Amplification In Breast Cancer Research Articles

Abstract 5142: VEGFA gene amplification and protein expression in breast cancer

Abstract Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p<0.0001). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5142. doi:10.1158/1538-7445.AM2011-5142

Validation of the Multiplex Ligation-dependent Probe Amplification (MLPA) Technique for the Determination of HER2 Gene Amplification in Breast Cancer

Multiplex ligation-dependent probe amplification (MLPA) is a polymerase chain reaction-based assay that can assess HER2 gene copy number relative to control genes. Institutional ethics board approval was obtained. Using commercially available kits, 208 consecutive invasive breast cancers undergoing routine in situ hybridization testing (chromogenic in situ hybridization and fluorescence in situ hybridization) were also tested independently by MLPA. The American Society of Clinical Oncology/College of American Pathologists guidelines were applied for the reporting of ISH results. In accordance with earlier studies, MLPA results were reported as amplified when the HER2 gene copy number per cell was ≥ 4.0. At the conclusion of all ISH testing 25 of 208 cases (12.0%) were regarded as amplified, 182 (87.5%) as nonamplified, and 1 case (0.5%) as undetermined owing to insufficient tissue. This case is excluded from further analysis. Of the 182 cases categorized as not amplified by ISH, all were also negative by MLPA. Of the 25 cases amplified by ISH, 23 (92.0%) were amplified by MLPA, but the 2 remaining ISH-amplified cases were negative by MLPA. Using ISH as the reference test, MLPA has the following diagnostic indices: sensitivity 92.0%, specificity 100%, positive predictive value 100%, negative predictive value 98.9%, and overall accuracy 99.0%. The high level of concordance between MLPA and ISH results, exceeding the American Society of Clinical Oncology requirements for validation of a new test, supports the use of MLPA for assessment of HER2-amplification. These results merit further consideration of MLPA as a possible alternative or additional platform for HER2 testing.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract P6-05-03: Analysis of College of American Pathologists Recommendations for Reporting HER2 Intra-Tumoral heterogeneity (ITH) on 1329 Breast Cancers

Abstract Background: The College of American Pathologists (CAP) recently published recommendations for reporting intratumoral heterogeneity for HER2 gene amplification in breast cancers. These guidelines recommend reporting cases with between 5-50% of cells with HER2:CEP17 ratios > 2.2 (or > 6 HER2 signals/cell) as “heterogeneous for HER2 gene amplification.” Because these recommendations lack biological and clinical foundation, we applied them to a large number of historical breast cancers and compared the ITH classification to routine FISH Her2 results. Materials and Methods: We collected Her2 and CEP17 counts in 32,116 tumor cells from 1329 consecutive breast cancer cases analyzed by FISH. Data from Excel counting sheets were imported to a SQL Server database for analysis and statistics; histograms and analyses were also performed in Excel. We specifically computed concordances between guidelines for traditional and intra-tumoral heterogeneity (ITH) reporting. Results: 309 of 1,329 cases (23%) met the criteria for heterogeneous HER2 gene amplification by HER2:CEP17 ratio, but > 79% of these were non-amplified by standard FISH reporting criteria. Using Her2 signals per cell, ITH was found in 87 (6.5%) of cases and most of these were not amplified or equivocal by standard criteria. Discussion: A significant proportion of breast cancers contain ITC heterogeneity based on CAP/ASCO reporting recommendations. The majority of these cases are non-amplified without consideration of ITH. Because it is not known whether these patients (who would not currently be treated) would benefit from HER2 targeted therapies, it may be premature to use the ITH reporting recommendations for clinical decision-making. More validation of ITH criteria and biological significance are needed. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-05-03.

Abstract P3-10-24: Fibroblast Growth Factor Receptor 1 Amplification and Overexpression in Breast Cancer Tissue Microarrays Using Chromogenic In Situ Hybridization and Immunohistochemistry

Abstract Fibroblast growth factor receptors 1 (FGFR1) belongs to a subfamily of receptor tyrosine kinases (RTKs), which are involved in proliferation and apoptosis. FGFR1 amplification and mRNA overexpression occurs in ∼10% of breast cancers and has been associated with poor outcome. However, studies assessing FGFR1 amplification and protein overexpression in a large collection of breast tumors are lacking. In this prospective, we examined a series of primary breast cancers samples derived from the BCIRG trials assembled into 15 tissue microarrays. FGFR1 gene amplification was studied using chromogenic in situ hybridization (CISH) and evaluated with respect to association with level of protein expression and clinicopathological parameters. FGFR1 gene amplification was significantly associated with high protein levels as determined by immunohistochemistry (P<0.0001, 3294 pts with available matched CISH and IHC records). Overall, the incidence of FGFR1 amplification found was 9.7% (160 out of 1646 pts. with available clinicopathological records) without a statistically significant difference between Her2- (78 out 720 pts., 11%) and Her2+ (82 out of 926 pts., 9%) cancers. In both cohorts, the hormone receptor-positive (ER+/PR+) cancers showed statistically significant higher levels of FGFR1 amplification compared to hormone receptor-negative tumors. In the analysis of the association of FGFR1 and the presence of PIK3CA mutations, the incidence of FGFR1 amplificationwas greatly reduced in mutant vs. wt PI3KCA tumors. In these cohorts, a clear relationship between FGFR1 amplification status and clinical outcome was not detected. Data from this large study confirms recently reported incidences of FGFR1 amplification in breast cancer and shows for the first time an association between FGFR1 gene amplification and protein overexpression. Moreover, the lower incidence of FGFR1 amplification in PIK3CA mutated cancers suggests that these are largely exclusive molecular events that could benefit from different targeted therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-24.

Establishment of the Australian In Situ Hybridization Program for the Assessment of HER2 Amplification in Breast Cancer

In August 2006, the Australian government announced a decision to subsidize trastuzumab therapy for early breast cancer, to commence 6 weeks later. It was mandated that HER2 gene amplification, determined by in situ hybridization (ISH), be shown, and that the sponsor company, Roche Products Pty Ltd, should fund this testing. This announcement potentially required provision of ISH testing for HER2 for every newly diagnosed breast cancer, where previously HER2 testing had been performed by immunohistochemistry with support from a single fluorescence ISH (FISH) reference laboratory for indeterminate cases. The Australian HER2 Testing Advisory Board, an independent expert group, responded to the challenge of rapidly providing accurate nationwide ISH testing. Bright-field ISH was selected as the testing platform and a decentralized testing model, with support from a central FISH laboratory, was adopted. An implementation plan was developed addressing standards for training, accreditation, and quality assurance. Within 6 weeks, 8 pathology laboratories were accredited for ISH testing and by September 2008, 2 years after the announcement, 22 ISH testing laboratories were taking part in the national program and almost 20,000 ISH tests had been performed. This article describes the design and rapid implementation of a nationwide program of bright-field ISH as the first-line testing platform for HER2 status in early breast cancer. We believe that this model for the coordinated and large-scale implementation of a new biomarker test has wide application, given that accurate assessment of a range of novel biomarkers is being used increasingly to determine eligibility for new targeted treatment modalities.

Gene amplification in breast cancer – looking for the molecular mechanisms

Amplification of genes in breast cancer cells was shown to be coordinated in the majority of cases assuming the presence of common undisclosed mechanism underlying intrachromosomal gene amplification process. Overexpression and amplification of the HER-2/neu gene is observed in 20–30% of breast cancer cases and predicts more frequent relapse and shorter survival time. However, the initial events and molecular mechanisms of chromosomal aberrations in the region around HER2/neu as well as the precise boundaries of amplified regions remain to be obscure. This study was an attempt to define the boundaries of amplified chromosome 17q12-q21 region around HER2/neu gene. Structural peculiarities of amplification boundary regions were investigated by various techniques. Gene dosage analysis was performed for several genes located around HER2/neu gene in 154 breast cancer tissue samples. The copy number of the ppparbp, her2/neu, znfn1a3 Casc3 and top2a genes were analyzed by comparative TaqMan real time PCR. Fifty six samples (36 %) were found to be her2/neu-amplified (NHER2/neu ≥ 1,5), the boundaries for HER2-containing amplicons were mapped. Observed results suggest that similar positions of amplified region boundaries in different breast cancer samples are not accidental and may be related with the DNA structural and/or sequence peculiarities of boundary regions. Trying to find “special” sequences at the amplicon boundaries, we performed the analysis of sequence flexibility in the region TBC1D3– HER2/neu – TOP2A using the TwistFlex program. The analysis revealed several high flexible sequences in SOCS7, FBX047, HER2/neu and ZNFN1A3 genes. The locations of amplified region boundaries in 30% of cases were inside ZNFN1A3 gene. Organization of DNA sequence and nucleotide composition of FlexZNF-1 and FlexZNF-2 were similar to AT-rich islands of common fragile site (FS). Sequences of FlexZNF-1 and FlexZNF-2 have strong potential to form secondary structures according to the analysis by Mfold software (GCG Wisconsin PackageTM). Thus, the analysis of DNA sequence in TBC1D3– HER2/neu – TOP2A region revealed the existence of two flexible sequences in the ZNFN1A3 gene sequence with the strong potential to form stable secondary structures which may affect normal DNA replication at these sites and result in DNA double strand break appearance. The location of FlexZNF-1 and FlexZNF-2 strongly coincided with the location of amplified around HER2/neu gene fragment boundaries observed by real time PCR analysis. This observation indicates involvement of Flex sites in the process of intrachromosomal DNA amplification.

Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer

Background:Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods.Methods:To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administration-approved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n=75) and lung (n=71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients’ primary and metastatic cancer.Results:The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (P<0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (P<0.0001). Recovered CTCs were relatively pure (60–70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs.Conclusion:The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour.

Real-Time RT-PCR Analysis for Evaluating the Her2/neu Status in Breast Cancer

Objective: The Her2/neu status is of great clinical value in breast tumor patients. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques are the test of choice for many practicing pathologies. The main objective of this retrospective study was to investigate the relationship between Her2/neu breast cancer amplification and overexpression (DNA, mRNA and protein). Methods: To accomplish this goal, we evaluated Her2/neu mRNA expression by real-time quantitative RT-PCR, gene amplification by FISH and protein expression by IHC. Results: An excellent correlation between FISH and IHC Her2/neu results was observed, confirming that protein levels were directly related to DNA amplification. Polysomy 17 was frequently found in tumors showing Her2/neu overexpression. However, we did not find any statistically significant correlation among DNA, mRNA and protein levels, suggesting that Her2/neu could be post-transcriptionally regulated. Conclusions: There was a highagreement between Her2/neu gene amplification and protein overexpression but not mRNA expression levels. Nevertheless, IHC3+ and FISH-positive tumors indicated higher expression levels of Her2/neu mRNA by RT-PCR than those observed in IHC and FISH-negative tumors. These findings question the relevance of quantitative RT-PCR in routine assessment of Her2/neu overexpression in human breast cancer in the clinical laboratory setting.

Simultaneous detection of TOP2A and HER2 gene amplification by multiplex ligation-dependent probe amplification in breast cancer

HER-2/neu gene amplification, found in certain subtypes of (breast-) cancers, is an independent prognostic factor of poor outcome and determines eligibility for systemic treatment with trastuzumab. TopoIIα (TOP2A) gene amplification seems to be predictive of response to a class of cytostatic agents called TopoII inhibitors, which include the anthracyclines. The observed increased efficacy of anthracyclines in HER2-positive tumors is thought to arise from the close proximity of both genes on chromosome 17, where the TopoII amplification status will determine the anthracycline sensitivity. This study aimed to validate a new polymerase chain reaction-based test, called multiplex ligation-dependent probe amplification (MLPA), as a simple and quick method to simultaneously assess HER-2/neu and TopoIIα gene amplification status in paraffin-embedded breast cancer samples. To this end, MLPA results were compared with TopoIIα, HER2 chromogenic in situ hybridization (CISH). We also assessed TopoIIα protein expression by immunohistochemistry. Of 353 patients, 9% showed TopoIIα amplification by MLPA and 13% of patients were HER2 amplified. TopoIIα amplification was seen in 42% of HER2-amplified cases and showed no high level amplification without HER2 amplification. Eleven patients displayed TopoIIα loss (3%). Concordance between MLPA and CISH was 91% for TopoIIα and 96% for HER2. Correlation between amplification and overexpression of TopoIIα was significant (P=0.035), but amplification did not always predict protein overexpression. Loss of the TopoIIα gene was almost never associated with loss of its protein. In conclusion, MLPA is an easy and accurate method to simultaneously detect breast cancer HER-2/neu and TopoIIα copy number status in paraffin-embedded tissue, and thus an attractive supplement or alternative to CISH.

29. c-MYC gene amplification is associated with a poor prognosis in invasive ductal carcinoma

Background c-MYC is a proto-oncogene located on 8q24 with well-established critical roles in the development and progression of breast cancer. However, there is relatively little information available on the frequency of its amplification in breast cancer, with widely varying estimates in the literature and only two studies using the gold-standard of fluorescent in situ hybridisation (FISH). Moreover, there is little consensus as to a clinically relevant c-MYC/CEP8 (centromeric region of chromosome 8) ratio in determining patient outcome or response to therapy. Methods We investigated the frequency of c-MYC gene amplification using FISH in formalin fixed, paraffin processed tissues in 16 tissue microarrays from a cohort of 262 patients with operable invasive ductal carcinoma. C-MYC gene and CEP8 region copy number were determined in 20 representative tumour nuclei and a ratio c-MYC/CEP8 calculated. Two different ratios were tested using Kaplan-Meier survival analysis for their prognostic strength; 2.2 as used to determine HER2 gene amplification, and a newly described ratio of 1.3 which co-segregated with outcome. Chi squared analysis was then used to investigate the clinicopathological associations of c-MYC gene amplification Results A c-MYC/CEP8 ratio of 1.3 identified a poor prognostic group in the cohort (HR 2.28, p = 14; 0.006), while a ratio of 2.2 showed only a trend towards significance (p = 14; 0.087). Using a ratio of 1.3, 22.1% of cancers showed c-MYC gene amplification, while 61% were diploid and 16.9% were polysomic. c-MYC gene amplification was associated with larger, grade 3, ER negative, Ki67 high tumours (all p 5 0.05). Furthermore there was a highly significant association with the basal-like phenotype (p = 14; 0.004). Conclusion We have validated that a c-MYC/CEP8 ratio of 1.3 is able to identify a poor prognosis group of breast cancer patients. c-MYC gene amplification is associated with a number of high risk clinicopathological variables.

Multiplex Ligation-Dependent Probe Amplification to Detect HER2 Amplification in Breast Cancer: New Insights in Optimal Cut-Off Value

Multiplex Ligation-Dependent Probe Amplification to Detect HER2 Amplification in Breast Cancer: New Insights in Optimal Cut-Off Value

Comparison of Pathvysion and Poseidon HER2 FISH assays in measuring HER2 amplification in breast cancer: a validation study

AimsThe current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial Poseidon HER2 fluorescence in situ hybridisation (FISH) assay....

Fibroblastic Growth Factor Pathway

Fibroblastic Growth Factor Pathway

Comparison of fluorescence in situ hybridization and immunohistochemistry for assessment of HER-2 status in breast cancer patients

The accurate assessment of a proto-oncogene, human epidermal growth factor receptor-2 gene (HER-2), is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry (IHC) is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization (FISH) has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases (34.6%) by FISH and the HER-2 protein over-expression (score 3+) in 15 cases (28.8%) by IHC. Immunohistochemically, 28.6% of the cases scored as 2+ and 93.3% of the cases scored as 3+ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer (P<0.005). No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.

The Correlation Between Dual-color Chromogenic In Situ Hybridization and Fluorescence In Situ Hybridization in Assessing HER2 Gene Amplification in Breast Cancer

Fluorescence in situ hybridization (FISH) is regarded as the gold standard method for detecting HER2 gene amplification. Chromogenic in situ hybridization (CISH) is a promising alternative to FISH because CISH has the advantages of being a method evaluated by bright-field microscopy and the generated chromogenic signals are also stable. This study presents a dual color CISH for simultaneous detection of the HER2 gene and chromosome 17. The CISH method performs a chromogenic detection "on top" of the Food and Drug Administration (FDA)-approved HER2 FISH pharmDx method, where the fluorochrome-labeled probes are detected using enzyme-labeled antibodies and visualized by chromogenic enzymatic reactions. The HER2 status (amplified/not amplified and HER2 ratios) was evaluated by the CISH method and compared with results obtained by the FDA-approved FISH method. Of the 72 successfully investigated invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative to the FISH method.

Bright-field In Situ Hybridization for HER2 Gene Amplification in Breast Cancer Using Tissue Microarrays

HER2 gene amplification or overexpression occurs in 15% to 25% of breast cancers and has implications for treatment and prognosis. The most commonly used methods for HER2 testing are fluorescence in situ hybridization (FISH) and immunohistochemistry. FISH is considered to be the reference standard and more accurately predicts response to trastuzumab, but is technically demanding, expensive, and requires specialized equipment. In situ hybridization is required to be eligible for adjuvant treatment with trastuzumab in Australia. Bright-field in situ hybridization is an alternative to FISH and uses a combination of in situ methodology and a peroxidase-mediated chromogenic substrate such as diaminobenzidine [chromogenic in situ hybridization (CISH)] or multimer technology coupled with enzyme metallography [silver-enhanced in situ hybridization (SISH)] to create a marker visible under bright-field microscopy. CISH was introduced into diagnostic testing in Australia in October 2006. SISH methodology is a more recent introduction into the testing repertoire. An evaluation of CISH and SISH performance to assess patient outcome were performed using tissue microarrays. Tissue microarrays were constructed in duplicate using material from 593 patients with invasive breast carcinoma and assessed using CISH and SISH. Gene amplification was assessed using the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria (single probe: diploid, 1 to 2.5 copies/nucleus; polysomy >2.5 to 4 copies/nucleus; equivocal, >4 to 6 copies/nucleus; low-level amplification, >6 to 10 copies/nucleus and high-level amplification >10 copies/nucleus; dual probe HER2/CHR17 ratio: nonamplified <1.8, equivocal 1.8 to 2.2, amplified >2.2). Results were informative for 337 tissue cores comprising 230 patient samples. Concordance rates were 96% for HER2 single probe CISH and SISH and 95.5% for single probe CISH and dual probe HER2/CHR17 SISH. Both bright-field methods correlated with immunohistochemistry results and with breast cancer-specific survival. HER2 SISH testing combines the advantages of automation and bright-field microscopy to facilitate workflow within the laboratory, improves turnaround time, and correlates with patient outcome.

Association of the level of HER2/neu (HER2) gene amplification in breast cancer and the magnitude of antigen specific T-cell immunity achieved after HER2 vaccination

3059 Background: Studies have demonstrated that the level of HER2 gene amplification in breast cancer, assessed by fluorescence in situ hybridization (FISH), correlates with favorable clinical response after treatment with trastuzumab. We questioned whether HER2 gene amplification impacted the development of HER2-specific T-cell immunity following immunization with a HER2 vaccine. Methods: Patients with HER2+ stage III or IV breast cancer, treated to complete remission or stable bone only disease, were enrolled in one of two concurrent clinical trials of HER2-specific vaccines. Eligibility criteria between the two studies were similar. Patients received either a plasmid DNA-based vaccine encoding the HER2 intracellular domain or a peptide-based vaccine composed of 3 HER2 class II epitopes. Peripheral blood was assessed for HER2-specific T-cell responses by interferon gamma (IFN-g) ELISPOT prior to, immediately after, and 6 months to 1 year after the end of vaccinations. Both immune response and FISH data were available on 31 patients. Results: Correlation of FISH levels to IFN-g spots/well in evaluable patients revealed the level of HER2 gene amplification was not related to the presence of pre-existent HER2-specific T-cell immunity prior to vaccination (p=0.43), the generation of a HER2-specific immune response after vaccination (p=0.35), or the persistence of the HER2-specific T-cell response (p=0.33). However, the magnitude of the T-cell response achieved was less as HER2 gene amplification increased (p=0.05). Conclusions: The level of HER2 gene amplification in the primary tumor can adversely impact the magnitude of HER2-specific T-cell immunity achieved after vaccination. No significant financial relationships to disclose.

An Alternative and Reliable Real-time Quantitative PCR Method to Determine HER2/neu Amplification in Breast Cancer

The overexpression of HER-2/neu is an independent prognostic factor of clinical outcome of breast cancer, therefore determination of HER-2/neu status is now an integral part of the clinicopathologic workup. The ways of measuring the copy number of the HER-2/neu gene in tumor cells comprise in situ hybridization techniques and real-time polymerase chain reaction (PCR). Quantitative real-time PCR is a relatively new technique for assessing HER-2/neu gene amplification with high sensitivity. However, the HER-2/neu Quantification Kit developed by Roche designed for a LightCycler 1.5 platform had been withdrawn from the commercial market; therefore, we were encouraged to design an alternative LightCycler-based method that offers the desired level of reliability. One hundred breast cancer cases with known HER-2/neu status have been examined with the original Roche developed HER-2/neu Quantification kit and the custom real-time PCR assay. The newly developed, custom PCR showed sensitivity of 91.43%, specificity of 90.63%, and accuracy of 90.91% taking fluorescence in situ hybridization results as the end point. We have described a novel real-time PCR technique for the relative quantification of the HER2/neu gene on a LightCycler 1.5 platform. We have determined that our method is eligible and ideal for the supplement of regular fluorescence in situ hybridization reactions, concerning its high sensitivity and reliability.

Her-2/neu gene overexpression in breast carcinoma and its association with clinicopathological characteristics of the disease.

To assess the frequency of Her-2/neu gene amplification in breast cancer and its association with clinicopathologic characteristics of the disease. Cross-sectional comparative study. Study was carried out at Surgical Unit I, Holy Family Hospital, Rawalpindi, from January 2005 to December 2006. A total of 50 patients diagnosed of having breast cancer on biopsy of breast lump were included in the study. All patients underwent mastectomy and samples were submitted to AFIP (Armed Forces Institute of Pathology, Rawalpindi) for histopathology and determination of estrogen and progesterone receptors and Her-2/neu gene amplification. Moreover, association of Her-2/neu amplification to other characteristics like tumour size, node involvement, histological grade and presence of estrogen and progesterone receptors were also studied. Out of 50 patients, 18 (36%) were positive for Her-2/neu overexpression. Among the various prognostic factors, age showed a statistically significant relation to Her-2/neu positivity (p=0.001). The Her-2/neu positive cancers were larger in size (mean size of 7.9 +/- 3.615 and 5.4 +/- 1.906 cm in Her-2/neu positive and negative cases respectively; p=0.002). Her-2/neu positive cases had statistically significant relation to number of involved lymph nodes (mean number of 9.9 and 4.4 lymph nodes in Her-2/neu positive and negative cases respectively; p=0.001). Tumour grade and nuclear grade had no statistically significant relation to Her-2/neu status (p=0.19 and 0.434 respectively). Both estrogen and progesterone receptors are inversely related to Her-2/neu positivity (p=0.028 and 0.006 respectively). The pathological and clinical characteristics of Her-2/neu positive and negative subsets differ, and Her-2/neu overexpression was associated with other poor prognostic factors of age, tumour size and number of nodes involved. There was an inverse relation of Her-2/neu status and hormone receptor.