HER2 Gene Copy Research Articles

Genomic aberrations in normal tissue adjacent to HER2-amplified breast cancers: field cancerization or contaminating tumor cells?

Field cancerization effects as well as isolated tumor cell foci extending well beyond the invasive tumor margin have been described previously to account for local recurrence rates following breast conserving surgery despite adequate surgical margins and breast radiotherapy. To look for evidence of possible tumor cell contamination or field cancerization by genetic effects, a pilot study (Study 1: 12 sample pairs) followed by a verification study (Study 2: 20 sample pairs) were performed on DNA extracted from HER2-positive breast tumors and matching normal adjacent mammary tissue samples excised 1–3 cm beyond the invasive tumor margin. High-resolution molecular inversion probe (MIP) arrays were used to compare genomic copy number variations, including increased HER2 gene copies, between the paired samples; as well, a detailed histologic and immunohistochemical (IHC) re-evaluation of all Study 2 samples was performed blinded to the genomic results to characterize the adjacent normal tissue composition bracketing the DNA-extracted samples. Overall, 14/32 (44 %) sample pairs from both studies produced genome-wide evidence of genetic aberrations including HER2 copy number gains within the adjacent normal tissue samples. The observed single-parental origin of monoallelic HER2 amplicon haplotypes shared by informative tumor–normal pairs, as well as commonly gained loci elsewhere on 17q, suggested the presence of contaminating tumor cells in the genomically aberrant normal samples. Histologic and IHC analyses identified occult 25–200 μm tumor cell clusters overexpressing HER2 scattered in more than half, but not all, of the genomically aberrant normal samples re-evaluated, but in none of the genomically normal samples. These genomic and microscopic findings support the conclusion that tumor cell contamination rather than genetic field cancerization represents the likeliest cause of local clinical recurrence rates following breast conserving surgery, and mandate caution in assuming the genomic normalcy of histologically benign appearing peritumor breast tissue.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-012-2290-3) contains supplementary material, which is available to authorized users.

Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models

BackgroundTrastuzumab is currently approved for the clinical treatment of breast and gastric cancer patients with HER-2 positive tumors, but not yet for the treatment of esophageal carcinoma patients, whose tumors typically show 5 ~ 35% HER-2 gene amplification and 0 ~ 56% HER-2 protein expression. This study aimed to investigate the therapeutic efficacy of Trastuzumab in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.MethodsPDECX models were established by implanting patient esophageal squamous cell carcinoma (ESCC) tissues into immunodeficient (SCID/nude) mice. HER-2 gene copy number (GCN) and protein expression were determined in xenograft tissues and corresponding patient EC samples by FISH and IHC analysis. Trastuzumab anti-tumor efficacy was evaluated within these PDECX models (n = 8 animals/group). Furthermore, hotspot mutations of EGFR, K-ras, B-raf and PIK3CA genes were screened for in the PDECX models and their corresponding patient’s ESCC tissues. Similarity between the PDECX models and their corresponding patient’s ESCC tissue was confirmed by histology, morphology, HER-2 GCN and mutation.ResultsNone of the PDECX models (or their corresponding patient’s ESCC tissues) harbored HER-2 gene amplification. IHC staining showed HER-2 positivity (IHC 2+) in 2 PDECX models and negativity in 3 PDECX models. Significant tumor regression was observed in the Trastuzumab-treated EC044 HER-2 positive model (IHC 2+). A second HER-2 positive (IHC 2+) model, EC039, harbored a known PIK3CA mutation and showed strong activation of the AKT signaling pathway and was insensitive to Trastuzumab treatment, but could be resensitised using a combination of Trastuzumab and AKT inhibitor AZD5363. In summary, we established 5 PDECX mouse models and demonstrated tumor regression in response to Trastuzumab treatment in a HER-2 IHC 2+ model, but resistance in a HER-2 IHC 2+/PIK3CA mutated model.ConclusionsThis study demonstrates Trastuzumab-induced tumor regressions in HER-2 positive tumors, and highlights PIK3CA mutation as a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models.

HER family receptor and ligand status in thymic carcinoma

Overexpression and gene amplification of the HER family of receptors and their ligands are important prognostic factors in many solid tumors and treatment targeting these molecules has recently become available. The role of this group of receptors has only rarely been described in thymic epithelial neoplasms and never before in a series of cases consisting exclusively of thymic carcinoma.Twenty-four primary squamous cell carcinomas of the thymus were examined for immunohistochemical expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR (pEGFR), HER2, phosphorylated HER2 (pHER2), HER3, phosphorylated HER3 (pHER3) and their ligands epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin and epiregulin. Fluorescence in situ hybridization (FISH) analysis for amplification of the EGFR and HER2 genes was performed including assessment of the copy numbers of EGFR and HER2 gene per cell and the ratio of EGFR and HER2 to centromere 7 and 17, respectively.Significant immunohistochemical expression was observed for EGFR (33.3%), pEGFR (33.3%), HER2 (58.3%), HER3 (45.8%), TGF-α (54.1%), amphiregulin (25.0%) and epiregulin (91.7%). A single case showed HER2 gene amplification by FISH. Increased EGFR and HER2 gene copy numbers were observed in 2 (8.4%) and 18 cases (75%), respectively. Eight cases (33.3%) showed an increased HER2:CEP17 ratio.The results of this study indicate that EGFR and HER2 amplification is a rare event in thymic carcinoma, however, protein expression for HER receptors as well as their ligands is a common finding indicating that targeted therapy directed against these molecules may be considered in the treatment of these tumors.

Actionable targets in pancreatic cancer detected by immunohistochemistry (IHC), microarray (MA) fluorescent in situ hybridization (FISH), and mutational analysis.

4013 Background: A great need exists for new therapeutic approaches for patients with pancreatic cancer. Methods: The study cohort included 1029 patients analyzedfor a.) up to 29 different immunohistochemical biomarkers – (e.g., COX-2, MGMT, PGP, RRM1, TOPOI, TOPOII, SPARC etc.)b.); in up to 450 patients’ specimens a whole genome expression analysis was performed using HumanHT-12 v4 beadChips ( Illumina Inc.,San Diego, CA) c.) in up to 695 patients FISH for c-Myc, EGFR, HER2 and TOPO2A gene copy amplifications; and d.) in up to 783 patients sequencing for KRAS, EGFR, PI3CA and BRAF, was performed. Results: IHC identified actionable targets included; 74% high COX-2; 57% negative ERCC1; 8% negative MGMT; 22% negative MRP1; 47% negative PGP; 77% low RRM1, 44% high SPARC; 30% high TOPO2A; 61% high TOPOI and 73% negative TS. Other biologically important findings by IHC for possible new therapeutics included 27% negative PTEN; and 20% high PDGFR. Microarray results presented multiple overexpressed targets for consideration including 36% of specimens with overexpressed adenosine deaminase; 28% asparagine synthase; 17% BCL2; 20% survivin; 23% carboxylesterase; 67% DNMT1; 40% thymidine phosphatase; 49% EPHA2 (and others in the src family of kinases); 57% FOLR2; 41% HDAC1; 62% HiF1α; 23% IL2RA (CD25); 46% NFkB1; 48% OGFR; 32% RARA; 26% VEGFR; and 43% vitamin D receptor. FISH yielded 2% amplified EGFR and 10% amplified Her2neu. Sequencing noted 73% mutated KRAS and 3% mutated PIK3CA. Conclusions: Examining actionable targets in patients’ pancreatic cancers (a)reiterates the commonality and importance of KRAS mutations in this disease (needs renewed targeting effort) (b)suggests that TOPO2 inhibitors (particularly if transport into tumor can be improved) should be examined in this disease (c)suggests other pathways to target including DNA repair, epigenetic, Src and inflammation (d) suggests protein turnover, amino acid targets and folate receptor2 as fresh areas to explore against the disease. Supported in part by a Stand Up To Cancer Dream Team Award.

Significance of chromosome 17 polysomy in breast cancer.

e12002 Background: Human epidermal growth factor receptor 2 (HER-2) overexpression occurs in up to 30% of breast cancers and has been associated with poor clinical outcomes that have improved with advances in HER-2 directed therapy. The HER-2 gene resides on the long arm of chromosome 17 and amplification is typically assessed via immunohistochemistry or fluorescence in situ hybridization (FISH); HER-2 is interpreted as amplified, non-amplified, or equivocal. The effect and clinical impact of chromosome 17 polysomy in specimens interpreted as HER-2 non-amplified has not been well-established in breast cancer patients. This subgroup may represent a unique set of patients who may benefit from HER-2 directed therapy given the increase in HER-2 gene copy number. If tumors with polysomy 17 share biologic similarities with those positive for HER-2 amplification this may significantly influence the approach to treating these patients. In a single institution study we reviewed all breast cancer cases diagnosed in 2008 which were reported as HER-2 equivocal or non-amplified via FISH and with chromosome 17 polysomy in order to extrapolate disease-free and overall survival data within this subset. Methods: HER-2 expression via FISH from patients diagnosed with breast cancer in 2008 was reviewed. In those patients with equivocal or non-amplified HER-2 expression we selected those with chromosome 17 polysomy defined as a chromosome 17 centromere copy number of > 3 per tumor cell. A total of 9 patients were identified. Results: The median age was 74 (36-88). Median tumor size was 1.6 cm (0.7-4.9) and 8 patients (88%) had both estrogen and progesterone positive tumors. Stage distribution was as follows: stage IA: 4 (44%); stage IIA: 2 (22%); stage IIB: 2 (22%) and stage IIIA: 1 (11%). The actuarial 3 year disease free survival was 67%. Conclusions: Breast cancers with equivocal or non-amplified HER-2 expression with chromosome 17 polysomy represent a unique subset of tumors with a biology that is not well-understood. Previously published studies have yielded conflicting results regarding the prognostic significance of this genotype. Our study reflects a three year survival of 67% which suggests that these patients represent an aggressive subset.

Post-progression off-label Trastuzumab treatment of HER2 amplified metastatic gastric cancer - Case study

Background: A change in the HER2 gene copy number can be detected at 16–20% cent of gastric cancer patients. ToGA trial proved that chemotherapy combined with targeted HER-2 antibodies improved survival of this group by 20%, which prompted a registration of Trastuzumab as the first-line treatment of this patient group; its administration is indicated until the onset of progression.

P1-12-17: Fluorescent In Situ Hybridization Evaluation of HER2 Status in Tumors with Chromosome 17 Polysomy.

Abstract Introduction: According to the American Society of Clinical Oncology-College of American Pathologists recommendations for HER2 testing, a positive fluorescent in situ hybridization (FISH) result is defined as >6 HER2 gene copies/nucleus (for test systems with no internal control probe [single-color]) or as HER2 gene/chromosome 17 centromere (CEP17) ratio > 2.2 (for systems with an internal control probe [dual-color]). Although an increase in CEP17 copy number (average ≥ 3.0 copies/nucleus) is commonly considered to represent polysomy of chromosome 17, it can also be a result of gains of 17q with centromere involvement, or amplification of the centromeric region. The classification of HER2−positive tumors according to the HER2/CEP17 ratio may therefore misclassify a fraction of truly amplified cases as polysomic. We prospectively evaluated tumors with chromosome 17 polysomy but no HER2 amplification to assess HER2 status using the above two FISH classifications and immunohistochemistry (IHC). Materials and methods: Tumors were tested for gene amplification by FISH with probes to HER2/neu and CEP17 using the PathVysion HER-2 DNA Probe Kit (Vysis). Classification was based on the HER2/CEP17 ratio (amplified when > 2.2) and average HER2 gene copy number/nucleus (amplified when > 6 copies). Both polysomic and equivocal cases (HER2/CEP17 ratio 1.8 - 2.2) were further studied by IHC using the HercepTest (Dako) with 0–3 scoring system (overexpression when 3+). Results: From March 2010 to May 2011 we evaluated 31 primary breast cancers showing chromosome 17 polysomy. Median HER2/CEP17 ratio was 1.3 (range 0.5−1.9), median HER2 copy number was 5.4 (range 2.6−13.8), and median CEP17 copy number was 4.2 (range 3.2−8.0). Thirteen (42%) had an average HER2 gene copy number > 6/nucleus (median 6.8, range 6.1−13.8) and would therefore be considered as amplified if classified according to the absolute HER2 gene copy number. Nine (75%) of these were 3+ at IHC and the remaining 4 were 2+, whereas among the 18 cases with an average HER2 gene copy number < 6/nucleus, one was 2+, ten were 1+, and 7 scored 0. Twenty-nine cases showed negative HER2/CEP17 ratios (< 1.8) and three cases equivocal HER2/CEP17 ratios (between 1.8 and 2.2). Using HER2/CEP17 ratio as first assessment and IHC only in equivocal cases, only one of the 31 “polysomic” cases would have been classified as HER2−positive. However, 8 polysomic cases with HER2/CEP17 ratio < 1.8 showed 3+ immunostaining (all with average HER2 gene copy number > 6/nucleus), while 3 other cases had an average HER2 gene copy number > 6/nucleus with 2+ immunostaining. Conclusions: Our results show that both FISH evaluation criteria and IHC can modify the percentage of polysomic tumors classified as HER2−positive. However, the number of gene copies/nucleus appears more frequently associated with 3+ IHC than the HER2/CEP17 ratio. The predictive impact of the former method on response to anti-HER2 treatments warrants further investigation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-17.

P1-07-19: Analysis of HER2−Status in Breast Cancer by Mass Spectrometry in Archival, Formalin-Fixed Tissues.

Abstract HER2 (ERBB2) is overexpressed in about 25% of breast cancers and predicts clinical benefit from trastuzumab, as well as response to anthracycline-based chemotherapy. Fluorescence in situ hybridization (FISH) to detect HER2 gene copy number and immunohistochemistry (IHC) to detect HER2 protein levels are approved by the FDA to identify HER2−positive (H2) tumors. However, the 2007 ASCO/CAP report concluded that approximately 20% of HER2 testing may be inaccurate. Further, the available data did not clearly demonstrate clear superiority of either IHC or FISH as a predictor of benefit from anti-HER2 therapy. Discordance between these methods is as high as 5%. Thus, novel complementary quantitative methods for interrogating HER2 expression in tumors are needed. Targeted protein analysis by multiple reaction monitoring mass spectrometry (MRM-MS) offers a powerful approach to configure assays for specific proteins without using antibodies. Our studies using this platform have demonstrated applicability to formalin-fixed, paraffin-embedded (FFPE) specimens. In the current studies, we used this approach to measure signals from two tryptic peptides specific to HER2, one each from the extracellular and intracellular domains, selected from among 28 candidates based on their signal intensity and sharpness of their chromatographic profiles. Preliminary studies with a HER2−overexpressing BT474 xenograft in mice demonstrated quantitation and detected previously reported HER2 ectodomain shedding. Subsequent analysis of FFPE tissue from five H2 and five triple-negative (TN) tumors yielded measurement of at least 1 femptomole of receptor for H2 tumors and less than 0.2 femptomole of receptor for TN tumors per microgram of digest analyzed. If we assume 200 picograms of protein per cell, the results suggest 110,00 to 468,000 receptors per cell in the H2 tumors and only 2,000 to 14,000 receptors per cell in the TN tumors. Despite significant biological variability in receptor levels measured among the specimens of each type, a clear separation of the H2 and TN tumors was achieved based on the peptide quantitation. This preliminary study demonstrates the potential of MRM-MS in FFPE tissue to provide an alternate approach to IHC-based protein analysis. MRM-MS offers the potential for more, accurate and robust HER2 quantification in clinical breast cancer tissues. The next phase of this work will encompass a larger sample set, including tumors with equivocal and negative FISH and/or IHC test results. Correlation with response to anti-Her2 therapy will be performed in samples with available follow-up data. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-19.

ES3-3: Adjuvant Therapy in Patients with a Borderline HER-2 Status.

Abstract Adjuvant trastuzumab is indicated in individuals with early breast cancer which has HER2 gene amplification and/or HER2 protein overexpression. In the pivotal phase III adjuvant trials which tested the addition of trastuzumab to chemotherapy, positive HER2 status was defined as strong and complete cell membrane staining by immunohistochemistry (IHC) of >10% of invasive tumor cells or gene amplification determined by fluorescence in-situ hybridisation (FISH) ratio of HER2 gene copy number to chromosome 17 centromeres (CEP17) of >2.0. The decision to use trastuzumab is based on a binary categorization of HER2 as positive or negative. As such, the definition of HER2 positivity is critical, as it dictates who will or will not receive potentially efficacious treatment. The challenge in creating guidelines for anti-HER2 treatment is that an HER2 result is described as positive or negative, but HER2 exists as a continuum of gene copy number and protein expression. A source of uncertainty in the management of patients is the discordance between the diagnostic thresholds for HER2 adopted in the adjuvant trastuzumab trials and those specified in the subsequently published American Society of Clinical Oncology and the College of American Pathologists (ASCO-CAP) guidelines. According to ASCO-CAP, HER2 positivity is defined by uniform intense and complete membrane staining by IHC in ≥30% of cells or HER2/ CEP17 FISH ratio≥2.2. The definition is stricter than thresholds applied in the adjuvant trials, identifying a narrower population as HER2 positive. A further source of uncertainty is discordance between IHC and FISH. There is generally high concordance between the two methods, as increased HER2 protein is generally attributable to HER2 amplification. Discordant results may occur if one assay is correct and the other is incorrect, due to pre-analytic, analytic, and/or post-analytic error. True discordance may be attributable to intra-tumoral heterogeneity or polysomy chromosome 17. Within a tumor, HER2 amplification and/or overexpression may be detected in discrete focal HER2 amplified clones (FHAC) or in individual cells diffusely scattered on a dominant background of HER2 negative/equivocal expression. FHAC have been reported in association with discordance between IHC and FISH. Cells with polysomy chromosome 17 have extra copies of the HER2 gene. In such cases, tumors may be IHC positive although the HER2:CEP17 FISH ratio is not elevated. Notably, available data are inconsistent and uncertainty remains as to whether polysomy 17 without HER2 amplification is associated with protein overexpression During this session, clinical situations with borderline HER2−status will be presented and discussed. Biological heterogeneity within the same tumor, polysomy chromosome 17, and moderate HER2 positivity may contribute to determination of an uncertain HER2 status, making unclear the benefit of adjuvant trastuzumab in these cases. Clinical considerations highlighting pros/cons of adjuvant trastuzumab in these cases, as well as updated biological information and clinical perspectives, will be presented. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr ES3-3.

Assessing the Impact of Polysomy-17 on HER2 Status and the Correlations of HER2 Status With Prognostic Variables (ER, PR, p53, Ki-67) in Epithelial Ovarian Cancer

Although HER2 overexpression and Her2 amplification have been noted in breast and a variety of human cancers, we report here for the first time the impact of polysomy-17 on HER2 status and the correlations between HER2 status and other prognostic factors in patients with epithelial ovarian cancers (EOC).We analyzed HER2, estrogen receptor (ER), progesterone receptor (PR), p53, and Ki-67 protein overexpressions by immunohistochemistry (IHC) and determined Her2 gene amplification by fluorescence in situ hybridization (FISH) in 27 tissue microarray samples from EOC patients.We achieved 100% positive concordance (3/3) and 100% negative concordance (19/19) between HER2 testing by IHC and FISH. Both the total Her2 gene copies and FISH scores increased significantly in a stepwise order through the negative, equivocal, and positive HER2 IHC result categories in all 27 cases (P=0.001, P=0.001), and still increased significantly in 18 nonpolysomy-17 cases (P=0.007 and 0.013) after the exclusion of 9 polysomy-17 cases. HER2 protein expression is inversely correlated with both ER (P=0.002) and PR expressions (P=0.046). Her2 gene amplification is inversely correlated with ER expression (P=0.007) but not with PR expression (P=0.106).This study showed extremely high positive and negative concordances between Her2 FISH and HER2 IHC assays. Polysomy-17 is insufficient for causing a significant impact on the relationship between HER2 testing by IHC and FISH in EOC. ER and PR expressions were inversely correlated with HER2 protein expression. In addition, ER but not PR expression is inversely correlated with Her2 gene amplification.

Dual-color silver-enhanced in situ hybridization for assessing HER2 gene amplification in breast cancer

Amplification of the human epidermal growth factor receptor 2 (HER2) gene occurs in 20–25% of breast cancers, and is recognized as a prognostic and predictive marker. HER2 gene amplification, evaluated as a change in protein expression or gene copy number, can be identified by a number of methods. Fluorescence in situ hybridization (FISH) is considered the gold standard for HER2 gene copy number determination; however, a number of impediments prevent its wider use in a clinical setting. The aims of our study were to compare dual-color silver-enhanced in situ hybridization (SISH) with single-color SISH and FISH on formalin-fixed, paraffin-embedded sections, and to validate its use as a routine method for assessing HER2 status in breast cancers. A total of 146 invasive breast carcinoma cases were assessed for HER2 gene amplification by FISH and dual-color SISH. Dual-color SISH and FISH results exhibited a concordance rate of 97% (κ=0.912). A comparison of the single-color SISH method with dual-color SISH showed that 142 of 146 cases were in agreement (97%, κ=0.930). Our results showed that dual-color SISH is a viable alternative to FISH that offers a number of advantages in a clinical setting.

Evaluation of HER2 gene status by qPCR in breast cancer samples with nonconclusive FISH.

612 Background: Trastuzumab therapy is indicated to breast cancer patients with Her-2 gene amplification/overexpression. Fluorescence in situ hybridization (FISH) assay is commonly used for Her-2 gene copy number detection. However, in our consecutive cohort, 136/2608 (5.2%) patient samples could not be tested by FISH due to poor quality of tissue. The aim of this study was to develop alternative DNA based method which can detect Her-2 gene amplification in poor quality samples. Methods: Three duplex quantitative real-time PCR (qPCR) reactions were designed for assessment of Her-2 gene in Formalin-Fixed, Paraffin-Embedded (FFPE) breast cancer tissues. Three genes rarely amplified in breast cancer, glucosidase 1 (GCS1), deoxycytidine kinase (DCK) and epsin 2 (EPN2), were used as a reference. Her-2 gene true amplification was defined by ratio of Her-2/reference gene >2.2 at least in 2 of 3 reference genes, grey zone amplification was characterized by ratio 1.8 to 2.2. Sensitivity of the method was determined using concentration range of Her-2 amplified CALU3 (20 copies/nucleus) and MDA-MB-231 cells with physiologic Her-2 gene copy number. Specificity of the method was assessed in prospective consecutive cohort of 192 FFPE samples examined by FISH and qPCR in parallel. Furthermore, the qPCR method was used to evaluate Her-2 gene status in 136/2,608 samples not testable by FISH. Results: The qPCR method was highly reproducible and detected Her-2 gene amplification in samples containing at least 10% of positive cells. Concordance (positivity vs. negativity) was observed in 91% of cases. Further, we have evaluated by qPCR 136 tissues, in which FISH method failed, and found 72/136 (52.9%) breast cancers with normal status of Her-2 gene, grey zone and true amplifications were detected in 9/136 (6.6%) and 18/136 (13.2%) of patient samples respectively. qPCR was not conclusive in 27.2% (37/136) of cases. Conclusions: Our study describes a new alternative qPCR based method for Her-2 evaluation in poor quality samples, where FISH assay failed. On the basis of this examination, additional 27/2,608 (1.03%) women from total breast cancer population, were indicated for trastuzumab therapy.

HER2 Protein and Gene Variation between Primary and Metastatic Breast Cancer: Significance and Impact on Patient Care

To analyze HER2 status in primary breast cancer (PBC) compared with correspondent metachronous metastases and to investigate whether BC phenotype may be predictive of change in HER2 expression. HER2 was investigated by immunohistochemistry, silver in situ hybridization (SISH), and FISH, in a series of 137 tumors, building up a tissue microarray to concurrently analyze each single PBC and metastatic (MBC) on the same slide. HER2 status was discordant in 14 cases (10%): 12 negative in PBC and positive in metastases and two positive in PBC and negative in metastases (P = 0.04). These findings were confirmed by a PCR based test termed Multiplex Ligation-dependent Probe Amplification (MLPA). HER2 status changed in hormone receptor-positive BC more frequently than in negative ones (P = 0.002). In addition, we evaluated HER2 gene and chromosome 17 copy number by SISH in the 123 cases with unchanged HER2 status during progression. We found consistent HER2 gene copy number stability in the 100 nonamplified cases. Conversely, of the 23 amplified PBC, 13 (57%) demonstrated a significant increase in the HER2 gene and chromosome 17 copy number in their paired metastases (P = 0.01), as defined by SISH (k = 0.54, P < 0.0001) and MLPA. Patients who changed HER2 status from negative to positive, presented significant longer time to progression when treated with trastuzumab compared to those who were untreated (P = 0.04). When feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from primary hormone receptor-positive BC.

Breast cancer evaluation by fluorescent dot detection using combined mathematical morphology and multifractal techniques

BackgroundFluorescence in situ hybridization (FISH) is very accurate method for measuring HER2 gene copies, as a sign of potential breast cancer. This method requires small tissue samples, and has a high sensitivity to detect abnormalities from a histological section. By using multiple colors, this method allows the detection of multiple targets simultaneously. The target parts in the cells become visible as colored dots. The HER-2 probes are visible as orange stained spots under a fluorescent microscope while probes for centromere 17 (CEP-17), the chromosome on which the gene HER-2/neu is located, are visible as green spots.MethodsThe conventional analysis involves the scoring of the ratio of HER-2/neu over CEP 17 dots within each cell nucleus and then averaging the scores for a number of 60 cells. A ratio of 2.0 of HER-2/neu to CEP 17 copy number denotes amplification. Several methods have been proposed for the detection and automated evaluation (dot counting) of FISH signals. In this paper the combined method based on the mathematical morphology (MM) and inverse multifractal (IMF) analysis is suggested. Similar method was applied recently in detection of microcalcifications in digital mammograms, and was very successful.ResultsThe combined MM using top-hat and bottom-hat filters, and the IMF method was applied to FISH images from Molecular Biology Lab, Department of Pathology, Wielkoposka Cancer Center, Poznan. Initial results indicate that this method can be applied to FISH images for the evaluation of HER2/neu status.ConclusionsMathematical morphology and multifractal approach are used for colored dot detection and counting in FISH images. Initial results derived on clinical cases are promising. Note that the overlapping of colored dots, particularly red/orange dots, needs additional improvements in post-processing.

EGFR/erB-1, HER2/erB-2, CK7, LP34, Ki67 and P53 expression in preneoplastic lesions of bronchial epithelium: an immunohistochemical and genetic study

A prognostic interpretation of preneoplastic lesions would have impact in bronchial carcinoma early diagnosis and through the study of Erb-B family receptors as they have an important role in lung carcinogenesis. The existence of drugs as tyrosine kinase inhibitors stressed the importance of studying gene alterations for selected chemoprevention schemes and characterization of carcinogenesis. Bronchial preneoplastic lesions were characterized by immunohistochemistry using the antibodies LP34 (high weigh molecular cytokeratin), CK7, chromogranin A, Ki67, p53, C-erbB-2 and EGFR. HER2 and EGFR gene copy number was also evaluated by fluorescent in situ hybridization in those lesions. The expected results defined the origin cell for basal cell hyperplasia and squamous metaplasia as adaptative lesions and dysplasia. By known experiences and published data, beyond the stem cell, the spectral evolution of bronchial preneoplastic lesions was demonstrated by characterizing basal cells (LP34) and their neoplastic potentiality. Dysplasias showed a higher expression of EGFR, Ki67 and p53 with a stepwise increase with the gravity of the respective grading. C-erbB-2 immunohistochemical overexpression was a rare event in preneoplastic lesions. Polysomy was the main mechanism for EGFR and HER2/neu higher gene copy number and together with increased proliferation index (Ki67) will account to preview bronchial carcinogenesis.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. FISH detection of HER2 gene amplification rate was 16.9% (10/59), HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% (29/59). HER2 protein expression was 42.4% (25/59), HER2 gene amplification rates in patients with +++, ++ HER2 protein expression were 3/3 and 5/8, while in patients with + HER2 protein expression, it was 2/14, there was significant difference (P 0.05). HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

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.

Chromosome 17 Polysomy without HER2 Amplification Does Not Predict Response to Lapatinib in Metastatic Breast Cancer—Letter

We have read with great interest the article by Downey et al. on chromosome 17 polysomy without HER2 amplification and prediction of response of metastatic breast cancer patients to lapatinib (1). The authors report that in a group of 405 HER2 FISH-negative patients, patients with chromosome 17 polysomy (n = 44, Chr17 ≥ 2.2 or 2.0) did not show a significant benefit from HER2-directed therapy compared with nonpolysomic patients. The authors used the current definition of chromosome 17 polysomy accepted by the Food and Drug Administration and American Society of Clinical Oncology/College of American Pathologists. They comment that it is unclear if chromosome 17 polysomy increases HER2 mRNA and protein product. The authors did, however, not mention the recent observation that chromosome 17 polysomy is far less common in breast cancer than previously suggested (2–4) and may, in fact, be very rare. These 3 independent studies using comparative genomic hybridization, FISH, and multiplex ligation-dependent probe amplification report that chromosome 17 genes show complex patterns of gains and losses unrelated to the copy number status of the centromere (CEP17). This could be another explanation for the fact that the study of Downey et al. could not confirm an association between “polysomy 17” and an improved outcome with the addition of lapatinib to paclitaxel, previously observed by Kaufman et al. (5).The conflicting results of Downey et al. and Kaufman et al., but also the recent study by Bartlett et al. (6), indicate that large prospective clinical trials are necessary to validate whether patients showing increased CEP17 indeed show a better response to HER2-targeted therapies and anthracyclines, also in the absence of HER2 or topoisomerase 2 (TOP2A) amplification. In addition, these studies should assess chromosome 17 status in a less simplistic way than merely looking at CEP17 and HER2 gene copy number, to help elucidate the mechanism by which CEP17 or other pericentromeric chromosome 17 genes could possibly influence trastuzumab/lapatinib and anthracycline sensitivity.See the Response, p. 6178No potential conflicts of interest were disclosed.

HER2 expression in breast cancer: Comparisons of fluorescence in situ hybridization and silver in situ hybridization, and immunohistochemical staining using monoclonal antibody and polyclonal antibody

ABSTRACT Background and aim: The human epidermal growth factor receptor 2 (HER2) gene is an important tumor marker in breast cancer. The silver in situ hybridization (SISH) has been recently introduced for measuring the HER2 amplification status. We evaluated the concordance between HER2 gene amplification in invasive breast cancer as determined by the fluorescence in situ hybridization (FISH) and SISH techniques and we compared the results to that of immunohistochemical (IHC) staining with using polyclonal c‐erbB‐2 antibody and monoclonal HER2 antibody. Methods: A total of 90 cases were analyzed by direct‐labeled manual FISH and bright field automated SISH. All the specimens underwent IHC staining using c‐erbB‐2 and PATHWAY 4B5. The evaluation was performed by following the recommendation of the manufacturer and the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Results: The overall concordance rate between FISH and SISH was 98.8% (Kappa index: 0.96). There was no significant difference in estimating the HER2 status using polyclonal and monoclonal antibody. Conclusions: The 98.8% concordance of FISH fulfills the ASCO/CAP requirement of greater than 95% concordance for the amplified cases versus the non‐amplified cases. The HER2 gene copy status can be reliably determined by SISH.

The HER2 gene copies per tumor cell either before or after correction for chromosome-17 correlated significantly with HER2 IHC results in epithelial ovarian cancer in a tissue microarray study

HER2 gene amplification and HER2 protein overexpression are important factors in predicting clinical sensitivity to anti-HER2 monoclonal antibody therapy in breast cancer patients. The purpose of this study is to evaluate the correlation between HER2 protein expressions and the HER2 gene copies per tumor cell either before or after chromosome-17 correction in epithelial ovarian cancer (EOC). Adopting 2007 ASCO/CAP guideline recommendations for HER2 testing, 27 tissue microarray (TMA) samples from EOC patients were analyzed by immunohistochemistry (IHC) using Dako, c-erb-B2 antibody and subsequently examined by fluorescence in situ hybridization (FISH) using Abbott/Vysis, PathVysion HER2 DNA Probe Kit. The overall concordance revealed 81.5% between HER2 IHC and HER2 FISH results. Additionally, HER2 gene copies prior to chromosome-17 correction increased significantly in a stepwise order through the negative, equivocal, and positive IHC result categories (P = 0.026), as did the HER2 gene copies after chromosome-17 correction (P = 0.028). On the other hand, HER2 IHC results correlated significantly with both chromosome-17 uncorrected HER2 gene copy numbers (ρ = 0.430, P = 0.025) and chromosome-17 corrected HER2 gene copy numbers (ρ = 0.524, P = 0.027). We demonstrated that both chromosome-17 corrected and uncorrected HER2 gene copies correlated significantly with HER2 IHC result categories; and tests for the HER2 gene copies per tumor cell either before or after correction for chromosome-17 can be applied as a potentially valuable tool in analyzing the HER2 status in EOC.