Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn’s disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own.

Rising prevalence rates of high-risk human papillomaviruses (hrHPV) infection in oropharyngeal carcinoma (up to 80 %) have been reported in North America and Scandinavia. We have analysed 424 German and 163 Brazilian head and neck squamous cell carcinomas (HNSCC) from the oral cavity (OSCC), oropharynx (OPSCC) and hypopharynx (HPSCC) using p16 immunohistochemistry, HPV DNA PCR and sequencing, hrHPV DNA in situ hybridisation (ISH) and hrHPV E6/E7 RNA ISH. In the German series, 52/424 cases (12.3 %) were p16-positive/hrHPV-positive (OSCC 3.8 % [10/265], OPSCC 34.4 % [42/122], HPSCC 0 % [0/37]). In addition, there were 9 cases that were p16-positive/hrHPV-negative (5 OPSCC and 4 OSCC). In the Brazilian series, the overall hrHPV DNA prevalence by PCR was 11.0 % ([18/163]; OSCC 6 % [5/83], OPSCC 15.5 % [11/71], HPSCC 22.2 % [2/9]). Ten of these cases were hrHPV-positive/p16-positive. The remaining 8 hrHPV-positive/p16-negative cases were also negative in both ISH assays. Furthermore, 5 p16-positive/hrHPV-negative cases (2 OPSCC and 3 OSCC) were identified. In both series, HPV16 was by far the most common HPV type detected. We confirm that regardless of geographical origin, the highest hrHPV prevalence in HNSCC is observed in oropharyngeal carcinomas. The proportion of HPV-associated OPSCC was substantially higher in the German cohort than in the Brazilian series (34.4 vs. 15.5 %), and in both groups, the prevalence of hrHPV in OPSCC was much lower than in recent reports from North America and Scandinavia. We suggest, therefore, that it may be possible to define areas with high (e.g. USA, Canada, Scandinavia), intermediate (e.g. Germany) and low (e.g. Brazil) prevalences of HPV infection in OPSCC.

Detecting human papillomavirus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) is clinically relevant, but there is no agreement about the most appropriate methodology. We have studied 64 oropharyngeal carcinomas using p16 immunohistochemistry, HPV DNA in situ hybridisation (ISH) and HPV DNA polymerase chain reaction (PCR) followed by pyrosequencing. We have also evaluated a new assay, RNAscope, designed to detect HPV E6/E7 RNA transcripts. Using a threshold of 70 % labelled tumour cells, 21 cases (32.8 %) were p16 positive. Of these, 19 cases scored positive with at least one HPV detection assay. Sixteen cases were positive by HPV DNA-ISH, and 18 cases were positive using the E6/E7 RNAscope assay. By PCR and pyrosequencing, HPV16 was detected in 15 cases, while one case each harboured HPV33, 35 and 56. All p16-negative cases were negative using these assays. We conclude that p16 expression is a useful surrogate marker for HPV infection in HNSCC with a high negative predictive value and that p16-positive cases should be further evaluated for HPV infection, preferably by PCR followed by type determination. Using RNase digestion experiments, we show that the RNAscope assay is not suitable for the reliable discrimination between E6/E7 RNA transcripts and viral DNA.

Identification of human papillomavirus (HPV) association in head and neck squamous cell carcinoma (HNSCC) is important to identify patients with favorable disease course. However, molecular HPV detection is not universally available. p16 has been proposed as a surrogate marker for HPV infection in HNSCC but, use on its own may result in wrong assignment of some cases to the group of HPV-associated tumors. We have therefore studied 424 HNSCC cases with known p16 and HPV DNA polymerase chain reaction (PCR) status for expression of retinoblastoma protein (pRb) and CyclinD1 by immunohistochemistry using 6-tiered scales (0 to 5) and a combined score (0 to 10). Sixty-one of 424 cases showed overexpression of p16. Of these, 52 cases were HPV DNA-PCR-positive. HPV association strongly correlated with low expression scores for pRb and CyclinD1 individually (scores ≤2) or combined (score sum ≤4), whereas HPV-negative carcinomas showed widely distributed expression scores. High expression scores for pRb or for pRb/CyclinD1 were observed exclusively in HPV DNA-PCR-negative cases. Three of 9 p16-positive/HPV DNA-PCR-negative cases showed high expression of pRb and displayed a high combined pRb/CyclinD1 score. We conclude that HPV-positive HNSCC are characterized by p16 overexpression and low scores for pRb, CyclinD1, and a low combined pRb/CyclinD1 score. High pRb or combined pRb/CyclinD1 scores are strong indicators for HPV-negativity and may justify excluding these cases from further molecular HPV testing. Furthermore p16-positive/HPV DNA-PCR-negative cases show heterogeneous expression of pRb and CyclinD1, including high pRb or high combined pRb/CyclinD1 scores suggesting that at least some of these cases are truly HPV negative.

Macrophages are important factors in the pathogenesis and prognosis of malignant tumors and represent a possible target for therapeutic intervention. Depending on the tumor entity and the prevalent polarization status, macrophages can be associated with a favorable or unfavorable clinical outcome. It is becoming clear, however, that the conventional definitions of M1 polarized tumor inhibitory and M2 polarized tumor promoting macrophages do not adequately reflect the heterogeneity and plasticity of macrophages. Macrophages can support tumor growth through direct interactions with the neoplastic cells, by promoting tissue remodeling and angiogenesis and by inhibiting local immune reactions. To achieve comparability of clinical studies, it will be necessary to reach a consensus nomenclature of macrophage polarization. Furthermore, methods for the quantitative characterization of macrophage populations in malignant tumors will have to be standardized. It is unlikely that single marker immunohistochemistry will be adequate in this context. In any case it is necessary to provide unequivocal information regarding the markers or marker combinations used.