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Gastric cancer is one of the worldwide leading cancers which is highly prevalent in Eastern Asia. In Hong Kong, there were 1101 new cases and 687 cancer death in year 2011. So far gastric cancer is known to have poor prognosis and high relapse rate, for which a more effective therapy is sorely needed. With increasing knowledge in the role of HER2 in gastric cancer, a novel targeting agent, Trastuzumab, in combination with chemotherapy has become a promising personalized therapy for advanced gastric cancer and gastro-oesophageal junction cancer patients, particularly effective in HER2 positive patient subgroup. The clinical efficacy and safety of Trastuzumab therapy were ascertained by a recent worldwide phrase III randomized controlled trial called ToGA study in 2010. The assessment of the HER2 status has become crucial in selecting the right patient that will benefit most for the trastuzumab therapy. The aim of our study is to evaluate the local HER2 amplification rate in a group of gastric cancer patients and the associated tumour characteristics as well as sharing our technical experience in optimizing/standardizing the Silver ISH protocol including problem shooting in some difficult cases. From Oct 2012 to Apr 2014, we have accessed 68 requests of gastric cancer for HER2 status determination using Ventana Silver ISH assay, 61 were from stomach or gastro-oesophageal junction, 40 of them were endoscopic biopsy and 21 were resection specimens. Among them the majority were poorly differentiated (65.8%) and of intestinal sub-type (59.1%) according to Lauren classification. Thirteen out of 61 (21.3%) samples were found to be HER2 amplified. Majority of the HER2 positive cases were found in intestinal type and moderately differentiated adenocarcinoma. Eighty percent of the IHC 3+ cases and 29.6% of the IHC 2+ equivocal cases that required reflex tested with SISH were found to be HER2 amplified. There exists a great variation in the reported HER2 amplification rates in previous studies, which are apparently due to the difference in methodology and scoring algorithm apart from the different population groups. Recently, the introduction of the Hofmann’s modification on the scoring algorithm of HER2 assay dedicated for gastric cancer, and the increasing popularity in using DISH/SISH as a reliable alternative to FISH in detecting the HER2 status, have standardized the test and hence narrowed the variation. In order to achieve a more reliable staining result and accurate interpretation, optimization/ standardization of the analytical procedures, as well as the pre-analytical procedures become necessary. The standardization of the formalin fixation time seems to be a crucial factor in this regard. Therefore, the establishment of gastric cancer specimen handling guideline hospital-wide, and specific guideline recommendations for gastric cancer from ASCO/CAP are warranted. / published_or_final_version / Pathology / Master / Master of Medical Sciences

The human epidermal growth factor receptor (HER-2) is over-expressed in 20-30% of breast carcinomas and is a prognostic marker for poor patient outcome. We previously identified the transcription/translation factor Y-box binding protein-1 (YB-1) to be a novel substrate of AKT which binds to epidermal growth factor receptor (EGFR) and HER-2 promoters once phosphorylated (Wu J et al. 2006). YB-1 is over-expressed in approximately 40% of breast cancers; its expression is strongly correlated with HER-2 and is associated with poor patient survival. In order to gain a deeper understanding of the functional role of YB-1 in HER-2 over-expressing breast cancer, we silenced the expression of this factor in BT474-m1 and MDA-MB-453 cells. The loss of YB-1 inhibited the growth of BT474-m1 and MDA-MB-453 cells in monolayer and/or in soft agar. Consistent with this, we found a decrease in the expression of YB-1 responsive gene egfr and/or her-2 in BT474-m1 and MDA-MB-453 cells, which could begin to explain how growth is promoted by this factor. Furthermore, loss of YB-1 expression induced apoptosis in BT474-m1 cells. Beyond its role in tumor growth, YB-1 is also strongly linked to drug resistance. We therefore addressed whether it could play a part in Herceptin sensitivity. Herceptin is currently being used to treat patients with HER-2 positive breast cancer; however, only 30% of the patients respond to the therapy and many of them develop resistance within the first year of treatment. Therefore, it is of utmost importance to understand the biology of HER-2 over-expressing breast cancer to develop novel therapies that can benefit more patients. First we established that Herceptin inhibited BT474-m1 cell growth in anchorage-independent conditions whereas MDA-MB-453 cells were resistant to this treatment. We subsequently demonstrated that knock-down of YB-1 increased sensitivity of BT474-m1 cells to Herceptin while MDA-MB-453 cells failed to respond to the combination treatment. The mechanism for Herceptin resistance in MDA-MB-453 cells still remains elusive and requires further investigation. Thus far, we conclude that YB-1 is needed for the growth and survival of HER-2 positive BT474-m1 and MDA-MB-453 breast cancer cells by inducing members of the HER family.

The human epidermal growth factor receptor (HER-2) is over-expressed in 20-30% of breast carcinomas and is a prognostic marker for poor patient outcome. We previously identified the transcription/translation factor Y-box binding protein-1 (YB-1) to be a novel substrate of AKT which binds to epidermal growth factor receptor (EGFR) and HER-2 promoters once phosphorylated (Wu J et al. 2006). YB-1 is over-expressed in approximately 40% of breast cancers; its expression is strongly correlated with HER-2 and is associated with poor patient survival. In order to gain a deeper understanding of the functional role of YB-1 in HER-2 over-expressing breast cancer, we silenced the expression of this factor in BT474-m1 and MDA-MB-453 cells. The loss of YB-1 inhibited the growth of BT474-m1 and MDA-MB-453 cells in monolayer and/or in soft agar. Consistent with this, we found a decrease in the expression of YB-1 responsive gene egfr and/or her-2 in BT474-m1 and MDA-MB-453 cells, which could begin to explain how growth is promoted by this factor. Furthermore, loss of YB-1 expression induced apoptosis in BT474-m1 cells. Beyond its role in tumor growth, YB-1 is also strongly linked to drug resistance. We therefore addressed whether it could play a part in Herceptin sensitivity. Herceptin is currently being used to treat patients with HER-2 positive breast cancer; however, only 30% of the patients respond to the therapy and many of them develop resistance within the first year of treatment. Therefore, it is of utmost importance to understand the biology of HER-2 over-expressing breast cancer to develop novel therapies that can benefit more patients. First we established that Herceptin inhibited BT474-m1 cell growth in anchorage-independent conditions whereas MDA-MB-453 cells were resistant to this treatment. We subsequently demonstrated that knock-down of YB-1 increased sensitivity of BT474-m1 cells to Herceptin while MDA-MB-453 cells failed to respond to the combination treatment. The mechanism for Herceptin resistance in MDA-MB-453 cells still remains elusive and requires further investigation. Thus far, we conclude that YB-1 is needed for the growth and survival of HER-2 positive BT474-m1 and MDA-MB-453 breast cancer cells by inducing members of the HER family.

Thesis (M.S.)--Northern Michigan University, 2009. / Bibliography: leaves 35-36.

Thesis (M.S.)--Northern Michigan University, 2009. / Bibliography: leaves 114-116.

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisors: Babatunde Ogunnaike, Dept. of Chemical Engineering; and Boris N. Kholodenko, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University. Includes bibliographical references.

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Made available in DSpace on 2014-06-12T23:04:27Z (GMT). No. of bitstreams: 2 arquivo8877_1.pdf: 1460374 bytes, checksum: c08f2c8b72d50467c3068b2ce9762c97 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / A proteína cerbB-2 tem sido bastante estudada não só por sua importância como fator prognóstico dos carcinomas da mama, mas sobretudo por ser um indicador para terapias com esquemas quimioterápicos. Para eliminar a subjetividade da interpretação do método imunohistoquímico convencional, este estudo tem como objetivo quantificar, morfometricamente, a imunomarcação da proteína cerbB-2 expressa em tumores de mama. Fragmentos de tecido mamário normal (n=10) e com doença tumoral (carcinoma ductal invasivo, CDI, n=51; fibroadenoma, n=11) foram fixados em formalina, submetidos à rotina histológica para inclusão em parafina. Cortes histológicos (4mm), corados em hematoxilina e eosina foram examinados para confirmar o diagnóstico. Os cortes foram incubados com solução de anticorpos por uma por uma hora em temperatura ambiente. A marcação foi visualizada após incubação com diaminobenzidina (DAB) e peróxido de hidrogênio. A análise morfométrica foi realizada utilizando uma estação de análise digital de imagens através do software de análise OPTIMAS®. A partir dos resultados obtidos pode-se concluir que a superexpressão do cerbB-2 em casos de CDI é um fenômeno condizente com o estágio de proliferação das células neoplásicas e quando analisados os casos de fibroadenoma, este marcador não exibiu qualquer correlação ou padrão específico, ao contrário apresentaram resultados semelhantes ao tecido mamário normal. Não houve diferenças significativas entre os diferentes scores qualitativos e a análise morfométrica digital, o que no mínimo demonstra a necessidade de estudos mais acurados a fim de resolver esta dificuldade de interpretação