Manuel F. Fresno

Expression of cyclin D1 and Ki-67 in squamous cell carcinoma of the oral cavity: clinicopathological and prognostic significance.

The prognostic and clinicopathologic significance of cyclin D1 and Ki-67 expressions was studied in oral squamous cell carcinomas. We performed an immunohistochemical study to determine the level of expression of cyclin D1 and Ki-67 labelling index in tumor specimens obtained from 35 patients, of whom 14 died as a result of recurrent disease, and 20 were free of recurrence at the end of the follow-up period. Overexpression of cyclin D1 was significantly associated with regional lymph node metastases (P=0.00005) and advanced tumor stage (P=0.0007). The relative risk for nodal metastases in the cases that overexpressed cyclin D1 was 2.6. The Ki-67 labelling index was significantly (P=0.001) higher in tumors with poor histologic grade of differentiation. Our results showed that cyclin D1 is a useful prognostic factor, and suggested it could be a marker to help determine the appropriate treatment for patients with oral squamous cell carcinoma. Cyclin D1 and Ki-67 overexpression were positively correlated.

Distinctive clinicopathological associations of amplification of the cortactin gene at 11q13 in head and neck squamous cell carcinomas.

Amplification of the 11q13 region is a prevalent genetic alteration in head and neck squamous cell carcinoma (HNSCC). We investigated the clinical significance of cortactin (CTTN) and cyclin D1 (CCND1) amplification in both malignant transformation and tumour progression. CTTN and CCND1 amplification was analysed by differential and real‐time PCR in a prospective series of laryngeal/pharyngeal carcinomas and archival premalignant tissues. CTTN mRNA and protein expression were respectively determined by real‐time RT‐PCR and immunohistochemistry, and correlated with gene status. Molecular alterations were associated with clinicopathological parameters and disease outcome. CTTN and CCND1 amplifications were respectively found in 75 (37%) and 90 (45%) tumours. Both correlated with advanced disease; however, only CTTN amplification was associated with recurrence and reduced disease‐specific survival (p = 0.0022). Strikingly, CTTN amplification differentially influenced survival depending on tumour site (p = 0.0001 larynx versus p = 0.68 pharynx) and was an independent predictor of reduced survival in the larynx (p = 0.04). CCND1 amplification was detected in early tumourigenesis and increased with the severity of dysplasia. Importantly, CTTN amplification was only found in high‐grade dysplasias that progressed to invasive carcinoma. CTTN gene status strongly correlated with mRNA and protein expression. Furthermore, CTTN overexpression correlated significantly with reduced disease‐specific survival (p = 0.018). Taken together, these data indicate that CTTN may serve as a valuable biomarker to identify patients with laryngeal tumours at high risk of recurrence and poor outcome. Copyright

Localization of metallothionein in breast carcinomas. An immunohistochemical study

Metallothionein (MT) is a cysteine-rich, low molecular weight protein that binds zinc, copper, and cadmium. It is present in a number of normal cells including hepatocytes particularly during fetal and early postnatal life. It has been suggested that developmental profile of MT is similar to other oncofetal gene products and hence, it could be used as a marker for aggressive tumour behaviour. In order to test that hypothesis, we used a monoclonal antibody to MT and immunohistochemically evaluated formalin-fixed, paraffin-embedded tissues from 79 breast carcinomas. In non-neoplastic breast tissue, a strong nuclear and cytoplasmic staining was observed in myoepithelial cells. Positive staining for MT was present in 35 (44%) of breast carcinomas. In most positive cases, nuclear, or both nuclear and cytoplasmic staining was seen. All positive tumours were invasive ductal carcinomas, including a medullary and a metaplastic carcinoma. None of the mucinous, lobular, or intraductal papillary carcinomas reacted for MT. A statistically significant association was found between MT immunostaining and histological grade (P<0.01) as well as with nuclear grade (P<0.01). We also observed an inverse relationship between MT staining and oestrogen receptor content of tumours (P<0.01). Similarly, a statistically significant association was found between moderate and strong MT immunostainig and decreased overall survival and shorter disease-free survival (P<0.01). MT immunostaining was also predective of a worse prognosis in the subgroup of lymph node negative (P<0.001) and oestrogen receptor negative patients (P<0.01). No statistically significant association was found between MT staining and size of tumour or the presence of lymph node metastasis. We conclude that MT staining may be a useful marker of less differentiated and more aggressive carcinomas of the breast.

Initial thermal heat hypoalgesia and delayed hyperalgesia in a murine model of bone cancer pain.

The recent development of rodent models of bone cancer pain has started to provide the basis for demonstrating the particular neurochemical and behavioral entity of cancer pain. Behaviourally, both spontaneous pain and hyperalgesia related to mechanical, but not thermal, noxious stimuli have been described in cancer-bearing animals. We have carried out a histological and behavioural study focused on the reactivity to noxious heat in C3H/HeJ mice receiving an intratibial injection of 10(5) NCTC 2472 cells. These cells, able to induce an osteosarcoma, break through bone into soft tissues 2 weeks after cell inoculation, producing a macroscopical increase of the limb size from the fourth week. Thermal reactivity is diminished during the first 2 weeks after cell implantation, this hypoalgesia being reversed by the administration of naloxone (10 mg/kg). In contrast, during the fourth and fifth weeks after NCTC 2472 cell implantation, an increased nociceptive heat reactivity, instead of hypoalgesia, was obtained. This thermal hyperalgesia was prevented by the systemic administration of morphine (15 mg/kg). Throughout the whole period studied, mice showed signs of spontaneous pain behaviour that reached its maximum 3 weeks after inoculation. In conclusion, we show that the presence of thermal heat hyperalgesia is preceded by an initial opioid-mediated hypoalgesic state, in this murine model of bone cancer pain.

Podoplanin expression in the development and progression of laryngeal squamous cell carcinomas.

BackgroundPodoplanin expression is attracting interest as a marker for cancer diagnosis and prognosis. We therefore investigated the expression pattern and clinical significance of podoplanin during the development and progression of laryngeal carcinomas.ResultsPodoplanin expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal premalignancies and 53 patients with laryngeal squamous cell carcinomas. We found podoplanin expression extending from the basal to the suprabasal layer of the epithelium in 37 (44%) of 84 dysplastic lesions, whereas normal epithelium showed negligible expression. Patients carrying podoplanin-positive lesions had a higher laryngeal cancer incidence than those with negative expression reaching borderline statistical significance (51% versus 30%, P = 0.071). Podoplanin expression in laryngeal carcinomas exhibited two distinct patterns. 20 (38%) cases showed diffuse expression in most tumour cells and 33 (62%) focal expression at the proliferating periphery of tumour nests. High podoplanin expression was inversely correlated with T classification (P = 0.033), disease stage (P = 0.006), and pathological grade (P = 0.04). There was a trend, although not significant, towards reduced disease-specific survival for patients with low podoplanin levels (P = 0.31) and diffuse expression pattern (P = 0.08).ConclusionsPodoplanin expression increases in the early stages of laryngeal tumourigenesis and it seems to be associated with a higher laryngeal cancer risk. Podoplanin expression in laryngeal squamous cell carcinomas, however, diminishes during tumour progression. Taken together, these data support a role for podoplanin expression in the initiation but not in the progression of laryngeal cancers.

A molecular risk score based on 4 functional pathways for advanced classical Hodgkin lymphoma

Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P < .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

Annexin A2 localizes to the basal epithelial layer and is down-regulated in dysplasia and head and neck squamous cell carcinoma☆

Annexin A2 is a highly expressed gene with important roles in cell membrane physiology and is frequently dysregulated in cancer. The objective of this study was to determine the pattern of expression and prognostic significance of annexin A2 protein in head and neck squamous cell carcinoma. We assessed both quantitative changes and qualitative distribution of annexin A2 mRNA and protein expression in normal and diseased tissues by immunohistochemistry, immunofluorescence and in situ hybridization. Annexin A2 expression was confined to the basal and suprabasal cells of normal epithelium and the protein cellular location was consistently observed at the cell membrane. Expression levels correlated with histopathological grade, showing significant suppression in moderately and poorly differentiated tumours. We conclude that annexin A2 exhibits a characteristic pattern of expression, distinct from other annexins and suggestive of a cell-specific functional role. The marked reduction of annexin A2 in poorly differentiated tumours and dysplastic tissue is expected to result in a loss of function aimed at the coordination of membrane signalling enzyme complexes, actin polymerization and extracellular matrix proteolysis. The phenotypic consequences may become manifest in an alteration of epithelial tissue growth and remodelling with secondary influence on tumour development, progression and metastasis.

Complete spontaneous regression of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a very aggressive primary cutaneous neoplasm most often occurring on the head and neck of the elderly. Spontaneous regression of MCC was first described in 1986. A 79-year-old woman with MCC on the right cheek underwent spontaneous regression of the malignancy, documented by photographic follow-up, computed tomography, and histologic studies. A review of the literature is presented. Complete clinical and histologic regression of MCC was observed in the present case. Although the literature documents 11 similar cases, only 6 can be regarded as complete spontaneous regressions following exclusive performance of a biopsy (primary complete spontaneous regression). Primary complete spontaneous regression of MCC is infrequent, and most case reports describe this phenomenon in women with MCC on the cheek. The reasons underlying regression are unknown.

Variability in the Degree of Expression of Phosphorylated IκBα in Chronic Lymphocytic Leukemia Cases With Nodal Involvement

Purpose: Based on previous preliminary observations, we hypothesize that the molecular and clinical variability of chronic lymphocytic leukemia (CLL) reflects differences in the degree of nuclear factor (NF)-κB activation, as determined by the expression of phosphorylated IκBα (p-IκBα). Experimental Design: The expression profile (mRNA and protein expression) was analyzed with the Centro Nacional de Investigaciones Oncológicas Oncochip, a cDNA microarray containing 6386 cancer-related genes, and a tissue microarray (TMA). The results were correlated with the IgVH mutational status, ZAP-70 expression, cytogenetic alterations, and clinical outcome. Results: We found correlations between the presence of p-IκBα, a surrogate marker of NF-κB activation, and changes in the expression profile (mRNA and protein expression) and clinical outcome in a series of CLL cases with lymph node involvement. Activation of NF-κB, as determined by the expression of p-IκBα, was associated with the expression of a set of genes comprising key genes involved in the control of B-cell receptor signaling, signal transduction, and apoptosis, including SYK, LYN, BCL2, CCR7, BTK, PIK3CD, and others. Cases with increased expression of p-IκBα showed longer overall survival than cases with lower expression. A Cox regression model was derived to estimate some parameters of prognostic interest: IgVH mutational status, ZAP-70, and p-IκBα expression. The multivariate analysis disclosed p-IκBα and ZAP-70 expression as independent prognostic factors of survival. Conclusions: A variable degree of activation of NF-κB, as determined by the expression of p-IκBα, is an identifiable event in CLL, and is correlated with changes in the expression profile and overall survival.

Large B-cell lymphoma presenting in the spleen: Identification of different clinicopathologic conditions

Only a few series of splenic large B-cell lymphoma have been previously reported, including limited immunophenotypic studies and clinical data. Here we review the clinical data, morphology, and immunophenotype of series of 33 cases of large B-cell lymphoma presenting in the spleen. Three main groups of tumors are identified. Group A was characterized by macronodular tumors (20 cases), with predominantly stage I disease and a favorable clinical outcome. All cases were bcl6 positive. Group B was characterized by a micronodular pattern (nine cases), including a subset with T-cell-rich B-cell lymphoma features. Most of the patients in this group were diagnosed at advanced clinical stages and died of the disease. All cases were bcl6 positive. Group C was characterized by diffuse red pulp infiltration (four cases) and advanced clinical stages and showed an aggressive behavior. All but one case were bcl6 positive. The results of this series define a characteristic type of large B-cell lymphoma presenting in the spleen as a tumoral mass, associated with a relatively favorable clinical course. Additionally, they provide evidence that clinical presentation as a tumor confined to the spleen and the hilar lymph nodes is associated with lower aggressivity.