Real-Time PCR Method as Diagnostic Tool for Detection of Periodontal Pathogens in Patients with Periodontitis.

The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent® semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for A. actinomycetemcomitans was 97.5%, for P. gingivalis 96.88%, and for P. intermedia 95.24%. The sensitivity for Tannerella forsythia and T. denticola was 100%. The Spearman correlation factor of two different measurements was 0.976 for A. actinomycetemcomitans, 0.967 for P. gingivalis, 0.949 for P. intermedia, 0.966 for Tannerella forsythia, and 0.917 for T. denticola. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.

Refining the Global Phylogeny of Mitochondrial N1a, X, and HV2 Haplogroups Based on Rare Mitogenomes from Croatian Isolates.

Mitochondrial DNA (mtDNA) has been used for decades as a predominant tool in population genetics and as a valuable addition to forensic genetic research, owing to its unique maternal inheritance pattern that enables the tracing of individuals along the maternal lineage across numerous generations. The dynamic interplay between evolutionary forces, primarily genetic drift, bottlenecks, and the founder effect, can exert significant influence on genetic profiles. Consequently, the Adriatic islands have accumulated a subset of lineages that exhibits remarkable absence or rarity within other European populations. This distinctive genetic composition underscores the islands' potential as a significant resource in phylogenetic research, with implications reaching beyond regional boundaries to contribute to a global understanding. In the initial attempt to expand the mitochondrial forensic database of the Croatian population with haplotypes from small isolated communities, we sequenced mitogenomes of rare haplogroups from different Croatian island and mainland populations using next-generation sequencing (NGS). In the next step and based on the obtained results, we refined the global phylogeny of haplogroup N1a, HV2, and X by analyzing rare haplotypes, which are absent from the current phylogenetic tree. The trees were based on 16 novel and 52 previously published samples, revealing completely novel branches in the X and HV2 haplogroups and a new European cluster in the ancestral N1a variant, previously believed to be an exclusively African-Asian haplogroup. The research emphasizes the importance of investigating geographically isolated populations and their unique characteristics within a global context.

Inherited Thrombophilia and Risk of Thrombosis in Children with Cancer: a Single-center Experience.

OBJECTIVES: Thrombosis is an increasingly recognized complication of childhood malignancy and its treatment. The incidence and etiology of pediatric cancer-related thrombosis is still not well understood. The aim of this study was to evaluate the prevalence of common prothrombotic genetic conditions in children with cancer, the frequency of thrombosis, and the role of inherited thrombophilia in the development of thrombosis in a pediatric oncology population. PATIENTS AND METHODS: Forty-seven children (36 treated for hematological malignancies and 11 for solid tumors) with a median age of 8.8. years (range 0.4 - 19.3 years) were included in the study. Genetic polymorphisms of Factor V Leiden (G1691A), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were determined by real-time polymerase chain reaction-based DNA analysis. RESULTS: Four (8.5%) patients were heterozygous for Factor V Leiden, 3 (6.4%) were heterozygous for prothrombin G20210A mutation, and 3 (6.4%) were homozygous for MTHFR C677T mutation. All patients had implanted central venous catheters. Four (8.5%) children had documented thrombosis, three of which were in the upper venous system. Two of the four patients with thrombosis had Factor V Leiden heterozygosity. CONCLUSIONS: Thrombosis is an important complication of childhood cancer. The risk of thrombosis may be increased in patients with Factor V Leiden. In the absence of consensus guidelines, our results support the recommendation for thrombophilia screening in children with cancer.

PD-L1 expression is regulated by microphthalmia-associated transcription factor (MITF) in nodular melanoma.

Malignant melanoma (MM) is known to avoid the host's immune response. Studies on in vitro melanoma cell lines link the microphthalmia-associated transcription factor (MITF) with the regulation of the PD-L1 expression. It seems that MITF affects the activation of the gene responsible for PD-L1 protein expression. Several proteins, including Bcl-2 and Cyclin D1, play major roles in malignant melanoma cell cycle regulation and survival. Our study aims to assess the relationship between MITF, Bcl-2, and cyclin D1 protein expression and the expression of the PD-L1 molecule. Additionally, we examined the association of BRAF mutation, MITF, and CCND1 gene amplification with PD-L1 protein expression. We performed immunohistochemical staining on fifty-two tumour samples from patients diagnosed with nodular melanoma (NM). BRAF V600 mutation, MITF, and CCND1 gene amplification analyses were analyzed by the Sanger sequencing and QRT-PCR methods, respectively. Statistical analyses confirmed the significant inverse correlation between cyclin D1 and PD-L1 expression (p = 0.001) and correlation between PD-L1 and MITF protein expression (p = 0.023). We found a statistically significant inverse correlation between the present MITF gene amplification and PD-L1 (p = 0.007) and MITF protein expression (p = 3.4 ×10-6), respectively. Our study, performed on clinical NM materials, supports the in vitro study findings providing a rationale for the potential MITF-dependent regulation of PD-L1 expression in malignant melanoma.

Matrix metalloproteinases MMP-1, MMP-2, and MMP-13 are overexpressed in primary nodular melanoma.

BACKGROUND: The spread and invasion of malignant melanoma cells involve degradation and reorganization of the extracellular matrix by the activation of several matrix metalloproteinases (MMPs). This study analyzed the expression of MMP-1, MMP-2, and MMP-13 proteins in primary nodular melanoma (NM) and dysplastic nevi (DN) as a significant risk factor for melanoma development. The secondary goal was to analyze the correlation of MMPs protein expression in NM with tumor invasion, BRAF V600 mutation status, and overall survival. METHODS: Immunohistochemistry for MMP-1, MMP-2, and MMP-13 was performed on nodular melanoma (n = 52) and dysplastic nevi (n = 28) on tissue microarray (TMA). BRAF V600 mutation analysis on NM samples was performed by the Sanger sequencing method. RESULTS: A high level of MMPs expression in NM samples (>30%) compared with DN (<8%) was statistically significant (P < 0.001). BRAF V600 mutations were detected in 15 of 39 (38.5%) NM samples. This study revealed an interesting finding that MMP-1 and MMP-13 protein expression in the BRAF V600 mutated melanomas were significantly lower than in the BRAF V600 wild type (P < 0.05). CONCLUSION: Cox analysis revealed that Clark categories, Breslow thickness, and MMP-1 high protein expression are predictive factors for shorter overall survival (P < 0.05).

Ungual fibroma in 12-year-old boy with hypomelanotic macules, intellectual disability and attention deficit hyperactivity disorder-possible tuberous sclerosis.

OBJECTIVE: To report a case of a 12-year-old boy with intellectual disability and attention deficit hyperactivity disorder, who came to surgery for an examination due to a minor bulge on the left thumb, which had been growing for the previous month. His mother denied any trauma. METHODS: After the removal of the clinically ambiguous bulge and a pathohistological confirmation that it was a periungual fibroma, complete patient analysis was performed due to the presence of hypomelanotic macules and a suspected tuberous sclerosis. RESULTS: Considering the presence of hypomelanotic macules, as one of the main criteria, possible TS diagnosis was set. CONCLUSION: Early detection of the symptoms of TS enables a timely provision of protocols for further patient monitoring, which affects the patient's morbidity and mortality.

Genetic heritage of Croatians in the Southeastern European gene pool-Y chromosome analysis of the Croatian continental and Island population.

OBJECTIVES: The research objective of this study is to enlarge and deepen the Y chromosome research on the Croatian population and enable additional insights into the population diversity and historic events that shaped the current genetic landscape of Croatia and Southeastern Europe (SEE). MATERIALS AND METHODS: A high-resolution phylogenetic and phylogeographic analysis of 66 biallelic (SNPs) and 17 microsatellite (STRs) markers of the Y chromosome was performed using 720 Croatian samples. The obtained results were placed in a wider European context by comparison with ∼4450 samples from a number of other European populations. RESULTS: A high diversity of haplogroups was observed in the overall Croatian sample, and all typical European Y chromosome haplogroups with corresponding clinal patterns were observed. Three distinct genetic signals were identifiable in the Croatian paternal gene pool - I2a1b-M423, R1a1a1b1a*-M558, and E1b1b1a1b1a-V13 haplogroups. DISCUSSION: The analyses of the dominant and autochthonous I2a1b-M423 lineage (>30%) suggest that SEE had a significant role in the Upper Paleolithic, the R1a1a1b1a*-M558 lineage (19%) represents a signal from present day Slavic populations of Central Europe in the Croatian population, and the phylogeography of the E1b1b1a1b1a-V13 clade (around 9%) implies cultural diffusion of agriculture into Europe via the Balkan Peninsula. Am. J. Hum. Biol., 2016. © 2016 Wiley Periodicals, Inc. Am. J. Hum. Biol. 28:837-845, 2016. © 2016Wiley Periodicals, Inc.

Cellular Blue Nevus Diagnosed following Excision of Melanoma: A Challenge in Diagnosis.

A case of a 41-year-old woman with a history of nodular melanoma (NM), associated with an indurated dome-shaped blue-black nodule with a diameter of 1.2 cm in the gluteal region, is presented. Clinical diagnosis of the lesion, present from birth, was blue nevus. Recently, the nodule has been showing a mild enlargement and thus complete resection was performed. Histological analysis revealed a pigmented lesion with an expansive pattern of extension into the dermis and the subcutaneous adipose tissue. The lesion displayed an alveolar pattern as well as a pigmented dendritic cell pattern. The histology was consistent with cellular blue nevus (CBN); however, the history of NM which was excised one year earlier, as well as the clinical information about the slow growing lesion, included a differential diagnosis of CBN, borderline melanocytic tumor, and malignant blue nevus. Additional immunohistochemical (HMB-45, p16, and Ki-67) and molecular (BRAF V600E mutation) analyses were performed on both lesions: the CBN-like and the previously excised NM. Along with lesion history and histological analyses, p16 staining and BRAF were useful diagnostic tools for confirming the benign nature of CBN in this case.

EGFR and cyclin D1 in nodular melanoma: correlation with pathohistological parameters and overall survival.

Considering that nodular melanoma (NM) has the potential to show an early distant metastasis, there is an urgent need for the discovery and evaluation of new diagnostic and prognostic biomarkers. We aimed to investigate the protein expression of membrane and nuclear epidermal growth factor receptor (EGFR), cyclin D1, and the corresponding gene status in NM samples and correlate the results obtained with clinicopathological parameters and overall survival of patients. Immunohistochemical and fluorescence in-situ hybridization analyses were carried out on tissue microarrays constructed from 110 NM samples, 30 compound nevi, and 38 dysplastic nevi. NM samples showed 24% strong cyclin D1 and 37% strong Ki67 protein expression compared with 3 and 0% strong cyclin D1 and Ki67 expression in the control group. Membrane EGFR expression was detected in 50% of NM cases, whereas EGFR gene amplification was detected in only 4% of NM cases. Multiple NM samples presented simultaneous membrane and nuclear EGFR expression. We found a negative correlation between tumor thickness and membrane EGFR expression. It was also observed that membrane EGFR 3+ NM samples presented ulceration significantly more often than membrane EGFR-negative (0) NM samples. In univariate analysis, carried out on 44 patients with follow-up data, both nuclear and membrane EGFR overexpression showed a correlation with a shorter overall survival. Nuclear EGFR (++, +++) showed 3.06 and membrane EGFR (2+, 3+) showed 2.76 higher risk of mortality compared with patients with low and negative nuclear and membrane EGFR expression (P<0.05).

Osteopontin expression is an independent factor for poor survival in oral squamous cell carcinoma: a computer-assisted analysis on TMA sections.

INTRODUCTION: Osteopontin (OPN) is non-collagenous extracellular matrix protein involved in various physiological and pathological events, including tumor progression. The aim of this study was to analyze the expression of OPN in normal oral mucosa and oral squamous cell carcinoma (OSCC) and to assess its prognostic significance. METHODS: The expression of OPN was immunohistochemicaly analyzed in 86 OSCC and compared with clinicopathological variable such as tumor size, nodal stage, WHO clinical stage, Ki-67 proliferation index, and patients' outcome. OPN mRNA was analyzed using quantitative real-time PCR and compared with protein OPN expression and clinical outcome in 18 OSCC samples. RESULTS: The expression of OPN protein was found in OSCC tumor cells (t-OPN) and various stromal cells (s-OPN). High level of t-OPN expression was associated with higher nodal stage (P = 0.045), higher WHO clinical stage (P = 0.033), and poor clinical outcome (P = 0.022). In multivariate analysis, t-OPN emerged as an adverse independent factor for survival (P = 0.049). Although correlated with t-OPN (P = 0.005), s-OPN was not significantly associated with clinical parameters, including patients' outcome. Also, there was no association between OPN and clinical parameters at the mRNA level. CONCLUSION: OPN is upregulated in tumor and stromal OSCC cells. Tumor cell-derived OPN is involved in tumor progression and can independently predict the clinical outcome. Stromal-derived OPN probably has a different function compared with OPN secreted from tumor cells.

JAK2-v617F mutation is associated with clinical and laboratory features of myeloproliferative neoplasms.

The aim of this study is to investigate the differences of clinical and laboratory parameters between patients with JAK2-V617F positive myeloproliferative neoplasms (MPNs) and JAK2 wild type MPNs. DNA was isolated from peripheral blood granulocytes of 106 patients treated at Rijeka University Hospital Center: 41 with polycythemia vera (PV), 43 with essential thrombocythemia (ET), 9 with primary myelofibrosis (PMF) and 13 with myeloproliferative neoplasm--unclassifiable (MPN-u). The JAK2-V617F mutation was detected using allele specific PCR. Laboratory and clinical parameters were obtained from patient's medical records. The JAK2-V617F mutation was detected in 69% (73/106) patients with MPNs. The results revealed significantly different prevalence of JAK2-V617F mutation, between MPNs entities: 88% in PV 58% in ET, 56% in PMF and 54% in MPNs-unclassified disorders. The JAK2-V617F mutation significantly correlated with higher leukocyte count and alkaline phosphatase co re in ET group and with higher platelets count, leukocyte alkaline phosphatase score and serum lactate dehydrogenase in PV group. Vascular events were associated with elevated platelets count in whole MPNs group, with higher platelets and leukocyte count in ET and with splenomegaly in PVpatients. Clinical and laboratory data revealed significant contribution ofJAK2-V617F mutation to the development of clinical phenotype in patients with distinct subgroups of MPNs.

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma.

BACKGROUND: The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival. METHODS: The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining. RESULTS: Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage. CONCLUSION: In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

Gene amplification of epidermal growth factor receptor in atypical glottic hyperplasia.

The study searched for epidermal growth factor receptor (EGFR) gene amplification in hyperplastic glottis lesions. After classical pathohistological findings of hematoxylin-eosin (HE) slides and quantitative immunohistochemical (IHC) analysis, fluorescent in situ hybridization (FISH) was used on tissue microarrays of laryngeal hyperplastic tissue ranging from normal mucosa to abnormal and atypical hyperplastic lesions. FISH analysis of two atypical hyperplastic lesions discovered the amplification of EGFR gene while it was not found in simple and abnormal hyperplastic lesions. The results may indicate that EGFR gene amplifications could possibly correlate with the histopathologic picture. Tissue samples burdened with specific oncogen signatures like EGFR gene amplification could be detected in precancerous lesion. This might improve follow-up and treatment protocols of glottic lesions which are an everyday problem for ENT practitioners. Further research is mandatory to confirm our findings.

mRNA expression of bone morphogenetic proteins and their receptors in human renal cell carcinoma.

INTRODUCTION: Bone morphogenetic proteins (BMPs) have been studied in several cancers, but only limited information is available about renal cell carcinomas (RCCs). We determined the expression of mRNA of several BMP ligands and BMP receptors (BMPRs) in healthy kidney tissue and RCCs, and data were compared to clinicopathological parameters. MATERIAL AND METHODS: Sixty-four samples of RCCs and healthy renal tissues were prospectively examined. The expression of BMP2, BMP4, BMP6, BMP7, BMPRIA, BMPRIB and BMPRII mRNA was determined using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The expression levels of different BMP ligands and BMPRs were considerably higher in RCCs than in normal kidney tissue. BMP ligands showed elevated expression in clear-cell RCCs, whereas all three BMPRs showed higher expression levels in non-clear-cell RCCs. In clear-cell RCCs, the expression levels of BMP2 progressively increased and expression levels of BMP6, BMP7 and BMPRIB were lost with higher tumor stage. CONCLUSIONS: All BMPs and their receptors have stronger expression levels in RCC. The expression level of BMP2 is strongly elevated in kidney cancer.

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma.

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.

Genetic polymorphisms of 15 STR loci in the population of the island of Cres (Croatia).

BACKGROUND: The population of the island of Cres presents one of the few persisting Eastern Adriatic isolates and is thereby suitable for human population differentiation analyses. AIM: The aim of this study was to analyse the genetic structure of the island of Cres with respect to its eight sub-populations and to compare the genetic variation of the island of Cres with other Eastern Adriatic islands and the Croatian mainland. SUBJECTS AND METHODS: Fifteen AmpFlSTR identifiler loci were analysed in a sample group of 122 unrelated autochthonous individuals from the island of Cres, Croatia. RESULTS: Analysis of STR polymorphisms revealed genetic homogeneity among sub-populations of the island of Cres and small but significant levels of genetic heterogeneity among geographically distant Eastern Adriatic islands. CONCLUSION: Despite a considerable degree of genetic homogeneity among the studied Eastern Adriatic islands, small but significant differentiation between distant islands indicates geographic sub-structuring which follows the isolation by distance model. This study is supportive of the notion that STR markers are useful for genetic differentiation between larger and geographically more distant regions.

Macrophage chemotactic protein-1 mRNA levels in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common malignancies whose incidence increases, and the treatment results are not satisfactory. The aim of this study was to determine the capacity of NHL to produce MCP-1, chemokine that induces chemotaxis of macrophages and lymphoid cells. The mRNA expression and protein MCP-1 expression were determined in the samples of 20 patients with NHL and 8 reactive tonsils. MCP-1 mRNA was detected in 8/8 tonsils and in 19/20 patients with NHL by real-time PCR analysis. In addition, the amount of detected MCP-1 cDNA was significantly higher in patients with limited stage, good IPI, normal level of fibrinogen and LDH. Finally, in patients with aggressive NHL, the level of MCP-1 cDNA was higher than in indolent tumours. Immunohistochemical analysis revealed that majority of stromal elements such as macrophages, endothelial and smooth muscle cells in reactive as well as in neoplastic lymphoid tissue showed strong cytoplasmic MCP-1 expression. Moderate cytoplasmic MCP-1 expression was also observed in reactive lymphocytes, while tumour cells of indolent NHL were mostly pale in comparison with aggressive lymphomas which predominantly demonstrated intense MCP-1 staining. These intriguing preliminary results emphasize the need for further investigations that must be conducted on the representative sample with concordant measurement of serum MCP-1 level.

Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis.

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).

A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis.

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.