by Ho, Wai Fun. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 79-87). / LIST OF TABLES --- p.iv / LIST OF FIGURES --- p.vi / ACKNOWLEDGEMENTS --- p.vii / ABSTRACT --- p.viii / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- METABOLIC DERANGEMENTS AND CACHEXIA IN CANCER --- p.1 / Chapter 1.2 --- PROTEIN METABOLISM IN MALIGNANCY --- p.4 / Chapter 1.3 --- REVIEW OF REPORTS ON AMINO ACID DISTURBANCES IN MALIGNANCY --- p.5 / Chapter 1.4 --- AMINO ACID ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY --- p.10 / Chapter 1.4.1 --- Amino Acid Analysis by Ion-Exchange HPLC --- p.11 / Chapter 1.4.2 --- Amino Acid Analysis by Reversed-Phase HPLC --- p.13 / Chapter 1.4.3 --- Derivatizing Agents --- p.15 / Chapter 1.5 --- CHOICE OF CANCER PATIENTS AND METHODOLOGY FOR THIS STUDY --- p.19 / Chapter 1.5.1 --- Choice of Cancer Patients --- p.19 / Chapter 1.5.2 --- Methodology Chosen and Its Principle --- p.20 / Chapter 2. --- OBJECTIVES --- p.23 / Chapter 3. --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- STUDY SUBJECTS --- p.24 / Chapter 3.1.1 --- Patients --- p.24 / Chapter 3.1.2 --- Control Subjects --- p.25 / Chapter 3.2 --- CLINICAL FEATURES --- p.25 / Chapter 3.3 --- BLOOD COLLECTION --- p.25 / Chapter 3.4 --- GENERAL BIOCHEMICAL TESTS --- p.26 / Chapter 3.5 --- PLASMA AMINO ACID ANALYSIS BY HPLC --- p.26 / Chapter 3.5.1 --- Apparatus --- p.26 / Chapter 3.5.2 --- Reagents --- p.27 / Chapter 3.5.3 --- Reagent Preparation --- p.28 / Chapter 3.5.3.1 --- Mobile phase --- p.28 / Chapter 3.5.3.2 --- Derivatizing reagent --- p.29 / Chapter 3.5.4 --- Standard Preparation --- p.29 / Chapter 3.5.4.1 --- Internal standard solution --- p.29 / Chapter 3.5.4.2 --- Composite standard solution --- p.30 / Chapter 3.5.4.3 --- Composite standard-internal standard mixture --- p.32 / Chapter 3.5.5 --- Sample Preparation --- p.32 / Chapter 3.5.5.1 --- Protein removal --- p.32 / Chapter 3.5.5.2 --- Addition of internal standard --- p.32 / Chapter 3.5.6 --- Preparation of Samples for the WISP Sample Processor --- p.33 / Chapter 3.5.7 --- Sample Queue for the WISP Sample Processor --- p.33 / Chapter 3.5.8 --- Automated Derivatization Procedure --- p.36 / Chapter 3.5.9 --- Chromatographic Conditions --- p.36 / Chapter 3.6 --- STATISTICAL STUDIES --- p.38 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- ANALYTICAL PERFORMANCES --- p.40 / Chapter 4.1.1 --- Chromatograms --- p.40 / Chapter 4.1.2 --- Precision --- p.47 / Chapter 4.1.3 --- Linearity --- p.47 / Chapter 4.1.4 --- Analytical Recovery --- p.51 / Chapter 4.2 --- DATA DISTRIBUTION STUDIES --- p.53 / Chapter 4.3 --- PATIENTS' ANTHROPOMETRIC DATA AND BLOOD BIOCHEMISTRY --- p.55 / Chapter 4.4 --- FREE PLASMA AMINO ACID CONCENTRATIONS IN NORMAL CONTROLS --- p.59 / Chapter 4.5 --- FREE PLASMA AMINO ACID CONCENTRATIONS IN CANCER PATIENTS --- p.59 / Chapter 5. --- DISCUSSION --- p.68 / Chapter 5.1 --- METHOD ESTABLISHMENT --- p.68 / Chapter 5.2 --- NORMAL CONTROLS --- p.68 / Chapter 5.3 --- CANCER PATIENTS --- p.71 / Chapter 5.3.1 --- Nasopharyngeal Cancer --- p.71 / Chapter 5.3.2 --- Lung Cancer --- p.71 / Chapter 5.3.3 --- Breast Cancer --- p.72 / Chapter 5.3.4 --- Colorectal Cancer --- p.74 / Chapter 5.4 --- SUMMARY OF THE PLASMA AMINO ACID PROFILES IN CANCER --- p.75 / Chapter 5.5 --- FURTHER STUDIES --- p.76 / Chapter 6. --- CONCLUSION --- p.78 / Chapter 7. --- REFERENCES --- p.79

by Chan, Kim Hung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 76-89). / Abstract also in Chinese. / Chapter 1. --- INTRODUCION --- p.1 / Chapter 1.1 --- amino acid analysis by high performance liquid chromatography --- p.1 / Chapter 1.1.1 --- History and Development --- p.1 / Chapter 1.1.2 --- Separation mechanism --- p.2 / Chapter 1.1.3 --- Derivatization --- p.4 / Chapter 1.1.4 --- Dqproteinization --- p.8 / Chapter 1.1.5 --- Ion-exchange or Reversed-phase HPLC --- p.9 / Chapter 1.2 --- amino acid pattern in cancer patient --- p.11 / Chapter 1.2.1 --- Cancer cachexia --- p.11 / Chapter 1.2.2 --- Causes of cancer cachexia --- p.11 / Chapter 1.2.3 --- Cytokines --- p.12 / Chapter 1.2.4 --- Metabolic Alteration in cancer cachexia --- p.13 / Chapter 1.2.5 --- Amino Acid Studies --- p.14 / Chapter 1.3 --- methodology chosen --- p.19 / Chapter 1.4 --- patient sample chosen --- p.21 / Chapter 2. --- OBJECTIVES --- p.22 / Chapter 3. --- MATERIALS AND METHOD --- p.23 / Chapter 3.1 --- apparatus --- p.23 / Chapter 3.1.1 --- HPLC System --- p.23 / Chapter 3.1.2 --- Column --- p.23 / Chapter 3.1.3 --- Detector --- p.23 / Chapter 3.1.4 --- ChemStation --- p.24 / Chapter 3.2 --- reagents --- p.24 / Chapter 3.2.1 --- Reagent and Chemical source --- p.24 / Chapter 3.2.2 --- Mobile phase --- p.24 / Chapter 3.2.3 --- Derivatization Reagent --- p.25 / Chapter 3.2.4 --- Standard preparation --- p.26 / Chapter 3.2.5 --- Internal standard --- p.28 / Chapter 3.3 --- sample preparation --- p.28 / Chapter 3.4 --- chromatographic conditions --- p.29 / Chapter 3.4.1 --- Column Temperature --- p.29 / Chapter 3.4.2 --- Injector Program --- p.29 / Chapter 3.4.3 --- Time Table for gradient elution and flow program --- p.32 / Chapter 3.5.1 --- OP A and sample Ratio and Volume --- p.32 / Chapter 3.5.2 --- Derivatization Concentration --- p.33 / Chapter 3.5.3 --- Derivatization time --- p.33 / Chapter 3 6 --- analytical performance --- p.34 / Chapter 3.6.1 --- Linearity testing --- p.34 / Chapter 3.6.2 --- Recovery studies --- p.34 / Chapter 3.6.3 --- Precision --- p.34 / Chapter 3.6.4 --- Sample storage --- p.35 / Chapter 3.7 --- clinical sample studies --- p.35 / Chapter 3.8 --- statistical analysis --- p.36 / Chapter 4 --- RESULT --- p.37 / Chapter 4.1 --- chromatographic separation --- p.37 / Chapter 4.2 --- optimization --- p.40 / Chapter 4.2.1 --- OPA and sample Ratio and Volume --- p.40 / Chapter 4.2.2 --- Derivatization time --- p.43 / Chapter 4.2.3 --- OPA Concentration --- p.43 / Chapter 4.3 --- analytical performance --- p.46 / Chapter 4.3.1 --- Linearity --- p.46 / Chapter 4.3.2 --- Recovery studies --- p.46 / Chapter 4.3.3 --- Precision Studies --- p.50 / Chapter 4.3.4 --- Sample storage studies --- p.53 / Chapter 4.4 --- clinical sample study --- p.55 / Chapter 5. --- DISCUSSION --- p.64 / Chapter 5.1 --- analytical --- p.64 / Chapter 5.2 --- clinical --- p.71 / Chapter 5.2.1 --- Normal controls --- p.71 / Chapter 5.2.2 --- Colorectal Cancer --- p.71 / Chapter 5.2.3 --- Lung Cancer --- p.72 / Chapter 5.2.4 --- Nasopharyngeal Cancer --- p.73 / Chapter 5.2.5 --- Summary --- p.74 / Chapter 6. --- CONCLUSION --- p.75 / Chapter 7. --- REFERENCES --- p.75

Intermittent blood flow in tumour microvasculature is believed to contribute to heterogeneity in tumour oxygen delivery; transient vessel nonperfusion is thought to result in acutely hypoxic cells resistant to conventional radiotherapy. This thesis describes three main areas of work: (1) the development of a histologic method capable of detecting intermittent blood flow in experimental tumours at the single vessel level; (2) the quantification and characterization of tumour blood flow fluctuations in the murine SCCVII carcinoma; and (3) the modification of tumour blood flow and the reduction of flow heterogeneity using vasoactive drugs. A double staining technique involving the sequential intravenous injection of two fluorescent vascular markers was used to detect transient episodes of tumour vessel nonperfusion. The stains employed were Hoechst 33342 and the carbocyanine dye, DiOC₇(3), both of which have short (< 3 minutes) circulation half-lives and preferentially stain cells adjacent to perfused blood vessels. When injections of the vascular markers are separated by some interval, each stain defines only those tumour vessels which were perfused during the few minutes immediately post-injection; thus, two "pictures" of tumour microvascular flow are obtained and tumour vessels subject to periods of nonperfusion can be easily visualized in frozen sections since they are outlined by one stain but not the other. Using the double staining technique, in which Hoechst 33342 and then DiOC₇(3) are administered intravenously 20 minutes apart to unrestrained C3H/He mice, staining mismatch (indicative of transient vessel nonperfusion) is regularly observed in subcutaneous SCCVII carcinoma. Vessels stained with DiOC₇(3) only (reperfusion of previously nonperfused vessels) or with H33342 only (nonperfusion of previously perfused vessels) are observed in approximately equal numbers. The percentage of tumour vessels subject to intermittent flow is a function of SCCVII tumour size: tumours ≤100 mg do not exhibit statistically significant amounts of mismatch. At sizes > 100 mg, overall staining mismatch is significantly increased over background levels and maximum mismatch is observed at tumour sizes >400 mg (8.6 ±2.9%). In most tumours, transient vessel nonperfusion is more pronounced in central tumour regions. In addition to mismatch observed in individual vessels, large "patches" of unequal staining are also seen. Anaesthesia or restraint do not significantly influence intermittent blood flow. The above information suggests that transient episodes of tumour vessel nonperfusion occur as a consequence of flow reduction in a feeding vessel; vessels in central regions of large tumours may be susceptible to collapse as a result of elevated tumour interstitial pressure. In the SCCVII tumour, a small number of peripheral vessels possess vascular smooth muscle and thus may be capable of vasomotor activity. The importance of perfusion pressure in the control of tumour microcirculatory flow was examined using vasoactive drugs. Hydralazine, a vasodilator which lowers blood pressure, causes a profound reduction in tumour RBC flow to 8.7 + 6.4% of pretreatment values in unanaesthetized mice. The drug causes collapse of central tumour vessels: following a dose of 10mg/kg intravenously, 36±16% of vessels are completely nonperfused, as detected using the double staining technique. Conversely, elevation of blood pressure using the vasoconstrictor angiotensin II results in a 2-3x increase in tumour blood flow. In addition, angiotensin II infusion significantly reduces the number of tumour vessels subject to transient nonperfusion from 8.1 % to 2.0%. However, intermittent blood flow in the SCCVII carcinoma can also be influenced by nonvasoactive drugs: nicotinamide, the amide form of vitamin B3, reduces episodes of transient nonperfusion. In summary, intermittent blood flow has been characterized in a transplanted murine squamous cell carcinoma using a novel fluorescent double staining method which allows the detection of flow fluctuations in solid tumours at the microvascular level. If transient episodes of nonperfusion occur in human tumours and result in impaired oxygen or drug delivery, then such flow fluctuations may be an important factor limiting tumour cure or local control by radiotherapy or chemotherapy. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 6 uppsatser.

O crescimento e a progressão tumorais estão associados à indução da angiogênese. O objetivo deste estudo foi avaliar a imunoexpressão do VEGF e a densidade de microvasos em adenocarcinomas gástricos. Espécimes cirúrgicos de 89 pacientes submetidos à ressecção gástrica com dissecção linfonodal a D2 foram analisados quanto à densidade microvascular tumoral. Testes imunohistoquímicos foram realizados utilizando-se os anticorpos CD31, CD34, Fator VIII e VEGF através do método da streptavidina-biotina. Os vasos foram contados nas áreas de maior vascularização tumoral e o VEGF foi graduado de 0 a 3, de acordo com a intensidade da imunocoloração. Os resultados imunohistoquímicos foram comparados com os dados patológicos e de extensão loco-regional da doença. Sessenta pacientes (67,4%) eram do sexo masculino e a média de idade foi 59,9 (±13,8) anos. Os tumores foram classificados em tipo intestinal em 62 casos (69,7%) e em difuso em 27 (30%). Onze pacientes (12,4%) apresentaram tumores precoces. A densidade microvascular apresentou média de 67,8 (±31,5) para o anticorpo CD31 e 94,2 (±39,0) para o CD34. A média do número de vasos corados pelo Fator VIII foi 9,2 (±8,2). Houve uma correlação entre os resultados imunohistoquímicos para CD31 e CD34, com r=0,57 e p=0,01. Segundo o VEGF os tumores foram: grau 0 = 16 (18%), I = 48 (53,9%), II = 16 (18%) e III = 9 (10,1%). Não houve associação entre a DMV e os dados patológicos: tipo histológico, grau de diferenciação, tamanho do tumor, invasão da parede gástrica, metástases linfonodais e metástase à distância. A imunoexpressão do VEGF foi associada com alta DMV (p=0,03) e com o estádio tumoral - TNM (p=0,003). Os resultados deste estudo permitiram concluir que a coloração imunohistoquímica com o Fator VIII não é segura como um marcador de células endoteliais; que os anticorpos CD31 e CD34 foram úteis na avaliação da DMV; e que a imunoexpressão do VEGF pode identificar um comportamento biológico mais agressivo / Tumor growth and progression, particularly in aggressive and malignant tumors, are associated with the induction of angiogenesis. The aim of this study was to evaluate the role of VEGF immunoexpression and microvascular density in gastric adenocarcinomas. Specimens from eightynine patients who underwent gastric resection with DII lymph node dissection were analyzed regarding microvascular density of the tumors. Immunohistochemistry for CD31, CD34, Factor VIII and VEGF were performed using the streptavidin-biotin-method. The vessels were counted in a hot spot area of the tumors and VEGF was categorized as grade 0 to III, according to the intensity. Immunohistochemical results were compared to pathological data and loco-regional extension of the disease. In the results of 89 patients, sixty (67.4%) were men, and the mean age was 59.9 ± 13.8 years. The tumors were classified as intestinal type in 62 (69.7%) and diffuse in 27 (30.3%). Eleven (12.4%) were early gastric tumors. Microvascular density showed a mean of 67.8 (± 31.5) for CD31 and 94.2 (± 39) for CD34 vessels. The mean number of vessels revealed by the antibody Fator VIII was 9.2 (± 8.2). There was a correlation between immunohistochemistry for CD31 and CD34, r = 0.57, p < 0.01. The tumors were classified for VEGF as: grade 0 = 16 (18%); I = 48 (53,9%); II = 16 (18%) and III = 9 (10,1%). There was no association between immunohistochemistry and pathological data including: histologic type, grade of differentiation, tumor size, tumor depth, lymph node metastasis and distant metastasis. However, VEGF immunoexpression was associated with high microvascular density (p = 0,03) and there was an association between the immunoexpression of VEGF and the tumor stage - TNM (p = 0,003). It was concluded that immunohistochemical staining for anti-Factor VIII was not reliable as a microvascular density marker; CD31 and CD34 were useful to demonstrate microvascular density and VEGF immunoexpression may identify tumors with more aggressive biological behavior

O crescimento e a progressão tumorais estão associados à indução da angiogênese. O objetivo deste estudo foi avaliar a imunoexpressão do VEGF e a densidade de microvasos em adenocarcinomas gástricos. Espécimes cirúrgicos de 89 pacientes submetidos à ressecção gástrica com dissecção linfonodal a D2 foram analisados quanto à densidade microvascular tumoral. Testes imunohistoquímicos foram realizados utilizando-se os anticorpos CD31, CD34, Fator VIII e VEGF através do método da streptavidina-biotina. Os vasos foram contados nas áreas de maior vascularização tumoral e o VEGF foi graduado de 0 a 3, de acordo com a intensidade da imunocoloração. Os resultados imunohistoquímicos foram comparados com os dados patológicos e de extensão loco-regional da doença. Sessenta pacientes (67,4%) eram do sexo masculino e a média de idade foi 59,9 (±13,8) anos. Os tumores foram classificados em tipo intestinal em 62 casos (69,7%) e em difuso em 27 (30%). Onze pacientes (12,4%) apresentaram tumores precoces. A densidade microvascular apresentou média de 67,8 (±31,5) para o anticorpo CD31 e 94,2 (±39,0) para o CD34. A média do número de vasos corados pelo Fator VIII foi 9,2 (±8,2). Houve uma correlação entre os resultados imunohistoquímicos para CD31 e CD34, com r=0,57 e p=0,01. Segundo o VEGF os tumores foram: grau 0 = 16 (18%), I = 48 (53,9%), II = 16 (18%) e III = 9 (10,1%). Não houve associação entre a DMV e os dados patológicos: tipo histológico, grau de diferenciação, tamanho do tumor, invasão da parede gástrica, metástases linfonodais e metástase à distância. A imunoexpressão do VEGF foi associada com alta DMV (p=0,03) e com o estádio tumoral - TNM (p=0,003). Os resultados deste estudo permitiram concluir que a coloração imunohistoquímica com o Fator VIII não é segura como um marcador de células endoteliais; que os anticorpos CD31 e CD34 foram úteis na avaliação da DMV; e que a imunoexpressão do VEGF pode identificar um comportamento biológico mais agressivo / Tumor growth and progression, particularly in aggressive and malignant tumors, are associated with the induction of angiogenesis. The aim of this study was to evaluate the role of VEGF immunoexpression and microvascular density in gastric adenocarcinomas. Specimens from eightynine patients who underwent gastric resection with DII lymph node dissection were analyzed regarding microvascular density of the tumors. Immunohistochemistry for CD31, CD34, Factor VIII and VEGF were performed using the streptavidin-biotin-method. The vessels were counted in a hot spot area of the tumors and VEGF was categorized as grade 0 to III, according to the intensity. Immunohistochemical results were compared to pathological data and loco-regional extension of the disease. In the results of 89 patients, sixty (67.4%) were men, and the mean age was 59.9 ± 13.8 years. The tumors were classified as intestinal type in 62 (69.7%) and diffuse in 27 (30.3%). Eleven (12.4%) were early gastric tumors. Microvascular density showed a mean of 67.8 (± 31.5) for CD31 and 94.2 (± 39) for CD34 vessels. The mean number of vessels revealed by the antibody Fator VIII was 9.2 (± 8.2). There was a correlation between immunohistochemistry for CD31 and CD34, r = 0.57, p < 0.01. The tumors were classified for VEGF as: grade 0 = 16 (18%); I = 48 (53,9%); II = 16 (18%) and III = 9 (10,1%). There was no association between immunohistochemistry and pathological data including: histologic type, grade of differentiation, tumor size, tumor depth, lymph node metastasis and distant metastasis. However, VEGF immunoexpression was associated with high microvascular density (p = 0,03) and there was an association between the immunoexpression of VEGF and the tumor stage - TNM (p = 0,003). It was concluded that immunohistochemical staining for anti-Factor VIII was not reliable as a microvascular density marker; CD31 and CD34 were useful to demonstrate microvascular density and VEGF immunoexpression may identify tumors with more aggressive biological behavior

A radiação ionizante prévia promove alterações actínicas em tecidos peritumorais,o que poderia influenciar a demarcação do linfonodo sentinela.O presente estudo desenvolveu modelo experimental para demarcação do linfonodo sentinela do reto do rato e para definição da dose de radiação (curva de calibração). O objetivo foi avaliar a influência da radiação ionizante pré-operatória sobre a marcação, com corante azul patente, do linfonodo sentinela do reto de ratos. A amostra foi constituída por 40 ratos machos Wistar e dividida em 2 grupos:Grupo 1 (controle não irradiado; n = 20) e Grupo 2 (irradiado com 1200cGy e demarcado 2 dias após; n = 20). Foi observado aumento linear do tempo de coloração do linfonodo no Grupo 2. Concluindo,a irradiação pré-operatória não influiu na demarcação do linfonodo sentinela do reto do rato / Previous ionizing radiation induces actinic alterations in peritumoral tissues and thus might influence the localization of the sentinel lymph node. The present study developed an experimental model for the localization of the sentinel lymph node of the rectum of the rat and for the definition of the dose of radiation (calibration curve). The objective was to evaluate the influence of preoperative ionizing radiation on the staining of a patent blue dye in the sentinel lymph node of the rectum in rats.The sample was composed of 40 male Wistar rats and was divided in two groups: Group 1 (non-irradiated control; n = 20 ) and Group 2(irradiated with 1200cGy and stained 2 days afterwards; n = 20). It was observed a linear increase in the time for the staining of the lymph in Group 2.In conclusion, preoperative irradiation did not influence the staining of the sentinel lymph node of the rectum in rats

A radiação ionizante prévia promove alterações actínicas em tecidos peritumorais,o que poderia influenciar a demarcação do linfonodo sentinela.O presente estudo desenvolveu modelo experimental para demarcação do linfonodo sentinela do reto do rato e para definição da dose de radiação (curva de calibração). O objetivo foi avaliar a influência da radiação ionizante pré-operatória sobre a marcação, com corante azul patente, do linfonodo sentinela do reto de ratos. A amostra foi constituída por 40 ratos machos Wistar e dividida em 2 grupos:Grupo 1 (controle não irradiado; n = 20) e Grupo 2 (irradiado com 1200cGy e demarcado 2 dias após; n = 20). Foi observado aumento linear do tempo de coloração do linfonodo no Grupo 2. Concluindo,a irradiação pré-operatória não influiu na demarcação do linfonodo sentinela do reto do rato / Previous ionizing radiation induces actinic alterations in peritumoral tissues and thus might influence the localization of the sentinel lymph node. The present study developed an experimental model for the localization of the sentinel lymph node of the rectum of the rat and for the definition of the dose of radiation (calibration curve). The objective was to evaluate the influence of preoperative ionizing radiation on the staining of a patent blue dye in the sentinel lymph node of the rectum in rats.The sample was composed of 40 male Wistar rats and was divided in two groups: Group 1 (non-irradiated control; n = 20 ) and Group 2(irradiated with 1200cGy and stained 2 days afterwards; n = 20). It was observed a linear increase in the time for the staining of the lymph in Group 2.In conclusion, preoperative irradiation did not influence the staining of the sentinel lymph node of the rectum in rats