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Abstract

Patients with neck squamous cell carcinoma from unknown primary tumor (NSCCUP) present with lymph node metastases without evidence for a primary tumor. Most patients undergo an aggressive multimodal treatment, which induces severe toxicity. Primary tumors of NSCCUP can be hidden in the oropharynx. Human papillomavirus (HPV) is causally involved in a subgroup of oropharyngeal squamous cell carcinomas (OPSCC) associated with early lymph node metastasis and good prognosis. Detection of markers for HPV transformation in NSCCUP could allow focusing on the oropharynx in primary tumor search and could be of value for choice and extent of treatment. In this retrospective multicenter study analyzing 180 NSCCUP cases from Heidelberg, Treviso and Barcelona, a substantial proportion (16%) was driven by HPV, mainly by HPV16 (89%). The prevalence of HPV-driven NSCCUP varied by geographical region, ranging from 10% in Barcelona to 20% in Heidelberg, and increased with year of diagnosis from 9% during 1988-2004 to 23% during 2005-2014 (p=0.007). Compared to HPV mRNA as gold standard to identify HPV-driven tumors, sensitivity and specificity of HPV DNA, p16INK4a overexpression or the combination of both markers in NSCCUP ranged from 81-100% and 89-100%, respectively, with the lowest concordance for p16INK4a (kappa=0.7) and the highest for the combination (kappa=0.95). HPV seropositivity was a promising diagnostic marker for HPV-driven NSCCUP, since the detection of HPV antibodies in serum from NSCCUP patients had a sensitivity of 91% and specificity of 100% compared to HPV mRNA detected in the metastasis (kappa=0.93). HPV-driven NSCCUP were molecularly different from non-HPV-driven NSCCUP, because they presented with a distinct DNA methylation pattern in five gene promoters and they did not harbor disruptive TP53 mutations, which were common in non-HPV-driven NSCCUP (52% vs. 0%, p=0.0002). Patients with HPV-driven, as well as HPV-seropositive NSCCUP had significantly better overall and progression-free survival rates (p≤0.002). Based on the observed survival benefit, HPV mRNA status assessment should be included in NSCCUP diagnosis. Besides an extended diagnostic work-up of the oropharynx in patients with HPV-driven NSCCUP, de-intensification of radiotherapy concentrating on the oropharynx appears a promising therapeutic strategy, the efficacy of which should be assessed in prospective trials. Analysis of twelve pairs of HPV16-driven OPSCC and corresponding lymph node metastases revealed consistent presence of HPV DNA and mRNA. However, heterogeneity was observed regarding HPV integration status and DNA methylation. Viral-cellular junctions identified in the primary tumor were present in only 43% of corresponding metastases, while new viral-cellular junctions were detected in 14%. Metastases had overall lower methylation levels in the five gene promoters included in the assessed HPV-associated methylation signature compared to primary tumors.