Mahmood Manavi

Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue.

AbstractcDNA arrays provide a powerful tool to identify gene expression pattern that are potentially associated with tumor invasion and metastasis. However, genes work at the protein level and, since the transcriptional activity of a gene does not necessarily reflect cellular protein expression, the identification and quantification of proteins is essential for the understanding of molecular events leading to malignant transformation. We have therefore employed a high-throughput protein microarray system which contains 378 well-characterized monoclonal antibodies in order to compare the gene expression pattern of malignant and adjacent normal breast tissue in a patient with primary breast cancer. Using this technique, we have identified a number of proteins that show increased expression levels in malignant breast tissues such as casein kinase Ie, p53, annexin XI, CDC25C, eIF-4E and MAP kinase 7. The expression of other proteins, such as the multifunctional regulator 14-3-3e was found to be decreased in malignant breast tissue, whereas the majority of proteins remained unchanged when compared to the corresponding non-malignant samples. The protein expression pattern was confirmed by immunohistochemistry, in which antibodies against 8 representative proteins known to be involved in carcinogenesis were employed in paraffin-embedded normal and malignant tissue sections deriving from the same patient. In each case, the results obtained by IHC matched the data obtained by antibody microarray system. Taken together, we have described for the first time a tumor cell specificity protein expression pattern by use of a novel commercially available antibody microarray system. We have thus demonstrated the feasibility of high-throughput protein arrays in the proteomic analysis of human breast tissue. We hypothesize that the use of protein arrays will not only increase our understanding of the molecular events, but could prove useful in evaluating prognosis and in determining optimal antineoplastic therapy.

Co-expression of ErbB-family members in human breast cancer: Her-2/neu is the preferred dimerization candidate in nodal-positive tumors

Over-expression of members of the ErbB-receptor family has been associated with malignant transformation. The amplification of Her-2/neu in tumor tissue is now an established prognostic factor in breast cancer. In order to initiate signal transduction, ErbB-receptor monomers need to form homo- or heterodimers. The composition of these dimers is thought to influence both quality and quantity of downstream signaling pathways, and to determine the biological response. We have investigated the protein expression pattern of the four ErbB-receptors EGFR, Her-2/neu, Her-3 and Her-4, and correlated it with their putative ligands EGF, TGF-α and HRG in 74 women with invasive breast cancer. Using western blot-analysis on cell membrane isolates, we detected the co-expression of all four ErbB-family members in 79.7% of cases, and of all of the three investigated ligands in 82.4%. We did not observe a correlation between EGFR and Her-2/neu or Her-4 protein expression, EGFR and Her-3 (p = 0.005), and Her-3 and Her-4 (p = 0.05) were clearly co-expressed. The strongest overall correlation, was found between Her-2/neu and Her-3 (p < 0.001) and between Her-2/neu and Her-4 (p = 0.001). This was particularly true in nodal-positive tumors (p <0.001 and p = 0.002) whereas in nodal-negative tumors the co-expression was either less significant (Her-2/neu and Her-3; p = 0.01) or not significant (Her-2/neu and Her-4). The co-expression of EGFR/Her-3 was associated with the expression of all ligands, whereas the Her-2/neu/Her-3 was correlated with HRG (p = 0.002), thereby indicating a functional relation between specific receptor-dimer combinations and putative ligands. Taken together, we have performed the first comprehensive survey of ErbB-system expression in breast cancer, and have demonstrated the presence of a co-regulated receptor/ligand system in vivo. We have further shown that Her-2/neu is the preferred co-expression partner in nodal-positive tumors and thus the most likely dimerization candidate in malignant breast tumors.

Presence of nanobacteria in psammoma bodies of ovarian cancer: evidence for pathogenetic role in intratumoral biomineralization

Aims:  The presence of laminated, calcified extracellular debris known as psammoma bodies is a well‐known histomorphological feature of ovarian adenocarcinomas and other human malignancies. Biomineralization has recently been found to be associated with a group of extremely small Gram‐negative bacteria capable of precipitating calcium salts. The aim of the present study was to evaluate a possible pathogenic link between the development of psammoma bodies and nanobacteria infection.

Her-2/ neu- triggered intracellular tyrosine kinase activation: in vivo relevance of ligand-independent activation mechanisms and impact upon the efficacy of trastuzumab-based treatment

Proteolytic cleavage of the Her-2/neu extracellular domain (ECD) has been shown to initiate receptor phosphorylation representing Her-2/neu activation in vitro. The present investigation was performed to evaluate the clinical relevance of ECD cleavage for Her-2/neu activation and the consequences of active intracellular Her-2/neu signalling reflected by tyrosine kinase phosphorylation in patients treated with the anti-Her-2/neu antibody trastuzumab. Sera from 62 patients receiving trastuzumab-based treatment for Her-2/neu overexpressing metastatic breast cancer were assessed for pretreatment ECD levels using an enzyme-linked immunosorbent assay. In parallel, Her-2/neu activation status of tumour specimens was assessed by immunohistochemistry using a Her-2/neu phosphorylation state specific antibody (PN2A) and correlated with the patients’ ECD levels and clinical course of disease. Serum ECD levels were significantly higher in 15 (24%) patients with tumours exhibiting activated Her-2/neu as compared to those without detectable Her-2/neu phosphorylation (median 148.2 vs 28.5 ng ml−1, P=0.010). Whereas response rate only showed a trend to be higher in patients with Her-2/neu-phosphorylated breast cancer (47 vs 34%, P=0.197), both uni- and multivariate analyses revealed that the median progression-free survival under trastuzumab-based treatment was significantly longer in patients with Her-2/neu-phosphorylated breast cancer–11.7 (95% CI 5.2–18.3) months–when compared to the progression-free survival of 4.5 (95% CI 3.4–5.6) months observed in patients with tumours lacking phosphorylated Her-2/neu (P=0.001). Proteolytic cleavage of the ECD represents a biologically relevant ligand-independent mechanism of Her-2/neu activation in vivo. The influence of Her-2/neu activation status upon the outcome of trastuzumab-based therapies merits further investigation in larger prospective trials.

Her‐2/neu and EGFR tyrosine kinase activation predict the efficacy of trastuzumab‐based therapy in patients with metastatic breast cancer

Her‐2/neu overexpression in human breast cancer leads to an aggressive biological behavior and poor prognosis. Although the anti‐Her‐2/neu antibody trastuzumab (Herceptin®) has become a valuable therapeutic option for patients with Her‐2/neu‐overexpressing breast cancer, many patients do not benefit from this therapy. To evaluate the effect of receptor activation on tumor response, we have investigated the phosphorylation status of Her‐2/neu and EGFR in 46 Her‐2/neu‐overexpressing tumor samples from trastuzumab‐treated metastatic breast cancer patients by immunohistochemistry. Activated (p)tyr‐1248 Her‐2/neu was detected in 9 of 46 breast cancers (20%), and activated (p)tyr‐845 and (p)tyr‐1173 EGFR were both present in 6 tumors (13%) while EGFR was present in 16 cases (35%). ptyr‐1248 Her‐2/neu showed a trend to correlate with increased response to trastuzumab (p = 0.063), while ptyr‐845, ptyr‐1173 EGFR and EGFR did not. The presence of ptyr‐1248 Her‐2/neu and ptyr‐845 or ptyr‐1173 EGFR, however, was a strong predictor of both response to trastuzumab‐based treatment (OR = 8.0, p = 0.021 and OR = 8.0, p = 0.021) and clinical benefit (OR = 5.47, p = 0.041 and OR = 6.22, p = 0.028 multivariate logistic regression analysis). Furthermore, ptyr‐845 EGFR and ptyr‐1248 Her‐2/neu were both independent predictors of progression‐free survival (RR = 0.21, p = 0.01 and RR = 0.45, p = 0.026, multivariate analysis). Patients with ptyr‐845 EGFR positive tumors also tended toward increased overall survival (RR = 0.17, p = 0.082). Taken together, we have demonstrated that the determination of activated EGFR improves the utility of ptyr‐1248 Her‐2/neu staining in predicting the clinical outcome of patients undergoing trastuzumab treatment. We hypothesize that the activation state of both Her‐2/neu and EGFR are key determinants for trastuzumab efficacy.

Human papilloma virus DNA : A factor in the pathogenesis of mammary Paget's disease ?

The paraffin sections from 20 nipples with Pagets disease (10 central intraductal and 10 invasive ductal carcinomas) were analyzed for human papilloma virus (HPV) DNA of the low- and intermediate/high-risk groups. Polymerase chain reaction (PCR) and dot (slot) blot hybridization were used for the detection of HPV DNA types 6/11/16/18/31/33/35. In addition, we examined the c-erbB-2 oncogene expression in the specimens to differentiate benign cells in the surface epithelium of the nipple and areola from Paget cells. We found that the oncogene expression of the c-erbB-2 displayed a strong signal in the Paget cells. Using PCR and dot (slot) blot hybridization, we could not detect the HPV DNAs that are specific for the low- and intermediate/high riskgroups in the 20 cases of Pagets disease. Our results showed for the first time that this type of virus did not contribute to the pathogenesis of Pagets disease.

Does T1, N0-1 vulvar cancer treated by vulvectomy but not lymphadenectomy need inguinofemoral radiation?

PURPOSE The objective of our study was to demonstrate differences in relapse rates, total survival times, and complication rates between inguinofemoral radiation and its absence in cases of invasive vulvar carcinoma without lymph node involvement (FIGO Stages T1, N0-1). METHODS AND MATERIALS From 1974 to 1990, 135 patients with invasive vulvar carcinoma in Stage T1 without clinical evidence of inguinal lymph node involvement underwent simple vulvectomy performed by hot-knife resection without lymphadenectomy. Although 65 patients (Group 1) received postoperative inguinofemoral radiation therapy, 70 patients (Group 2) did not, and none received local vulva irradiation. RESULTS The 5-year survival rates were 93.7% in Group 1 and 91.4% in Group 2 (p = NS). Although clitoris involvement was significantly more prevalent in the irradiation group (p = 0.04), inguinal relapse was found less frequently in Group 1 (4.6% or 3 out of 65 patients) than in group 2 (10% or 7 out of 70 patients) (p = 0.32). The complication rates were, 7.7% in Group 1 and 2.9% in Group 2, 2.7% for vaginal stenosis (two patients in each group), 1.5% for inguinal pain (one patient in Group 1), 1.5% for rectovaginal fistula (one patient in Group 1), 1.5% for vulvar infection (one patient in Group 1). CONCLUSION No statistically significant differences in the relapse rates and survival times were found. Risk factors were equally distributed in both study groups except for clitoris involvement. The 5-year survival rates in both groups were similar to those reported in the literature for radical vulvectomy and inguinal lymph-node dissection (83-96%). Morbidity in our study was low. Although our data showed similar results in both groups, we are not recommending at this time to omit groin radiation in general, but it may be justified in low-risk cases.

Simvastatin reduces serum level of vascular endothelial growth factor in hypercholesterolemic patients.

Vascular endothelial growth factor plays a pivotal role in the progression of atherosclerotic lesions and causes instability of atherosclerotic plaques by inducing neoangiogenesis inside the current plaque. The pro-inflammatory cytokine interleukin (IL-) 6 induces vascular endothelial growth factor in smooth muscle cells (SMC). HMG-CoA reductase inhibitors (statins), display beside their lipid-lowering potency various pleiotropic effects. Such pleiotropic effects include improvement of endothelial dysfunction, increased nitric oxide bioavailability, antioxidant properties, inhibition of inflammatory responses, and stabilization of atherosclerotic plaques. In this study we investigate the influence of statin treatment on the serum levels of VEGF in hypercholesterolemic patients. One hundred and seven hypercholesterolemic patients were treated with 20 (n = 52) or 40 mg (n = 55) simvastatin daily. Six weeks of treatment resulted in a significant decrease of VEGF from 1017.1 ± 297.8 pg/mL at baseline to 543.5 ± 317.4 pg/mL after 6 weeks (−47.7%) and to 211.8 ± 155.3 pg/mL after 6 months (−79.7%; all P < 0.001). IL-6 induced the expression of vascular endothelial growth factor in human SMC as analyzed by rt-PCR and flow cytometry. Statins decreased the stimulatory effect of IL-6 on mRNA and protein levels. This effect could be inhibited by co-incubation with mevalonate acid. This study contributes in understanding the pleiotropic effects of statins particularly with regard to their use in treatment and prevention of cardiovascular disease.

Oncogenic Potential of c-erbB-2 and Its Association with c-K-ras in Premalignant and Malignant Lesions of the Human Uterine Endometrium

The aim of this study was to detect activated c-K-ras by gene point mutation and to find c-erbB-2 gene amplification with p185 expression in association with the c-K-ras gene product p21 in the human endometrium. Specimens obtained from 25 normal, 31 hyperplastic and 72 malignant samples of the human endometrium were examined for point mutation in codons 12, 13 and 61 of the c-K-ras by direct sequencing and c-erbB-2 gene amplification with p185 and p21 expression by differential polymerase chain reaction (DPCR) and immunohistochemistry. Neither the normal endometrium nor endometrial hyperplasias were found to have mutations in the c-K-ras gene, although a double mutation of codons 12 and 13 as a single-point mutation was observed in one case of endometrioid carcinoma (2.8%). In each of two other cases of endometrioid carcinoma (2/72), two single-point mutations of codon 13 (5.6%) were shown. Using DPCR, we found c-erbB-2 to be amplified in 15 premalignant (48%) and 45 malignant (63%) samples. We noticed that nonamplification of the c-erbB-2 gene was associated with the absence of immunoreactivity. Our data indicate that, while c-erbB-2 plays a role in the early development of endometrioid carcinomas, c-K-ras gene activation by point mutation does not.

Salpingitis caused by Chlamydia trachomatis and its significance for infertility

Between July and November 1991. 32 women (mean age 24.8 years) were examined laparoscopically in our department for suspected tubal sterility. All women had smears taken from cervix, vagina, and urethra, and all were negative regarding an infection with Chlamydia trachomatis. All women had open fallopian tubes, however, with inflammatory changes that varied in degree. Fifteen women reported pains and were classified as PID (pelvis inflammatory disease)‐positive, as compared to the PID‐negative group of 17 women without pain. In the group of the 15 PID‐positive women, we could detect Chlamy dia trachomatis in the form of salpingitis in II cases in the direct demonstration of the infectious agent. IgA antibodies were delected in the serum of all of these women. in 12 of them IgA +IgG antibodies. In the group of the 17 PID‐negative women, three were positive in the direct detection of the infectious agent. and IgA and/or IgG antibodies were detected in five cases. 38% of the women in the PID‐positive group and 68% in the PID‐negative group conceived within a period of one year after having completed a treatment with antibiotics.