I solemnly declare that this dissertation titled “ HER2NEU AND KI67EXPRESSION AS IMMUNOLOCALISATION IN COLORECTALCARCINOMA ” submitted by me for the degree of M.D, is the record work carried out by me during the period of 2015-2018 under the guidance of Prof. Dr. K. SWAMINATHAN, M.D, Professor of Pathology, Department of Pathology, Tirunelveli Medical College, Tirunelveli. The dissertation is submitted to The Tamilnadu Dr. M.G.R. Medical University, Chennai, towards the partial fulfilment of requirements for the award of M.D. Degree (Branch III) Pathology examination to be held in MAY 2018.
Out of the 40 cases 33 patients were above 65 years of age and 7 patients were below 65 years of age. The median age was 55 years (Age Range 30-80 Years). According to the various data on age wise distribution of urothelial carcinoma is more common in older age groups (Ranu Roy Biswas et al., 2013; David J Grignon, 2009). In conjunction to these studies our findings also show relatively more number of patients in the older age group. Gender distribution of our study showed that the majority of the patients comprised of males (90%). According to the literatures male predilection in urothelial carcinomas was also observed (male=86%, female=14%) (KalpanaBeniwal et al., 2015). In our study, among the clinical parameters painless hematuria was most common in all patients independent of grade and stage of the tumor which was also observed in one study (Ranu Roy Biswas et al., 2013). Smoking habit was present in 70% of the patients in our study group which indicate significant association with urothelial tumor which was in concordance with several previous literatures (Ranu Roy Biswas et al., 2013; KalpanaBeniwal et al., 2015). Assessment of Immunohistochemicalmarkers Ki67 and Her2neu overexpression was correlated with urothelial tumors and its clinicopathological parameters. Many studies on individual marker’s association with urothelial tumors has been conducted on both Ki67 and Her2neu but literatures on both
The HER-2/neu gene was originally called ‘neu’ as it was first derived from rat neuro/glioblastoma cell lines. Coussens and coworkers named it HER2 because its primary sequence was very similar to Human Epidermal Growth Factor receptor (EGFR or ERBB or ERBB1). (86) This human proto-oncogene, also known as c-erbB2, ErbB-2 is a 185- kilodalton transmembrane receptor tyrosine kinase located at chromosome 17q. These proteins belong to subclass I of the super-family of receptor tyrosine kinases. They are expressed in many tissues of epithelial, mesenchymal, and neuronal origin and are critical for cell proliferation and tissue differentiation. The clinical significance of HER- 2/neu has already been evaluated in colorectal, breast, stomach, lung, head and neck, pancreatic, urothelial carcinoma, and gliomas and prostate cancers; patients with elevated HER-2/neu demonstrate poor survival compared with patients with lower level of HER-2/neu. (46,47,48)
Colorectalcarcinoma is one of the most deadly cancer with increased morbidity and mortality in the word. (1) Incidence of colorectalcarcinoma is 9% and this being the third most common cancer world wide. (2) Classic adenocarcinomas accounts for 90% of colorectal carcinomas and it is the most common type. Mucinous adenocarcinoma, signet ring cell carcinoma, medullary carcinoma, serrated carcinoma, adenosquamous carcinoma and small cell carcinomas account for the remainder cases. In a study by Manmeet Kaur Gill et al (2011) showed that conventional adenocarcinoma consitituted about 77.5% followed by mucinous adenocarcinoma 17.5% and signet ring cell carcinoma 2.5%. (3)
Despite a reduction in operative mortality and morbidity through the availability of better antibiotics and methods of bowel preparation, the 5 year survival rate of colorectal cancer following surgery has remained between 20-50% in the past three decades. An important reason for this lack of progress is the extent of tumour dissemination at the time of diagnosis so that eradication of the disease is still not attainable. Various radical surgical techniques have been developed over the years but have not not been proved to increase survival. Recently, there has been a renewed interest in the the no-touch technique in the prevention of tumour dissemination but this is not widely practised and fails to show benefit in survival. Resection of the mesorectum may prevent recurrent rectal carcinoma and increase survival but more supportive evidence is required. Patients with hepatic metastases generally have a poor prognosis but there is evidence to suggest that surgery can increase the survival in some of these patients. Unfortunately, only a small number of patients are suitable for hepatic resection of colorectal cancer metastases, as only those with less than 4 tumour deposits and who at the same time do not have extra-hepatic disease are likely to do well. It is clear that following surgery on the primary cancer systemic therapy is needed to treat occult dissemination. Various adjuvant therapies have been developed, but the results of these treatments have been disappointing in terms of survival, although they may delay the onset of recurrent disease. To date there is still no effective chemotherapeutic agent against colorectal cancer.
Estrogen and progesterone receptors predict response to hormonal therapy. 80% of women with ER/PR positive tumors respond to hormone therapy compared to 10% response in ER/PR negative subgroup. 28 Her2neu is a transmembrane epidermal growth factor receptor protein also known as c erb2. Its overexpression was reported to have poorer outcome and it predicts the response to trastuzumab, lapatinib and anthracycline based chemotherapy. Recently gene expression profiling has been shown to predict survival, recurrence free interval and to identify appropriate therapy for individual patients to which they respond better.
Oestrogen and progesterone receptors form an important part of breast cancer management. Oestrogen and progesterone receptor examination is recommended for prediction of response to hormonal treatment. Disease- free and overall survival decrease with less receptors in most cases. However some studies have shown that ER positive patients showed poor prognosis similar to ER negative patients [17, 18]. An interesting phenomenon has been observed with co expression of HER-2/neu with oestrogen receptors. Tumours with positive ER and HER- 2/neu do not response to tamoxifen treatment due to interaction of tyrosine kinase pathway with hormonal pathway .
Objective: HER-2/neu status in gastric carcinomas (GCs) has not been studied before in the Egyp- tian population. Materials and Methods: Seventy-three GCs were evaluated for the expression of HER-2/neu and Ki-67 using immunohistochemistry (IHC). Fluorescence in situ Hybridization (FISH) was done for HER2/neu IHC score 2+ cases. Results: Out of the 73 gastric carcinomas, 23 (31.5%) were score 0, 17 (23.3%) were score 1+, 23 (31.5%) were score 2+, and 10 (13.7%) were score 3+. FISH analysis revealed that the HER-2/neu gene was amplified in 11 out of the 23 cases (47.8%) scored 2+ by IHC. Therefore, the overall HER-2/neu positivity rate was 28.8% and it was signifi- cantly associated with higher T-stage and lymphovascular invasion (LVI). The Ki-67 expression rate ranged between 10% and 100%, with 84.9% of the cases (n = 62) featuring high Ki-67 scores. High Ki-67 score was significantly associated with male sex, tumor grade, and number of positive nodes. HER-2/neu protein expression correlated significantly with Ki-67 score. Twenty tumors showed combined HER-2/neu positivity and high Ki-67 score and were significantly associated with higher T-stage and occurrence of LVI, implying a more aggressive behavior. Conclusions: The rate of HER-2/neu positive GCs in our series simulates universal rates, thereby mandating routine evaluation of HER-2/neu status in all GCs submitted to our laboratory to benefit from trastuzumab therapy. Further studies on a larger number of GCs are required to prove that the concurrent HER-2/neu positivity and high Ki-67 score are markers of worse prognosis.
In conclusion, the positive expression of these biomar- kers is associated with biologically aggressive tumors and poor prognostic profile. Although the samples were taken from an area where the exposure to depleted ura- nium is a risk, the incidence of co-expression of both p53 and HER-2/neu markers does not differ from simi- lar cancer samples in areas that have not been exposed to depleted uranium, though, the greater immunoex- pression of Her-2/neu in breast cancer in this popula- tion with risk for DU exposure, compared with findings on other populations not at risk, requires further inves- tigation as it may reflect the possible role of DU in the induction or acceleration of network signaling between different Her-2 receptors. New lines of treatment which includes genetic modulation of the signaling pathway of both genes should be considered in patients’ medical follow up. Unfortunately for DU, knowledge of the exposure time, dose absorbed, route, length of exposure and its health consequences on the Iraqi population is still lacking. This is chiefly due to restricted access of scientists required to conduct such study and should form the basis for future investigations.
multivariate analyses were performed, the latter adjusting for the NPI score and the treatment received (tamoxifen/ chemotherapy/no treatment). As the NPI is based on nodal involvement, on tumour size and on grade, patients with histologies for whom grade is undefined (‘other’ cancers) were excluded from the regression analyses. Thirteen patients (all either ductal carcinoma, lobular carci- noma or mixed histology) had no grade information recorded in the data and one patient had no tumour size recorded. These patients were included in the analysis using multiple imputation methods  to estimate the missing values.
In the present study as per CAP grading, HER2/neu positivity was found in 50% of well-differentiated and 36.4% of moderately differentiated tumors with none of the poorly differentiated tumors expressing HER2/neu and this was statistically highly significant with p value <0.01 and indicating that better-differentiated tumors showed HER2/neu overexpression. The findings of the present study are in concordance with the other studies in that HER2/neuexpression is more in well and moderately differentiated tumors in comparison to poorly differentiated tumors (Table 7). An exception to this is the study by Tewari M et al, which shows the total absence of HER2/neuexpression in well-differentiated tumors. 19 This could probably be explained by the fact
HER-2/neu over-expression or mutation results in quantitative and qualitative alteration in the membrane proteins which is the basis of its detection. , Even though clinical correlation had not been established, many series had suggested that amplification or over-expression of the oncogene might be a marker of poor prognosis in cancers of the ovary, endometrium, breast etc. ,, There were reports in the literature that HER-2/neu over-expression correlates with reduced benefit of adjuvant therapy as in cases of carcinoma breast with tamoxifen therapy. Many studies have shown that regression of HER-2/neu suppresses the malignant phenotypes of cancer cells over-expressing this oncoprotein, which may serve as an excellent target for developing anti-cancer agents specific for HER-2/neu over- expression.  HER-2expression can be estimated either by immunohistochemistry or by fluorescent in situ hybridization (FISH). FISH can be used for doubtful cases in IHC to confirm, HER2/neuexpression. This combination is not necessary for low (0-1+) or high (3+) grades of immunohistochemical stain as the correlation with gene amplification status is high.
Microarray data analysis and clustering. DAT files were analyzed by MAS 5.0 to generate background-normalized image data (CEL files). Probe set intensities were obtained by means of the robust multiar- ray analysis method (19). The full data set was normalized accord- ing to the invariant set method (20). The funnel-shaped procedure described by Saviozzi et al. (21) was then applied. The resulting 5,482 probe sets were analyzed by combining two statistical approaches implemented in significance analysis of microarrays (22): two-class unpaired sample method and the multiclass response test (detailed description of the procedure is available at http://www.bioinfor- matica.unito.it/bioinformatics/Forni/additional_info/ (23). This analysis produced a total of 2,179 probe sets differentially expressed in at least one of the three experimental groups. The validity of the method was demonstrated by real-time RT-PCR evaluation of the expression of several cancer-related genes (see additional informa- tion, ref. 23). The 2,179 probe sets were converted in virtual two-dye experiments comparing all replicates of each experimental group with wk2prg replicates (i.e., wk10ntj/wk2prgi; wk22nti/wk2prgi; wk22pbi/wk2prgi, where j = 1 → 2 and i = 1 → 3). Principal compo- nents analysis (PCA) (24) and hierarchical clustering were performed on virtual two-dye experiments with a TIGR MultiExperiment View- er (http://www.tigr.org/software/). Two-dimensional (2D) hierar- chical clustering (25) of PCA results was used to identify groups of genes specifically modulated in wk22pb only. We used a complete hierarchical clustering together with various distance metrics. The best solution was obtained by Euclidean distance, and genes specif- ically modulated only in wk22pb are readily apparent by inspection of the cluster dendrogram (see Figure 5C). Gene ontology classifi- cation (26) was performed with the DAVID/EASE annotation tool (http://david.niaid.nih.gov/david/).
HER-2/neu is over-expressed and amplified in patients with metastatic breast cancer. An increase serum level (>15 ng/mL) of its shed extracellular domain (sECD- HER2/neu) is indicative of the potential presence and associated progression of this disease. In this report a newly developed ALYGNSA system has been shown to detect an order of magnitude lower level of sECD-HER2/neu than the commercial ELISA kit counterpart. Pre- vention of HER-2/neu overexpression has been exten- sively demonstrated using novel HER-2/neu-blocking agents [8,15,16]. Monoclonal therapeutic antibodies known to bind to extracellular domains of HER-2/neu have been well characterized [17 and references therein]; they include: pertuzumab/Omnitarg which acts upon Domain II and is believed to hinder receptor dimeriza- tion; cetuximab/Erbitux which blocks essential struc- tural transitions of Domain III; and perhaps the most well known and of greatest interest here, trastuzu- mab/Herceptin which binds to Domain IV blocking the action of sheddase and inhibiting sECD-HER2/neu formation. Trastuzumab was the first FDA approved HER-2/neu monoclonal antibody used in the treatment of HER2/neu overexpression in breast cancer patients. sECD-HER2/neu (>15 ng/mL) is currently used to se- lect patients for therapy with trastuzumab.
Older age is associated with elevation of Ki67, where aging is a process that cannot be altered, and cancer is a disease which are more frequently seen in elderly. Aging and cancer have many similarities such as genome instabilities, although cancer cells are often benefited from mutation, other cell accumulates mutation damage, resulting in physiology decline and aging. Telomere attrition is also a common characteristics, but cancer cells can avoid cell cycle defenders by activating telomerase enzyme. Inhibitor enzyme which is responsible in cell’s proliferation can also experience kovalem modification against DNA and histone. Proteostasis is disturbed in aging and cancer cells, protein accumulative aggregations and toxic effect were general characteristics of several disease which linked with age. Cancer avoid this process with regulating chaperone activity, proteasome, and lysosome. 16
Abstract The Wnt/β-catenin pathway plays an important role in the genesis of familial adenomatous polyposis, the most common form of inherited colorectalcarcinoma (CRC). Also, the inflammatory bowel diseases (IBDs) predispose to cancer development; and cyclooxygenase 2 (COX-2) seems to be pivotal in their pathogenesis. This study aimed to investigate the relationship between the expression of COX-2 protein and β-catenin in colorectal cancer. The study enrolled 45 patients, all of whom underwent surgery and immunohistochemical staining of tissue specimens for COX-2 and β-catenin was done. Correlation between the two modulators and their relationship with clinicopathological features were examined. In 34 cases (75.56%) of the tumor samples; β-catenin immunoreactivity was found in the cytoplasm and/or membrane compartment. On the other hand, COX-2 immunoreactivity was weakly and/or strongly positive in 32 cases (71.11%) and negative in 13 (28.89%). Positivity was detected in the cytoplasm and in the perinuclear area. Increased expression of β-catenin was correlated to Duke stage (P=0.009). Furthermore, nuclear β-catenin localization showed a correlation to the Duke stage (P=0.029) and insignificant correlation with distant metastases (P = 0.336). Positive COX-2expression showed a significant relation to, liver metastases (P = 0.042), and Duke stage (P = 0.011) and insignificant correlation to lymph node invasion (P=0.25). These data indicate that cytoplasmic/membrane β-catenin over-expressions as well as positive COX-2 expressions are associated with a more aggressive behavior of the disease.
Background: Breast cancer is the most common of all cancers and is the leading cause of cancer deaths in women worldwide, accounting for >1.6% of deaths and case fatality rates are highest in low-resource countries. Prognostic factors include the type of tumor, size of the tumor, tumor grade, number of involved lymph nodes, Ki67 status (cellular marker for proliferation), and the patient's age. Numerous studies have been conducted on these prognostic factors and their relationships with one another, however, the studies have not reported proper association of Ki67 proliferative index with other clinicopathological parameters. Breast cancer aggressiveness can be correlated with proliferation status of tumor cells, which can be obtained with Ki67 marker. the present study is considered to analyze the association of Ki67 with tumor size, lymph node involvement, histological grade, ER, PR, HER2 status in breast carcinoma.These predictive and prognostic factors help us to stage, plan and choose an appropriate mode of management that increases the patients longevity and improves their quality of life.
BALB/cnAnCr (BALB/c) mice were from Charles River Laboratories (Calco, Italy); mice with the Ig µ heavy-chain gene KO (16) (BALB- µ IgKO) were kindly provided by Thomas Blankenstein (Freie University, Berlin, Ger- many); mice with the FcγRI/III gene KO (44) (BALB- FcγRI/IIIKO) were from Taconic Laboratories (German- town, New York, USA); mice with the β 2-microglobulin gene KO (BALB-β2mKO) (45) and the IFN-γ gene KO (BALB-IFNγKO) (46) were from Jackson Laboratories (Bar Harbor, Maine, USA); mice with the CD1 gene KO (47) (BALB-CD1KO) were kindly provided by Luc Van Kaer (Vanderbilt University, Nashville, Tennessee, USA); mice with the MCP-1 gene KO (48) were kindly provided by Barrett Rollins (Dana Farber Institute, Boston, Mass- achusetts, USA); mice with the perforin (pfp) gene KO (BALB-pfpKO) and the IFN-γ gene double KO (49) (BALB-IFNγ-pfpKO) were from the Peter MacCallum Cancer Institute (East Melbourne, Australia). Mice were treated properly and humanely in accordance with Euro- pean Community guidelines. When required, 22, 18, 14, 8, and 5 days before tumor challenge (day 0) or 2, 5, 8, 11, and 13 days after, a few mice received intraperitoneal injections of 0.2 ml of HBSS containing a 1:20 dilution of antiasialo GM1 rabbit anti-serum (Wako Chemicals GmbH, Dusseldorf, Germany) or 200 µg of anti-CD4 (GK1.5 hybridoma, L3T4, American Type Culture Col- lection, Rockville, Maryland, USA), anti-CD8 (TIB-105 hybridoma, Lyt 2 ATCC), or anti-polymorphonuclear cell (PMN) mAb (RB6-8C5 hybridoma kindly provided by R.L. Coffman, DNAX Inc., Palo Alto, California, USA). Flow cytometry of residual blood and spleen cells collected 3 days after the last injection showed that target leukocytes were selectively decreased to below 1 per 5000 cells.
Results were evaluated statistically using ANOVA. Data were transformed to a natural logarithmic scale. Analyses of variance using the post hoc Tukey test were performed to compare serum HER-2/neu levels, menopausal status and stage of disease. Student’s t-test was used to compare serum HER-2/neu levels between patients with different age groups, node status and hormone receptor (ER, PgR) status. Multivariate logistic regression was used to find association of risk factors with the elevated serum HER-2/neu levels. Result estimation was performed using SPSS (v 16.0; SPSS Inc, Chicago, IL).