measurable circulating HER-2/neu levels. In contrast, reports using the FDA-cleared serum HER-2/neu test reported that all individuals tested, both normal (male and female) and cancer patients, had some level of circulating serum HER-2/ neu. In a 1999 report , using the same enzyme-linked immunosorbent assay based on mAb 4D5, baseline serum concentrations of the circulating HER-2/neu were below the detectable concentration in 73 out of 191 patients (38%). The article concluded that no significant correlation could be demonstrated between shed HER-2/neu concentrations and patient response status. However, the conclusions were based on data from a nonvalidated immunoassay with no standardization, and no references demonstrating the specificity of the research assay were presented. However, an explanation for these results can be derived from a report by Wong and Mass  presented at the ASCO 2000 annual meeting. In the report they compared the homebrew mAb 4D5 assay with the FDA-cleared test. The poster compared serum samples from the same MBC patients using both the mAb 4D5-based assay and the FDA-cleared assay. The authors concluded that the mAb 4D5 assay was not as sensitive as the FDA-cleared assay, which helps explain the results reported by the above-cited studies as well as other studies using the homebrew mAb 4D5 assay [25,26]. In a similar abstract published in 2004 at the annual ASCO meeting , Leyland-Jones and coworkers reported on a study in which they measured HER-2/neu levels in 366 cancer patients, again using the homebrew mAb 4D5 assay. Included was a combination of stage 2 and stage 3 breast cancer patients and patients with non-small-cell lung carcinoma (NSCLC). In the study it was concluded that no obvious relationship existed between baseline HER-2/neu levels and patient response, and in all cases the levels dropped with antiproliferative therapy. Of the 366 patients included in the 2004 poster, 103 patients were from the NSCLC study. Combining breast cancer and NSCLC with stage 2 and 3 cancer studies does not seem appropriate in light of the FDA cleared indication for patients with MBC. Despite the abstract and poster presented in 2000 by Wong and coworkers  showing that the 4D5 assay had less analytical sensitivity than the FDA-cleared assay, the authors of the 2004 ASCO poster used the homebrew 4D5 assay. In addition, the FDA assay is cleared for monitoring patients with values above 15 ng/ml. In the 2004 poster by Leyland- Jones and coworkers , conclusions were also based on plotting values below 15 ng/ml. To date, these data have not been published in a peer-reviewed journal.
Measured levels of the sECD-HER2/neu protein greater than 15 ng/ml were indicative of the potential presence and the associated progression of primary tu- mors to metastatic breast cancer [4,6,7]. Most commer- cially available assays use this concentration as a focal point leaving smaller amounts undetected. Since the over expression of HER-2/neu oncogene is a useful tool as a prognostic and predictive marker for breast cancer, de- velopment of a more sensitive biomarker assay appeared ideal for monitoring the progression, the early recurrence of metastatic breast cancer, and the response to therapy [5,8,9]. A more sensitive assay would also allow for the establishment of an individual’s baseline level of sECD- HER-2/neu protein through which more accurate detec- tion of unexpected increase might be achieved. Finally, an improved assay may be useful to monitor the normal signaling activity of the Her-2/neu pathway.
Cardiotoxicity is a significant concern among patients treated with trastuzumab who were previously treated with anthracyclines . In first-line treatment of advanced stage breast cancer, trastuzumab in combination with anthracycline and cyclophosphamide (AC; n = 143) resulted in 27% and 16% incidences of any cardiac dysfunction and New York Heart Association class III-IV heart failure, respectively, as compared with 7% and 5% with trastuzumab alone, and 7% and 3% with anthracycline and cyclophosphamide alone . Although the precise mechanism of trastuzumab-induced cardiotoxicity is unknown, HER-2 appears to serve as a survival factor for cardiac myocytes . Recently, an increased incidence of cardiotoxicity was demonstrated in patients receiving imatinib , which targets members of the abl kinase family, raising questions as to whether small molecule TKIs, particularly those targeting HER-2, might also have cardiotoxic effects. Lapatinib appears to carry lower risk for cardiotoxicity compared with trastuzumab . For example, in a randomized phase III clinical trial comparing the combination of lapatinib plus capecitabine with capecitabine alone in women with relapsed HER-2 positive breast cancer previously treated with an anthracycline and trastuzumab , there were four asymptomatic cardiac events in the lapatinib/capecitabine arm (n = 163). All lapatinib trials excluded patients with left ventricular ejection fraction of 50% or less, or below the lower limit of institutional normal levels, potentially biasing the data by selecting those individuals who are at lower risk for developing cardiotoxicity.
Seidman et al. examined the use of herceptin and pacli- taxel in patients with metastatic disease irrespective of HER-2 status . The original intention of the study was to compare roughly equal numbers of HER-2-posi- tive and HER-2-negative patients and to make compar- isons of response rate according to different assay techniques. Overall, the combination was associated with response rates of 80% in patients who were HER- 2-positive and only 43% in patients who were HER-2- negative. Response was probably better defined by the use of the monoclonal antibody, TAB 250, rather than the now more widely used HercepTest kit. Given the rel- ative lack of efficacy of herceptin in HER-2-negative patients, it seems unlikely that this will be the subject of much further study. Whether the weekly schedule of paclitaxel in combination with herceptin is superior to the 3-weekly schedule in those patients whose tumours overexpress HER-2 will clearly need to be tested in the context of a randomised study.
were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to analyze the difference between the transfection groups and the negative control group. One-way ANOVA was used to compare the means of multiple groups. The q test was used for intergroup pairwise comparisons. The chi-squared test was used to analyze the differences in the positive expression rates of CD44st and HER-2 mRNA or protein in breast cancer tissues. The K–S test showed that the P values for the average Δ Ct values of CD44st and HER-2 were 0.12 and 0.19, which indicated that the data met normal distribution. The correlation between CD44 protein expres- sion and the average Δ Ct value of CD44st mRNA expression was evaluated using Spearman correlation analysis. The correlation between the expression of CD44st mRNA and HER-2 mRNA was also analyzed. P , 0.05 was considered
The Her-2 protein (p185, Her-2/neu, ErbB-2) has a molecular weight of 185- KDa transmembrane tyrosine kinase receptor and belongs to the family of EGFRs . This family is consists of four types: HER1, HER2, HER3, HER4.These receptors consists of an ligand binding domain situated extracellularly, a small transmembrane domain, and a domain with tyrosine kinase activity present intracellularly. The binding of different substances to the extracellular domain initiates a series of events that resulted in proliferation of cancer cells, cell death, cell to cell adhesion, cellular differentiation and migration.
Upstream (5 0 -TGGGAGCCTGGCATTTCTG-3 0 ) and downstream (5 0 -TCCGGCC ATGCTGAGATGTA-3 0 ) primers were designed based on HER-2/neu cDNA se- quence obtained from GenBank. For cloning, HindIII/ XbaI restriction endonuclease sites were inserted flanking the target gene primers. Primers were synthesized by TaKaRa Biotechnology Co., Ltd. Total RNA was isolated from Ishikawa cells using TRIzol reagent (TaKaRa, China) according to the manufacturer’s instructions. HER-2/neu cDNA was reverse-transcribed using the One Step RNA PCR Kit (TaKaRa) according to the manufacturer’s recom- mendations. PCR conditions included denaturation at 94°C for 5 min, 25 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 1 min, and extension at 72°C for 6 min, with a final extension at 72°C for 10 min. PCR products were separated on 1% agarose gel and eluted. The PCR product was sent to TaKaRa for sequencing.
In recent years, much attention has been paid to the anti-tumor effect of emodin, with the main focus on neuroectodermal tumors, liver cancer, lung squamous cell carcinoma, merkel cell skin cancer, stomach cancer, and leukemia . Emodin inhibits K562 leukemia cells and prolongs survival . A possible mechanism is the inhibition of cancer cell DNA, RNA, and protein biosynthesis. AE (1,8-dihydroxy-3-(hydroxymethyl)-9,10- anthracenedione) is a bioactive anthraquinone compound extracted from rhubarb roots. It effectively inhibits the proliferation of human colon cancer cell lines and induces apoptosis . However, the inhibition of growth ability and mechanism of AE in HER-2-overexpressing cell lines are unclear. Therefore, we investigated the molecular mechanisms of the anthraquinone compounds inducing apoptosis of HER-2-overexpressing human breast cancer cell lines. In particular, we compared three anthraquinones, namely emodin, AE, and rhein, for HER- 2 inhibitory activity and the optimal AE concentration (Figure 1A–1D). We confirmed that AE treatment mainly reduced cancer cell viability by inducing apoptosis (Figure 2H–2J).
The HER-2 overexpressed human breast cancer SKBr-3 cells were cultured in medium containing 5 μ g/mL trastu- zumab continuously for 6 months, which resulting in the acquisition of trastuzumab resistance. Compared with the parental SKBr-3 cells, MTT assay showed that the tras- tuzumab-resistant cells displayed a significantly higher viability or proliferative capacity in the presence of every different concentration of trastuzumab (Figure 1A). Besides, the resistant SKBr-3 cells displayed dramatically increased colony formation ability under the treatment of trastuzumab (Figure 1B). We next assessed EGFR and HER-2 expression in the parental cells and trastuzumab-resistant cells, and found that both the mRNA and protein levels of EGFR and HER-2 were increased in the trastuzumab-resistant cells (Figure 1C and D). Results also showed that the phosphoryla- tion levels of EGFR and HER-2 as well as the key kinases of their downstream pathways which including STAT3, Akt, and ERK were significantly enhanced (Figure 1D and E).
found SRC1 to be related to HER-2 expression, but they did not test the correlation between SRC1 and Ki-67, which indi- cated that SRC1 was related not only to the HER-2 expres- sion status but also to the proliferation of breast cancer cells. However, we have not found a proper interpretation for the observation that SRC1 was related to Ki-67 expression but did not increase the primary cancer growth. Double staining of SRC1 and NANOG in breast cancer cell lines should be done and the potential mechanism of them regulating the metastasis of breast cancer should be found out in the future study. The former study reported that SRC1 knockdown inhibited Ets-2-mediated HER-2 expression and Akt activa- tion in the mammary tumors. 7
While there was a significant relationship between HER-2 expression and metastasis, especially BM, there was no apparent relation- ship between the position of the primary lesion (colon or rectum) and that of the metastasis. One study suggested that BM from colon can- cer always occurred in the supratentorial brain, while that from rectal cancer occurred in the subtentorial part of the brain . In the cur- rent study, colon cancer BM was supratentorial more often than rectal cancer BM (72.73% vs. 50.00%) (Table 7), consistent with this previous result, although the difference did not reach significance due to the small sample size. HER-2, NT-3, and survival of CRC BM
9. Pegram MD, Slamon DJ. Combination therapy with trastuzumab (Herceptin) and cisplatin for chemoresistant metastatic breast cancer: evidence for receptor-enhanced chemosensitivity. Semin Oncol. 1999; 26(4 Suppl 12):89-95. 10. Jimenez RE, Hussain M, Bianco FJ, Jr., Vaishampayan U, Tabazcka P, Sakr WA, Pontes JE, et al. Her-2/neu overexpression in muscle-invasive urothelial carcinoma of the bladder: prognostic significance and comparative analysis in primary and metastatic tumors. Clin Cancer Res. 2001;7(8):2440-2447.
In this study, we first developed a method to functionalize G5 PAMAM dendrimers with anti HER- 2-monoclonal antibody (Herceptin), DOTA-Gd, and Alexa Fluor 647 (AF647) dye, as well as entrap AuNPs to prepare the multifunctional conjugate Au-G5-Gd- Herceptin-AF647, 7. Next, we evaluated the ability of 7 to bind and internalize into cell lines overexpressing HER-2, which to the best of our knowledge has not been previously demonstrated. HER-2 of the human epidermal growth factor receptor family was selected because HER-2 is often overexpressed in certain types of epithelia cancer including breast cancer and lung cancer [27, 28]. Indeed, the anti-HER-2 antibody has been utilized for cancer therapy as well as employed within the scope of targeted cancer cells and drug delivery [29–31].
This is to certify that the dissertation entitled “CORRELATION OF ER, PR AND HER-2/neu WITH HISTOLOGICAL VARIANTS OF BREAST CARCINOMA” is a record of bonafide work done by DR. R.D. Puvitha, Post graduate student in the Department of Pathology, Coimbatore under the supervision of DR. R. VIMALA, M.D., Professor and Head, Department of Pathology, Coimbatore Medical College and submitted in partial fulfillment of the regulations of the Tamilnadu Dr.M.G.R. Medical University towards the award of M.D. Degree in Pathology.
The Cancer Genome Atlas (TCGA) is a large database of sequencing results generated from studies involving genome analysis in a rigorous and consistent manner . This allowed us to perform a direct comparison between the TCGA data and the results from our PCR-array plasma profiling study of TNBC and DPBC. We evaluated a panel of miRNAs related to BC and we identified the most specific miRNAs for TNBC and DPBC. The validation was done in a new independent patient cohort with the help of qRT-PCR technology. Furthermore, by overlapping the miRNA patterns, we identified either common or specific miRNA signatures for the two selected subtypes of Her-2 negative BC. Based on the ex- pression level of the transcripts, miRNAs survival curves were generated. The results revealed the prognostic po- tential of some miRNAs, as well as their interdependence with some metastasis related genes.
This is to certify that the dissertation entitled “HER2 neu expression in colorectal adenocarcinoma and its correlation with clinicopathologic variables” is a record of bonafide work done by Dr.N.VANI in the Department of Pathology, Coimbatore Medical College, Coimbatore under the guidance and supervision of Dr.V.PRABA, M.D., Associate Professor, Department of Pathology, Coimbatore Medical College and submitted in partial fulfilment of the requirements for the award of M.D. Degree (Branch III) in Pathology by The Tamilnadu Dr. MGR Medical University, Chennai.
Immunohistochemistry was used to detect ER, PR and HER-2 in breast cancer tissues before and after neoadjuvant chemotherapy.ER, PR and HER2 antibodies were purchased from Ventana, USA. The film was read by a professional pa- thology chief physician through an optical microscope, and the results were judged based on the percentage of positive cells. According to the results of HE staining, the tumor tissue is distinguished from the normal breast tissue, and the cancer nest is distinguished from the cancerous periphery. The evaluation crite- ria for ER and PR: 1) ≥50% of infiltrating cancer cells were labeled with nuclear staining (+++); 2) 20% - 50% of infiltrating cancer cells were labeled with nuc- lear staining (++); 3) 1% - 20% of invasive cancer cells are labeled with nuclear staining (+); 4) labeled negative if not stained. The recommended criteria “Her- ceptin Test” was used for determining HER-2 results.ER, PR, and HER2 are changed from +++ to − - ++, ++ to + - −, and + to − are determined to be down-regulated. Otherwise, it shall be determined to up-regulated.
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).
Materials. The following peptides used in this study, either for immunization or in vitro use, were HLA-A2 flu matrix peptide (pFlu), GILGFVFTL (19); HLA-A2 cytomegalovirus (CMV) peptide, NLVPMVATV (20); and HER-2/neu peptides, p369-384, KIFGSLAFLPESFDGDPA (21), p688-703, RRL- LQETELVEPLTPS (21), p971-984, ELVSEFSRMARD- PQ (21), p369-377, KIFGSLAFL (6), p689-697, RLLQETELV (7), and p971-979, ELVSEFSRM (18). All peptides used for in vitro immunological monitoring were manufactured either by United Biochemical Inc. (Seattle, Washington, USA) or Multiple Peptide Systems Inc. (San Diego, California, USA), and all were greater than 95% pure as assessed by HPLC and mass-spectro- metric analysis. Peptides used in vaccine preparations were manufactured by Multiple Peptide Systems (kind- ly provided by Corixa Corp., Seattle, Washington, USA) and approved for use in humans. Ficoll-Hypaque was purchased from Amersham Pharmacia Biotech (Upp- sala, Sweden). RPMI-1640, HBSS, and PBS were pur- chased from Life Technologies (Rockville, Maryland, USA) and EHAA-120 from Biofluids (Rockville, Mary-
I solemnly declare that this dissertation titled “ HER2 NEU AND KI67 EXPRESSION AS IMMUNOLOCALISATION IN COLORECTAL CARCINOMA ” 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.