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.
significantly lower rate of liver and bone metastases. 9 There is a slight decrease in expression of these bio-markers in the metastatic tumours. This effect may be due to tumour heterogeneity, a well-known fact, in anticancer chemo sensitivity, and may be reflected in hormonal receptor status of metastatic breast carcinoma. Neoplastic cells from high-grade tumours may also loose estrogen and progesterone receptors during the process of metastasis. HER-2/neu expression, however, remains almost same in primary and metastatic breast carcinomas. 10,11 Metastatic breast cancer is an important area of research for both researchers and clinicians because MBC has a poor prognosis. The present study was conducted to compare the expression of these bio-markers between primary and metastatic breast carcinoma.
In the other phase I study, 19 patients with stage IV breast or ovarian cancer were immunized with three HER2/neu peptides plus GM-CSF . The peptides, corresponding to sequences HER2 369-384, HER2 688-703 and HER2 971-984, each contained a putative HLA-A2 binding motif. After immunization, 83% of the patients had prolifer- ative responses to at least one of the peptides and some of them also showed proliferative responses to recombi- nant parts of HER2/neu. In some patients there was also an increase in the number of T cell precursors specific for the nonamer contained within the immunizing peptides. However, limited cytotoxicity of target cells expressing the antigen was observed (18% killing of SKOV3-A2 with clone anti-HER2 369-377 peptide, and 25% killing of Epstein-Barr virus-transformed lymphoblastoid B cells transfected with HER2/neu versus 12% in untransfected cells). Clinical response to treatment was not reported. Clinical relevance of immune responses induced against HER2/neu peptides
negative), score 1+ (negative; just perceptible staining of the membrane in > 10% of the malignant cells), score 2+ (moderate staining of the partial membrane in > 10% of the malignant cells) and score 3+ (strong circumferential staining of the entire membrane creat- ing a fish-net pattern in > 10% of the malignant cells) . The intensity of p53 nuclear stain was classified into score 0 (negative), score 1+ (weak or mild stain- ing, with 5-10% tumor cells staining positive), score 2+ (moderate staining with less than 25% of tumor cells staining positive), score 3+ (strong staining, with 25- 50% of tumor cells staining positive) and score 4+ (highly strong staining with over 50% of tumor cells staining positive) . All biopsies were classified into three grades: Grade I, Grade II and Grade III, accord- ing to the modified Bloom Richardson Grading System . The results were statistically evaluated with a Chi-squared test (at a significant level of p <0.05) and correlation-regression analysis (at a significance level of R = 0.3) using SSPS software.
prognosis. The expression of angiogenesis factors such as vascular endothelial growth factor has been observed in a subset of gastric cancers, indicating the potential role of angiogenesis inhibitor therapy. Membrane-type matrix metalloproteinase is preferentially expressed in some gastric cancer cells with co-localization and activation of the zymogens proMMP-2. Advanced gastric cancer patients express increased activation of plasminogen. Specific alterations such as these need true prevalence determination and further characterization in gastric cancer before genetic tests can be designed for clinical use.
Breast cancer, which is the most diagnosed form of can- cer in women, will account for 27% of cancer cases in 2009 according to the American Cancer Society. The human epidermal growth factor HER-2 oncogene (c- erbB-2) has been implicated in this disease [1-4]. The HER-2/neu oncoprotein is amplified and over-expressed in 25 to 30 percent of patients with aggressive breast cancer. Further, the full-length receptor (p185HER2) undergoes a proteolytic cleavage resulting in the release of the soluble shed ECD/HER2 (sECD-HER2/neu) fragment. The resultant truncated intracellular form con- taining the kinase domain is associated with enhanced signaling activity and consequently contributes to me- tastatic breast cancer . The sECD/HER2/neu frag- ment from the surface of breast cancer cells once shed into the blood of individuals can be quantified, making a
statistical evaluation of patients at different time points. Kaplan–Meier curves and log-rank (Mantel-Cox) test were used for the evaluation of progression-free survival (PFS). Cut off limits for preexisting (R0) AE36 peptide- specific T cell frequencies (IFNγ producing cells in re- sponse to stimulation with AE36 at baseline) and LR at first vaccine (LR1) were calculated by cut off finder, http://molpath.charite.de/cutoff/index.jsp. In cases where the number of samples to be analyzed was <20 and thus the cut off finder could not be applied, X-tile software was used for defining the cut off for low- and high frequencies of tumor antigen-specific (dextramer-positive) CD8 + T cell populations . Rmax represents the time point during vaccinations with the highest frequency of antigen specific T cells detected. The ratio Rmax/R0 was considered high when value was >2, i.e. when Rmax was at least twofold above R0 frequency detected. Statistically significant dif- ferences were considered when the P value was ≤ 0.05.
Lymph-vascular invasion is a phenomenon which is more prevalent in certain molecular subtypes of breast cancers and is associated with lymph-node status (Pinder et al., 1993; Simpson and Page, 1994) and the expression of HER-2 / neu, as it is published in some studies (Kim et al., 2006; Millar et al., 2009) and as shown results in our series. Immunohistochemical parameters reported in our series showed a positive correlation with the expression of HER-2 / neu. According to the results, as it was expected, the high mitotic index reflects the strong expression of proliferative index determined by monoclonal antibody Ki67 in this particular group of patients, especially that in most studies this parameter is treated as an independent prognostic factor regardless of the type of breast cancer (Bhargava et al., 2009; Reed et al., 2000; Millar et al., 2009). Expression of the protein
Numerous epidemiological and experimental studies have shown the strong relationship between HER-2/neu- positivity and lack of hormone receptor expression in breast tumours [2,10,18,22]. In our study, HER-2/neu positive tumours were weakly related with the absence of estrogen receptors, although this was not statistically sig- nificant (see table 2). Because different ER status can result in different correlations between risk factors and HER-2/ neu+ breast cancer, it is always important to examine these interactions under ER stratification . Since antiestro- gens can lower HER-2/neu levels in ER negative tumours, it is possible that an excess of estrogens can stimulate HER-2/neu in these tumours [8,18]. This mechanism could explain the stronger association between obesity (a situation with an overload of estrogens as mentioned above) and HER-2/neu-positivity among ER negative patients that was found in the present study (see table 5). The interview was conducted during the subjects' first visit to the unit and before clinical examination or any other intervention took place. This constitutes an advantage, because there was no chance that the subjects (both cases and controls) would be influenced by the diagnosis and might therefore falsely inflate the relative risk. Thus, the
Many patients with metastatic breast cancer witness prolonged survival which can be associated with the develop- ment of multiple sites of metastasis with differing biology. This study reveals the impact of tumor discordance which could occur during the evolution of metastasis. We studied hormone receptor and HER-2/neu discordance in primary, first, and second metastatic disease, and we noted the best survival was experienced by patients whose tumors remained ER positive at all three time points whereas significantly worse survival was found in those patients whose tumors remained ER negative at all three time points. Patients with
When examining the techniques used for the assessment of the HER-2/neu status, gene-based assays almost uni- formly confirmed the negative prognostic impact of HER-2/ neu in node-negative patients. Especially in the past few years, FISH has gained considerable interest as a reliable and valid method for determining the HER-2/neu status, confirming its prognostic utility [18,26]. Compared with other gene-based assays such as Southern blotting or polymerase chain reaction, FISH is not hampered by dilu- tional artefacts possibly resulting from a mixture of different cell populations. Similarly to IHC it allows for the specific detection of the alteration in individual cells within the important architectural context. However, in comparison with IHC, FISH is rather time-consuming and leads to sub- stantial costs . It is nevertheless considered the gold standard for assessing HER-2/neu status. A potential advance in the practicability of in situ hybridisation could be the more recently described chromogenic in situ hybrid- isation, which has shown a good correlation with FISH  and an independent prognostic importance in patients with node-negative breast cancer .
In the present study the oncogenes c-myc and HER-2/ neu were examined with regard to their ability to predict DFS, OS and rate of recurrence, as well as mortality. All patients were randomly selected from one department (Frauenklinik Klinikum Ibbenbueren, Ibbenbueren, Germany). C-myc amplification was identified in 21.5% and HER-2/neu amplification in 30.4%. Berns and coworkers [12–14] reported amplification of c-myc in 20% and of HER-2/neu in 24% using a standard southern blot technique. In a selected high-risk cohort of NNBC patients, Roux-Dosseto et al.  applied the same method and found prevalence rates for c-myc and HER-2/ neu amplification of 25% and 31%, respectively. Those oncogenes were assessed in the present study using a double differential PCR technique [17,18,40,43]. Using this method, Brandt et al.  found c-myc to be amplified in 19.7% and HER-2/neu in 16.7% of breast cancers, without consideration of nodal status; coamplification of those oncogenes was present in 7%. In the present study, simultaneous amplification of both oncogenes was observed in 12.2%. As in the present investigation, Roux- Dosseto et al.  found that c-myc amplification among NNBC patients was significantly associated with earlier recurrence in univariate and multivariate analyses. However, HER-2/neu-amplified NNBC patients did not have the same outcome. Accordingly, c-myc amplification appears to be an independent prognostic marker, which has greater predictive power than does oestrogen recep- tor status and tumour grade. As early as 1992, Berns and coworkers [11,12,14] reported that patients with c-myc- amplified breast cancers had an unfavourable prognosis. The first study to mention possible prognostic importance of HER-2/neu gene amplification was reported in 1987 by Slamon et al. . That study included 187 patients with NNBC and node-positive breast cancer; by univariate and multivariate analyses, it revealed that HER-2/neu amplifi- cation correlated very closely with shorter DFS and OS in a subgroup of 87 node-positive patients. In 1993, in an analysis of 210 patients, Press et al.  found that amplifi- cation of HER-2/neu correlated with unfavourable progno- sis with respect to DFS.
Colorectal carcinoma is one of the most deadly cancer with increased morbidity and mortality in the word. (1) Incidence of colorectal carcinoma 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)
immunohistochemical expression of tumor markers (ER, PR, HER- 2/neu, P53 and Ki67) in patients with breast cancer was different from other studies. This difference may be due to genetic differences, however other factors such as threshold for positivity are responsible for at least some of the differences. 2 One study showed that 75% of
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.
After three washes in TBS, the slides were incubated with a 1:25 dilution of mouse anti- ER α monoclonal primary antibody (clone: 1D5; M7047; DakoCytomation, Denmark), 1:25 dilu- tion of mouse anti-PgR monoclonal primary antibody (clone: PgR 636; M3569; DakoCytomation, Denmark), 1:25 dilu- tion of mouse anti-HER-2/neu monoclonal primary antibody (clone: CB11; NCL-L-CB11; Visionbiosystems Asia Pacific), 1:800 Ki-67 (clone MIB-1 DakoCytomation, Denmark), 1:50 Bcl-2 (clone 124), and 1:50 EGFR (clone H11) in TBS for 1 hour at room temperature. After three more washes in TBS, secondary antibody (K0355; DakoCytomation, Denmark) biotinylated goat antibody (LINK) to mouse/rabbit immu- noglobulin, dilute antibody (1:100) in TBS was applied for 1 hour at room temperature. After an additional three washes, streptavidin–biotin/horse radish peroxidase complex (Enzyme Label) (K0355; DakoCytomation, Denmark) dilute antibody (1:50) in TBS was applied for 1 hour at room temperature. After an additional three washes, the staining was visualized by adding diaminobenzidine (DAB kit; K3467; DakoCytoma- tion, Denmark) for 5 minutes at room temperature. The slides were washed well in tap water and counterstained with Harris’s hematoxylin for 10 seconds to 1 minute and then dehydrated, cleared, and mounted in Distrene Plasticiser Xylene (DPX). Positive and negative controls were performed with each batch
In contrast to breast cancer where IHC for HER-2/neu is usually homogenous, tumour heterogeneity is more common in gastric cancer. Heterogeneous staining can sometimes be seen within one gland. The most important reason for heterogeneous staining, however, is that up to one third of gastric cancers are of mixed intestinal/diffuse type. Strong staining is often seen in areas of an intestinal type of differentiation, while areas of diffuse types of differentiation are negative. (34) Signet ring cell carcinomas are almost always negative. An identical heterogeneous pattern of staining is identified at the DNA level (amplification). Because of heterogeneity, the 10% cut-off level for positivity, which is required in breast cancer, is omitted in gastric cancer. Positivity in gastric cancer specimens is thus independent of the percentage of stained cells and it is sufficient to have a cohesive group of cells displaying HER-2/neu positivity. The heterogeneity of HER-2/neu overexpression / amplication is difficult to explain from the biologic point of view the mechanisms leading to silencing HER-2/neu expression in an area of a tumor with homogeneous HER-2/neu amplification, is at present unknown. (32)
of patients with HER-2/neu negative tumors compared to those with HER-2/neu positive tumors suggests the presence of high affinity T cells in the former. Weak T cell responses were associated with weak and scattered nuclear and cytosolic IFN-g Ra while stronger T cell responses in patients with HER-2/neu- tumors was asso- ciated with an intense and uniform nuclear IFN-g Ra. Our findings suggest that the increased rate of HER-2/ neu positive DCIS compared with breast cancer may reflect the loss of HER-2/neu during tumorigenesis in premalignant cells where IFN-g signaling pathway is active. This possibility is also supported by a number of observations reported by other groups. For instance, induction of HER-2/neu-specific IFN-g producing T cell responses in patients with DCIS resulted in loss of HER-2/neu expression . It was also reported that overexpression of HER-2/neu in DCIS lesions predicts the presence of invasive foci in patients with DCIS . Others also have suggested that the low frequency of HER-2/neu expression (20-25%) in invasive breast can- cer implies that HER-2/neu loss is an epiphenomenon of disease progression .
CD4 T-cell help is required during the generation and maintenance of effective antitumor CD8 T cell–mediated immunity. The goal of this study was to determine whether HER-2/neu–specific CD8 T-cell immunity could be elicited using HER-2/neu–derived MHC class II “helper” peptides, which contain encompassed HLA-A2–binding motifs. Nineteen HLA-A2 patients with HER-2/neu–overex- pressing cancers received a vaccine preparation consisting of putative HER-2/neu helper peptides p369–384, p688–703, and p971–984. Contained within these sequences are the HLA-A2–binding motifs p369–377, p689–697, and p971–979. After vaccination, the mean peptide-specific T-cell pre- cursor frequency to the HLA-A2 peptides increased in the majority of patients. In addition, the pep- tide-specific T cells were able to lyse tumors. The responses were long-lived and detectable for more than 1 year after the final vaccination in select patients. These results demonstrate that HER-2/neu MHC class II epitopes containing encompassed MHC class I epitopes are able to induce long-lasting HER-2–specific IFN-γ–producing CD8 T cells.