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2005
Zekri, A. - R. N., A. A. Bahnassy, M. Hafez, A. M. El-Shehaby, G. M. Sherif, H. M. Khaled, and N. Zakhary, "Alterations of the fragile histidine triad gene in hepatitis C virus-associated hepatocellular carcinoma.", Journal of gastroenterology and hepatology, vol. 20, issue 1, pp. 87-94, 2005 Jan. Abstract

BACKGROUND AND AIM: The present study was conducted to address whether homozygous deletion (HZD) or transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCC).

METHODS: Homozygous deletion of the FHIT gene at exons 3-9 was assessed as well as mRNA FHIT expression using reverse transcription polymerase chain reaction. The study included 23 samples of HCC, 11 on top of cirrhosis and 12 non-cirrhotic, in addition to five cases with chronic active hepatitis (CAH), as well as seven morphologically normal tissues distant to the tumor (NDT) and 10 normal liver samples from liver transplantation donors.

RESULTS: Homozygous deletion was found in 18 of 23 HCC cases. The highest incidence of deletion was detected in exon 9 (52.0%) and the lowest in exon 7 (4.3%). Ten of the 18 cases (55.5%) showed deletion in more than one exon, eight in two exons, one in three exons and one in five exons. There was a significant association between HZD of exons 5 and 9 and HCC arising on top of cirrhosis (P = 0.041 and 0.006, respectively) as well as between exons 8 and 9 and the presence of CAH (P = 0.029 and 0.034, respectively). Aberrant FHIT transcripts were detected in 15 HCC cases (65.2%), 13 of them showed complete reduction of the mRNA transcripts and two showed abnormal bands. Sequence analysis of abnormal-sized transcripts revealed that they were generated by the fusion of exons 5 and 7 as well as exons 7 and 9. In contrast, six of the seven NDT samples tested (85.6%) showed HZD in one or more exons. None of the normal liver samples from liver transplantation donors showed any changes. The highest incidence of HZD was detected in exon 9 (five of six cases representing 83.3%) and the lowest was in exon 4 (one of six cases representing 16.7%). Four cases showed the same aberrant FHIT HZD in both NDT and matched HCC.

CONCLUSIONS: The results of the present study indicate that the FHIT gene is a frequent target in hepatitis C virus-associated HCC and that alterations affecting this gene could be an early event in this type of neoplasm as they were detected in cirrhotic and CAH patients. However, this should be confirmed by a larger, extended study including more cases of cirrhotic and CAH patients as well as matched tumor and normal samples.

g Zekri, A. - R. N. a, A. A. c Bahnassy, M. A. Hafez, A. M. R. f El-Shehaby, G. M. d Sherif, H. M. e Khaled, and N. b Zakhary, "Alterations of the fragile histidine triad gene in hepatitis C virus-associated hepatocellular carcinoma", Journal of Gastroenterology and Hepatology (Australia), vol. 20, no. 1, pp. 87-94, 2005. AbstractWebsite

Background and Aim: The present study was conducted to address whether homozygous deletion (HZD) or transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCC). Methods: Homozygous deletion of the FHIT gene at exons 3-9 was assessed as well as mRNA FHIT expression using reverse transcription polymerase chain reaction. The study included 23 samples of HCC, 11 on top of cirrhosis and 12 non-cirrhotic, in addition to five cases with chronic active hepatitis (CAH), as well as seven morphologically normal tissues distant to the tumor (NDT) and 10 normal liver samples from liver transplantation donors. Results: Homozygous deletion was found in 18 of 23 HCC cases. The highest incidence of deletion was detected in exon 9 (52.0%) and the lowest in exon 7 (4.3%). Ten of the 18 cases (55.5%) showed deletion in more than one exon, eight in two exons, one in three exons and one in five exons. There was a significant association between HZD of exons 5 and 9 and HCC arising on top of cirrhosis (P = 0.041 and 0.006, respectively) as well as between exons 8 and 9 and the presence of CAH (P = 0.029 and 0.034, respectively). Aberrant FHIT transcripts were detected in 15 HCC cases (65.2%), 13 of them showed complete reduction of the mRNA transcripts and two showed abnormal bands. Sequence analysis of abnormal-sized transcripts revealed that they were generated by the fusion of exons 5 and 7 as well as exons 7 and 9. In contrast, six of the seven NDT samples tested (85.6%) showed HZD in one or more exons. None of the normal liver samples from liver transplantation donors showed any changes. The highest incidence of HZD was detected in exon 9 (five of six cases representing 83.3%) and the lowest was in exon 4 (one of six cases representing 16.7%). Four cases showed the same aberrant FHIT HZD in both NDT and matched HCC. Conclusions: The results of the present study indicate that the FHIT gene is a frequent target in hepatitis C virus-associated HCC and that alterations affecting this gene could be an early event in this type of neoplasm as they were detected in cirrhotic and CAH patients. However, this should be confirmed by a larger, extended study including more cases of cirrhotic and CAH patients as well as matched tumor and normal samples. © 2005 Blackwell Publishing Asia Pty Ltd.

g Zekri, A. - R. N. a, A. A. c Bahnassy, M. A. Hafez, A. M. R. f El-Shehaby, G. M. d Sherif, H. M. e Khaled, and N. b Zakhary, "Alterations of the fragile histidine triad gene in hepatitis C virus-associated hepatocellular carcinoma", Journal of Gastroenterology and Hepatology (Australia), vol. 20, no. 1, pp. 87-94, 2005. AbstractWebsite

Background and Aim: The present study was conducted to address whether homozygous deletion (HZD) or transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of hepatitis C virus-associated hepatocellular carcinoma (HCC). Methods: Homozygous deletion of the FHIT gene at exons 3-9 was assessed as well as mRNA FHIT expression using reverse transcription polymerase chain reaction. The study included 23 samples of HCC, 11 on top of cirrhosis and 12 non-cirrhotic, in addition to five cases with chronic active hepatitis (CAH), as well as seven morphologically normal tissues distant to the tumor (NDT) and 10 normal liver samples from liver transplantation donors. Results: Homozygous deletion was found in 18 of 23 HCC cases. The highest incidence of deletion was detected in exon 9 (52.0%) and the lowest in exon 7 (4.3%). Ten of the 18 cases (55.5%) showed deletion in more than one exon, eight in two exons, one in three exons and one in five exons. There was a significant association between HZD of exons 5 and 9 and HCC arising on top of cirrhosis (P = 0.041 and 0.006, respectively) as well as between exons 8 and 9 and the presence of CAH (P = 0.029 and 0.034, respectively). Aberrant FHIT transcripts were detected in 15 HCC cases (65.2%), 13 of them showed complete reduction of the mRNA transcripts and two showed abnormal bands. Sequence analysis of abnormal-sized transcripts revealed that they were generated by the fusion of exons 5 and 7 as well as exons 7 and 9. In contrast, six of the seven NDT samples tested (85.6%) showed HZD in one or more exons. None of the normal liver samples from liver transplantation donors showed any changes. The highest incidence of HZD was detected in exon 9 (five of six cases representing 83.3%) and the lowest was in exon 4 (one of six cases representing 16.7%). Four cases showed the same aberrant FHIT HZD in both NDT and matched HCC. Conclusions: The results of the present study indicate that the FHIT gene is a frequent target in hepatitis C virus-associated HCC and that alterations affecting this gene could be an early event in this type of neoplasm as they were detected in cirrhotic and CAH patients. However, this should be confirmed by a larger, extended study including more cases of cirrhotic and CAH patients as well as matched tumor and normal samples. © 2005 Blackwell Publishing Asia Pty Ltd.

Hortobagyi, G. N. a, J. b de la Garza Salazar, K. c Pritchard, D. d Amadori, R. e Haidinger, C. A. f Hudis, H. g Khaled, M. - C. h Liu, M. i Martin, M. j Namer, et al., "The global breast cancer burden: Variations in epidemiology and survival", Clinical Breast Cancer, vol. 6, no. 5, pp. 391-401, 2005. AbstractWebsite

Breast cancer is the most common type of cancer and the most common cause of cancer-related mortality among women worldwide. However, the burden is not evenly distributed, and, according to the best available data, there are large variations in the incidence, mortality, and survival between different countries and regions and within specific regions. Many complex factors underlie these variations, including population structure (eg, age, race, and ethnicity), lifestyle, environment, socioeconomic status, risk factor prevalence, mammography use, disease stage at diagnosis, and access to high-quality care. We review recent breast cancer incidence and mortality statistics and explore why these vary so greatly across the world. Further research is needed to fully understand the reasons for variations in breast cancer outcomes. This will aid the development of tailored strategies to improve outcomes in general as well as the standard of care for underserved populations and reduce the burden of breast cancer worldwide.

Bahnassi, A. A. a, A. - R. N. b g Zekri, S. a El-Houssini, N. M. a Mokhtar, A. O. c Abdel-Aziz, G. M. d Sherif, A. M. e El-Mishad, and H. M. f Khaled, "Hepatitis C virus-NS3P in relation to p53, p21waf, mdm2, p21-ras and c-erbB2 in hepatocarcinogenesis", Journal of Gastroenterology and Hepatology (Australia), vol. 20, no. 11, pp. 1731-1740, 2005. AbstractWebsite

Background: The non-structural protein 3 (NS3P) of hepatitis C virus (HCV) genome was linked to the neoplastic transformation of normal hepatocytes in chronically infected patients. However, the exact mechanisms involved in this process are unidentified yet, especially in the Egyptian population where the commonest type is genotype 4. Methods: We investigated 32 HCV reverse transcriptase-polymerase chain reaction (RT-PCR) positive hepatocellular carcinoma (HCC) cases and 18 morphologically normal hepatic tissues distant to tumors (MNT) for the correlation between HCV-NS3P, p53, p21waf, mdm2, p21ras and c-erbB2 and DNA content by immunohistochemistry and image analysis. Results: The NS3P expression was lower in HCC (65.6%) than in MNT (94.4%) patients. The expression level of studied genes in HCC was: p53 (56.25%), p21waf (43.7%), mdm2 (59.4%), p21-ras (73.3%) and c-erbB2 (75%). Whereas in MNT, it was 22.2, 61.1, 44.4, 41.2 and 77.8%, respectively. The NS3P expression showed a significant correlation with the presence of cirrhosis, chronic active hepatitis (CAH) and tumor grade (P < 0.05). c-erbB2 overexpression and p21waf loss were higher in MNT than in HCC patients, however, this did not reach a statistically significant level. There was a statistically significant correlation between NS3P, c-erbB2 and p21 waf (P < 0.01). There was also a significant correlation between p21waf loss and CAH (P = 0.01) as well as between mdm2, c-erbB2 and cirrhosis (P = 0.025 and 0.001) in HCC cases. There was a statistically significant difference in the ploidy status between HCC and MNT, but there was no significant relationship between the ploidy status and other clinicopathological features. Conclusion: The carcinogenic effect of NS3P is probably exerted at an early stage of HCC possibly through a pathway involving c-erbB2 and p21waf alterations. In contrast, p53, p21ras and mdm2 alterations are late events in hepatocarcinogenesis and are usually associated with an aggressive phenotype. © 2005 Blackwell Publishing Asia Pty Ltd.

Khaled, H. a, O. b El Hattab, D. A. a Moneim, H. A. c Kassem, A. c Morsi, G. b Sherif, T. a Darwish, and R. a Gaafar, "A prognostic index (bladder prognostic index) for bilharzial-related invasive bladder cancer", Urologic Oncology: Seminars and Original Investigations, vol. 23, no. 4, pp. 254-260, 2005. AbstractWebsite

{Purpose: Bladder cancer is still the most common solid tumor among adult males in Egypt because of the prevalence of bilharzial infestation, especially in the countryside. In this prospective study, we have recorded the prognostic factors for 180 patients with invasive bladder cancer for whom standard radical cystectomy had been performed to develop a prognostic index (bladder prognostic index) that defines high risk patients who are more vulnerable to disease relapse after surgery and who may benefit from additional therapy. Patients and Methods: The study was performed between January 1997 and December 1999, in which 180 patients with histopathologically proved invasive bladder cancer associated with bilharziasis underwent radical cystectomy or anterior pelvic exenteration. After surgery, patients were regularly followed for a minimum of 2 years. Results: Our patients included 141 males and 39 females. Squamous cell carcinoma was the most common type (53.3%), and most of the tumors were grade II (61.1%). A total of 173 patients had their tumors operable, while 7 were inoperable. We had 5 (2.8%) operative related mortalities. At 5 years postoperatively, free and overall survival rates for the whole group of patients were 31.44% ± 5.9% and 32.5% ± 6.8%, respectively. Tumor pathologic stage, grade, and nodal affection were the only significant factors with impact on survival (P = 0.008, 0.051, and 0.004, respectively). These 3 prognostic indexes were used to design a model to predict an individual patient's risk factor for recurrence. Patients were then assigned to one of the 4 risk groups according to the score achieved in this prognostic index (0 = low risk

Yang, H. a, K. a Yang, A. a Khafagi, Y. a Tang, T. E. b Carey, A. W. c Opipari, R. c d Lieberman, P. A. e Oeth, W. f Lancaster, H. P. g Klinger, et al., "Sensitive detection of human papillomavirus in cervical, head/neck, and schistosomiasis-associated bladder malignancies", Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 21, pp. 7683-7688, 2005. AbstractWebsite

We assayed for the presence of human papilloma virus (HPV) DMA in serum and/or peripheral blood fraction (PBF) of individuals with cervical, head/neck, or bladder cancer due to schistosomiasis. Using mass spectroscopy coupled with competitive PCR, HPV DNA was detected at the individual molecule level by using "Mass-ARRAY" assays. The resultant sensitivity was superior to real-time fluorescent PCR-based assays, while specificity was maintained. Our principal findings were: (i) Virtually all tested cervical cancers and schistosomiasis-associated bladder cancers, and a plurality of head/neck cancers, are associated with HPV DNA in the tumor. (ii) All 27 bladder cancers due to schistosomiasis were associated with the presence of HPV-16 DNA, which can be detected in tumor and serum but not in PBF. In contrast, no serum HPV-16 DNA signal was detected in seven individuals with schistosomiasis-associated bladder cancers after surgical removal of the tumor. (iii) Among the head/neck cancers we studied, anterior tumors were more often associated with HPV DNA in tumor, serum, and/or PBF than posterior tumors. (iv) In cervical cancer, where all tumors contain HPV DNA, viral DNA could be detected often in serum and/or PBF. Further, HPV-16 DNA was detected in serum and/or PBF of most patients with untreated high-grade cervical dysplasia but disappeared if the dysplasia was eliminated. The sensitive, specific, and quantitative MassARRAY technique should make it feasible to monitor cancer occurrence and treatment and recurrence of malignancies and dysplasias associated with HPV DNA. © 2005 by The National Academy of Sciences of the USA.

Yang, H. a, K. a Yang, A. a Khafagi, Y. a Tang, T. E. b Carey, A. W. c Opipari, R. c d Lieberman, P. A. e Oeth, W. f Lancaster, H. P. g Klinger, et al., "Sensitive detection of human papillomavirus in cervical, head/neck, and schistosomiasis-associated bladder malignancies", Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 21, pp. 7683-7688, 2005. AbstractWebsite

We assayed for the presence of human papilloma virus (HPV) DMA in serum and/or peripheral blood fraction (PBF) of individuals with cervical, head/neck, or bladder cancer due to schistosomiasis. Using mass spectroscopy coupled with competitive PCR, HPV DNA was detected at the individual molecule level by using "Mass-ARRAY" assays. The resultant sensitivity was superior to real-time fluorescent PCR-based assays, while specificity was maintained. Our principal findings were: (i) Virtually all tested cervical cancers and schistosomiasis-associated bladder cancers, and a plurality of head/neck cancers, are associated with HPV DNA in the tumor. (ii) All 27 bladder cancers due to schistosomiasis were associated with the presence of HPV-16 DNA, which can be detected in tumor and serum but not in PBF. In contrast, no serum HPV-16 DNA signal was detected in seven individuals with schistosomiasis-associated bladder cancers after surgical removal of the tumor. (iii) Among the head/neck cancers we studied, anterior tumors were more often associated with HPV DNA in tumor, serum, and/or PBF than posterior tumors. (iv) In cervical cancer, where all tumors contain HPV DNA, viral DNA could be detected often in serum and/or PBF. Further, HPV-16 DNA was detected in serum and/or PBF of most patients with untreated high-grade cervical dysplasia but disappeared if the dysplasia was eliminated. The sensitive, specific, and quantitative MassARRAY technique should make it feasible to monitor cancer occurrence and treatment and recurrence of malignancies and dysplasias associated with HPV DNA. © 2005 by The National Academy of Sciences of the USA.

Khaled, H. M., "Systemic management of bladder cancer in Egypt: revisited.", Journal of the Egyptian National Cancer Institute, vol. 17, no. 3, pp. 127-131, 2005. AbstractWebsite

Bladder cancer is still the most frequent malignant tumor among Egyptian males. It has a peculiar biologic, clinico-pathologic features and responsiveness to chemotherapy profile than that observed in Western countries. The current review aims to demonstrate the present state-of- art in using systemic therapy as part of the management options available to treat such patients at different stages of their disease. Individualizing therapy for these patients based on more rationale basis is the challenge that oncologists must face in the near future.

f Zekri, A. R. N. a, H. M. A. a El-Din, A. A. b Bahnassy, A. M. R. c El-Shehabi, H. a El-Leethy, A. d Omar, and H. M. e Khaled, "TRUGENE sequencing versus INNO-LiPA for sub-genotyping of HCV genotype-4", Journal of Medical Virology, vol. 75, no. 3, pp. 412-420, 2005. AbstractWebsite

Hepatitis C virus genotypes and subtypes determination is an important factor for understanding the epidemiology of the virus, in the pre-treatment evaluation of the patients and in defining better treatment strategies. In the present study, we compared two commercially available assays for HCV genotyping: the reverse hybridization based Innogenetics INNO-LiPA HCV II and the direct sequencing by TRUGENE assay. The study included 31 HCV-RNA positive Egyptian patients; 18 patients with chronic active hepatitis, 8 with HCC, and 5 with cirrhosis. Using the TRUGENE genotyping test, all the samples had genotype 4 (100%) and subtyped as 4a in 18/31(58%), 4c in 10/31 (32%), 4e in 1/31 (3%), 4a/c in 1/31 (3%), and 4g in 1/31 (3%). Using the INNO-LiPA assay, 30 samples had genotype 4 (97%), and 1 sample had genotype 1e (3%). One sample showed mixed infection with type 4f and type 1. Only six samples were subtypable by INNO-LiPA, three were genotype 4c/d, and the other three were 4f, 4e, and 1e. Seven samples gave reactivity in the INNO-LiPA of lines 5, 6, 16, 17, 18, which are considered untypable by the interpretation chart but considered to be a rare HCV genotype 4 by the manufacturer. At the genotype level, there was a 97% concordance between TRUGENE sequencing and INNO-LiPA, but at the subtype level the concordance rate was 3% only. We conclude that the TRUGENE genotyping assay is a reliable test for HCV genotyping for the detection of major types and subtypes detection, while IN NO-LiPA is a good test at the genotype level but unreliable for subtyping especially in the Egyptian population. This is mainly due to the high diversity of genotype 4, which is the most prevalent genotype in Egypt. © 2005 Wiley-Liss, Inc.

f Zekri, A. R. N. a, H. M. A. a El-Din, A. A. b Bahnassy, A. M. R. c El-Shehabi, H. a El-Leethy, A. d Omar, and H. M. e Khaled, "TRUGENE sequencing versus INNO-LiPA for sub-genotyping of HCV genotype-4", Journal of Medical Virology, vol. 75, no. 3, pp. 412-420, 2005. AbstractWebsite

Hepatitis C virus genotypes and subtypes determination is an important factor for understanding the epidemiology of the virus, in the pre-treatment evaluation of the patients and in defining better treatment strategies. In the present study, we compared two commercially available assays for HCV genotyping: the reverse hybridization based Innogenetics INNO-LiPA HCV II and the direct sequencing by TRUGENE assay. The study included 31 HCV-RNA positive Egyptian patients; 18 patients with chronic active hepatitis, 8 with HCC, and 5 with cirrhosis. Using the TRUGENE genotyping test, all the samples had genotype 4 (100%) and subtyped as 4a in 18/31(58%), 4c in 10/31 (32%), 4e in 1/31 (3%), 4a/c in 1/31 (3%), and 4g in 1/31 (3%). Using the INNO-LiPA assay, 30 samples had genotype 4 (97%), and 1 sample had genotype 1e (3%). One sample showed mixed infection with type 4f and type 1. Only six samples were subtypable by INNO-LiPA, three were genotype 4c/d, and the other three were 4f, 4e, and 1e. Seven samples gave reactivity in the INNO-LiPA of lines 5, 6, 16, 17, 18, which are considered untypable by the interpretation chart but considered to be a rare HCV genotype 4 by the manufacturer. At the genotype level, there was a 97% concordance between TRUGENE sequencing and INNO-LiPA, but at the subtype level the concordance rate was 3% only. We conclude that the TRUGENE genotyping assay is a reliable test for HCV genotyping for the detection of major types and subtypes detection, while IN NO-LiPA is a good test at the genotype level but unreliable for subtyping especially in the Egyptian population. This is mainly due to the high diversity of genotype 4, which is the most prevalent genotype in Egypt. © 2005 Wiley-Liss, Inc.

2004
Khaled, H. M., M. S. Aly, and N. Mokhtar, "Chromosomal aberrations in Cis and Ta bilharzial bladder cancer: a theory of pathogenesis.", Urologic oncology, vol. 22, issue 6, pp. 443-7, 2004 Nov-Dec. Abstract

BACKGROUND: Bladder cancer manifests many different forms ranging from superficial to aggressive muscle invasive stages, which suggests that various genetic alterations are involved. Several attempts have been made to establish correlations between specific genetic alterations and various stages of the disease. At the National Cancer Institute (NCI), Cairo, bladder cancer constitutes 30.3% of all cancers. Bladder cancer observed among Egyptians has a clinico-pathological profile that differs from transitional cell carcinoma (TCC) seen in the western world.

PATIENTS AND METHODS: We used fluorescence in situ hybridization to detect numerical chromosome changes in 25 patients presenting with carcinoma in situ and Ta lesions. Twenty-four cases had transitional cell carcinoma and one case had squamous cell carcinoma.

RESULTS: Five out of 24 TCC cases had diploid chromosome count with all the probes. Numerical chromosome aberrations were detected in 19 cases (79%). In eight cases, a loss of chromosome 9 was observed. In one case, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 17, whereas another three cases showed a gain of chromosome 7. Loss of chromosome Y was observed in nine of the 22 male cases studied (40.9%). The only case with SCC had normal diploid chromosome count with all the probes used.

CONCLUSION: When the genetic basis of bilharzial related bladder cancer is fully understood, new diagnostic and therapeutic strategies will be developed, which in turn may promote better clinical management by pathologists and urologists. A theory of bilharzial related bladder cancer pathogenesis is suggested.

Aly, M. S., and H. M. Khaled, "Detection of C-erb B2 gene amplification in bilharzial associated bladder cancer using fluorescence in situ hybridization.", Urologic oncology, vol. 22, issue 6, pp. 448-52, 2004 Nov-Dec. Abstract

BACKGROUND: Gene amplifications are common events in different tumor types and may confer diagnostic, prognostic, or therapeutic information for patient management. Fluorescence in situ hybridization (FISH) represents a standard methodologic approach for testing for this genetic alteration, as it is rapid, reproducible and extremely reliable in detecting presence of C-erb-B2 gene amplification for clinical utility.

PATIENTS AND METHODS: In this study, FISH is used in a series of archival human bilharzial bladder cancer specimens to evaluate for the presence of cerbB-2 gene alterations in the most common malignant tumor in bilharzial endemic areas, e.g., Egypt and some other countries. The study included 40 cases, 30 males and 10 females. Their ages ranged between 30 years and 76 years (median: 51 years). Twenty-one cases had squamous cell carcinoma, 16 had transitional cell carcinoma, two had adenocarcinoma, and one case had undifferentiated carcinoma.

RESULTS: Thirteen out of 40 tumor samples (32.5%) show evidence of true C-erb-B2 gene amplification. Of the remaining samples, 24 (60%) show no gene amplification and three (7.5%) fall into the borderline category with a ratio between one and two C-erb-B2 genes/cell relative to chromosome 17 centromeres. No evidence of chromosome 17 polysomy was found in any cases scored as single copy with the C-erb-B2 probe.

CONCLUSION: No significant association was found between gene amplification and any of the tested clinicopathologic parameters or tumor recurrence except for tumor grade where higher tumor grades tended to be associated with more C-erb-B2 gene amplification (P = 0.01) thus reflecting more tumor aggressiveness. So, the amplification of C-erb-B2 in bilharzial associated bladder cancer is probably not independently related to clinical outcome of patients.

Gaafar, R. M., R. Hamza, H. M. Khaled, M. Elserafi, O. Mansour, N. A. Karim, D. Abdelmoneim, I. El Attar, and S. Soliman, "Gemcitabine and cisplatin in the treatment of advanced non-small cell lung cancer: National Cancer Institute Cairo experience.", Journal of the Egyptian National Cancer Institute, vol. 16, issue 1, pp. 1-7, 2004 Mar. Abstract

AIM OF THE WORK: The aim of the present study is to document the antitumor activity of the combination of gemcitabine and cisplatin for the treatment of advanced NSCLC, asses the nature and severity of the side effects and elicit the impact of the combination chemotherapy on progression free survival and overall survival.

PATIENTS AND METHODS: From August 1997 to August 2001, we conducted a phase II study of gemcitabine and cisplatin in 60 chemonaive patients (21 stage IIIB and 39 stage IV). For the first 34 cases, gemcitabine was given at a dose of 1,000 mg/m2 IV on days 1, 8 and 15 with cisplatin 100 mg/m2 on day 15, every 28 days. In the following 26 patients, the regimen was modified to gemcitabine 1,250 mg/m2 days 1 and 8 and cisplatin 80 mg/m2 day 1, every 21 days.

RESULTS: Patients included 53 males and 7 females [median age, 52 years (range, 28-69)]. Twenty-nine had adenocarcinoma, 18 large-cell carcinoma and 13 squamous-cell carcinoma. Thirty-one patients had a performance status (PS) of 2 and 22 presented with weight loss. All patients were evaluable for response. Three patients achieved a complete response (CR) and 22 had partial response (PR), giving an overall response of 41.7%, with a median duration of 10 months (range, 4-46 months). The time to progression (TTP) was 8 months (range, 2-46 months), with a median overall survival of 9 months (range, 2-46 months). The one-year survival rate was 30.3% for the entire study population, 44% for responders, and statistically improved in patients with a PS of I and those with no weight loss. A total of 255 cycles were administered (median, four cycles/patient). Myelosuppression was significant (but manageable) with grade 3/4 neutropenia in 32.6% of cases, anemia in 18.6% and thrombocytopenia in 20.4%. Nonhematologic toxicity was limited to grade 3/4 nausea and vomiting in 28.8% of cases and impaired liver enzymes in 13.6%.

CONCLUSION: Inspite of the relatively poor prognostic characteristics in the study population, gemcitabine and cisplatin, was an effective combination with tolerable, manageable toxicity in advanced NSCLC.

Ismail, M. S., W. Wynendaele, J. L. E. Aerts, R. Paridaens, R. Gaafar, N. Shakankiry, H. M. Khaled, M. - R. Christiaens, H. Wildiers, S. Omar, et al., "Detection of micrometastatic disease and monitoring of perioperative tumor cell dissemination in primary operable breast cancer patients using real-time quantitative reverse transcription-PCR.", Clinical cancer research : an official journal of the American Association for Cancer Research, vol. 10, issue 1 Pt 1, pp. 196-201, 2004 Jan 1. Abstract

PURPOSE: We previously found a statistically significant number of cytokeratin 19 (CK19)+ cells in peripheral blood (PB) of stage IV breast cancer (BC) patients compared with those of healthy volunteers, using a quantitative real-time reverse transcription-PCR. We aimed to apply the technique on bone marrow (BM) of primary operable BC patients. Pre- and postoperative PB samples of these patients were further analyzed to investigate possible shedding of CK19+ cells during the operation.

EXPERIMENTAL DESIGN: In 54 primary operable BC patients, we analyzed 50 BM samples taken preoperatively and 297 PB samples. PB samples were collected before surgery; immediately after surgery; on the first, second, and fifth day postoperatively; and one month postoperatively.

RESULTS: In BM of controls and BC patients, we detected a median of 28 and 568 CK19+ cells/5 x 10(6) leukocytes, respectively (P < 0.001). In preoperative blood (B-1) samples, we measured a median of 109 CK19+ cells. Using the upper limit of 95% confidence interval of controls as cutoff, 74% and 52% of BM and (B-1), respectively were considered CK19+. There was no significant correlation between CK19+ cells in BM and (B-1) and classical prognostic factors. We found no significant difference between blood samples at different time points with respect to the average CK19+ cells.

CONCLUSIONS: In primary BC patients, we detected high numbers of CK19+ cells in BM and PB (B-1) samples compared with controls. However, no significant correlation between the presence of CK19+ cells in BM and PB and classical prognostic factors was found. We detected no statistically significant influence of surgical manipulation on CK19+ cells.

El-Din, H. A. M., M. A. Attia, M. R. Hamza, H. M. Khaled, A. - H. M. Thoraya, and S. A. Eisa, "Hepatitis C Virus and related changes in immunological parameters in non Hodgkin's lymphoma patients.", The Egyptian journal of immunology / Egyptian Association of Immunologists, vol. 11, issue 1, pp. 55-64, 2004. Abstract

Viral hepatitis is a common and important problem in immunocompromised cancer patients. The present study was conducted to investigate changes in some cellular and humoral immunological parameters as a consequence of HCV infection in non Hodgkin's lymphoma patients (NHL). The study included 40 NHL patients: 20 anti-HCV antibody positive (Gr. I ), and 20 anti-HCV antibody negative (Gr.II ). In addition, forty non-cancer controls (NCCs) were included: 20 of them were anti-HCV antibody positive (Gr. III) and 20 anti-HCV antibody negative (Gr. IV). The studied immunological parameters included serum levels of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), and soluble tumor necrosis factor receptors (s-TNFr) measured by ELISA, as well as assessment of T and B lymphocyte subsets by PAP immunostaining method. Mean IL-1 level (pg/ml) was significantly higher in Gr. 1 (14 +/- 6) and Gr. III (20 +/- 12) as compared to those in Gr. II (7 +/- 5) and Gr. IV (9 +/- 6). Mean IL-2 level (pg/ml) was also significantly higher in Gr. I (132 +/- 101) and Gr. III (135 +/- 59) compared to those in Gr. II (36 +/- 29) and Gr. IV (31 +/- 48). On the other hand, level of IL-6 showed no significant difference between groups. The mean level of sTNF-r, (ng/ml) was only significantly higher in Gr. I (2.9 +/- 1.7) when compared to that in Gr. IV (1.9 +/- 2.2). In group IV, the average percentage of CD3 (70 +/- 4%) and CD4 (44 +/- 5%) were significantly higher than in those of Gr. I (CD3 = 51 +/- 11%, CD4 = 30 +/- 12%), Gr. II (CD3 = 52 +/- 7%, CD4 = 30 +/- 8%), and Gr. III (CD3 = 52 +/- 9%, CD4 = 26 +/- 8%). From all the above immunological and virological features two main tips could be inferred: (1) HCV leads a mild course of infection in NCCs evidenced by normal ALT level in all but 20% of subjects, normal IL-6, sTNF-r, lower counts of CD4+ T cells and hence a mild hepatocellular injury, and (2) In the immunocompromised NHL patients the virus leads potentially more aggressive course as evidenced by higher viremia, as well as significant elevation in sTNF-r, and CD8+ depression.

Khaled, H. M. a, M. S. c Aly, and N. b Mokhtar, "Chromosomal aberrations in Cis and Ta bilharzial bladder cancer: A theory of pathogenesis", Urologic Oncology: Seminars and Original Investigations, vol. 22, no. 6, pp. 443-447, 2004. AbstractWebsite

Background: Bladder cancer manifests many different forms ranging from superficial to aggressive muscle invasive stages, which suggests that various genetic alterations are involved. Several attempts have been made to establish correlations between specific genetic alterations and various stages of the disease. At the National Cancer Institute (NCI), Cairo, bladder cancer constitutes 30.3% of all cancers. Bladder cancer observed among Egyptians has a clinico-pathological profile that differs from transitional cell carcinoma (TCC) seen in the western world. Patients and Methods: We used fluorescence in situ hybridization to detect numerical chromosome changes in 25 patients presenting with carcinoma in situ and Ta lesions. Twenty-four cases had transitional cell carcinoma and one case had squamous cell carcinoma. Results: Five out of 24 TCC cases had diploid chromosome count with all the probes. Numerical chromosome aberrations were detected in 19 cases (79%). In eight cases, a loss of chromosome 9 was observed. In one case, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 17, whereas another three cases showed a gain of chromosome 7. Loss of chromosome Y was observed in nine of the 22 male cases studied (40.9%). The only case with SCC had normal diploid chromosome count with all the probes used. Conclusion: When the genetic basis of bilharzial related bladder cancer is fully understood, new diagnostic and therapeutic strategies will be developed, which in turn may promote better clinical management by pathologists and urologists. A theory of bilharzial related bladder cancer pathogenesis is suggested. © 2004 Elsevier Inc. All rights reserved.

Khaled, H. M. a, M. S. c Aly, and N. b Mokhtar, "Chromosomal aberrations in Cis and Ta bilharzial bladder cancer: A theory of pathogenesis", Urologic Oncology: Seminars and Original Investigations, vol. 22, no. 6, pp. 443-447, 2004. AbstractWebsite

Background: Bladder cancer manifests many different forms ranging from superficial to aggressive muscle invasive stages, which suggests that various genetic alterations are involved. Several attempts have been made to establish correlations between specific genetic alterations and various stages of the disease. At the National Cancer Institute (NCI), Cairo, bladder cancer constitutes 30.3% of all cancers. Bladder cancer observed among Egyptians has a clinico-pathological profile that differs from transitional cell carcinoma (TCC) seen in the western world. Patients and Methods: We used fluorescence in situ hybridization to detect numerical chromosome changes in 25 patients presenting with carcinoma in situ and Ta lesions. Twenty-four cases had transitional cell carcinoma and one case had squamous cell carcinoma. Results: Five out of 24 TCC cases had diploid chromosome count with all the probes. Numerical chromosome aberrations were detected in 19 cases (79%). In eight cases, a loss of chromosome 9 was observed. In one case, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 17, whereas another three cases showed a gain of chromosome 7. Loss of chromosome Y was observed in nine of the 22 male cases studied (40.9%). The only case with SCC had normal diploid chromosome count with all the probes used. Conclusion: When the genetic basis of bilharzial related bladder cancer is fully understood, new diagnostic and therapeutic strategies will be developed, which in turn may promote better clinical management by pathologists and urologists. A theory of bilharzial related bladder cancer pathogenesis is suggested. © 2004 Elsevier Inc. All rights reserved.

Gutiérrez, M. I. a, A. K. a Siraj, H. b Khaled, N. c Koon, W. c El-Rifai, and K. a d e Bhatia, "CpG island methylation in Schistosoma- and non-Schistosoma-associated bladder cancer", Modern Pathology, vol. 17, no. 10, pp. 1268-1274, 2004. AbstractWebsite

Urothelial carcinomas (TCC) constitute the vast majority of bladder cancers in most of the world. On the other hand, squamous cell bladder carcinoma, a rare subtype in the Western world, is a common subtype in areas with endemic Schistosoma infection. Although schistosomal infection has been reported to influence DNA methylation, the pattern and extent of CpG island hypermethylation in squamous cell carcinomas remain unknown. In this study, we used methylation-specific PCR to characterize 12 cancer-related genes in 41 bladder cancer samples from Egypt (31 squamous cell carcinomas (SCC), 21 of them associated with Schistosoma and 10 TCC, five of which were Schistosoma- associated). The genes analyzed included E-cadherin, DAP-Kinase, O 6MGMT, p14, p15, p16, FHIT, APC, RASSF1A, GSTP1, RARβ and p73. Methylation of at least one gene was detected in all squamous cell tumors except two, and 45% of samples had at least three methylated genes. The average methylation index was 0.24, corresponding to three of the 12 analyzed genes. Schistosoma-associated tumors had more genes methylated than non-Schistosoma tumors (average MI: 0.29 vs 0.14) (P = 0.027). Although the extent of methylation in TCC (average MI: 0.16) was lower than in squamous cell carcinomas (SCC), the overall profile of methylation was similar, with Schistosoma-associated cases having a higher methylation index. Our results suggest that schistosomal involvement associates with a greater degree of epigenetic changes in the bladder epithelium.

Gutiérrez, M. I. a, A. K. a Siraj, H. b Khaled, N. c Koon, W. c El-Rifai, and K. a d e Bhatia, "CpG island methylation in Schistosoma- and non-Schistosoma-associated bladder cancer", Modern Pathology, vol. 17, no. 10, pp. 1268-1274, 2004. AbstractWebsite

Urothelial carcinomas (TCC) constitute the vast majority of bladder cancers in most of the world. On the other hand, squamous cell bladder carcinoma, a rare subtype in the Western world, is a common subtype in areas with endemic Schistosoma infection. Although schistosomal infection has been reported to influence DNA methylation, the pattern and extent of CpG island hypermethylation in squamous cell carcinomas remain unknown. In this study, we used methylation-specific PCR to characterize 12 cancer-related genes in 41 bladder cancer samples from Egypt (31 squamous cell carcinomas (SCC), 21 of them associated with Schistosoma and 10 TCC, five of which were Schistosoma- associated). The genes analyzed included E-cadherin, DAP-Kinase, O 6MGMT, p14, p15, p16, FHIT, APC, RASSF1A, GSTP1, RARβ and p73. Methylation of at least one gene was detected in all squamous cell tumors except two, and 45% of samples had at least three methylated genes. The average methylation index was 0.24, corresponding to three of the 12 analyzed genes. Schistosoma-associated tumors had more genes methylated than non-Schistosoma tumors (average MI: 0.29 vs 0.14) (P = 0.027). Although the extent of methylation in TCC (average MI: 0.16) was lower than in squamous cell carcinomas (SCC), the overall profile of methylation was similar, with Schistosoma-associated cases having a higher methylation index. Our results suggest that schistosomal involvement associates with a greater degree of epigenetic changes in the bladder epithelium.

Aly, M. S. a, and H. M. b Khaled, "Detection of C-erb B2 gene amplification in bilharzial associated bladder cancer using fluorescence in situ hybridization", Urologic Oncology: Seminars and Original Investigations, vol. 22, no. 6, pp. 448-452, 2004. AbstractWebsite

Background: Gene amplifications are common events in different tumor types and may confer diagnostic, prognostic, or therapeutic information for patient management. Fluorescence in situ hybridization (FISH) represents a standard methodologic approach for testing for this genetic alteration, as it is rapid, reproducible and extremely reliable in detecting presence of C-erb-B2 gene amplification for clinical utility. Patients and Methods: In this study, FISH is used in a series of archival human bilharzial bladder cancer specimens to evaluate for the presence of cerbB-2 gene alterations in the most common malignant tumor in bilharzial endemic areas, e.g., Egypt and some other countries. The study included 40 cases, 30 males and 10 females. Their ages ranged between 30 years and 76 years (median: 51 years). Twenty-one cases had squamous cell carcinoma, 16 had transitional cell carcinoma, two had adenocarcinoma, and one case had undifferentiated carcinoma. Results: Thirteen out of 40 tumor samples (32.5%) show evidence of true C-erb-B2 gene amplification. Of the remaining samples, 24 (60%) show no gene amplification and three (7.5%) fall into the borderline category with a ratio between one and two C-erb-B2 genes/cell relative to chromosome 17 centromeres. No evidence of chromosome 17 polysomy was found in any cases scored as single copy with the C-erb-B2 probe. Conclusion: No significant association was found between gene amplification and any of the tested clinicopathologic parameters or tumor recurrence except for tumor grade where higher tumor grades tended to be associated with more C-erb-B2 gene amplification (P = 0.01) thus reflecting more tumor aggressiveness. So, the amplification of C-erb-B2 in bilharzial associated bladder cancer is probably not independently related to clinical outcome of patients. © 2004 Elsevier Inc. All rights reserved.

Aly, M. S. a, and H. M. b Khaled, "Detection of C-erb B2 gene amplification in bilharzial associated bladder cancer using fluorescence in situ hybridization", Urologic Oncology: Seminars and Original Investigations, vol. 22, no. 6, pp. 448-452, 2004. AbstractWebsite

Background: Gene amplifications are common events in different tumor types and may confer diagnostic, prognostic, or therapeutic information for patient management. Fluorescence in situ hybridization (FISH) represents a standard methodologic approach for testing for this genetic alteration, as it is rapid, reproducible and extremely reliable in detecting presence of C-erb-B2 gene amplification for clinical utility. Patients and Methods: In this study, FISH is used in a series of archival human bilharzial bladder cancer specimens to evaluate for the presence of cerbB-2 gene alterations in the most common malignant tumor in bilharzial endemic areas, e.g., Egypt and some other countries. The study included 40 cases, 30 males and 10 females. Their ages ranged between 30 years and 76 years (median: 51 years). Twenty-one cases had squamous cell carcinoma, 16 had transitional cell carcinoma, two had adenocarcinoma, and one case had undifferentiated carcinoma. Results: Thirteen out of 40 tumor samples (32.5%) show evidence of true C-erb-B2 gene amplification. Of the remaining samples, 24 (60%) show no gene amplification and three (7.5%) fall into the borderline category with a ratio between one and two C-erb-B2 genes/cell relative to chromosome 17 centromeres. No evidence of chromosome 17 polysomy was found in any cases scored as single copy with the C-erb-B2 probe. Conclusion: No significant association was found between gene amplification and any of the tested clinicopathologic parameters or tumor recurrence except for tumor grade where higher tumor grades tended to be associated with more C-erb-B2 gene amplification (P = 0.01) thus reflecting more tumor aggressiveness. So, the amplification of C-erb-B2 in bilharzial associated bladder cancer is probably not independently related to clinical outcome of patients. © 2004 Elsevier Inc. All rights reserved.

b Ismail, M. S. a, W. a Wynendaele, J. L. E. a Aerts, R. a c Paridaens, R. b Gaafar, N. b Shakankiry, H. M. b Khaled, M. - R. a Christiaens, H. a Wildiers, S. b Omar, et al., "Detection of Micrometastatic Disease and Monitoring of Perioperative Tumor Cell Dissemination in Primary Operable Breast Cancer Patients Using Real-Time Quantitative Reverse Transcription-PCR", Clinical Cancer Research, vol. 10, no. 1 I, pp. 196-201, 2004. AbstractWebsite

Purpose: We previously found a statistically significant number of cytokeratin 19 (CK19)+ cells in peripheral blood (PB) of stage IV breast cancer (BC) patients compared with those of healthy volunteers, using a quantitative real-time reverse transcription-PCR. We aimed to apply the technique on bone marrow (BM) of primary operable BC patients. Pre- and postoperative PB samples of these patients were further analyzed to investigate possible shedding of CK19+ cells during the operation. Experimental Design: In 54 primary operable BC patients, we analyzed 50 BM samples taken preoperatively and 297 PB samples. PB samples were collected before surgery; immediately after surgery; on the first, second, and fifth day postoperatively; and one month postoperatively. Results: In BM of controls and BC patients, we detected a median of 28 and 568 CK19+ cells/5 × 106 leukocytes, respectively (P < 0.001). In preoperative blood (B-1) samples, we measured a median of 109 CK19+ cells. Using the upper limit of 95% confidence interval of controls as cutoff, 74% and 52% of BM and (B-1), respectively were considered CK19+. There was no significant correlation between CK19+ cells in BM and (B-1) and classical prognostic factors. We found no significant difference between blood samples at different time points with respect to the average CK19+ cells. Conclusions: In primary BC patients, we detected high numbers of CK19+ cells in BM and PB (B-1) samples compared with controls. However, no significant correlation between the presence of CK19+ cells in BM and PB and classical prognostic factors was found. We detected no statistically significant influence of surgical manipulation on CK19+ cells.

b Ismail, M. S. a, W. a Wynendaele, J. L. E. a Aerts, R. a c Paridaens, R. b Gaafar, N. b Shakankiry, H. M. b Khaled, M. - R. a Christiaens, H. a Wildiers, S. b Omar, et al., "Detection of Micrometastatic Disease and Monitoring of Perioperative Tumor Cell Dissemination in Primary Operable Breast Cancer Patients Using Real-Time Quantitative Reverse Transcription-PCR", Clinical Cancer Research, vol. 10, no. 1 I, pp. 196-201, 2004. AbstractWebsite

Purpose: We previously found a statistically significant number of cytokeratin 19 (CK19)+ cells in peripheral blood (PB) of stage IV breast cancer (BC) patients compared with those of healthy volunteers, using a quantitative real-time reverse transcription-PCR. We aimed to apply the technique on bone marrow (BM) of primary operable BC patients. Pre- and postoperative PB samples of these patients were further analyzed to investigate possible shedding of CK19+ cells during the operation. Experimental Design: In 54 primary operable BC patients, we analyzed 50 BM samples taken preoperatively and 297 PB samples. PB samples were collected before surgery; immediately after surgery; on the first, second, and fifth day postoperatively; and one month postoperatively. Results: In BM of controls and BC patients, we detected a median of 28 and 568 CK19+ cells/5 × 106 leukocytes, respectively (P < 0.001). In preoperative blood (B-1) samples, we measured a median of 109 CK19+ cells. Using the upper limit of 95% confidence interval of controls as cutoff, 74% and 52% of BM and (B-1), respectively were considered CK19+. There was no significant correlation between CK19+ cells in BM and (B-1) and classical prognostic factors. We found no significant difference between blood samples at different time points with respect to the average CK19+ cells. Conclusions: In primary BC patients, we detected high numbers of CK19+ cells in BM and PB (B-1) samples compared with controls. However, no significant correlation between the presence of CK19+ cells in BM and PB and classical prognostic factors was found. We detected no statistically significant influence of surgical manipulation on CK19+ cells.

el-Din, H. M., M. A. Attia, M. R. Hamza, H. M. Khaled, M. A. Thoraya, and S. A. Eisa, "Hepatitis C Virus and related changes in immunological parameters in non Hodgkin's lymphoma patients.", Egypt J Immunol, vol. 11, no. 1, pp. 55-64, 2004. AbstractWebsite

{Viral hepatitis is a common and important problem in immunocompromised cancer patients. The present study was conducted to investigate changes in some cellular and humoral immunological parameters as a consequence of HCV infection in non Hodgkin's lymphoma patients (NHL). The study included 40 NHL patients: 20 anti-HCV antibody positive (Gr. I ), and 20 anti-HCV antibody negative (Gr.II ). In addition, forty non-cancer controls (NCCs) were included: 20 of them were anti-HCV antibody positive (Gr. III) and 20 anti-HCV antibody negative (Gr. IV). The studied immunological parameters included serum levels of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), and soluble tumor necrosis factor receptors (s-TNFr) measured by ELISA, as well as assessment of T and B lymphocyte subsets by PAP immunostaining method. Mean IL-1 level (pg/ml) was significantly higher in Gr. 1 (14 +/- 6) and Gr. III (20 +/- 12) as compared to those in Gr. II (7 +/- 5) and Gr. IV (9 +/- 6). Mean IL-2 level (pg/ml) was also significantly higher in Gr. I (132 +/- 101) and Gr. III (135 +/- 59) compared to those in Gr. II (36 +/- 29) and Gr. IV (31 +/- 48). On the other hand, level of IL-6 showed no significant difference between groups. The mean level of sTNF-r, (ng/ml) was only significantly higher in Gr. I (2.9 +/- 1.7) when compared to that in Gr. IV (1.9 +/- 2.2). In group IV, the average percentage of CD3 (70 +/- 4%) and CD4 (44 +/- 5%) were significantly higher than in those of Gr. I (CD3 = 51 +/- 11%

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