Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma

Abstract

Background: Human papillomavirus (HPV) is a causal factor in virtually all cervical and a subset of oropharyngeal squamous cell carcinoma (OP-SCC), whereas its role in laryngeal squamous cell carcinoma (L-SCC) is unclear.

Methods: Formalin-fixed paraffin-embedded (N=154) and deep-frozen tissues (N=55) of 102 L-SCC patients were analysed for the presence of 51 mucosal HPV types. HPV DNA-positive (HPV DNA+) cases were analysed for E6*I mRNA transcripts of all high risk (HR)/probably/possibly (p)HR-HPV identified, and for HPV type 16 (HPV16) viral load. Expression of p16(INK4a), pRb, cyclin D1 and p53 was analysed by immunohistochemistry.

Results: Ninety-two patients were valid in DNA analysis, of which 32 (35%) had at least one HPV DNA+ sample. Among the 29 single infections, 22 (76%) were HPV16, 2 (7%) HPV56 and 1 each (4%) HPV45, HPV53, HPV70, HPV11 and HPV42. Three cases harboured HPV16 with HPV33 (twice) or HPV45. Only 32% of HPV DNA+ findings were reproducible. Among HPV16 DNA+ L-SCC, 2 out of 23 (9%) had high viral loads, 5 out of 25 (21%) expressed E6*I mRNA and 3 out of 21 (14%) showed high p16(INK4a) and low pRb expression (all three HPV16 RNA-positive), immunohistochemical marker combination not identified in any other HPV DNA+ or HPV DNA-negative (HPV DNA-) L-SCC, respectively.

Conclusion: HPV type 16 has a causative role in a small subgroup of L-SCC (<5% in this German hospital series).

Figure 1

Study flow chart. Overall flow chart showing the grouping of L-SCC in relation to HPV DNA, viral load, E6*I mRNA expression and immunohistochemical (IHC) protein pattern defining HPV involvement as extrapolated from CxCa and OP-SCC. HPV16_high—high, HPV16_low—low HPV16 viral load as determined by HPV16 qPCR. ^aFor two samples, HPV16 viral load could not be quantified because of β-globin negativity in qPCR. ^bFor two samples RNA was not analysable, that is, both HPV RNA and ubiquitin C RNA were negative. ^cFor 13 patient cases defined as HPV DNA−, ^done RNA− and ^eone RNA+ were of insufficient quality/quantity for IHC analysis. Combination of p16_high/pRb_low expression was found in 3 HPV DNA+/RNA+ tumours, but in none of the 23 HPV DNA+/RNA− or 47 HPV− tumours. This difference was statistically significant (χ²-test, P=0.002 and P<.0001). These three tumours were defined as HPV-driven L-SCC.

Figure 2

Examples of IHC staining for p16^INK4a, pRb and p53 in CxCa, L-SCC and NOM, respectively. HPV16-driven CxCa (FFPE section, (A) p16_high, (E) pRb_low, (I) p53_low); HPV16-driven L-SCC (FFPE section, (B) p16_high, (F) pRb_low, (J) p53_low); HPV DNA− L-SCC (FFPE core on TMA, (C) p16_low, (G) pRb_high, (K) p53_high); and NOM FFPE core on TMA, (D) p16_normal, (H) pRb_normal, (L) p53_normal.