Ita Hadžisejdić

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).

Hypoxia inducible factor-1α correlates with vascular endothelial growth factor A and C indicating worse prognosis in clear cell renal cell carcinoma

BackgroundThe role of angiogenesis in the pathogenesis of renal cell carcinoma is well recognized, however, the influence of tumor cells in this activity has not yet been fully clarified. The aim of this study was to analyze the expression of hypoxia inducible factor-1α (HIF-1α), a regulatory factor of angiogenic switch, in comparison to vascular endothelial growth factor A and C (VEGF-A and VEGF-C), recognized to be involved in blood and lymph vessel neoangiogenesis, with potential association in the prognosis of patients with renal cell carcinoma.MethodsNinety-four patients with diagnosis of clear cell renal cell carcinomas (CCRCC), all clinicopathological characteristics and overall survival were unrolled in this study. Immunohistochemicaly VEGF-A, VEGF-C, HIF-1α and Ki67 were detected on tumor cells and the staining was performed on tissue microarrays (TMA). The staining was evaluated as a percentage of cytoplasmic or nuclear positive tumor cells.ResultsVariable expression of all three proteins was confirmed. Both angiogenic factors demonstrated perimembranous or diffuse cytoplasmic staining, with diffuse pattern positively associated (p < 0.001). Nuclear HIF-1α expression (nHIF-1α) showed inverse correlation with diffuse cytoplasmic VEGF-A (p = 0.002) and VEGF-C (p = 0.053), while cytoplasmic HIF-1α expression (cHIF-1α) showed positive correlation with diffuse staining of both angiogenic factors (p < 0.001; p < 0.001, respectively). In comparison to clinicopathological characteristics, a higher nuclear grade (p = 0.006; p < 0.001, respectively), larger tumor size (p = 0.009; p = 0.015, respectively), higher stage (p = 0.023; p = 0.027, respectively) and shorter survival (p = 0.018; p = 0.024, respectively) were associated with overexpression of cHIF-1α and diffuse cytoplasmic VEGF-A expression. In contrary, overexpression of nHIF-1α was associated with better diagnostic parameters i.e. lower nuclear grade (p = 0.006), smaller tumor size (p = 0.057), and longer survival (p = 0.005).ConclusionOverexpression of VEGF-A and cHIF-1α in tumor cells highlights a more aggressive subtype of CCRCC that might have some clinical implications. The significance of nHIF-1α expression associated with better differentiated tumors should be further elucidated.

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.

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

BackgroundThe 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.MethodsThe 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.ResultsMembrane 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 patients survival, while there was no connection with pathological stage.ConclusionIn 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.

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.

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.

Human Papillomavirus Infection in Croatian Men: Prevalence and HPV Type Distribution

Human papillomavirus (HPV) infection has been identified as a major risk factor for cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (zur Hausen, 2002; Bosch et al., 2002; Munoz et al., 2003). HPV infection is one of the most common sexually transmitted infections, generally asymptomatic, with the worldwide prevalence in women with normal cytology of 11.4% (11.3-11.5%; 95%CI) (WHO/ICO Information Centre on HPV and Cervical Cancer. Summary report 2010). Epidemiological studies in the USA have reported that 75% of the 15-50 year-old population is infected with genital HPV during their lifetime. Among those, 60% are with transient infection, 10% with persistent infection (confirmed by detection of HPV-DNA in genital samples), 4% with mild cytological signs, and 1% with clinical lesions (WHO/ICO Information Centre on HPV and Cervical Cancer. Papillomavirus and Related Cancers in United States of America. Summary Report 2010). To date, more than 100 genotypes of HPV have been identified, with more than 40 anogenital types, at least 15 of which are oncogenic (Munoz et al. 2003; Clifford et al., 2005). Anogenital HPV types have been further classified into low-risk types (lrHPV, e.g., 6 and 11), which are associated with anogenital warts and mild dysplasia, and high-risk types (hrHPV, e.g., 16, 18, 31, and 33), which are associated with high-grade dysplasia and anogenital cancers, such as cervical and anal carcinoma. (Bosch et al., 2002; Smith et al., 2007). In Croatia, HPV testing is widely used as a secondary test to triage borderline cytology and as a follow-up after treatment of severe cervical lesions, in addition to conventional cytological screening (Grce et al., 2007). Grahovac et al. (2007) investigated the HPV prevalence and type distribution among 361 women regularly attending gynecological examinations, and showed 67.9% overall prevalence of hrHPV in women with abnormal PAP smears compared to 35.6% in women with normal cytology. Study done by Hadžisejdic et al., (2006) on prevalence of HPV genotypes in cervical cancer also revealed high prevalence of HPV-DNA in cervical lesions; 93% in CIN III, 92.6% in squamous cell carcinoma (SCC) and 92.5% in adenocarcinoma (ADC).

The Prognostic Importance of Nuclear Factor κB and Hypoxia-inducible Factor 1α in Relation to the Breast Cancer Subtype and the Overall Survival

Breast cancer shows extensive clinical and molecular heterogeneity. Prognostic factors are very important for outcome estimation in individual patients. Nuclear factor &kgr;B (NF-&kgr;B) and hypoxia-inducible factor 1&agr; (HIF-1&agr;) are transcriptional factors involved in cancerogenesis and in the metastatic spread of tumor cells. The aim of this study was to evaluate the expression of NF-&kgr;B and HIF-1&agr; and to correlate the immunohistochemical expression of these markers with the breast cancer subtype and the patient outcome. The retrospective study included 208 cases of ductal invasive breast cancers stratified by the molecular subtype according to the St. Gallen 2011 classification. The Kaplan-Meier survival curve showed that an increased mortality risk was associated with tumors belonging not to the luminal A subtypes but to the Her-2-enriched and luminal B-Her-2-positive subtypes instead (P<0.001). Activation of NF-&kgr;B was associated with estrogen-negative tumors (P=0.005). We found a better overall survival in NF-&kgr;B-positive tumors in the luminal A subtype (P=0.021). This may be explained as a consequence of a possible tumor-suppressing effect of NF-&kgr;B. HIF-1&agr; was related to the overall survival as a poor prognostic factor (P=0.036). In our opinion, the practical relevance of NF-&kgr;B and HIF-1&agr; expression as prognostic indicators and potential targets for specific therapies deserve further investigation.

Thoracic aortic aneurysm rupture into the esophagus.

A 73-year-old female with a history of mild hematemesis was transported to the hospital emergency department. Although she was under the influence of alcohol, the patient remained alert. She had hypotension (100/60 mmHg) and anemia (red cell count 2.80 9 10/L; hemoglobin 86 g/L; hematocrit 0.265 L/L). A digital rectal examination performed after admission revealed black, formed stool. She had medical comorbidities of choleithiasis and a history of alcohol abuse. An emergency upper digestive endoscopy was performed immediately, which showed blood clots and fresh blood in the stomach. There was a protrusion near the gastro-esophageal junction but, despite intensive examination, the immediate place of bleeding could not be identified. As the exact site of bleeding was undeterminable diluted adrenaline was injected into the suspected area of hemorrhage at the fundus of the stomach. The patient was transferred to the intensive care unit where intravenous fluids and a blood transfusion were administered. The patient became stable upon aggressive fluid administration and a surgeon was requested for consultation. The surgeon advised the hospital staff caring for the patient to continue with the conservative therapy course (that included frequent monitoring of hemodynamic parameters) until the exact bleeding site could be identified. However, a few hours after admission the patient started to vomit blood vigorously and became hypotensive. A diagnosis of thoracic aortic aneurysm (TAA) with esophageal fistula was made. Emergency surgical treatment was ordered. The patient was transferred to the operating room, where she collapsed and became unconscious and asystolic. She died one hour later despite critical life support measures. Although a complaint of medical negligence was not raised in this case, an autopsy was performed in accordance with Croatian law, which requires that all patients who die within 24 h of hospital admission have to be examined by a certified pathologist. The autopsy revealed a large spherical aneurysm, 5 cm in diameter, of the descending thoracic aorta; 1.5 cm below the tracheal bifurcation (Fig. 1). Rupture into the esophagus was observed, with an aorto-esophageal fistula (AEF) around 2 cm in diameter (Fig. 1). On the aortic side of the fistula, the opening was covered with laminated thrombus that was firm on palpation. The rest of the intima of the thoracic and abdominal aorta did not exhibit severe atherosclerotic changes. The stomach was completely filled with blood and clots (about 800 mL); with bloody and tarry contents in the intestines. Tissue histology around the fistula (the aortic and esophageal wall) showed hemorrhage, inflammation and fibrosis. The aortic wall was severely infiltrated with neutrophils around the rupture site (Fig. 2). The laminated thrombus appeared to have acted as a lid covering the fistula opening. There was no histological evidence of cystic medial degeneration in the aortic wall. No significant pathology was observed in any other organs.

Antiphospholipid antibodies associated with nodal marginal zone lymphoma and its progression to diffuse large B-cell lymphoma–a case report

An association between autoimmune events, as well as the development of antiphospholipid (aPL) antibodies and lymphoproliferative disorders is well recognized. We present the patient with coagulation abnormalities and non-Hodgkin lymphoma (NHL), primarily diagnosed as nodal marginal zone B-cell lymphoma (NMZL), and in relapse as diffuse large B-cell lymphoma (DLBCL). In the follow-up period, the patient simultaneously developed different aPL antibodies. The presence of aPL antibodies in NHL is frequent but it is not common in the NMZL. The aim of the present case report is to highlight the possible underlying increase of aPL antibodies in NMZL patients with coagulation tests abnormalities.