MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large-scale tissue microarrays.

For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer, we applied Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to a tissue micro array containing formalin-fixed and paraffin-embedded tissue samples from 349 patients. Analysis of our MALDI-IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki-67 labeling index. Fifteen out of 27 IMS signals revealed a significant association with survival. For seven signals (m/z 654, 776, 788, 904, 944, 975 and 1013) the absence and for eight signals (m/z 643, 678, 836, 886, 898, 1095, 1459 and 1477) the presence were associated with decreased life expectancy, including five masses (m/z 788, 836, 904, 944 and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these five masses resulted in a three-step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis and cell proliferation. Our data demonstrate the suitability of combining IMS and large-scale tissue micro arrays to simultaneously identify and validate clinically useful molecular marker. Copyright © 2017 John Wiley & Sons, Ltd.

Partial PTEN deletion is linked to poor prognosis in breast cancer.

BACKGROUND: Deletions of chromosome 10q23, including the PTEN (phosphatase and tensin homolog) locus, are known to occur in breast cancer, but systematic analyses of its clinical relevance are lacking. METHODS: We thus analyzed a tissue microarray (TMA) with 2,197 breast cancers by fluorescence in-situ hybridization (FISH) using a PTEN-specific probe. RESULTS: PTEN deletions were detected in 19% of no special type, 9% of lobular, 4% of tubular cancers and 46% in carcinomas with medullary features. 98.7% of deletions were heterozygous and only 1.3% were homozygous. PTEN deletion was significantly linked to advanced tumor stage (p=0.0054), high-grade (p<0.0001), high tumor cell proliferation (Ki67 Labeling Index; p<0.0001), and shortened overall survival (p=0.0090). PTEN deletions were inversely associated with features of luminal type breast cancers (ER/PR positivity; p<0.0001 each, and CCND1 amplification; p=0.0020). PTEN deletions were also strongly linked to amplification of genes involved in the PTEN/AKT pathway such as MYC (p=0.0430) and HER2 (p=0.0065). Remarkably the combined analysis of MYC, HER2, CCND1 and PTEN aberrations suggested that aberrations of multiple PTEN/AKT pathway genes have a strong additive effect on breast cancer prognosis. While cancers with one of these aberrations behaved only marginally different from cancers with none, disease outcome was markedly worse in cancers with two or more aberrations as compared to those with only one aberration (p=0.0002). In addition, the particularly poor prognosis of patients with HER2 amplification and PTEN deletions challenges the concept of PTEN deletions interfering with trastuzumab therapy. CONCLUSION: PTEN deletion occurs in a relevant fraction of breast cancers, and is linked to aggressive tumor behavior. Reduced PTEN function cooperates with MYC and HER2 activation in conferring aggressive phenotype to cancer cells.

8p deletion is strongly linked to poor prognosis in breast cancer.

Deletions of chromosome 8p occur frequently in breast cancers, but analyses of its clinical relevance have been limited to small patient cohorts and provided controversial results. A tissue microarray with 2,197 breast cancers was thus analyzed by fluorescence in-situ hybridization using an 8p21 probe in combination with a centromere 8 reference probe. 8p deletions were found in 50% of carcinomas with no special type, 67% of papillary, 28% of tubular, 37% of lobular cancers and 56% of cancers with medullary features. Deletions were always heterozygous. 8p deletion was significantly linked to advanced tumor stage (P < 0.0001), high-grade (P < 0.0001), high tumor cell proliferation (Ki67 Labeling Index; P < 0.0001), and shortened overall survival (P < 0.0001). For example, 8p deletion was seen in 32% of 290 grade 1, 43% of 438 grade 2, and 65% of 427 grade 3 cancers. In addition, 8p deletions were strongly linked to amplification of MYC (P < 0.0001), HER2 (P < 0.0001), and CCND1 (p = 0.001), but inversely associated with ER receptor expression (p = 0.0001). Remarkably, 46.5% of 8p-deleted cancers harbored amplification of at least one of the analyzed genes as compared to 27.5% amplifications in 8p-non-deleted cancers (P < 0.0001). In conclusion, 8p deletion characterizes a subset of particularly aggressive breast cancers. As 8p deletions are easy to analyze, this feature appears to be highly suited for future DNA based prognostic breast cancer panels. The strong link of 8p deletion with various gene amplifications raises the possibility of a role for regulating genomic stability.

[Invasive breast cancer: the current WHO classification]. / Invasive Mammakarzinome: Die aktuelle WHO-Klassifikation.

The World Health Organization (WHO) classification of tumors of the breast defines the international standards for tumor categorization and nomenclature. The fourth edition, published in 2012, provides an update on the current knowledge concerning the classification, immunohistology profile, differential diagnosis and genetics of these lesions. Compared to the previous edition, some terms have been modified, some entities were reclassified and some current molecular data have been added. This article focuses on invasive carcinomas. Definitions for histological diagnosis are supplemented by clinical, macroscopic and molecular characteristics as well as prognostic and predictive features.

Prognostic relevance of AIB1 (NCoA3) amplification and overexpression in breast cancer.

UNLABELLED: AIB1 (amplified in breast cancer 1) is an estrogen receptorα (ERα) co-activator, known to be amplified and overexpressed in a fraction of breast cancers. It has been linked to prognosis and tamoxifen resistance. However, results have been ambiguous. The different functions of AIB1 in ERα-positive and -negative disease are poorly understood. Therefore, we analyzed the clinical significance of AIB1 in breast cancer with respect to ERα-status and characterized the subgroups. 2,197 breast carcinomas sampled on a pre-existing tissue microarray (TMA) were analyzed for AIB1 expression and amplification by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). RESULTS: AIB1 expression was detected in 60 % of the tumors. It was associated with tumor size (p = 0.003), high histological grade (p < 0.0001), poor disease-specific, and overall survival (p = 0.0018 and p = 0.003). There was a strong inverse relationship between AIB1 and ERα expression (p < 0.0001). AIB1 overexpression was associated with increased Ki67 labeling index (p < 0.0001), even if analyzed for different ER expression levels. AIB1 amplification was found in 11 % of the carcinomas. It was associated with high histological grade (p = 0.0012), lymph node involvement (p = 0.0163), and poor disease-specific survival (p = 0.0032) but not with overall survival (p = 0.1672) or ER status (p = 0.4456). If ER-positive tumors were stratified according to their AIB1 amplification status, there was a significant worse disease-specific survival in cases showing AIB1 amplification (p = 0.0017). AIB1 expression is associated with unfavorable prognosis and tumor phenotype. It seems to unfold its oncogenic potential at least in part independent from its role as an ERα co-activator. AIB1 has an impact on cell cycle regulation in ERα-positive as well as ERα-negative tumors. Furthermore, AIB1 amplification characterizes a subgroup of ERα-positive breast cancer with worse outcome. Therefore, AIB1 might be helpful to identify those ERα-positive breast cancers patients who are candidates for adjuvant chemotherapy.

[HER2 ASCO guidelines. The answer to everything?]. / HER2-ASCO-Guidelines. Der Weisheit letzter Schluss?

The HER2 gene is amplified and overexpressed in about 15%-20% of breast cancers. For every newly diagnosed breast cancer HER2 testing is a standard routine procedure. This article focuses on a number of issues raised in the context of current HER2 testing in breast cancer. It particularly points out issues arising in the recently published ASCO-CAP (American Society of Clinical Oncology/College of American Pathologists) guideline recommendations for clinical testing of HER2. Despite the significant correlation between HER2 status determination by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), standard considerations of laboratory testing, such as test accuracy, reproducibility and precision as well as current data, favor FISH methods over IHC assay methods for the determination of HER2 status. Biological and technical considerations of HER2 testing are also important in clinical practice. For example, HER2 gene amplification is directly linked to the protein expression level in breast cancer; however, the HER2 protein is not consistently analyzed on formalin fixed tissues due to variability in fixation methods/times and the impact of this fixation on HER2 protein antigenicity. FISH is significantly less dependent on tissue fixation artifacts. Hence, FISH is more reproducible between both central and peripheral laboratories than IHC and is more accurate for HER2 measurement, as well as being more strongly correlated with responsiveness to trastuzumab and lapatinib treatment. Until other methods are able to ensure similar test accuracy, reproducibility, precision and predictive value, FISH is recommended as the primary HER2 testing modality for women with breast cancer who are candidates for HER2-targeted therapies.

Gene amplification in ductal carcinoma in situ of the breast.

Multiple different biologically and clinically relevant genes are often amplified in invasive breast cancer, including HER2, ESR1, CCND1, and MYC. So far, little is known about their role in tumor progression. To investigate their significance for tumor invasion, we compared pure ductal carcinoma in situ (DCIS) and DCIS associated with invasive cancer with regard to the amplification of these genes. Fluorescence in situ hybridization (FISH) was performed on a tissue microarray containing samples from 130 pure DCIS and 159 DCIS associated with invasive breast cancer. Of the latter patients, we analyzed the intraductal and invasive components separately. In addition, lymph node metastases of 23 patients with invasive carcinoma were included. Amplification rates of pure DCIS and DCIS associated with invasive cancer did not differ significantly (pure DCIS vs. DCIS associated with invasive cancer: HER2 22.7 vs. 24.2%, ESR1 19.0 vs. 24.1%, CCND1 10.0 vs. 14.8%, MYC 11.8 vs. 6.5%; P > 0.05). Furthermore, we observed a high concordance of the amplification status for all genes if in situ and invasive carcinoma of individual patients were compared. This applied also to the corresponding lymph node metastases. Our results indicate no significant differences between the gene amplification status of DCIS and invasive breast cancer concerning HER2, ESR1, CCND1, and MYC. Therefore, our data suggest an early role of all analyzed gene amplifications in breast cancer development but not in the initiation of invasive tumor growth.

HER-2/neu analysis in breast cancer bone metastases.

BACKGROUND: HER-2 is the target for antibody-based treatment of breast cancer (trastuzumab), which is highly successful in both advanced disease and the adjuvant setting. HER-2 can be analysed by fluorescence in situ hybridisation (FISH) for gene amplification or immunohistochemistry (IHC) for protein overexpression. AIM: As both methods are known to be influenced by previous tissue processing, to analyse the applicability of both FISH and IHC to decalcified bone metastases of breast cancer. METHODS: A tissue microarray (TMA) was constructed from 149 breast cancer bone metastases. Consecutive TMA sections were analysed by FISH (PathVysion) and IHC (HercepTest). RESULTS: FISH analysis was interpretable in 113 (85.0%) cases. Amplification was seen in 14 (12.4%) interpretable metastases. HER-2 positivity on IHC analysis was 3+ in 9.8% of cases and 2+ in 11.3%. A comparison of the two techniques revealed high concordance. Of the 14 cases of amplification, 10 (71%) showed 3+ IHC staining, two (14%) showed 2+, one (7%) showed 1+, and one (7%) showed 0+. Three of the four amplified cases that did not show 3+ IHC staining had an equivocal FISH result, with a HER-2/centromere 17 ratio of 1.8-2.2. Of the 13 cases that showed IHC 3+ staining, amplification was present in 10 (77%). CONCLUSIONS: HER-2 FISH analysis has an excellent success rate in highly standardised EDTA-decalcified bone metastases, suggesting that this method is easily applicable to decalcified tissues. The high concordance between IHC and FISH suggests that HER-2 IHC may be equally applicable to EDTA-treated tissues as to the usual formalin-fixed tissues.