPV 16 is the most prevalent HPV genotype associated with CIN and ICC, its detection and control would clearly offer a cost effective long-term strategy to reduce the cervical cancer burden. HPV vaccine development holds great promise for reducing the incidence of ICC, and the addition of a vaccine against HPV 16 is projected to be a cost-effective use of health care resources [9]. However, a type-specific HPV vaccine may reduce but not eliminate the risk of ICC [10]. Therefore, cervical cancer screening recommendations are unlikely to change for females who receive the HPV vaccine. Because of inadequate data on its long-term effectiveness, the impact of type-specific vaccines for other HR-HPVs, and duration of immunity, it is unlikely that routine screening programs or other preventive measures will be replaced by HPV vaccines in the near future. Therefore, at this point, a vaccine approach may not reduce the health care cost associated with prevention efforts for cervical cancer. Because of this, development of cost effective tests that can be used to identify women at risk is more important than ever. Tests that may identify women infected with HPV 16 are of high importance because of its higher carcinogenicity. As discussed below, such tests are also important for providing HPV vaccines to the most appropriate individuals.

Ideally, HPV prophylactic vaccine should be administered before the onset of sexual activity and females who have not been infected with any vaccine HPV type would receive the full benefit of vaccination. Females who are already sexually active but not infected with vaccine HPV type would still get protection from the vaccine but currently, there is no cost-effective test available for clinical use to determine whether a female has had any or all of the four HPV types in the currently available vaccine (HPV 6, 11, 16, and 18). Therefore, development of cost-effective tests which are able to detect HPV infections, especially, HPV 16 will not only be useful for routine screening but also for identifying women who will benefit from HPV vaccines even after the onset of sexual activity.

Protein biomarkers have a great potential in elucidating the biology of disease progression. Biomarker discovery efforts have shown that proteins such as the cyclin-dependent kinase inhibitor p16 are differentially expressed in normal versus cervical cancer cells. The potential candidates described so far require in situ hybridization or immune histochemistry of tissue samples. Ideally, we would like to find biomarkers that are easily detected in non-invasively collected samples, such as urine, using an assay that could be adapted for high throughput clinical applications. The merits of MALDI MS profiling and capillary electrophoresis coupled with electrospray ionization (ESI) MS has been recently reviewed by Albalat et al. [12]. While each platform offers distinct advantages, MALDI MS, is a particularly rapid and simple technique for analyzing complex biospecimens such as urine, serum and plasma [13].

The aim of this work was to explore the usefulness of urine mass spectrometry to identify women infected with HPV 16 or diagnosed with CIN 2+. Since there is no standard way of analyzing MALDI MS profiling data, an additional aim of the study was to use several statistical and data mining techniques to compare the accuracy of results generated by different predictive modeling techniques.

Materials and Methods

Patient population

The study was based on the analysis of urine samples collected from 235 women referred for colposcopy because of abnormal cervical cytology. All women were diagnosed with abnormal pap and were enrolled in a prospective follow-up study funded by the National Cancer Institute (R01 CA105448, Prognostic Significance of DNA & Histone Methylation). The parent study has been described in a previous publication [7]. The study protocol and procedures were approved by the UAB Institutional Review Board. Among the 235 women, 87 were diagnosed with CIN 2+ (cases) and 148 were diagnosed with ≤CIN 1 (non-cases). The average age among the women was 24.5years (SD=5.1, Range=19-48); 58% (n=136) were African American (AA) and 42% (n=99) identified themselves as Caucasian American (CA). Additional participant characteristics by case status are shown in Table 1. Parity was significantly higher among cases compared to controls as previously reported for this study population [7]. Urine peptide/protein profiles from this sample were used to predict CIN 2+ status. Because 126 women out of the 235 tested positive for HPV16, the urine peptide/protein profiles were also used to predict HPV16 infection status.