Expression of substance P and neurokinin‐1‐receptor in laryngeal cancer: linking chronic inflammation to cancer promotion and progression

Sir: Substance P (SP) is an undecapeptide that belongs to the tachykinin family and has been implicated in a myriad of physiological processes. In recent years it has been demonstrated that SP acts through the neurokinin-1 receptor (NK1R) as a mitogen on several human cancer cell lines. It seems that the SP ⁄ NK1R system could play an important role in the development of cancer, as SP may be a universal mitogen in NK1R-expressing tumour cell types. However, in their pioneer work on SP receptors in human primary neoplasms, Henning and coworkers did not study squamous cell carcinoma. Although NK1R has been recently described in the Hep-2 laryngeal carcinoma cell line, this is, to our knowledge, the first study of SP and NK1R expression in laryngeal cancer. We reviewed 114 consecutive cases of laryngeal carcinoma treated surgically from the Department of Otolaryngology. Surgical procedure, site of the primary tumour and TNM staging were recorded. None of the patients had received radiotherapy and ⁄ or chemotherapy prior to surgery. Of the 114 patients, 78 (68%) remained disease free at the end of the follow-up period (average 40 months, range 12–96 months). Tumour differentiation was recorded according to World Health Organization criteria. Cases were also carefully staged to conform to the American Joint Committee on Cancer Staging and End Results Reporting. The presence of adjacent non-neoplastic epithelium was also recorded. Laryngeal non-neoplastic epithelium consisted of pseudostratified squamous epithelium. The histological and immunohistochemical analyses were performed without any knowledge of the clinical stage, treatment or further course of the disease. For the detection of SP and NK1R we used monoclonal antibodies against SP (Sigma, St Louis, MO, USA) and NK1R (Sigma-Aldrich, Madrid, Spain), as previously described. The number of tumour cells showing immunoreactivity (brown staining) and its location were noted. Groups were formed according to the percentage of positive cells (0–24, 25–49, 50–74 and >75%). SP and NK1R expression in non-neoplastic epithelium adjacent to tumour was also recorded in the same way. The presence of basal and suprabasal expression was also recorded. The relationship between SP and NK1R expression and clinical and pathological parameters was determined by v analysis and Fisher’s exact test. All P-values corresponded to two-sided significance testing. The relationship of survival to the expression of proteins was examined using the Kaplan–Meyer method and multivariate survival analysis. SP was immunoreactive in 93 out of 97 samples of non-neoplastic epithelium close to the tumour. Reactivity was found mainly in the basal layers (Figure 1A), with >75% reactive cells. Out of 114 cases, 111 studied showed homogeneous SP expression, with 90 tumours scored as ++++ (Figure 1B). Only three cases were classified as negative. Six were considered +, seven ++ and eight +++. All 17 metastases studied were immunoreactive for SP (Figure1C). All cases showed the same expression as the primary tumour (+++ ⁄ ++++). Out of 97 cases, the basal layers were reactive for NK1R in 72 (Figure 2A). Ninety of 97 samples were positive for NK1R, with >25% of tumour cells reactive (Figure 2B). Reactivity was detected both in the membrane and cytoplasm. We did not find any relationship between expression in primary carcinomas and clinicopathological parameters or survival (data not shown). All cases of metastases (17 out of 17) were found to express NK1R in most tumour cells (+++ ⁄ ++++) (Figure 2C). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such as mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We found both membranous and cytoplasmic SP immunoreactivity, which can be explained on the basis of NK1R internalization. Cytoplasmic reactivity has also been reported in lymphocytes of children who go on to develop acute lymphoblastic leukaemia. The ubiquitous presence of SP in laryngeal tumours may represent a common mechanism of tumour progression. In fact, an intriguing question is why some lymphocytes in normal mucosa express SP, whereas those in metastatic lymph nodes do not. We propose a SP link between chronic mucosal inflammation and cancer, with submucosal lymphocytes secreting SP, as has been described in neoplastic lymphocytes. In addition, it has been demonstrated that the activation of NK1R by SP or SP analogues induces