Recent references:

Estimation of Binding Affinities of FKBP12 Inhibitors Using a Linear
Response Method. M. L. Lamb, J. Tirado-Rives & W. L. Jorgensen,
Bioorg. Med. Chem., 7, 851-860 (1999).

Origin of the Selectivity of Celecoxib Analogs with COX-1 and COX-2
from Docking and Monte Carlo Simulations. M. L. Plount-Price and
W. L. Jorgensen, J. Am. Chem. Soc., 122, 9455 (2000).

Rationale for the Observed COX-2/COX-1 Selectivity of Celecoxib from
Monte Carlo Simulations. M. L. P. Price and W. L. Jorgensen,
Bioorg. Med. Chem. Lett. 11, 0000 (2001). In press.

Validation of a Model for the Complex of HIV-1 Reverse Transcriptase
with Sustiva through Computation oof Resistance Profiles. R. C. Rizzo,
D.-P. Wang, J. Tirado-Rives, and W. L. Jorgensen, J. Am. Chem. Soc.,
122, 12898 (2000).

Estimation of Binding Affinities for HEPT and Nevirapine Analogs with
HIV-1 Reverse Transcriptase via Monte Carlo Simulations. R. C. Rizzo,
J. Tirado-Rives, and W. L. Jorgensen, J. Med. Chem., 44, 145 (2001).

Prediction of Properties from Simulations. W. L. Jorgensen and E. M.
Duffy, J. Am. Chem. Soc., 122, 2878 (2000). Bioorg. Med. Chem. Lett.
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Role of Multi Drug Resistance (MDR) Phenotype as a Predictive Marker of Chemotherapy and as a Prognostic Marker Breast Carcinoma (Poster Presentation)

A. Srivastava, Ph.D., MPH, FRCS, S.R Chaudhary, M.S, M.C. Mishra, M.S,
O. Coshic, M.S., FRCS, S.N Das, M.D, and *A. Goyal, M.S
Department of Surgery, All India Institute of Medical Sciences, New Delhi, India.

Background: Patients who respond to chemotherapy have a longer survival and enjoy better quality of life. Development of tests to predict response to chemotherapy will help to accurately select patients who may or may not benefit from such therapy. P-glycoprotein expression has been strongly associated with aggressive biologic behaviour, poor treatment response and poor outcome in many tumors. P-glycoprotein acts as a molecular pump that can extrude a wide variety of drugs from the cell. This lowers the intracellular drug concentration at the target and hence, results in drug resistance.

Aims and Objectives: a) To carry out sequential assessment of MDR gene expression before and after first cycle of neoadjuvant chemotherapy and to evaluate its role as a predictive marker for breast cancer response. b) To evaluate the role of MDR gene expression as a prognostic marker for local and systemic recurrences in patients undergoing post-operative adjuvant chemotherapy for breast cancer.

Material and Methods: Group A included 26 patients of biopsy/FNAC- proven breast cancer with locally advanced stage (IIIA, IIIB). They were studied for the multidrug resistant gene/p-glycoprotein(Pgp) expression before neoadjuvant chemotherapy (CAF/CMF) (day 0) and after one cycle of chemotherapy (CAF/CMF) (day 21). Chemotherapy responses were clinically assessed after three cycles of chemotherapy using the UICC criteria.

Group B included 21 patients of early breast cancer (stageI,II) undergoing modified radical mastectomy/conservative surgery followed by adjuvant chemotherapy with CAF/CMF. Tumor tissues taken from the specimen after surgery was analysed for p-glycoprotein expression. Follow-up information on local and systemic recurrence was recorded. We tried to evaluate the role of p-glycoprotein expression as a prognostic marker for local and systemic recurrence in these patients.

Flow cytometric analysis of p-glycoprotein was done by fluoroscence activated cell staining, (FAC-SCAN) from the tissues taken by FNAC/operative specimen. Relation of MDR protein to known prognostic factors e.g lymph node status, menopausal status was assessed in both groups.

Results: Group A: Clinically, after three cycles of chemotherapy 10 patients (38.5%) had complete response, 9 had partial response(34.6 %) and 7 patients (26.2%) had no response. The mean Pgp expression on day 0(before chemotherapy) and day 21(after one cycle of chemotherapy) in patients with complete response were 18.73%(SD-19.9, 95%CI= 3.36-27.09) and 10.97%(SD-18.52, 95%CI= –1.95-24.09) respectively(difference not statistically significant on Kruskal-Walls H test). The mean expression on day 0 and day 21 in patients with partial response were 34.38%(SD-30.5, 95%CI=11.05-56.39) and 27.2%(SD-38.4, 95%CI= -2.32-56.72) respectively (difference not statistically significant). The mean expression on day 0 and day 21 in patients with no response were 45.82%(SD-19.8, 95%CI=17.19-63.86) and 45.94%(SD-35.94, 95%CI=12.69-79.18) respectively (difference not statistically significant).

Group B: Mean Pgp expression in T1, T2, and T3 were 6.2%, 33.8% and 19.2% respectively. In 1 year follow up, only 1 patient developed systemic recurrence. There was no significant correlation between p-glycoprotein expression on both the days with systemic metastasis, lymphnode status and menopausal status in patients of both groups.

Conclusions: P-glycoprotein may be a useful marker in predicting the response to neoadjuvant chemotherapy for breast cancer. No comment is possible on the association of p-glycoprotein expression and local and systemic recurrence due to the short period of follow up. Determinations of p-glycoprotein levels in patients at diagnosis or relapse may have a major role in the design of future treatment.

Recent Advances in Brachytherapy

*Dattatreyudu Nori,M.D.,F.A.C.R., Jaganmohan Poli, M.D., and Boris Spektor, M.D.
New York Presbyterian Hospital – Weill Cornell Medical College &
The New York Hospital Medical Center Queens.

The treatment of cancer with radioactive sources using topical molds and intracavitary radium showed the efficacy of brachytherapy long before external radiation therapy became common practice. Recent technological innovations resulted in significant advances in the practice of brachytherapy . A major change in brachytherapy is the development of high intensity sources and technological innovations in high dose rate (HDR) brachytherapy. Advantages of HDR brachytherapy include outpatient treatment, avoidance of general anesthesia for most patients, and elimination of complications resulting from prolonged bed rest. HDR brachytherapy can provide improved physical dose delivery because of short treatment time and fixed applicator positioning, and optimized dosimetry with a computer – controlled stepping source. HDR technology is the basis of pulse- dose- rate (PDR) brachytherapy that uses the high intensity source for brief fractions over a low dose rate treatment. PDR combines many of the physical advantages of a single – stepping source remote afterloading design with the radiobiological advantages of conventional LDR brachytherapy.

Progress in comuterization and calculation modeling has promoted optimization in brachytherapy. Optimization in brachytherapy entails calculating the source strengths or source times to satisfy a set of dose criteria to achieve the best dose distribution for a brachytherapy implant. Optimization can be achieved by :

use of variable source strengths so that higher intensity sources are used in the periphery and lower intensity sources in the center
by variable source spacing with closer spacing of the sources in the periphery and more distant spacing of the sources toward the center ;
by using a single high intensity stepping source of 192 iridium with variable dwell time as in HDR and PDR brachytherapy.
By using real-time optimization such as the ultrasound based real-time optimization program. This is a significant improvement in that it allows the brachytherapist to visualize a three-dimensional isodose distribution within the tumor volume, and allows the brachytherapist to perform optimum placement of radio active sources.

Technology to support brachytherapy continues to advance rapidly. The interaction between images from diverse modalities and the various phases of the treatment planning is the basis of enhanced treatment planning procedures. Conventional brachytherapy dose distribution is displayed in two dimensions. This does not adequately represent the complexities within an implanted volume. For a more accurate representation, one has to account for tissue inhomogeneity, the effects of neighboring radioactive sources, to compensate for the applicator/carrier material, and to display resultant dose distribution in three dimensions. Multiple CT scan images of the region of interest are transferred to a three- dimensional reconstruction computer, which displays the isodose distribution three dimensionally in relation to the anatomical structures outlined. The use of isodose distribution produced by the radioactive sources in relation to the tumor and critical normal tissues.

The dose distribution from brachytherapy is inhomogeneous. Areas of high doses ("hot spots") within the treatment volume contribute to complications if they are close to critical normal tissues. Conversely, areas of low doses ("cold spots") reduce tumor control. A dose volume histogram (DVH) more accurately describes this dose variation by specifying the volume irradiated at a certain dose level. DVH is used to critically evaluate competing treatment plans and too critically evaluate competing treatment plans and to optimize the therapeutic ratio of the treatment.

Endovascular brachytherapy is set to explode onto the radiation oncology scene. Recent novel approaches of using radiotherapy for prevention of restenosis in coronary and peripheral vessels have interested laboratory and clinical researchers. Restenosis of arteries is a major limitation of angioplasty. Endovascular brachytherapy may contribute to the success of interventional techniques by preventing restenosis.

With advances in computerization an robotic engineering, we foresee brachytherapy being delivered by robotic control to allow access to clinical sites with a precision that is not possible by manual control. This technique has immediate application for brain tumors.

Historical Technical Developments in Mammography

Arthur G. Haus, Ph.D.
Medical Physicist, Delaware, Ohio

In mammograqphy, it is most important to consistently produce high-contrast, high-resolution images at the lowest radiation dose consistent with high image quality. In recent years, there have been many significant technological improvements in mammographic x-ray equipment, image recording systems, and viewing conditions. Some of the major technical development milestones in mammographic imaging are shown in Table 1. Until the mid 1980’s, many x-ray units were used that were not dedicated to mammography. These x-ray units had tungsten target tubes that were designed originally for medical imaging procedures, such as chest radiography. Some of these units had compression devices that were home made; therefore, breast compression was less than optimal by today’s standards. Many of these units had very large focal spots or short focal spot-to-breast surface distances that could result in significant geometric blur (unsharpness). Direct exposure (industrial type) x-ray films were being used, which often required long exposure times (causing blur by motion) and which resulted in high radiation exposure. In addition, viewing conditions were inadequate.

Today, mammography is performed with dedicated mammographic x-ray equipment. These units have specially designed tube targets, smaller focal spots, and significantly improved breast compression devices, among other features. Cassettes and screen-film combinations are designed specifically for mammography. Film processing and viewing conditions also have improved significantly over the years. In 2000, the first digital mammography system was approved by the Food and Drug Administration (FDA) for clinical use.

The American College of Radiology (ACR) Mammography Accreditation Program introduced in 1987, the ACR Quality Control Manuals introduced in 1992, and the Mammography Quality Standards Act which was implemented in 1994, have also had a significant impact on the improvement of the technical quality of mammographic images in the United States.

In summary, today it is possible to obtain mammograms with higher image quality that require significantly lower radiation doses compared with mammograms dating back to the 1970s and early 1980s. Some of the important technological improvements and quality control programs that have let to todays high quality mammographic images are discussed in this presentation.

Table 1. Technical Advances in Mammography

Year	Development
Prior to 1969	Conventional tungsten target x-ray tubes with direct exposure industrial type films were used
1969	Dedicated mammographic unit with molybdenum target tube and compression cone introduced (CGR Senographe)
1971	Xeroradiography system introduced for mammography (Xerox)
1972	Screen-film system introduced for mammography (Du Pont Lo-dose system)
1976	Rare earth screen-film system and special cassette introduced for mammography (Kodak Min-R system)
1977	Mammography x-ray unit for magnification with microfocal spot introduced (Radiological Sciences Inc.)
1978	Mammography unit with grid introduced (Philips)
1987	American College of Radiology Mammography Accreditation Program (ACR MAP) begins
1992	American College of Radiology Mammography Quality Control Manual for Radiologists, Radiologic Technologists, and Medical Physicists, introduced
1994	The Food and Drug Administration (FDA) implements the Mammography Quality Standards Act (MQSA)
2000	Digital mammography system approved by the FDA for clinical use (GE Senographe 2000D)

References

American College of Radiology (ACR) Mammography Quality Control Manual for Radiologists, Radiologic Technologists and Medical Physicists. American College of Radiology, Reston, VA 1999.

Haus, AG Physical Principles and Radiation Dose in Mammography. In Feig, SA and McLelland, R, editors. Breast Carcinoma Current Diagnosis and Treatment. Masson Publishing, New York, NY 1983.

Haus, AG, Cullinan, JE, Screen-Film Processing Systems for Medical Radiography: A Historical Review: Radiographics 9, 1989

Haus, AG, Yaffe, MJ, Screen-film and Digital Mammography: Image Quality and Radiation Dose Considerations, in Feig, SA. Editor Breast Imaging-Radiologic Clinics of North America. W.B. Saunders 38-4, Philadelphia, PA July 2000

Haus, AG. Yaffe, MJ, editors Categorical Course in Diagnostic Radiology Physics: Physical Aspects of Breast Imaging Current and Future Considerations. Syllabus. Radiological Society of North America. Oak Brook, IL 1999

Rothenberg, LN, Haus, AG, Physicists in Mammography – A Historical Perspective, Medical Physics 22; 11, November 1995

Comparison of Ultrasonography and Scintimammography for the Diagnosis of Breast Lesions (Poster presntation)

*Qian Yu¹ Wang Jin2 Li Yi²
(1) Department of Breast, Tumor Hospital, Tianjin Medical University Tianjin, China 300060
(2) Department of Ultrasound, Tianhe Hospital, Tianjin, China 300072

Objective: To compare the results of ultrasonography and scintimammography for the detection of breast lesions, and determine whether scintimammography can provide effective assistance in sonographic diagnosis of breast lesions.

Methods: 120 patients with breast lesions underwent both sonography and scintimammography. All patients had histopathological confirmation of the nature of the breast lesions, 69 of which were malignant and 51 were benign.

Results: Sonography was positive in 72 patients (65 true positive, 7 false positive) and negative in 48 patients (44 true negative, 4 false negative). The corresponding values of scintimammography were 73 (57, 16) and 47 (35, 12). The sensitivity of sonography was 94.2%, the specificity was 86.3%, and the positive and negative predictive values were 90.3% and 91.6%. The corresponding values for scintimammography were 82.6%, 68.6%, 78.15% and 74.4% respectively. The difference between them was statistically significant. (c² test).

Conclusion: The accuracy of sonography is better than scintimammography. After sonography has yielded questionable results, combined sonography plus scintimammography can improve the detection of breast cancer at the expense of additional false positive results.

Study on the Distinctive Infrared Spectral Features in Breast Cancer Specimen (Poster presntation)

Li Liu¹, *Qian Yu², Minjin Yang¹
(1) Center of Biomedical Analysis, Nankai University, Tianjin 300070, China
(2) Dept. of Breast, Tumor Hospital, Tianjin Medical University, Tianjin 300060, China

Substantial differences were found in the spectral properties of surgical samples from 20 women patients with histologically confirmed normal and cancerous breast tissues. The most striking changes in the spectra were observed in the symmetric and asymmetric stretching bends of phosphodiester groups, which shifted to a shorter wavenumber ~3 cm^–1 in v_sPO^-₂ , and to a longer wavenumber, ~2 cm^–1, in vs . The ratio of A_{1 173} /A_{1 163} increased and that of A_{1 025}/ A_{1 082} decreased, the intensity of symmetric and asymmetric stretching bands of CH₃ decreased and those of CH₂ increased in all of the breast cancer samples. Our findings indicate that in breast cancer tissue, the degree of hydrogen-bonding of oxygen atoms in the backbone of nucleic acid increased; the content of glycogen decreased; the degree of hydrogen-bonding of OH groups in serine tyrosine, and threonine residues of cell proteins decreased; and also there were changes in the packing and the conformational structure of the methylene chains of membrane lipids.

Establishment of an Animal Model of Parotid Gland Squamous Cell Carcinoma Induced by DMBA (Poster presntation)

Xilin Xiang DDS
Department of Stomatology
Shanghai Sixth People's Hospital
Yishan RD 600#, Shanghai City 200233
P.R.China

Objective: To establish an animal model of parotid gland (PG) tumor of the squamous cell carcinoma (SCC) of salivary gland and use it to research carcinogenesis

Methods: A histopathological approach for carcinogenesis in rat PGs induced by using 9,10-dimethyl-1,2-benzanthracene (DMBA) was evaluated. A total of 60 male and female Sprague-Dawley (SD) rats of 8 weeks old and 180-200g weight were obtained from the Animal Center of Shanghai. Under pentobarbital sodium anesthesia, the right PGs were exposed by surgical procedure. A sponge pellet (1.5mm_1.5mm_1.5mm) was used as the carrier of the carcinogen. The sponge containing 2% DMBA (Fluka, Switzerland)/acetone solution was implanted into the glandular tissue of the right PGs. Six rats (3 males and 3 females) were killed every 2 weeks after the DMBA/sponge implantation. The same method of sponge implantation without DMBA was used at the left side of PG as a control. All rats left were killed after 20 weeks. The PGs were fixed in 10% formalin buffer solution for 24 hours, and embedded in paraffin, then 4_m-thick sections were made for the histopathological study.

Results: The earliest tumor occurred 4 weeks after implantation of the sponge, a total of 25 lateral tumors were induced (12 females, 13 males). No tumor was found in the controlled PG. The peak time of tumorogenesis was 8-12 weeks after implantation; all tumors induced were squamous cell carcinomas. The induced tumors grew slowly, which were present as nodular masses without capsules, and the borders were not clear. They were slightly hard when palpated. The process of carcinogenesis can be described as follows: squamous metaplasia of cyst-like structures occurred, then SCCs were induced and invaded surrounding tissues. No metastasis was observed in regional lymph nodes and other organs.

Conclusions: SCCs of PG can be induced by implantation of DMBA. The present study supports the conclusion that all duct segments undergo squamous metaplasia, and therefore may participate in the genesis of neoplasia during experimental carcinogenesis.

Key words: parotid gland, squamous cell carcinoma, animal model, 9,10-dimethyl-1, 2-benzanthracence

Towards Non-invasive Imaging of Gene Expression by Positron Emission Tomography.

A. Jacobs, MD, M. Hartung, PhD, A. Winkeler, PhD, C. Dittmar, PhD, W.D. Heiss, MD.

Max-Planck-Institute for Neurological Research, Center for Molecular Medicine (ZMMK) and Department of Neurology at the University of Cologne, Germany. E-mail: Andreas.Jacobs@pet.mpin-koeln.mpg.de.

Objective: Construction and functional evaluation of HSV-1 amplicon vectors mediating proportional co-expression of HSV-1-tk as PET marker gene and gfp (green fluorescent protein) and E.coli cd (cytosine deaminase) genes.

Background: For the assessment of the clinical efficiency of gene therapy vectors it is desirable to determine the location, magnitude and duration of vector mediated therapeutic gene expression. The tk (thymidine kinase) gene of HSV-1 has been shown to function as a ‘marker gene’ for the non-invasive in vivo localisation of tk expression by PET (Tjuvajev et al. 1995-1999; Gambhir et al. 1998-2000). Using double-gene expressing cassettes with tk as PET marker gene, PET analysis could be used to monitor the distribution of the tk gene, which would indirectly indicate the location and expression of a second linked and proportionally co-expressed gene.

Methods: Several double-/tripple-gene constructs expressing HSV-1-tk, gfp and E.coli cd genes were engineered based on gene fusion or the use of an internal ribosome entry site (IRES) and cloned into HSV-1 amplicons. Functional analyses (i. gfp-expression; ii. sensitivity to the pro-drugs GCV and 5-FC) and Western blot analysis were carried out after infection of rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicons.

Results: All vector constructs mediated GFP expression and sensitised 9L and Gli36 cells towards GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. However, the levels of GFP expression and the LD50s as well as the levels of protein expression as assessed by Western analysis varied depending on the location of the genes within the constructs, indicating the influence of the IRE site on the level of expression of the second gene. Most importantly, proportional co-expression of the PET marker gene HSV-1-tk and the linked gfp gene and the therapeutic E.coli cd gene was observed.

Conclusions: Double-gene constructs carrying the HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect non-invasive localisation of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency is highly desirable and could have a critical impact on the clinical application, management and assessment of patients undergoing gene therapy for gliomas.

Supported in part by ZMMK grant TV46 and MSWF grant 516-400 002 99.

Detection of Amplification of the Telomerase Reverse Transcriptase Gene in Human Tumors by Fluorescence in Situ Hybridization Combined with Spectral Karotyping Technique (Poster Presentation)

Dawei Xu, MD, PhD, Anju Zhang, MD, Chengyun Zheng, MD, Charlotta Lindvall, MD, Mi Hou, MD, Zhongqun Yan, MD, Marie Henriksson, PhD, Elisabeth Blennow, MD, PhD, Magnus Nordenskjold, MD, PhD, Anders Zetterberg, MD, PhD, Magnus Bjorkholm, MD, PhD and Astrid Gruber, MD, PhD
Department of Medicine, Division of Hematology, Department of Oncology and Pathology, Department of Molecular Medicine and Microbiology & Tumorbiology Center, Karolinska Hospital and Institutet, SE-171 76, Stockholm, Sweden

Activation of telomerase is a crucial step during cellular immortalization and malignant transformation of human cells, and requires the induction of the catalytic component, telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. It is poorly understood how the hTERT gene is activated in human cancer cells. In the present study, we used fluorescence in situ hybridization (FISH) combined with the novel spectral karotyping (SKY) technique to examine the hTERT gene copy number in human cancer cell lines and in primary tumor tissues. Amplification of the hTERT gene was observed in 8/26 (31%) tumor cell lines. In the neuroblastoma cell line Lan2 cells, the hTERT was amplified in double minutes as shown by FISH/SKY. In addition, 17/58 (30%) primary tumors examined (8/21 lung, 3/10 cervix and 5/19 breast carcinomas, and 1/8 neuroblastomas). Thirteen of 26 (50%) cell lines and 13 of 58 (22%) primary tumors displayed gain of hTERT gene copies with 3 to 4 per cell. The present findings imply that the hTERT locus may be a frequent target for amplification during tumorigenesis and that this genetic event probably contributes to dysregulation of telomerase activity occurring in human tumors.

Real Time Quantitative telomeric repeat amplification protocol Assay for Telomerase Activity (Poster Presentation)

Mi Hou, MD, Dawei Xu, MD, PhD, Magnus Björkholm, MD, PhD, and Astrid Gruber, MD, PhD
Department of Medicine, Division of Hematology, Karolinska Hospital and Institutet, SE-171 76 Stockholm, Sweden

Telomerase is a ribonucleoprotein enzyme associated with cellular immortalization and malignant transformation of human cells. The telomeric repeat amplification protocol (TRAP) is widely used for the detection of telomerase activity. The TRAP method, although highly sensitive and specific based on PCR amplification, is laborious and insufficient to provide a precise quantitative information. We have developed a real time quantitative TRAP (RTQ-TRAP) system by combining a real time PCR technique with the conventional TRAP protocol. Telomerase activity in human tumor cell lines was measured using the RTQ-TRAP assay and the method was found to be both accurate and reproducible over a wide range. Telomerase activity in 13 lymphoma samples as determined by the RTQ-TRAP was 9 fold lower than that measured by the conventional TRAP. The result suggests that the conventional TRAP assay frequently leads to an overestimate of telomerase activity in tumor samples. The RTQ-TRAP method is thus a powerful tool to rapidly and precisely quantify telomerase activity.