Studies on the variability of human papillomavirus (HPV) type 16 are based mostly on DNA sequencing of the viral oncogenes E6 and E7. In order to simplify variant identification, high resolution ...melting (HRM) analysis, which has been shown to distinguish amplicons differing in a single nucleotide, was employed.
Optimised HRM analysis was applied to 255 anogenital samples positive for HPV 16. The E6/E7 region of the HPV 16 genome was amplified using nested PCR with subsequent melting of the amplicons. Samples giving ambiguous melting profiles were melted again in the presence of reference HPV 16 DNA to define and confirm the novel melting profiles.
Out of 219 samples of Croatian origin, 65 reference variants, 119 E6-360G variants and 35 novel melting profiles were found. Samples containing unusual profiles were sequenced for identification. In addition, a subset of samples with two common variants, 23 reference and 34 E6-350G variants, was also sequenced to confirm the findings of high resolution melting. Concordance between the melting analysis and sequencing was 93.9%, while HRM sensitivity and specificity were 92.9% and 94.7%, respectively.
This study showed that HRM analysis can be useful for the identification of HPV 16 variants. The HRM method will be useful in low resource settings as it saves considerable time and resources compared to sequencing.
To evaluate the importance of epidermal growth factor receptor (EGFR) protein overexpression and gene amplification in carcinogenesis of glottic cancer.
In order to evaluate EGFR expression at ...protein and gene level, immunohistochemical (IHC) analysis and fluorescent in situ hybridization (FISH) were performed on tissue microarrays of laryngeal tissue (145 samples) -- 38 samples of normal mucosa, 46 samples of hyperplastic lesions, and 61 samples of cancerous lesions.
Membranous (mEGFR) and cytoplasmic (cEGFR) EGFR expression was significantly different between the analyzed groups. The differences were most striking in the suprabasal-transforming zone. IHC evaluation showed that high and low mEGFR staining contributed to the differentiation of dysplastic lesions, simple hyperplasia, and cancerous tissue, as well as between different degrees of atypia in hyperplastic lesions (P<0.050). EGFR gene amplification was not found in simple and abnormal hyperplastic lesions, but it was confirmed in 2/21 atypical hyperplasias, indicating that gene amplification can facilitate identification of malignant potential in hyperplastic lesions. In cancerous tissue, EGFR gene amplification was found in 8/50 samples. EGFR gene amplification was found in preinvasive cancer in one patient. In invasive carcinomas, gene amplification was not associated with stage or grade. Carcinomas with gene amplification showed significantly higher cEGFR expression (basal layer P=0.003; suprabasal layer P=0.002).
This study confirmed an increase in EGFR protein expression and gene amplification with the increase in biological aggressiveness of glottic lesions. A correlation between EGFR gene amplification and protein expression was established. Gene amplification proved to be an early event in glottic carcinogenesis, indicating its importance for glottic cancer prevention, early detection, and protocol selection.
To elucidate the mechanisms involved in apoptosis of psoriatic keratinocytes by examining the expression of pro-apoptotic (Bak, Bax) and anti-apoptotic (Bcl-2, Bcl-X) Bcl-2 family of proteins, as ...well as the expression of p53 and Ki-67 proteins in normal skin, and uninvolved and involved psoriatic skin.
A total of 90 skin samples (30 cases of involved and uninvolved psoriatic skin and normal skin) were examined immunohistochemically to determine the protein expression of p53, Ki-67, Bcl-2, Bcl-X, Bax, and Bak. The results were quantified and expressed as a percentage of positive keratinocytes.
There was a significant increase in Ki-67 (17.05 vs 3.65; P<0.001), Bcl-X (40.21 vs 13.97; P<0.001), Bak (89.46 vs 73.36; P<0.001), and Bax (50.00 vs 29.25; P<0.001) expression and a decrease in Bcl-2 (3.23 vs 6.25; P=0.008) expression in involved psoriatic skin, as well as an increase in Bcl-X (25.13 vs 13.97; P<0.001) expression in uninvolved psoriatic skin, when compared to normal skin. Samples with higher percentage of Ki-67 positive cells showed a higher percentage of p53 positive cells (correlation coefficient r=0.75 in involved psoriatic samples, P<0.001; r=0.88 in uninvolved psoriatic samples, P<0.001; and r=0.85 in normal skin samples, P<0.001). Samples with higher percentage of p53 positive cells expressed pro-apoptotic Bak and Bax in higher percentage of cells; the correlation coefficients were r=0.74 and r=0.68 in involved psoriatic samples (P<0.001 for both), r=0.75 and r=0.69 in uninvolved psoriatic samples (P<0.001, for both), and r=0.87 and r=0.70 in normal skin samples (P<0.001, for both).
Increased expression of Bcl-X protein was associated with psoriatic epidermal hyperplasia. Strong Bax and Bak expression in involved psoriatic skin are probably inhibitory mechanisms counteracting intensive proliferation.
Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of ...cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells.
Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples.
Statistical analysis showed an association of p65 NF-κB with OPN mRNA (
p
=
0.015) and protein (
p
<
0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (
p
=
0.006 and
p
=
0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.
The goal of this research was to determine the existence of the significant time differences in the identification of the recurrences and neck metastases in the patients surgically treated for the ...oral cavity cancer by comparing three postoperative follow up methods. The study included 286 patients surgically treated for oral and pharyngeal cancer in period from 1991 to 2007 at three different institutions, divided into three groups based on the different postoperative follow up protocol. In this study we were able to show that the period of identification of recurrences and neck metastases was significantly shorter in the group of patients whose follow up included neck ultrasound, along with methods of inspection and palpation of the oral cavity and the neck. In conclusion, implementation of more contemporary methods such as the neck ultrasound is needed along with usual follow up methods, such as inspection and palpation of the oral cavity and the neck. Also, follow up of the patients surgically treated for the oral cavity cancer should be conducted systematically. Ultrasound examination of the neck should be recommended due to its low cost, harmlessness, possible frequent usage, high quality visual imaging and possibility of combination with the fine needle aspiration cytology (FNAC) of the suspicious lymph nodes.
Skin melanoma is by far the most lethal skin cancer, it is unpredictable by nature and presents a severe diagnostic problem. One of the major issues in melanoma diagnostics is to differentiate it ...with confidence from a dysplastic nevus. Thus, the aim of this study was to evaluate hTERT expression on a spectrum of dermal lesions (from normal skin toprimary melanoma) in order to examine its possible role as a diagnostic marker in melanoma diagnostics. In this study we analyzed the expression of hTERT by real-time PCR on 58 freshly obtained biopsy samples (4 samples of normal skin, 12 dermal nevi, 23 dysplastic nevi, 19 primary melanomas). Our results showed slightly greater hTERT expression in dysplastic nevi than melanomas with major data overlap. Considering the given results, hTERT does not seem to be a reliable diagnostic marker for melanoma.
Objective The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of ...human papillomavirus (HPV) deoxyribonucleic acid (DNA). Study Design A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. Results In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the LINEAR ARRAY HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. Conclusion Together with extraction on the easyMAG instrument, the Amplicor HPV test, the LINEAR ARRAY HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.
Contrary to group A beta-hemolytic streptococcus as the most common cause of postinfectious glomerulonephritis (PIGN), Epstein-Barr virus (EBV) is only occasionally associated with acute renal ...involvement. We describe an 11-year-old boy who presented with clinical signs of infective mononucleosis and acute glomerulonephritis characterized by edema, hypertension and dark colored urine with diminished renal function. Serology tests confirmed streptococcal infection and acute EBV infection. Persistently depressed C3 complement and gross hematuria indicated renal biopsy which shows PIGN-type picture and, in addition, acute interstitial nephritis, both conclusive of streptococcal infection. We performed tissue DNA extraction by polymerase chain reaction (PCR) and demonstrated EBV-DNA from the kidney specimen supporting EBV involvement in renal tissue. This is the first reported case of PIGN with serologically-proven streptococcal and simultaneously, acute EBV co-infection. EBV-DNA extraction supported the EBV involvement in renal tissue suggesting that both etiologic agents might have contributed to renal inflammation. Adding serology evaluation for EBV in cases with typical clinical signs of infective mononucleosis and renal symptoms, EBV might be more commonly associated with PIGN than is currently appreciated.
Summary Generally, the cumulative UV exposure correlates with the site-specific incidence of skin cancer but more detailed analysis showed that the frequency of BCC of some facial regions cannot be ...explained with site-specific UV exposure. Apoptosis plays a central role in cancer development due to suppressed process of programmed cell death. Anatomic localization, UV and senescence can influence expression of apoptosis related molecules resulting in advantage of cancer cells development and tumor progression. Anti-tumor immune response, as an important factor in elimination of cancer cells, is also modified by UV radiation and aging, further contributing to cancer progression and its development. Hypothesis : UV radiation, senescence and proliferation intensity influence the expression of apoptosis related molecules in normal keratinocytes. Mutated cells express altered molecules with different degree of antigenicity. We propose that small differences in the expression of pro- and anti-apoptotic molecules in the keratinocytes in association with the local immune reaction (modulated also by UV radiation) determine the type of the non-melanoma skin cancer as well as their frequency (site-specific incidence). In vitro and in vivo models could explain how the modulation of UV can influence the apoptotic system and local immune reaction, while animal experiments could explain different site-specific incidence (frequency) of the same tumor. Better understanding of these mechanisms could lead to development of different therapeutic approaches for early elimination of cancer prone cells or cells in early stages of cancer development. The same model could be used to explain the site-specific localization of other types of cancer (i.e. melanoma) or benign tumors.
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