Perkins Mukunyadzi

The p16 (CDKN2a/INK4a) tumor-suppressor gene in head and neck squamous cell carcinoma: A promoter methylation and protein expression study in 100 cases

The p16 (CDKN2a/INK4a) gene is an important tumor-suppressor gene, involved in the p16/cyclin-dependent kinase/retinoblastoma gene pathway of cell cycle control. The p16 protein is considered to be a negative regulator of the pathway. The gene encodes an inhibitor of cyclin-dependent kinases 4 and 6, which regulate the phosphorylation of retinoblastoma gene and the G1 to S phase transition of the cell cycle. In the present study, p16 gene promoter hypermethylation patterns and p16 protein expression were analyzed in 100 consecutive untreated cases of primary head and neck squamous cell carcinoma by methylation-specific PCR and immunohistochemical staining. The p16 promoter hypermethylation and apparent loss of p16 protein expression were detected in 27% and 74% of head and neck squamous cell carcinoma, respectively. By χ2 test, history of alcohol or tobacco use was significantly correlated with the loss of p16 protein expression (P = .005 and.05, respectively). When patient follow-up data were correlated with various clinical and molecular parameters, tumor size and nodal and clinical stage were the strongest prognostic predictors for disease-free survival (tumor recurrence) and for cause-specific and overall survival in patients with head and neck squamous cell carcinoma. Neither p16 promoter hypermethylation nor apparent loss of p16 protein expression appears to be an independent prognostic factor, although loss of p16 protein may be used to predict overall patient survival in early-stage head and neck squamous cell carcinoma.

Induced Expression of Syndecan-1 in the Stroma of Head and Neck Squamous Cell Carcinoma

Syndecan-1 (CD138), a cell-surface heparan sulfate proteoglycan, is involved in cell–cell, cell-matrix interaction and growth factor binding. Loss of expression of syndecan-1 in tumor cells leads to decreased intercellular cohesion, increased potential for tumor invasiveness, and metastatic spread. Furthermore, induction of syndecan-1 expression in the tumor stroma has been postulated to promote tumor angiogenesis via its binding to growth factors such as basic fibroblast growth factor. Although syndecan-1 expression within tumor cells has been investigated in head and neck squamous cell carcinoma, stromal expression has not been studied in detail. We analyzed 38 cases of head and neck squamous cell carcinoma by immunohistochemical staining for syndecan-1 expression within the stroma. The expression of syndecan-1 within tumor cells of various histologic grades of differentiation, squamous cell carcinoma in situ cells, and benign squamous epithelium was also determined. Variable levels of diminished syndecan-1 expression were noted within the dysplastic cells of 9 of 16 (60%) squamous cell carcinoma in situ lesions and in all 38 (100%) invasive squamous cell carcinoma. In general, higher levels of syndecan-1 expression were observed in the well-differentiated tumors, in contrast to significant reduction of expression seen in poorly differentiated tumors. Syndecan-1 expression was observed within the stroma (in fibroblasts) surrounding infiltrating carcinoma cells in 28 of 38 (74%) cases. The intensity of syndecan-1 staining within the stroma showed generally an inverse correlation with the degree of tumor cell differentiation. Syndecan-1 expression was not detected in the stroma beneath normal squamous epithelium or adjacent to areas of squamous cell carcinoma in situ. We conclude that induced expression of syndecan-1 in the stroma surrounding tumor cells of invasive head and neck squamous cell carcinoma is a frequent event. The increased stromal syndecan-1 expression, coupled with its loss from the surface of carcinoma cells, may contribute to tumor cell invasion and the development of metastases.

Tissue effects of salivary gland fine-needle aspiration. Does this procedure preclude accurate histologic diagnosis?

Recent reports have alluded to various tissue effects secondary to fine-needle aspiration (FNA), particularly infarction observed in resected salivary gland masses, precluding accurate histologic diagnosis. Our experience with the use of 25-gauge needles indicates otherwise. We retrospectively reviewed 94 resected salivary gland masses previously sampled by FNA, looking for infarction, hemorrhage, needle track tumor seeding, and fibrosis. We assessed the significance of these complications and their impact on the histologic diagnosis. The median interval from FNA to excision was 25 days. Variable degrees of infarction and hemorrhage were present in 7 cases (7%) and 9 cases (10%), respectively. Infarction ranged from 5% to 80% (average, 20%), while hemorrhage averaged less than 20% of the material on the tissue sections. Significant infarction was present in acinic cell carcinomas (3/7), but histologic diagnosis was not compromised, and tissue alterations were absent. We conclude that FNA of salivary gland lesions using 25-gauge needles is safe and does not significantly alter the histologic diagnosis. The tissue effects observed did not preclude accurate diagnostic interpretation in any case.

The level of syndecan-1 expression is a distinguishing feature in behavior between keratoacanthoma and invasive cutaneous squamous cell carcinoma

Keratoacanthoma (KA) resolves spontaneously but is regarded by some as a variant of squamous cell carcinoma (SCC). However, others consider KA a totally benign entity. Syndecan-1 is one of the heparan sulfate proteoglycans that mediates intercellular and cell to matrix adhesion. Its expression appears to be inversely correlated with tumor aggressiveness and invasiveness. Previous studies have shown decreased levels of syndecan-1 expression in invasive cutaneous SCC, correlating with tumor de-differentiation. However, a similar study has never been done on KA. To investigate syndecan-1 expression in classic KA and compare the results with those of classic invasive SCC, 24 KAs were immunostained for syndecan-1 (CD 138) using the monoclonal antibody B-B4 on formalin-fixed paraffin-embedded tissue. Results were semi-quantitatively scored as either negative or positive (mild, moderate, or strong) and compared with those previously obtained on 23 invasive SCC and in situ lesions. All 24 KAs were positive for syndecan-1 expression. Staining intensity of 18 cases was comparable with that of SCC in situ or adjacent normal epidermis. By comparison, invasive SCC showed significantly diminished staining. Reduced staining in focal areas of cytologic atypia at the base was present in three KAs. Syndecan-1 expression in KA mirrors that of SCC in situ and normal epidermis, providing a molecular basis that biologically KA may be closely related to SCC in situ but distinctively different from invasive SCC.

Expression of tissue factor in prostate cancer correlates with malignant phenotype

Tissue factor (TF), apart from its established role in hemostasis, has been implicated in promoting angiogenesis and metastasis in a wide array of tumors including prostate cancer. Expression of TF was evaluated in freshly-resected prostate specimens obtained from patients with localized (n=9) and androgen ablated (n=6) disease using real-time reverse transcription-polymerase chain reaction and Western blot analysis. TF was detected in all specimens in both stages of the disease. We further analyzed for correlations between TF expression and those of several angiogenic growth factors and their receptors. TF RNA expression correlated significantly with expression of vascular endothelial growth factor-A in these specimens (s=0.621, P=0.013). Eighty-one prostate specimens from patients with benign prostatic hyperplasia (n=27), localized prostate cancer (ES, n=32), and advanced disease (n=22) were also evaluated using immunohistochemistry and findings were correlated with clinical parameters. TF expression was detected on epithelial cells of the malignant glands. Furthermore, its expression levels correlated significantly with Gleason score (s=0.58, P=0.0001) and with the stage of the disease (s=0.441, P=0.0001) in these specimens. These data support the role of TF in angiogenesis and disease progression.

Expression of peroxisome proliferator-activated receptor gamma in salivary duct carcinoma: Immunohistochemical analysis of 15 cases

Salivary duct carcinoma is a rare but highly aggressive tumor of the salivary glands that has poor prognosis. There is no effective cure for this tumor. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor family with diverse biological functions that include mediation of adipocyte differentiation, regulation of the monocyte–macrophage anti-inflammatory activity, and inhibition of tumor cell proliferation. Natural (prostaglandin J2, PG-J2) and synthetic (thiazolinediones) PPARγ ligands with anti-proliferative agonist activity have been identified. The expression of PPARγ has been demonstrated in human colorectal, pancreas, breast, and prostate cancers but has never been explored in salivary duct carcinoma. The aim of our study was to investigate the expression patterns of PPARγ in salivary duct carcinoma, a finding that may provide a mechanism for treating patients with this highly aggressive tumor. Archival formalin-fixed tissues from 15 salivary duct carcinoma cases were analyzed for PPARγ expression by an immunohistochemical staining method using a monoclonal antibody against the PPARγ. The tissue sections were subjected to antigen retrieval by a steam heat method. All the cases of salivary duct carcinoma originated from the parotid gland. Immunohistochemistry analyses showed positive expression of PPARγ in 12 (80%) cases, whereas 3 (20%) were negative. Of the positive cases, 9 (75%), 2 (17%) and 1 (8%) showed strong, moderate, and weak staining, respectively. All staining was cytoplasmic. Nuclear staining was not observed. We conclude that PPARγ is frequently (80%) expressed in salivary duct carcinoma, often at high levels, and is topographically located in the cytoplasm. The high-level expression of PPARγ may provide a potential molecular target for the treatment of salivary duct carcinoma using agonist ligands.

Promoter hypermethylation: an important epigenetic mechanism for hMLH1 gene inactivation in head and neck squamous cell carcinoma.

OBJECTIVE: The hMLH1 gene is one of the mismatch DNA repair genes. Inactivation of the hMLH1 gene has been implicated in the tumorigenesis of many types of human cancers. In most sporadic forms of human cancers, promoter hypermethylation is responsible for hMLH1 gene inactivation. Lack of hMLH1 protein expression has been found in a subset of head and neck squamous cell carcinomas (HNSCCs). The purpose of this study was to investigate whether promoter hypermethylation causes hMLH1 gene inactivation in HNSCCs. STUDY DESIGN: hMLH1 protein expression was determined by immunohistochemical staining in 62 cases, whereas hMLH1 gene promoter methylation was analyzed by methylation-sensitive restriction enzyme digestion, followed by polymerase chain reaction, in 35 cases of HNSCCs. RESULTS: Sixteen (26%) of 62 cases of HNSCCs showed near-complete loss of hMLH1 protein expression on immunohistochemical staining. Twelve (92%) of 13 cases that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 17 (77%) of 22 cases positive for the protein were free of promoter methylation. CONCLUSIONS: Promoter hypermethylation may be an important mechanism for hMLH1 gene inactivation in a subset of HNSCCs.

Diagnostic utility of renal cell carcinoma marker in cytopathology.

Renal cell carcinoma (RCC) not uncommonly presents with metastases and causes diagnostic difficulty to the cytopathologist who is involved in the initial diagnostic workup of tumors with an unknown primary site. RCC marker (RCC Ma) recognizes a human proximal tubule antigen and was shown to have high specificity and relatively low sensitivity in preliminary studies on routinely processed tissue sections. We investigated the diagnostic usefulness of RCC Ma immunohistochemically in fine-needle aspiration (FNA) samples. A total of 34 FNA samples obtained from the following carcinomas were used: 7 RCCs, 5 metastatic RCCs, 4 hepatocellular carcinomas, 2 non–small cell carcinomas of the lung, 3 metastatic non–small cell carcinomas of the lung, 4 invasive ductal carcinomas of the breast, 2 pancreatic ductal adenocarcinomas, 4 metastatic transitional cell carcinomas of the urinary bladder, and 3 metastatic colon carcinomas. Routinely processed cell block sections of FNA specimens were stained with RCC Ma by using routine immunohistochemistry. Presence and distribution of staining were evaluated. Two of 7 (29%) primary and 2 of 5 (40%) metastatic RCCs showed immunoreactivity in less than 50% of carcinoma cells. Staining was focal, cytoplasmic, and granular. Scattered positive cells were present in two of the four hepatocellular carcinomas. All breast, lung, pancreas, colon, and transitional cell carcinomas were negative. RCC antibody has a low sensitivity (33%), most likely because of its focal staining pattern, and a high specificity (91%) in FNA specimens. Immunoreactivity in metastatic carcinoma of an unknown primary site, especially as part of a panel of antibodies, is useful in diagnostic cytopathology. RCC antibody has not been studied in hepatocellular carcinoma, and the significance of positivity observed in some of our cases is unclear.

Concomitant loss of mitochondria and the DNA repair protein hOGG1 in clear cell carcinoma of the kidney.

The kidney is subjected to DNA oxidative damage from reactive oxygen species generated by free radicals and toxic metabolites, leading to formation of DNA base lesions. One such DNA lesion is 8-oxoguanine, which, if not sufficiently removed, is potentially mutagenic because it can cause G:C to T:A transversion in subsequent DNA replication. The human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene on chromosome 3, a region (3p25–26) that shows frequent loss of heterozygosity in clear cell renal cell carcinoma (CC-RCC), encodes for a DNA repair enzyme capable of excision repair of 8-oxoguanine. Of the known isoforms of the hOGG1 enzyme (types Ia, Ib, Ic, Id, and II), only 1, Ia, is found in the nucleus, whereas the rest show a mitochondrial distribution. We investigated, by an immunohistochemical staining method, the expression of hOGG1 protein in 40 cases of CC-RCC, using archival formalin-fixed tissue. To localize the hOGG1 enzyme in normal and tumor tissue, immuno-staining against cytochrome c, a specific mitochondrial enzyme, was also performed. The results showed marked reduction in hOGG1 expression in the majority of tumors, with complete loss of staining seen in 26 (65%) and moderate and weak positive staining present in 9 (22.5%) and 5 (12.5%) of the cases, respectively. Strong hOGG1 protein expression was present in normal tubular epithelium, located in the mitochondria. The results correlated with the expression patterns of cytochrome c. The findings indicate that loss of hOGG1 expression may have a role in development or progression of CC-RCC.

Cyclooxygenase-2 Expression in Prostate Cancer: An Inconsistent Therapeutic Target

Objectives: To determine cyclooxygenase-2 (COX-2) expression patterns in primary prostate tissue of patients undergoing androgen deprivation therapy and in patients with early stage disease. Methods: Gene and protein expression analysis were performed in freshly resected prostate cancer tissue obtained from patients with advanced stage cancer who presented with bladder obstruction Local stage cancer specimens were collected from patients undergoing radical prostatectomy (RP) Expression studies in fresh specimens included quantitative real time polymerase chain reaction, western immunoblotting and immunohistochemistry (IHC). Mean COX-2 gene expressions were compared in both stages. In addition to evaluating protein expression by IHC in fresh specimens, an unrelated archival specimen set of 23 additional clinically matched, advanced and 20 local stage cancer specimens were also analyzed. The pooled (fresh and archival) specimen set ( each) was then used for evaluating protein expression by IHC. Thirty non-c...