Prognostic significance of NDRG1 expression in oral and oropharyngeal squamous cell carcinoma

Human N-myc downstream-regulated gene 1 (NDRG1) is a metastasis suppressor gene with several potential functions, including cell differentiation, cell cycle regulation and response to hormones, nickel and stress. The purpose of this study was to investigate the immunoexpression of NDRG1 in oral and oropharyngeal squamous cell carcinomas searching for its role in the clinical course of these tumors. We investigated immunohistochemical expression of NDRG1 protein in 412 tissue microarray cores of tumor samples from 103 patients with oral and oropharyngeal squamous cell carcinomas and in 110 paraffin-embedded surgical margin sections. The results showed NDRG1 up-regulation in 101/103 (98.1 %) tumor samples, but no expression in any normal tissue sample. Western blot assays confirmed the immunohistochemical findings, suggesting that lower levels of NDRG1 are associated with a high mortality rate. NDRG1 overexpression was related to long-term specific survival (HR = 0.38; p = 0.009), whereas the presence of lymph-node metastasis showed the opposite association with survival (HR = 2.45; p = 0.013). Our findings reinforce the idea that NDRG1 plays a metastasis suppressor role in oral and oropharyngeal squamous cell carcinomas and may be a useful marker for these tumors.

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Aims: To evaluate the associations of excision repair cross complementing-group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2-19.7, P = 0.027], which was also high in patients with advanced stage (OR= 5.04, 95% CI: 1.07-23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40-33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07-23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.

Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Abstract Background To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). Patients and Methods We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFβ1, TGFβ2, TGFβ3 in oral SCC. Results Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFβ isoforms, we found that Smad2 mRNA and TGFβ1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFβ1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. Conclusion Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFβ signaling should be better clarified in the future.

Prognostic factors for lymph node metastasis from advanced squamous cell carcinoma of the skin of the trunk and extremities

Abstract Background Squamous cell carcinoma (SCC) of the skin of the trunk and extremities may present lymph node metastasis with difficult disease control and poor survival. The purpose of this study was to identify risk factors for lymph node metastasis and outcome. Patients/Methods Retrospective review of 57 patients with locally advanced SCC of the trunk and extremities was performed and several clinical variables including age, gender, ethnicity, previously injured skin (burns, scars, ulcers and others), patient origin (rural or urban), anatomic site and treatment were studied. Results Fifteen patients presented with previous skin lesions. Thirty-six were classified as T3 tumors and 21 as T4; 46 were N0, and 11, N1. Eleven N0 patients presented lymph node metastasis during follow up. Univariate analysis identified previous skin lesions (ulcers and scars) as risk factor for lymph node metastasis (p = 0.047). Better survival was demonstrated for T3 (p = 0.018) classification. N0 patients who presented lymph node metastasis during follow up (submitted to lymphadenectomy) had similar survival to patients without lymph node recurrence (p = 0.219). Conclusion Local advanced tumors are at risk of lymph node metastasis. Increased risk is associated to previous lesions at tumor site. T4 classification have worse prognosis. Lymph node recurrences in N0 patients, once treated, did not affect survival. For these patients, we propose close follow up and prompt treatment of lymph node metastasis. These results do not support indication for elective lymphadenectomy or sentinel node mapping. Further prospective studies must address this issue.

Immunohistochemical features of a papillary squamous cell carcinoma of the endometrium with transitional cell differentiation

An 84-year-old woman underwent hysterectomy due to a friable endometrial mass infiltrating almost half way through the myometrial wall. The tumor consisted of papillary structures with thin fibrovascular cores covered by several layers of pleomorphic cells. The deeply located neoplastic cells were ovoid with a pale eosinophilic cytoplasm resembling urothelial cells. A diagnosis of papillary squamous cell carcinoma of the endometrium with transitional cell differentiation was made. Although she recovered well after surgery, she died one year later because of disseminated disease. In an attempt to obtain new insights into the physiopathology of this very rare tumor, an immunohistochemical panel with 32 markers was performed. The neoplastic cells were positive for cytokeratin 5, vimentin, p63, p21, VEGF, Ki67, BAG1, and bcl-2. The expression of BAG-1 and bcl-2 may suggest that anti-apoptotic stimuli are preponderant in this neoplasm.

Inflammatory events during murine squamous cell carcinoma development

Background: Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. In SCC, tumour development is accompanied by an immune response that leads to massive tumour infiltration by inflammatory cells, and consequently, local and systemic production of cytokines, chemokines and other mediators. Studies in both humans and animal models indicate that imbalances in these inflammatory mediators are associated with cancer development. Methods: We used a multistage model of SCC to examine the involvement of elastase (ELA), myeloperoxidase (MPO), nitric oxide (NO), cytokines (IL-6, IL-10, IL-13, IL-17, TGF-β and TNF-α), and neutrophils and macrophages in tumour development. ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Results: ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Significantly higher levels of IL-6 and lower levels of IL-10 were detected at 4 weeks following 7,12-Dimethylbenz(a)anthracene (DMBA) treatment. Similar levels of IL-13 were detected in the precancerous microenvironment compared with control tissue. We identified significant increases in the number of GR-1+ neutrophils and F4/80+/GR-1- infiltrating cells in tissues at 4 and 8 weeks following treatment and a higher percentage of tumour-associated macrophages (TAM) expressing both GR-1 and F4/80, an activated phenotype, at 16 weeks. We found a significant correlation between levels of IL-10, IL-17, ELA, and activated TAMs and the lesions. Additionally, neutrophil infiltrate was positively correlated with MPO and NO levels in the lesions. Conclusion: Our results indicate an imbalance of inflammatory mediators in precancerous SCC caused by neutrophils and macrophages and culminating in pro-tumour local tissue alterations.

Isolated splenic metastasis from lung squamous cell carcinoma

Isolated splenic metastasis from lung cancer is a very rare occurrence with only a few reports available. Here, we report the case of a 82-year-old male who underwent a bilobectomy for a lung squamous cell carcinoma and 16 months later developed an isolated splenic metastasis. Additionally, previous reports are reviewed and discussed.

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

Intratumoral Lymphatic Vessel Density in Vulvar Squamous Cell Carcinomas: A Possible Association With Favorable Prognosis

Lymphatic vessels serve as major routes for regional dissemination, and therefore, lymph node status is a key indicator of prognosis. To predict lymph node metastasis, tumor lymphatic density and lymphangiogenesis-related molecules have been studied in various tumor types. To our knowledge, no previous studies have evaluated the role of intratumoral lymphatic vessel density (LVD) in the behavior of vulvar carcinomas. The aim of this study was to analyze intratumoral LVD in relation to patient survival and well-characterized prognostic factors for cancer. Thirty-five patients with vulvar squamous cell carcinoma underwent vulvectomy and dissection of regional lymph nodes. Clinical records were reviewed, in addition to histological grade, peritumoral lymphatic invasion, and depth of infiltration for each case. Tissue microarray paraffin blocks were created, and lymphatic vessels were detected using immunohistochemical staining of podoplanin (D2-40 antibody). Intratumoral LVD was quantified by counting the number of stained vessels. Higher values for intratumoral LVD were associated with low-grade and low-stage tumors, and with tumors without lymphatic invasion and reduced stromal infiltration. In a univariate analysis, high intratumoral LVD was associated with a higher rate of overall survival and a lower rate of lymph node metastasis. Our results suggest that increased intratumoral LVD is associated with favorable prognosis in vulvar squamous carcinomas.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5 gamma 2 chain as well as alpha 3, beta 1 subunits of alpha 3 beta 1 heterodimer and beta 4 subunit of integrin alpha 6 beta 4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of beta 1, beta 4 and alpha 3 integrins, and SISCCs lacked beta 1, beta 4 and alpha 3 integrins in the invasive front. AC cases were negative for the Ln-5 gamma 2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5 gamma 2 chain expression in the invasive front of the tumor. Integrin beta 1, beta 4 and alpha 3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5 gamma 2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. (C) 2012 Elsevier GmbH. All rights reserved.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Amino acids in squamous cell carcinomas and adjacent normal tissues from patients with larynx and oral cavity lesions

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)

Carcinoma de células escamosas em glândula mamária de gata doméstica

Background: In the feline species, 80% to 93% of neoplasias in the mammary gland are malignant, being the majority carcinomas. Among them, there is the mammary squamous cell carcinoma, which amounts to a very rare neoplasm in the domestic cat, with considerable potential for malignancy. This study aimed to report a case of squamous cell mammary carcinoma in the feline species. Case: A female cat, mixed breed, ten years old, presented history of skin lesion. The cat had been spayed two years before, but with previous administration of contraceptives. At the physical examination, it was observed ulcer between the caudal abdominal mammary glands. The occurrence of skin or mammary neoplasia was conceived. The following complementary tests were requested: complete blood count, serum biochemical profi le (renal and hepatic), chest radiographs, abdominal ultrasound, and incisional biopsy of the ulcerated region periphery, followed by classic histopathology. The lesion histopathology was compatible with squamous cell carcinoma of the mammary gland. Due to such a diagnosis, bilateral mastectomy was recommended. The material obtained during the surgical procedure was sent for anatomopathological analysis. Microscopically, surgical margins infi ltration and a regional lymph node were verifi ed. The owner was advised of the need for complementary therapies and medical monitoring of the cat. However, there was no return. It is noteworthy that the animal’s physical and laboratory examinations showed no neoplasia in other regions, being the squamous cell carcinoma of the mammary gland considered primary. Discussion: The malignant mammary neoplasia genesis in feline species, in general, seems to be related to steroid hormones. The ovariectomized females are less likely to develop the disease when compared to intact cats, but there is no protective effect of surgery on those spayed after two years of age regarding the appearance of the neoplasia. Thus, at the time the reported patient was ovariectomized, this effect no longer occurred. The synthetic progestins regularly used to prevent estrus increase by three times the risk of breast carcinomas onset. In humans, there is no clear defi nition of the etiology and pathogenesis of mammary squamous cell carcinoma. However, it has been suggested its association with extreme forms of squamous metaplasia present in pre-existing mammary adenocarcinoma, besides cysts, chronic infl ammations, abscesses and mammary gland adenofi bromas. In a hypothetical way, this etiology could also be related to the feline mammary carcinoma, although, for the case at issue, the exogenous and endogenous hormonal infl uence should not be excluded. It has been reported that mammary squamous cell carcinomas in cats are classifi ed in grades II and III (ie, moderately and poorly differentiated, respectively). Thus, they are considered tumors with more unfavorable prognosis. However, the monitoring of the clinical course, in order to evaluate possible recurrence of the neoplasia and metastases to distant sites, was not possible as the animal under discussion did not return. The squamous cell carcinoma is the most common skin tumor in feline species, despite the primary location in the mammary gland. It is, therefore, important to differentiate squamous cell carcinoma originated in the breast from histological types derived from skin. The description of this special and rare feline mammary carcinoma is important due to its particular characteristics and potential for malignancy.

Photodynamic therapy in actinic cheilitis: clinical and anatomopathological evaluation of 19 patients

BACKGROUND: Actinic cheilitis, a common disease caused by chronic solar exposure and tobacco use, is considered a premalignant lesion with potential to develop into squamous cell carcinoma. Some of the available treatments are invasive, have unaesthetic results and require multiple sessions. OBJECTIVE: To assess the efficacy of a therapy and its cosmetic results. METHODS: In this uncontrolled clinical trial a single photodynamic therapy (PDT) session using 16% methyl-aminolevulinate was performed on actinic cheilitis of the lower lip. A standardized questionnaire was applied in order to assess the clinical improvement from the patients' point of view and the satisfaction with the treatment. Anatomopathological evaluation was performed before the treatment and two months afterwards. RESULTS: The sample was composed of 19 patients (10 males and 9 females), phototypes I to III, with average age of 62 years. Main adverse effects were: sudden pain, scabs, herpes flare-up, and edema. The average score of pain during the procedure was 5,8+2,9. At the final assessment the patients reported improvement of 80% and satisfaction of 85% (p<0.01). Anatomopathological analysis showed a significant decrease of dysplasia (p=0.03) in spite of its presence in 84% of cases. There was no significant correlation between the level of dysplasia with either the subjective impression of clinical improvement (p=0.82) or with the patients' final satisfaction (p=0.96). CONCLUSION: PDT is effective in the treatment of actinic cheilitis, but it is associated with a significant level of pain. Due to the persistence of dysplasia, more research needs to be done in order to define the ideal number of sessions for the effective treatment of these lesions.