An important application of MALDI-TOF MS is the simultaneous analysis of multiple proteins to establish “fingerprint” profiles that discriminate disease from non-disease. This is an important approach, since no single biomarker or protein alone will improve the early detection/diagnosis of diseases, including cancer or pre-cancers. Body fluids, such as serum, are a source of putative protein biomarkers with the potential to elucidate organ-specific carcinogenic events. Because of its high sensitivity for proteins in the low molecular weight range and because of its capability of high throughput screening, MALDI-TOF MS has been used to distinguish healthy controls from patients diagnosed with several cancers including the colon, lung, ovary, breast and esophagus.11
Previous studies have also documented differences in serum protein profiles between cervical cancer and healthy controls. 16
Three differentially expressed potential biomarkers with relative molecular weights of 3974 Da, 4175 Da and 5906 Da identified in this study demonstrated ~90% sensitivity and specificity in disguising cervical cancer from controls. To our knowledge, the current study is the first to document the usage of MALDI-TOF-MS technology in the analysis of serum protein profiles of patients diagnosed with cervical pre-cancer and evaluated differences in sensitivity and specificity of protein peaks by race.
We focused on women infected with HPV 16 as this virus is the most frequent causative agent for developing cervical cancer world-wide.17
Even though only a fraction of women infected with HPV 16 develops CIN 2+, these lesions have the highest rate of progression to CC.18
Further, the recurrence rate of CIN 2+ after a loop electrosurgical excision procedure was shown to be significantly higher among those who were tested positive for HPV 16 before and after the procedure.19
Therefore, identification of this fraction of women and treatment of their lesions and closer follow-up after treatment are important unmet medical needs in the current management protocols. Currently available tests do not have adequate specificity for identifying women with HPV 16-associated CIN 2+.
In our population, 40% of women infected with HPV 16 were diagnosed with CIN grades higher than 2 (CIN 2+). Identification, treatment, and closer follow-up of these women would offer a cost-effective strategy to reduce the cervical cancer burden. A meta-analysis showed that detecting any HR-HPV by the Hybrid Capture 2 test among women with abnormal pap demonstrated 97.2% sensitivity for detecting CIN 2+ and 97.1% sensitivity for detecting CIN 3+. This analysis also demonstrated a pooled specificity of 30.6% and 26.1% when the outcome was CIN 2+ and CIN 3+ respectively. 20
A recent study demonstrated that the sensitivity of the HPV 16/18 genotyping test for detection of CIN 2+ was >93% while the specificity of the test for detection of CIN 2+ and CIN 3+ was 44.2% and 43%, respectively.21
Two protein peaks identified by our study demonstrated higher specificity for identifying CIN 2+ than these published studies. An increasing intensity of [m/z = 4459] was associated with a higher risk of being a case, regardless of race with a specificity of 58% for CIN 2 and a specificity of 75% for CIN 3. Further, to our knowledge for the first time, we also document interesting racial differences in the associations between peak intensities and higher risk of being diagnosed with CIN 2+. An increasing intensity of [m/z = 4154] was not only associated with a higher risk of being a case only among CAs, but also had an opposite effect among African AAs. With [m/z = 4459], the specificity was higher for AAs, but the sensitivity was higher for CAs suggesting that the predictive ability of the [m/z = 4459] peak intensities varies by race. With [m/z = 4154], on the other hand, the sensitivity was similar for the two races, but the specificity was higher for AAs, indicating that the peak had slightly better predictive ability among AA women.
This report suggests a capacity of serum protein profiles to differentiate between HPV 16 positive women free of true pre-neoplastic lesions (≤CIN 1) and women diagnosed with higher grades of CIN, especially CIN 3, in our population of women infected with HPV 16. In the statistical analyses, infection with other types of HR-HPVs was used as a predictor of case status, by itself and as an interaction with the m/z peaks, but no significant association was found. Therefore, these results suggest that it is unlikely that co-infections with other HPVs interfere with identifying CIN lesions in women infected with HPV 16. Further, the results also suggested that specific serum profiles might be useful for differentiating CIN cases from non-cases in AAs and CAs.
Identification of specific proteins associated with peaks that are significantly different between ≤CIN 1 and CIN 2+ by race may potentially lead to the development of new screening tests which are suitable in different racial groups. Identification of these serum proteins and development of antibody-based tests, such as ELISA, may lead to the development of cost-effective, non-invasive, sensitive, and simple to use tests. Further, serum based screening tests are likely to be more acceptable than cervical cell based tests in populations where the prevalence of obesity is high, as studies indicate lower rates of cervical cancer screening among obese compared with non-obese women due to embarrassment and perceived weight stigma.22
Also, lack of appropriately sized equipment for examination of obese women in some clinical settings may lead to poor quality cervical cell samples that result in unreliable or invalid test results. Therefore, serum based screening biomarkers are likely to be extremely useful in this group of women. Collectively, these results suggest the need for developing race-specific markers to maximize the usefulness of serum protein based tests as effective screening tools. Despite intense screening in the past decades, higher rates of cervical cancer still persist among some sub-groups of women, including AA women.23
Race-specific screening tests are likely to reduce these disparities.
Although these prediction results using serum biomarkers are promising, there was still a considerable amount of variability in the probabilities of case status that remained unexplained by the statistical models used in this study. A classical screening approach used in this study permitted the detection of the two individual peaks associated with case status. Only interactions between individual peaks and 4 participant characteristics (age, BMI, race, and infection with multiple HR-HPV types) were considered. However, peak by peak interactions (or peak by peak profiles) were not considered in either the classical screening approach or in the alternative LASSO and LAR peak selection procedures, due to the overwhelming number of possibilities. With 95 peaks, restricting the interactions or profiles to only a maximum of four peaks at a time, there are 4465 possible 2-way interactions, 138,415 possible 3-way interactions, and 3,183,545 possible 4-way interactions. Because interactions (or peak by peak profiles) cannot be ruled out from being associated with case status, further examination of these peak by peak profiles using larger sample sizes and data mining techniques is warranted in future studies.