Mass Spectrometric Comparison of HPV-Positive and HPV-Negative Oropharyngeal Cancer

Abstract

Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity (e.g., SLC3A2, cortactin, RBBP4, Numa1), offering explanations for the differential prognosis. The unequal expression of three proteins (SLC3A2, MCM2 and lamin B1) was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively.

Keywords: HPV; head and neck cancer; mass spectrometry; oropharynx.

Figure 1

Experimental workflow and patient survival. (A) 10 HPV-positive and 10 HPV-negative OPSCC were initially chosen for the analysis. Two extracts were excluded from the mass spectrometric analysis because of insufficient amounts of protein. A single HPV-negative sample was further excluded from the random forest analysis because its expression pattern severely impaired group separation. For reasons of comparability, the sample was also excluded from protein identification based on congruence with a comparable previous study (through p-value and fold change) although a control analysis demonstrated only a marginal influence of the sample in this approach. (B) Recurrence-free survival of the 17 patients whose tumors were included in the final analyses.

Figure 2

Proteins differentially expressed in HPV-positive and HPV-negative OPSCC. (A) Heat map depicting the differentially expressed proteins. Only four proteins were found to be expressed at a higher level in HPV-negative OPSCC. (B) Expression levels of proteins (almost) exclusively detected in HPV-positive OPSCC as assessed by LC–MS/MS intensity values. Note that for (A) and in the statistical analyses, random low-intensity values were assigned to proteins not detected in the LC–MS/MS measurement to avoid false-positive significance.

Figure 3

Expression of minichromosome maintenance proteins. All subunits of the MCM complex are, on average, expressed at a higher level in HPV-positive OPSCC, as assessed by LC–MS/MS intensity values. * not identified to be differentially expressed in our analyses.

Figure 4

Nuclear envelope proteins. (A) Expression and association of the expression levels of B-type lamins as assessed by LC–MS/MS intensity values. (B) Similar expression of LMNA in HPV-positive and –negative OPSCC. (C) Significant associations of the expression levels of lamin B1 and LAP2 and lamin B1 and Numa1, respectively.

Figure 5

Cytoskeleton regulators. Depicted are the individual expression levels of cytoskeleton organizing and regulating proteins with differential expression in HPV-positive and HPV-negative OPSCC as assessed by LC–MS/MS intensity values.

Figure 6

Validation of differential expression through IHC-analyses in an OPSCC tissue microarray. (A) Examples of staining intensities classified 0 (no), 1 (weak), 2 (moderate) and 3 (strong). (B) Semiquantitative expression scores (“maximum intensity” * “respective % of cells stained”) confirm significantly different expression of SLC3A2, MCM2 and lamin B1 dependent on p16-status (unpaired, one-sided t-test). Note that the semiquantitative scoring may underestimate expression differences, since factors of 1, 2 and 3 are cautious estimations when appraising the expression differences between weakly, moderately and strongly stained tumors. Depicted are median ± interquartile range.

Figure 7

Patient survival in dependence of the SLC3A2 or LRPPRC expression status. (A) Overall survival in dependence of SLC3A2 and p16 status. OPSCC were categorized as showing strong (3) vs. all other (0,1,2) staining intensities. (B) Overall survival in dependence of lamin B1 and p16 status. OPSCC were categorized as showing a low (0,1) vs. high (2,3) staining intensity.