Francisco Esteban

HLA class I gene expression on human primary tumours and autologous metastases: demonstration of selective losses of HLA antigens on colorectal, gastric and laryngeal carcinomas.

The expression of HLA class I antigens was studied in 99 primary tumour (colorectal, gastric and laryngeal carcinomas) and 57 autologous metastases using immunohistological techniques and monoclonal antibodies against class I monomorphic determinants, HLA-B isotypic determinants and HLA polymorphic determinants. Fourteen per cent of colorectal, 9.6% of gastric and 20% of laryngeal carcinomas completely lacked class I molecules. Selective losses of HLA-B antigens were also detected in 8.8, 3.4 and 5.8% of these tumours respectively. Taking into account complete and selective loss of HLA-B the average alteration in the class I molecules expression totalled 21%. The comparison of class I expression between primary tumours and autologous metastases showed differences in 24% of the patients. These differences consisted mainly in a decrease of class I expression by metastases. Nevertheless, four types of divergence were detected in laryngeal carcinomas, namely: +/-, +/+, -/+, -/-. In addition, a clear correlation between degree of differentiation and class I expression was observed in laryngeal tumours. Finally, when class I gene RFLPs were compared with DNA from 15 tumours and autologous normal mucosa or peripheral lymphocytes, no differences were detected between these samples.

Importance of tumour thickness measurement in prognosis of tongue cancer

Eighty-one patients who underwent surgery for cancer of the tongue were retrospectively studied to evaluate the influence on survival of some clinical and pathologic parameters. These parameters and data on the patients current status were gathered by the study of tissue sections, using haematoxylin-eosin staining, and from medical records. The 5-year survival rate was 68.5%. Univariate analysis showed that the parameters influencing survival were: T (P<0.01), pathologic T (P<0.01), N (P<0.05), pathologic N (P<0.05), extracapsular nodal spread (P<0.05), locoregional recurrence (P<0.01), and tumour thickness (P<0.05). Multivariate analysis showed that tumour thickness had the greatest influence on survival. Patients with tumour thickness of < or = 3 mm had a 5-year survival of 85.7%, significantly greater (P<0.05) than the rates of 58.3 and 57% for patients with tumour thickness of 4-7 mm and >7 mm, respectively. Wider studies are required to unify criteria for the measurement of this important prognostic parameter.

A role for substance P in cancer promotion and progression: a mechanism to counteract intracellular death signals following oncogene activation or DNA damage.

In the present review we discuss a central role for substance P (SP) in carcinogenesis. We suggest that one mechanism to induce mitogenesis of tumor cells is the activation of neurokinin-1 receptor (NK1R) through SP, linking cancer promotion and progression to a neurokinin-mediated environment. After reviewing the role of both SP and its receptor NK1R in normal and neoplastic cells we propose the use of neurokinin-1 receptor antagonists as a novel and promising approach for treating patients with cancer.

Lack of MHC class I antigens and tumour aggressiveness of the squamous cell carcinoma of the larynx

A series of 60 primary laryngeal and hypopharyngeal tumours, 24 lymph node metastases and normal tissue were evaluated in frozen sections for the expression of MHC class I antigens, using monoclonal antibodies and the APAAP technique. We found 13 tumours presenting total HLA-ABC loss, five with selective loss of HLA-A antigens and one with absence of HLA-B antigens. These losses were statistically associated with clinical and pathological parameters, such as T stage, degree of differentiation, scores according to the Jakobsson and Glanz grading systems and degree of leukocytic infiltration. Our results lead us to the following conclusions: (a) HLA class I losses were found in a group of tumours showing greater aggressiveness and worse prognosis; (b) these alterations in expression were not associated with an increased metastatic potential. Thus, the absence of HLA molecules in laryngeal tumours is related to greater local aggressiveness, and the loss of class I antigens seems to constitute an adaptive tumour mechanism to avoid the different anatomical and immunological barriers within the larynx.

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

Analysis of Ki-67 expression in oral squamous cell carcinoma: Why Ki-67 is not a prognostic indicator

OBJECTIVES To analyze the prognostic value of Ki-67 in oral cancer and its relationship with Ki-67 expression in precancerous epithelium. MATERIAL AND METHODS We studied 79 tumors from 65 patients. Immunohistochemistry study with Mib-1 monoclonal antibody was used to detect Ki-67 expression in tumor tissue and adjacent non-tumor tissue. The influence of different variables on survival was studied with univariate and multivariate analyses. RESULTS Ki-67 expression was significantly higher in well-differentiated versus poorly-differentiated carcinomas. The survival time of these patients was affected by the clinical presentation, T, N, stage, and surgical treatment. Ki-67 expression had no impact on survival. An association was found between the parabasal expression of Ki-67 in adjacent non-tumor epithelium and Ki-67 expression in the tumor. CONCLUSIONS Ki-67 lacks prognostic value, probably because it is a marker of the total fraction of proliferating cells, corresponding not only to cells in constant proliferation but also to proliferating cells destined for terminal differentiation.

Phenotypic expression of histocompatibility antigens in human primary tumours and metastases

HLA class I and II expression was studied on 244 (177 primary and 67 metastatic) solid human tumours of different origin. Alkaline immunophosphatase (APAAP) and immunoperoxidase were used on cryostatic sections to stain MHC antigens. Monomorphic MoAbs were used against class I heavy chain, β2-microglobulin, DR, DQ and DP molecules.Class I expression was homogeneous on colon, melanoma and epidermoidal primitive tumours. Loss of HLA class I antigens was more frequent on basal cell carcinomas and sarcomas and was related to tumour differentiation on larynx carcinoma. Class I expression was heterogeneous on breast, larynx and stomach primitive neoplasias. Class I negative tumours were more frequent on metastatic than on primitive melanomas. Divergence of class I between primary tumours and autologous metastases was observed on melanomas, larynx and colorectal carcinomas.Class II expression was heterogeneous on all tumours and in a large number of cases was associated with high intensity of leukocytic infiltrate. HLA-DR expression was higher than HLA-DP and HLA-DQ (DR>DP>DQ) and was related to tumour progression. Four human tumour cell lines were modulated with recombinant interferon-γ for HLA class I and II antigens. Different HLA profiles were obtained: increased class I and II expression, increased class II or a low response.Finally, class I genes from 22 tumours were compared with autologous normal cells by Southern blot analysis: 12 tumours were class I positive and 10 negative. No clear differences in RFLP were observed that could be associated with class I rearrangement. The results are discussed in relation to the role that histocompatibility antigens may play in tumour progression and invasiveness.

Cell proliferation associated with actions of the substance P/NK-1 receptor complex in keratocystic odontogenic tumours

The expression of substance P (SP) and its NK-1 receptor (NK-1R) in keratocystic odontogenic tumours (KOTs) was studied to determine whether the intrinsic growth potential of these lesions is related to a cell proliferation stimulus mediated by the SP/NK-1R complex. A total of 65 tissue samples of solitary non-recurrent KOTs, solitary recurrent KOTs, KOTs associated with nevoid basal cell carcinoma syndrome (NBCCS) and KOTs with chondroid wall were studied by immunohistochemistry, using anti-SP, anti-NK-1R and anti-Ki-67 monoclonal antibodies. Expression of these markers was analysed in infiltrating lymphocytes, in fibrous capsule, and in membrane, cytoplasm and nucleus of epithelial cells. SP expression in infiltrating lymphocytes was significantly associated with SP in fibrous capsule and epithelial cells. KOTs associated with NBCCS showed a significantly higher SP expression in all tissues and cell compartments compared with other KOT types. Finally, SP expression in epithelial cells was associated with positive Ki-67 expression in dysplastic epithelium. This first published report on SP and NK-1R expressions in KOTs demonstrates that actions of the SP/NK-1R complex may constitute a mechanism to stimulate epithelial cell proliferation in KOT. This pathway may be of special relevance in the multiple KOTs associated with NBCCS.

Ki‐67 expression in non‐tumour epithelium adjacent to oral cancer as risk marker for multiple oral tumours

OBJECTIVE The aim of this study was to determine whether the differential assessment of epithelial proliferation is useful to diagnose premalignant fields and assess the risk of multiple tumours. MATERIAL AND METHODS We analysed 83 oral carcinomas with associated non-tumour epithelium classified as distant or close according to its distance (> or <1 cm) from the invasion point, and as squamous hyperplasia, mild, moderate, severe dysplasia or carcinoma in situ. Twenty-five healthy oral mucosa samples were used as controls. An immunohistochemical technique was applied using Mib-1. Ki-67 in premalignant epithelium was assessed in basal layer, parabasal layer, medium and upper third. RESULTS Parabasal expression was significantly higher or showed a tendency to be higher in close and distant epithelia with any histological grade than in the controls. Parabasal Ki-67 significantly differed between distant epithelia associated with multiple vs single tumours (P < 0.001) and between distant epithelia associated with multiple tumours vs controls (P < 0.001). This difference was not observed between distant epithelia associated with single tumours and controls (P = 0.175). The cut-off point that differentiated epithelia associated with multiple tumours was >50% of Ki-67 + parabasal cells in distant epithelia, which yielded 0.88 sensitivity and 0.79 specificity. CONCLUSIONS The concept of a precancerous field may be linked to an increase in the proliferative activity of parabasal cells.

Ectopic pituitary adenoma in the sphenoid causing Nelson's syndrome

An ectopic functioning pituitary in the sphenoid is an extremely rare occurrence, and even rarer is pituitary adenoma causing symptoms of Nelsons syndrome. A case is presented of a young female diagnosed and treated in our clinic. The only functioning hypophyseal tissue was detected inside the sphenoid, as the pituitary gland had been radiated because of Cushings syndrome 10 years before and imaging studies revealed an empty sella.