TOP2A Amplification in Breast Cancer in the Absence of That of HER-2: Myth or Reality?

Confusion between topoisomerase IIα gene amplification and topoisomerase mRNA overexpression is proposed as possibly leading to misinerpretation in a study by Glynn et al. published in an earlier issue of The Oncologist.     

HER-2, TOP2A and chromosome 17 alterations in breast cancer

HER-2 amplification is a biomarker for identifying patients who respond to trastuzumab and has been evaluated as a factor predicting the response to anthracyclines. The relationship between HER-2 and response to anthracycline therapy may also be the result of the close localization of TOP2A on 17q. It has been a matter of debate whether these two genes, HER-2 and TOP2A, behave separately on different amplicons or act together thus making it possible to predict the TOP2A status from the HER-2 status. In this study TOP2A, HER-2 and chromosome 17 aneusomy were investigated by fluorescent in situ hybridization (FISH) in 50 consecutive breast cancer patients. HER-2 amplification was detected in 11 patients (22%) and TOP2A changes were seen in 6 patients (12%); two amplifications and two deletions were observed in HER-2-amplified cases and two deletions in HER-2-nonamplified cases. Three of the TOP2A-deleted cases had polysomy 17. HER-2 copy number was higher than the TOP2A copy number in one patient with co-amplification. Polysomy was observed in 9 cases (18%) and monosomy in 6 cases (12%). Aneusomy was the sole anomaly in 11 patients (22%). We conclude that the TOP2A status cannot be predicted from the HER-2 status and evaluation of the TOP2A status only in patients with HER-2 overexpression may lead to missing cases with TOP2A deletion with possible resistance to therapy. Other factors modulating topo IIα activity may also affect the response to therapy. Studies evaluating different parameters that can modulate topo IIα activity and the response to the drugs targeting the enzyme are necessary.     

TOP2A amplification in the absence of that of HER-2/neu: toward individualization of chemotherapeutic practice in breast cancer

Primary objective.

To investigate the relationship between human epidermal growth factor receptor (HER)-2/neu and the gene encoding topoisomerase IIα (TOP2A) in breast cancer, while elucidating their association with clinicopathological variables.

Methods.

Real-time quantitative polymerase chain reaction (RQ-PCR) was performed on a 96-patient study group to assess gene amplification, and levels were determined using the comparative cycle threshold approach and Taqman assays. An immunohistochemistry (IHC) microarray (n = 76) was then employed to check for correlation between gene amplification and protein expression levels.

Results.

Amplification levels of TOP2A did not differ significantly according to HER-2/neu status by either RQ-PCR or IHC microarray. Of the HER-2/neu⁻ patients, 29.1% demonstrated levels of TOP2A above the third quartile, whereas 22.9% of the HER-2/neu⁺ patients had values in the first quartile (log TOP2A <0.62), thereby indicating low-level amplification. Of the 60 patients characterized as HER-2/neu⁻ using IHC and fluorescence in situ hybridization (FISH), 22.9% were classified as TOP2A⁺ on the IHC microarray. Of the 14 patients deemed HER-2/neu⁺ using IHC and FISH, meanwhile, the majority (n = 10) were classified as TOP2A⁺.

Conclusions.

Our results indicate that amplification of TOP2A in breast cancer is not confined to those who are concomitantly HER-2/neu⁺, and suggest that a significant proportion of HER-2/neu⁻ patients exhibit high levels of TOP2A.     

HER2 overexpression in muscle-invasive urothelial carcinoma of the bladder: prognostic implications

The HER2 (c-erbB-2) receptor is overexpressed in a variety of human malignant tumors and, in breast carcinoma, has been identified as a target for anti-HER2-directed therapy with the monoclonal antibody (MAb) trastuzumab. The aim of this retrospective study was to evaluate immunohistochemic HER2 expression in a large cohort of muscle-invasive urothelial cell carcinomas of the urinary bladder and to compare the results to pathologic characteristics and survival. Paraffin-embedded tumor specimens from 138 patients undergoing radical cystectomy for muscle-invasive bladder carcinoma were studied immunohistochemically with the Food and Drug Administration (FDA)-approved HercepTest (Dako, Glostrup, Denmark). HER2 overexpression was observed in 57 of 138 tumors (41%) and occurred more frequently in high-grade carcinomas than in low-grade carcinomas (p = 0.036). No significant relationship with HER2 overexpression was registered for tumor staging and lymph node status. Kaplan-Meier curves showed a significantly worse disease-related survival (p = 0.034) in patients with HER2-overexpressing tumors compared to those without HER2 overexpression. In addition to lymph node status (p = 0.0001; relative risk [RR] = 2.93), HER2 status (p = 0.020; RR = 2.22) was identified as an independent predictor for disease-related survival in a multivariate analysis. These results suggest that HER2 expression might provide additional prognostic information in patients with muscle-invasive bladder carcinomas. Because many of these patients harbor HER2-overexpressing tumors, clinical trials evaluating the efficacy of trastuzumab in bladder carcinoma are warranted.     

敲降HER-2和TOP2A增强乳腺癌细胞放射敏感性研究

目的 研究HER-2和TOP2A对乳腺癌细胞系SK-Br-3的放射敏感性影响,并探讨二者联合作用,为临床研究奠定基础。方法 分别构建表达HER-2或TOP2A siRNA的重组质粒,并使用lipofectamine 2000将其转染至乳腺癌细胞SK-Br-3中,敲降HER-2和TOP2A的基因表达。2 d后利用蛋白印迹法检测干扰效果,利用流式细胞仪和MTT法分别检测放射处理后细胞凋亡和增殖变化,并检测凋亡和增殖相关蛋白Caspase-3、Bcl-2、Ki-67表达变化。克隆形成实验检测放射敏感性。结果在乳腺癌细胞SK-Br-3中敲降HER-2和TOP2A的表达,放射处理后细胞凋亡率升高,增殖率降低。凋亡标志蛋白活化的Caspase-3表达升高,凋亡抑制蛋白Bcl-2表达降低。细胞增殖标志蛋白Ki-67表达降低。同时敲降HER-2和TOP2A两个基因时促进凋亡和抑制增殖及克隆形成的作用增强,表明这两个基因存在协同效应。结论 敲降HER-2和TOP2A基因在乳腺癌细胞SK-Br-3中的表达后,乳腺癌细胞的放射敏感性提高,细胞凋亡率升高,细胞增殖率降低,克隆形成率降低,且2基因存在协同效应,作用机制可能与Caspase-3、Bcl-2有关的凋亡通路及细胞增殖蛋白Ki-67有关。     

No association between HER-2 gene polymorphism at codon 655 and a risk of bladder cancer

The amplification and overexpression of the human epidermal growth factor receptor 2 gene HER-2 (also known as c-erb-B2 or neu) have been shown to be associated with bladder cancer and its progression. Recent studies indicated an association between the Ile to Val polymorphism at codon 655 of HER-2 and susceptibility to breast cancer. To investigate the correlation between the Ile/Val polymorphism and the susceptibility and progression of bladder cancer, we analyzed the polymorphism in 232 patients with transitional cell carcinoma of the bladder and 408 normal controls. The frequencies of the Ile/Ile, Ile/Val and Val/Val genotype were 75.9%, 21.6% and 2.6%, respectively, in patients with bladder cancer and 75.7%, 23.0% and 1.2%, respectively, in controls. Statistical analyses of the genotype prevalence showed no significant difference between bladder cancer patients and normal controls (p = 0.419). Moreover, no significant differences in the genotype prevalence were observed when the patients were stratified according to the tumor grade, stage and smoking habits. When the Ile/Ile genotype was compared to the Ile/Val and Val/Val genotypes, a significant difference was found only between the patients with tumor stage Ta and those with T1-4 (age, gender and smoking habits-adjusted odds ratio = 2.13, 95% confidence interval = 1.09-4.15, p = 0.027). When the Ile/Ile + Ile/Val genotypes compared to the Val/Val genotype, no significant findings were observed. These results suggested that the HER-2 polymorphism at codon 655 is unlikely to be associated with the onset of bladder cancer. Furthermore, the findings suggest no association between this polymorphism and the disease progression in bladder cancer, although the possibility remains that the Ile/Ile genotype may be related to an increased risk of disease progression.     

Correlation between quantitative HER-2 protein expression and risk for brain metastases in HER-2+ advanced breast cancer patients receiving trastuzumab-containing therapy

Background.

Patients with human epidermal growth factor receptor (HER)-2⁺ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2⁺ advanced breast cancer patients treated with trastuzumab.

Methods.

The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2⁺ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark^® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy.

Results.

A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2⁺, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024).

Conclusions.

These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2⁺ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group.     

TOP2Ahigh is the phenotype of recurrence and metastasis whereas TOP2Aneg cells represent cancer stem cells in prostate cancer

Recurrence and metastasis are the main causes of death for prostate cancer patients and cancer stem cells (CSCs) are proposed to play important roles in cancer recurrence and metastasis. It is generally thought that genes upregulated in recurrent/metastatic disease are likely biomarkers of CSCs. Hence we analyzed multiple microarray datasets on prostate tumor tissues to identify upregulated genes associated with cancer recurrence/metastasis, and tried to explore whether those genes were true biomarkers of prostate CSCs. Our results indicated that TOP2A was the most highly upregulated gene in recurrent/metastatic prostate cancer, and its high expression was positively correlated with poor prognosis in patients. Using a promoter reporter system, we unexpectedly discovered enrichment of CSCs in TOP2A^neg cells. Compared to TOP2A^high cells, TOP2A^neg cells formed spheres and tumors more efficiently, and became enriched in the presence of stresses. Analysis of cell divisions by time lapse imaging indicated that more slow-cycling cells were observed in TOP2A^neg cells while the proportion of abnormal divisions was higher in TOP2A^high cells. Our studies demonstrate that TOP2A^high is the phenotype of recurrence/metastasis but TOP2A^neg cells show slow cycling and have CSCs properties in prostate cancer, which has significant implications for prostate cancer therapy.     

High-throughput tissue microarray analysis of CMYC amplificationin urinary bladder cancer

Alterations of chromosome 8, preferentially deletions of 8p and gains of 8q, belong to the most frequent cytogenetic changes in bladder cancer. CMYC on 8q24 is a candidate oncogene in this region. Little is known about the clinical significance of CMYC copy number changes in urinary bladder cancer because its frequency is low and a limited numbers of tumors were analyzed so far. To investigate the impact of CMYC alterations on tumor progression and patient prognosis in bladder cancer, we applied FISH to a tissue microarray containing 2317 bladder cancer samples. Presence of CMYC copy number increase was associated with advanced stage and high grade. CMYC amplifications were seen in 3 of 467 pTa (0.6%), 10 of 247 pT1 (4%) and 11 of 201 pT2-4 urothelial carcinomas (5.5%; p < 0.0001), as well as in 1 of 123 G1 (0.8%), 8 of 470 G2 (1.7%) and 17 of 365 G3 urothelial carcinomas (4.7%; p < 0.0001). CMYC gains were present in 49 of 467 pTa (10.5%), 39 of 247 pT1 (15.8%) and 43 of 201 pT2-4 urothelial carcinoma (21.4%; p < 0.0001), as well as in 7 of 123 G1 (5.7%), 56 of 470 G2 (11.9%) and 72 of 365 G3 urothelial carcinomas (19.7%; p < 0.0001). CMYC copy number changes were unrelated to prognosis of bladder cancer patients. We conclude that alterations of the CMYC gene, including copy number gains and amplifications, are linked to genetically unstable bladder cancers that are characterized by a high histologic grade and/or invasive growth. Patient prognosis was not affected by CMYC gene copy number changes.     

Trastuzumab plus paclitaxel or docetaxel in HER-2-negative/HER-2 ECD-positive anthracycline- and taxane-refractory advanced breast cancer

Trastuzumab is considered effective against human epidermal growth factor receptor (HER)-2-positive breast cancer as assessed by immunohistochemistry (IHC) and fluorescence or chromogenic in situ hybridization (FISH/CISH) on biopsy material. Trastuzumab is now approved in both the adjuvant and metastatic settings for this patient population. Because HER-2 extracellular domain (ECD) levels have been correlated with disease progression in the metastatic setting, we considered trastuzumab salvage therapy plus a taxane in heavily pretreated trastuzumab-naive relapsed breast cancer patients with high serum levels of HER-2 ECD (> or =15 ng/ml). All patients had previously failed at least two lines of anthracycline- and taxane-based regimens and were HER-2 negative by IHC and FISH/CISH prior to a centralized reanalysis, and were serum positive for HER-2 ECD (> or =15 ng/ml) at baseline. Regular serum accounts of HER-2 ECD were recorded and compared with response and survival outcomes. Twenty-two patients were finally eligible for salvage therapy. Minor responses were observed in five (23%) and stable disease (SD) was observed in 11 patients, leading to a clinical benefit rate of 73% (16 of 22 patients). The median time to progression and overall survival time were 5 (6.5 months in minor responders and SD) and 12 months, respectively; 11 and eight patients remained progression free for >6 and >12 months, respectively. Eleven and seven patients were alive at 12 and 15 months, respectively, after treatment start. Furthermore, in total, 13 (59.1%) patients obtained a biochemical response. In our study, patients with conventionally HER-2-negative disease but with expression of HER-2 ECD above the normal limit (> or =15 ng/ml) displayed a rapid response, both biochemically and clinically, to the trastuzumab-taxane combination. This is the first study assessing anti-HER-2-based treatment in HER-2-negative advanced breast cancer according to HER-2 ECD positivity; if our results are confirmed, additional patients with "hidden" HER-2-positive breast cancer might benefit from anti-HER-2 treatment.     

Aberrations of ERBB2 and TOP2A genes in breast cancer

Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co‐amplification of a whole amplicon containing both genes. Most gene signals are translocated to abnormal marker chromosomes. ERBB2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher ERBB2 ratio. This observation was confirmed by patient FISH data: among 276 (43% of all patients) abnormal tumors, 67% had different ERBB2 and TOP2A status. ERBB2 amplification with normal TOP2A status was found in 36% of the abnormal tumors (15% of all patients). Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while TOP2A deletion and ERBB2 amplification was observed in 16% of the abnormal cases (8% of all patients). A small number of tumors had TOP2A amplification (4%) or deletion (6%) without simultaneous changes of the ERBB2 gene. ERBB2 deletion was also observed (5%) but only in tumors with simultaneous TOP2A deletion. The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for ERBB2 in the amplified tumors (P<0.01). Amplification of the two genes may be caused by different mechanisms, leading to higher level of amplification for ERBB2 compared to TOP2A. In the majority of breast cancer patients, simultaneous aberration of ERBB2 and TOP2A is not explained by simple co‐amplification.     

Prognostic impact of phosphorylated HER-2 in HER-2+ primary breast cancer

Purpose.

Tyrosine 1248 is one of the autophosphorylation sites of human epidermal growth factor receptor (HER)-2. We determined the prognostic value of the expression level of tyrosine 1248–phosphorylated HER-2 (pHER-2) in patients with HER-2⁺ primary breast cancer using a reverse-phase protein array.

Patients and Methods.

The optimal cutoff value of pHER-2 for segregating disease-free survival (DFS) was determined by receiver operating characteristic (ROC) curve analysis. Five-year DFS for pHER-2 expression level was estimated with the Kaplan-Meier method using both derivation (n = 162) and validation (n = 227) cohorts.

Results.

Of the 162 patients in the derivation cohort, 26 had high HER-2 expression levels. The area under the ROC curve for pHER-2 level and DFS was 0.662. Nineteen of the 162 patients (11.7%) had high pHER-2 expression levels (pHER-2^high); 143 patients (88.3%) had low pHER-2 expression levels (pHER-2^low). Among the 26 patients with high HER-2 expression levels, the 17 pHER-2^high patients had a significantly lower 5-year DFS rate than the nine pHER-2^low patients (23.5% versus 77.8%). On multivariate analysis, only pHER-2^high independently predicted DFS in the Cox proportional hazards model. In the validation cohort, among 61 patients with high HER-2 expression, the difference in 5-year DFS rates between pHER-2^high (n = 7) and pHER-2^low (n = 54) patients was marginal (57.1% versus 81.5%).

Conclusion.

In patients with HER-2⁺ primary breast cancer, pHER-2^high patients had a lower 5-year DFS rate than pHER-2^low patients. Quantification of pHER-2 expression level may provide prognostic information beyond the current standard HER-2 status.     

Impact of HER-2 overexpression/amplification on the prognosis of gastric cancer patients undergoing resection: a single-center study of 1,036 patients

Background.

Opinions regarding the impact of human epidermal growth factor receptor (HER)-2 overexpression or HER-2 amplification on the prognosis of gastric cancer patients are mixed. The present study attempted to clarify this issue by investigating a large cohort of surgical patients.

Methods.

We investigated 1,036 gastric cancer patients undergoing curative-intent resection. Their surgical specimens were evaluated for HER-2 expression by immunohistochemistry (IHC), and those with HER-2 expression levels of 2+ were additionally subjected to fluorescence in situ hybridization (FISH). Data on demographic and clinicopathological features and relevant prognostic factors in these patients were analyzed.

Results.

HER-2 positivity was noted in 64 (6.1%) of 1,036 gastric cancer patients, including 46 patients whose HER-2 expression level was 3+ on IHC and 18 patients whose FISH results were positive. On univariate analysis, HER-2 positivity was more often associated with differentiated histology, intestinal type, and negative resection margins, whereas only differentiated histology was independently associated with HER-2 positivity in a logistic regression model. For stage I–IV gastric cancer, HER-2 was not a prognostic factor. In a subpopulation study, although HER-2 positivity emerged as a favorable prognostic factor for stage III–IV gastric cancer on univariate analysis, it failed to be an independent prognostic factor after multivariate adjustment.

Conclusions.

The prevalence of HER-2 positivity, determined using standardized assays and scoring criteria in a large cohort of gastric cancer patients after resection, was 6.1%. HER-2 positivity was phenotypically associated with differentiated histology. HER-2 is not an independent prognostic factor for gastric cancer.     

Evaluation of HER-2/neu Overexpression in Gastric Carcinoma using a Tissue Microarray

Background: Amplification and overexpression of human epidermal growth factor receptor 2 (HER2 /neu) oncogene has considerable prognostic value in breast and gastric cancers. This study aimed to evaluatethe frequency, overexpression pattern, clinical significance, and concordance between the results for proteinexpression and gene amplification of HER-2/neu in gastric and gastro-esophageal junction carcinomas.Materials and Methods: In this study, 101 gastric tissue samples which were included in tissue microarray wereimmunohistochemically examined for overexpression of HER2/neu. Chromogenic in situ hybridization (CISH)was used for HER-2/neu amplification. The correlation of HER2/neu amplification with clinicopathologicalparameters was also assessed. In addition, concordance between CISH and IHC was detected. Results: This studydemonstrated a significant difference in the overexpression of HER2/neu in gastric tumors. The overexpression ofHER2/neu was significantly higher in intestinal type, poorly differentiated grade, large size (5 cm≤) and positivenodal involvement tumors (p-value=0.041, 0.015, 0.038 and 0.071, respectively). Also, amplification of HER2/neuaccording to CISH test, had a significant positive correlation with tumor size and tumor type (p-value=0.018 and0.058, respectively).Concordance between CISH and IHC was 76.9% in 101 evaluable samples. Conclusions:IHC/CISH differences were attributed to basolateral membranous immunoreactivity of glandular cells resultingin incomplete membranous reactivity and/or a higher rate of tumor heterogeneity in gastric cancers comparedto breast cancers. Therefore, this can be a potential marker for targeted therapy of malignant gastric tumors.     

TOP2A在非小细胞肺癌中的高表达促进肿瘤细胞的增殖和侵袭能力

目的:探讨编码拓扑异构酶Ⅱα的基因TOP2A在非小细胞肺癌组织中的表达情况及其生物学功能。方法:采用荧光定量PCR方法检测102例非小细胞肺癌及其癌旁组织中TOP2A的mRNA表达水平,Western blot检测其中5对组织中TOP2A蛋白表达水平,分析TOP2A 表达与非小细胞肺癌主要临床病理特征的相关性。干扰非小细胞肺癌细胞株A549和HCC827中TOP2A的表达,MTT法检测其表达对肿瘤细胞增殖的影响,Transwell法检测TOP2A表达下调对肿瘤细胞侵袭能力的影响。应用流式细胞术检测TOP2A敲降后细胞的凋亡率和细胞周期。结果:TOP2A在非小细胞肺癌组织中的表达水平相较癌旁组织明显上升（P<0.05）,干扰TOP2A的表达会抑制非小细胞肺癌细胞的增殖和侵袭。流式细胞凋亡率实验结果显示转染siTOP2A后的非小细胞肺癌细胞早期凋亡率增加;流式周期分析显示siTOP2A转染后的细胞S期发生阻滞抑制细胞增殖。同时,TOP2A表达水平与非小细胞肺癌的肿瘤分期、淋巴结转移密切相关（P<0.05）。结论:TOP2A在非小细胞肺癌中表达上调,与肿瘤增殖和侵袭等生物学行为密切相关。     

Analysis of the expression of biomarkers in urinary bladder cancer using a tissue microarray

Dysregulation of Akt, PTEN, Drg-1, Cx-26, and L-plastin expression appear to be important in the progression of various cancers. Their expression in bladder cancer has not been well characterized. To assess the expression of these genes and their relationship to the outcome of bladder cancer, we used a bladder cancer tissue microarray (TMA) of 251 transitional cell carcinomas. We quantitated immunohistochemical staining of each protein using both automated and manual methods and correlated the expression levels with the clinicopathologic characteristics of the tumor and patient survival. Overall, the results from both automated and manual analyses were similar. We found a significant correlation between the expression of PTEN, Cx-26 and L-plastin with known clinically important pathologic features of bladder cancer (tumor grade, stage, and growth pattern). Aberrant localization patterns of Cx-26 and Drg-1 were observed in bladder tumors. There was also a significant correlation in expression among pAkt, PTEN, and L-plastin. Although the expression of these genes correlated with factors known to be associated with patient outcome, none of them was an independent predictor of progression-free or overall survival.     

Prognostic relevance of MAGE-A4 tumor antigen expression in transitional cell carcinoma of the urinary bladder: a tissue microarray study

TAAs of the MAGE family are mostly studied as targets of specific immune responses. Their potential relevance as tumor markers has also been underlined. We used a MAb, 57B, recognizing MAGE-A4 protein in paraffin-embedded sections, to evaluate its expression in bladder cancers by employing TMA including 2,317 samples from 1,849 patients. In 2,090/2,317 cases (90.2%), immunostaining yielded interpretable results. Since for some patients more than 1 sample was available, only interpretable first biopsies (n = 1,628) were considered. MAGE-A4 protein was expressed at significantly (p < 0.001) higher frequency in squamous (25/55, 45.5%) than in adeno (4/15, 26.7%), sarcomatoid (4/14, 28.6%), small cell (5/20, 25%) or transitional cell (281/1,522, 18.5%) carcinomas. In TCCs, overall MAGE-A4 positivity was significantly correlated with invasive phenotype (p < 0.001) and high tumor grade (p < 0.0001). Clinical data from 908 TCC patients were retrospectively evaluated, revealing that strong 57B staining was highly significantly associated with decreased tumor-specific survival (p < 0.0001). These data suggest that evaluation of MAGE-A4 protein expression is useful in the identification of groups of TCCs characterized by severe prognosis, thus possibly providing indications for early MAGE TAA-targeted immunotherapy.     

Coamplified and overexpressed genes at ERBB2 locus in gastric cancer

DNA copy number amplification at the chromosomal region of 17q is frequent in gastric cancer. Recently 17q21 was identified as the critical region for the amplicon formation because this region harbors the ERBB2 oncogene and several other targets, such as TOP2A and DARPP32. In our study, we characterized the amplification (52 cases) and expression (29 cases) levels of ERBB2, TOP2A and DARPP32 in gastric cancer samples. These 3 genes were concomitantly amplified in 17% of the intestinal type of gastric adenocarcinoma. However, the expression levels were independent, showing overexpression of DARPP32 (48%), TOP2A (17%) and ERBB2 (3%) studied by quantitative real-time PCR. The most frequently overexpressed gene, DARPP32, exhibited strong protein overexpression in 45% (30/66) of the cases in immunohistochemical study of gastric cancer tumor tissue array. Additional studies are required to thoroughly understand the biological significance of these genes in gastric cancer.     

Monosomy of chromosome 17 in breast cancer during interpretation of HER2 gene amplification

Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and <6 gene copy number at FISH.