Formalin-fixed Paraffin-embedded Breast Carcinoma Research Articles

Two Instrument Comparison of Reagents From a US FDA-Approved Assay for the Assessment of Ki-67 in High-Risk Early Breast Cancer.

The objective of this study was to measure concordance of results obtained from the US Food and Drug Administration–approved Ki-67 immunohistochemistry MIB-1 pharmDx assay performed on the Dako Omnis automated staining instrument (Omnis) versus results produced from the assay reagents applied using an optimized protocol on the more widely available Autostainer Link 48 (ASL48) platform. Tissue sections obtained from 40 formalin-fixed paraffin-embedded breast carcinoma samples, with available Oncotype DX Breast Recurrence Score (RS) results, were stained. Three certified pathologists scored slides at 3 timepoints, totaling 360 observations for each instrument (N=720 total) using the approved scoring approach. Using the ≥20% cutoff, agreement was calculated with corresponding 2-sided 95% percentile bootstrap confidence intervals (CIs). Pairwise comparisons (N=360) from the interinstrument evaluation, performed with all observers, resulted in 325 (90.3%) concordant outcomes (244 negative and 81 positive) and 35 (9.7%) discordant outcomes. The overall agreement was 90.3% (95% confidence interval, 85.6% to 94.4%). No significant systematic differences were observed between instruments. Specimens scored from the Omnis were on average <1% higher than ASL48, with high correlation and little bias between the continuous Ki-67 scores (concordance correlation coefficient=0.916). Most specimens with a Ki-67 score ≥20% had a RS >25. This study demonstrated that good concordance can be achieved with the reagents run on the ASL48 instrument when using an optimized protocol and standardized scoring.

Single-day HER2neu amplification assessment using chip-based digital PCR in formalin-fixed paraffin-embedded breast carcinoma tissue.

IntroductionHuman epidermal growth factor receptor 2 (HER2) amplification is present in almost 15%–20% of breast cancer tumors, making it an important parameter for testing. The present study was designed to evaluate a chip-based digital PCR (dPCR) system for assessing HER2 amplification from formalin-fixed paraffin-embedded breast carcinoma tissue and to compare this system with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).Materials and methodsA total of 84 breast carcinoma tissue samples were analyzed by IHC, FISH, and chip-based dPCR in a blinded manner.ResultsAll nine IHC-positive and 35 IHC-negative samples had equivalent results with dPCR, taking an amplification ratio threshold of 1.8 as a positive result. Of the 40 IHC equivocal samples, 10 were assessed as positive, 27 as negative, and three as equivocal by dPCR.ConclusionThese results demonstrate that chip-based dPCR is suitable for HER2 amplification detection in formalin-fixed paraffin-embedded samples in a clinical setting, providing the advantages of superior turnaround time, cost-effectiveness, and increased precision with absolute quantification compared with conventional tests such as FISH and IHC. This methodology was especially beneficial in tissue samples with low DNA concentration.

Detection of HER2 amplification in formalin-fixed paraffin-embedded breast carcinoma tissue with digital PCR using two TFF3 sequences as internal reference

PurposeDroplet digital PCR (ddPCR) is a highly accurate method to determine DNA concentration and detect copy number variations. We developed an approach to assess HER2 gene amplification status using ddPCR with two sequences of TFF3 as reference probes. Experimental design.76 templates of carcinoma DNA were prepared from formalin-fixed paraffin-embedded (FFPE) tissues. Digital PCR assay of the copy number of HER2 and TFF3 DNA was performed on the samples. The results were compared to prior fluorescent in-situ hybridation (FISH) assays performed on the same samples. ResultsThe ddPCR assay had high concordance with the conventionally used immunohistochemistry (IHC) and FISH methods. The ddPCR method returned fewer indeterminate results than IHC. Concordance between a ddPCR plus FISH method and IHC plus FISH can rise to 98.7% (75/76) after validation is carried out. ConclusionIt's potentially possible to improve the sensitivity and specifity of HER2 ddPCR assays using reference sequences not co-localized with HER2 on chromosome 17, and combining results from multiple sequences. Adopting an approach based on ddPCR HER2 assays plus FISH could lead to reduced costs, labour, and time consumption compared to current IHC plus FISH standard, while not losing precision.

Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study

BackgroundThe aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as a validation analysis of another previously published HeCOG study.MethodsRNA was extracted from 663 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples of high-risk early breast cancer patients enrolled in the randomized HE10/00 trial. Relative mRNA expression of all four HER family members was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).ResultsIn compliance with our previous study, the overall agreement between qRT-PCR and IHC/FISH for HER2 status determination was good (69%). Likewise, the overall concordance between qRT-PCR and IHC for EGFR status was high (81%). In line with our previously reported data, we demonstrated a positive association between HER2 and HER3 mRNA expression. Similarly, mRNA expression of HER3 and HER4 was positively associated with each other and negatively associated with EGFR. Regarding relationships with clinico-pathological parameters, our findings are also in agreement with our previous results. Generally, increased EGFR and HER2 mRNA expression was related to unfavorable, whereas high HER3 and HER4 mRNA expression was associated with favorable clinico-pathological parameters. In univariate analysis, no significant association between EGFR, HER2 and HER3 mRNA expression and overall survival (OS) or disease-free survival (DFS) was demonstrated. However, high EGFR protein expression was associated with significantly shorter OS (log-rank, p = 0.015). In compliance with our previously published data, increased HER4 mRNA expression had a significantly favorable prognostic value in terms of OS (p = 0.044) and DFS (p = 0.047). In multivariate analysis, among all HER receptors, only EGFR protein expression was found to affect OS (Wald’s p = 0.028) and DFS (p = 0.015) independently. Concerning the combined expression of all four HER family receptors, the combination of high EGFR, high HER2, low HER3 and low HER4 mRNA expression was associated with a trend for shorter OS (log-rank, p = 0.065) and significantly worse DFS (p = 0.033), compared with all other co-expression profiles.ConclusionsThese data indicate that qRT-PCR may represent a valid alternative method for evaluating the expression of HER family members in FFPE breast carcinoma tissue samples.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12609001036202

Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH

Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (κ=1.0, p<0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications.

Abstract C38: Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas.

Abstract Background: Mutations of the PIK3CA gene encoding for the p110α catalytic subunit of Phosphatidylinositol 3-kinases (PI3K) are found in approximately 25% of breast carcinomas. Most of PIK3CA mutations are reported as activators of the PI3K/AKT/mTOR pathway and lead to resistance to anti-HER2 therapies, and have an interest in molecular diagnosis for anti-mTOR and/or anti-PI3K therapies. Highly sensitive methods are required to detect PIK3CA mutations in paraffin-embedded tumor tissues. Methods: Forty-seven formalin-fixed paraffin embedded breast carcinomas tissues samples were assessed using two allele specific assays (Cobas® PIK3CA Mutation Test and PCR ARMS Scorpions®) and one non-specific real-time high resolution melting PCR assay (HRM) after macrodissection and DNA extraction. Cobas® allow the detection of 17 mutations on exons 1, 4, 7, 9 and 20 of PIK3CA, ARMS focuses on the detection on the four common hotspots (E542K, E545K/D, H1047R and H1047L) representing 90% of PIK3CA mutations and HRM allows the detection of all mutations located on the whole exons 9 and 20 of PIK3CA. Kappa statistics were used to compare the three assays. Results: Among the 47 samples, 18 (38.3%) were found to bear a PIK3CA mutation with Cobas®, 13 (27.7%) with ARMS and 19 (40.4%) with HRM. One sample was found to bear 3 mutations of PIK3CA (E542K, H1047X and H1049R). Calculated kappa were 0.893 (p&amp;lt;1.10-9) between Cobas® and HRM and 0.659 (p&amp;lt;1.10-9) between Cobas® and ARMS. Because the ARMS assay only focused on the 4 most common PIK3CA mutations, more than 10% of the samples were identified as false negative wild-type tumors although they had a mutation of PIK3CA as identified with Cobas® and HRM assays. Conclusion: Cobas® and HRM assays allowed to detect more extensively PIK3CA mutations than ARMS assay. ARMS assay only focusing on the 4 hotspots PIK3CA mutations seems to be inadequate with routine use since generating false negative PIK3CA wild-type results. Cobas® and HRM assays are significantly comparable for the detection of PIK3CA mutations, but Cobas® enables the characterization of the mutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C38. Citation Format: Alexandre Harlé, Maëva Lion, Marie Rouyer, Nicola Lozano, Jean-Louis Merlin. Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C38.

Quantitative measures of estrogen receptor expression in relation to breast cancer-specific mortality risk among white women and black women

IntroductionThe association of breast cancer patients’ mortality with estrogen receptor (ER) status (ER + versus ER-) has been well studied. However, little attention has been paid to the relationship between the quantitative measures of ER expression and mortality.MethodsWe evaluated the association between semi-quantitative, immunohistochemical staining of ER in formalin-fixed paraffin-embedded breast carcinomas and breast cancer-specific mortality risk in an observational cohort of invasive breast cancer in 681 white women and 523 black women ages 35-64 years at first diagnosis of invasive breast cancer, who were followed for a median of 10 years. The quantitative measures of ER examined here included the percentage of tumor cell nuclei positively stained for ER, ER Histo (H)-score, and a score based on an adaptation of an equation presented by Cuzick and colleagues, which combines weighted values of ER H-score, percentage of tumor cell nuclei positively stained for the progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) results. This is referred to as the ER/PR/HER2 score.ResultsAfter controlling for age at diagnosis, race, study site, tumor stage, and histologic grade in multivariable Cox proportional hazards regression models, both percentage of tumor cell nuclei positively stained for ER (Ptrend = 0.0003) and the ER H-score (Ptrend = 0.0004) were inversely associated with breast cancer-specific mortality risk. The ER/PR/HER2 score was positively associated with breast cancer-specific mortality risk in women with ER + tumor (Ptrend = 0.001). Analyses by race revealed that ER positivity was associated with reduced risk of breast cancer-specific mortality in white women and black women. The two quantitative measures for ER alone provided additional discrimination in breast cancer-specific mortality risk only among white women with ER + tumors (both Ptrend ≤ 0.01) while the ER/PR/HER2 score provided additional discrimination for both white women (Ptrend = 0.01) and black women (Ptrend = 0.03) with ER + tumors.ConclusionsOur data support quantitative immunohistochemical measures of ER, especially the ER/PR/HER2 score, as a more precise predictor for breast cancer-specific mortality risk than a simple determination of ER positivity.

Immunohistochemical analysis of the monoclonal antibody 4B5 in breast tissue expressing human epidermal growth factor receptor 4 (HER4)

A recent study examining the specificity of human epidermal growth factor receptor (HER) 2 pharmacodiagnostic antibodies demonstrated that CB11 and 4B5 stain both HER2-transfected and HER4-transfected cell lines. However, there has been no evidence showing that 4B5 has affinity for HER4 in clinically obtained tissues, and, if so, whether this has any impact on the assessment of HER2. We therefore sought to determine the expression of membrane-bound HER4 in clinical breast carcinomas, and evaluate its impact on the clinical utility of 4B5 in determining HER2 status. Breast carcinomas were assessed by immunohistochemistry (IHC) for membrane-bound HER4 using anti-HER4 clone E200. HER2 expression in these cases was then assessed using anti-HER2 clone 4B5, and a reference clone, SP3. In all 117 membrane HER4-positive cases (out of 241), 4B5 scored equal to or less than the reference anti-HER2 clone SP3. Eighteen cases were positive for membrane-bound HER4 by E200 and negative by 4B5, including a membrane HER4 level 3+ case. No cross-reactivity of 4B5 with membrane-bound HER4 was identified in the clinical IHC analysis of formalin-fixed paraffin-embedded breast carcinoma cases as evidenced by the HER4 antibody clone E200.

Quantitation of truncated HER2 in formalin-fixed paraffin-embedded breast carcinoma tissues.

e11130 Background: Trastuzumab treatment of Her2+ breast cancer is the benchmark success story for targeted cancer therapy, however, a significant number of patients are either non-responsive or experience disease progression. A novel mechanism of resistance in some patients is the expression of truncated Her2 (p95Her2), formed by proteolytic cleavage of the HER2 molecule. Truncated p95Her2 loses its extracellular trastuzumab binding epitope, but retains a constitutively active kinase domain predicted to be sensitive to kinase inhibitors, like lapatinib and neratinib. At present there are no diagnostic assays for this p95Her2. Methods: Using the Liquid Tissue-SRM (selective reaction monitoring) platform we have developed multiple mass spectrometric assays to quantify specific cytoplasmic or extracellular domain (ECD) peptides from Her2 directly from formalin fixed paraffin embedded (FFPE) clinical tissue. Results: We validated these assays using recombinant human Her2, where we saw equivalent intracellular and extracellular Her2 levels. In formalin fixed SkBr3 breast carcinoma cells, we also detected equivalent levels of the Her2 peptides, as expected. We initiated clinical validation of this assay by analyzing a dozen FFPE human breast carcinoma tumors where Her2 expression and gene amplification levels had been determined. In 9 of 12 tumors (IHC: 1+ to 3+) we measured equivalent levels of intracellular and ECD peptides. Most interesting, 3 tumors showed dramatic differences in the levels of Her2 peptides detected. In two 3+ tumors, the intracellular peptide was 50% higher than the ECD peptide and in the last tumor more than twice the expression of the ECD Her2 peptide. These data suggest that in these 3 human tumors Her2 is missing a significant percent of the ECD, and is truncated. Continuing our clinical validation work we are analyzing larger numbers of Her2 positive breast tumors from clinical cohorts capable of confirming the clinical usefulness of the p95Her2 assay. Conclusions: Measuring Her2 truncation in Her2 positive breast tumors will benefit patients by enabling clinicians to select patient more likely to benefit to TKI (lapatinib, neratinib) therapy and will help physicians to make earlier informed therapeutic decisions.

Abstract 1514: Comparative quantitation of Warburg Effect proteins in FFPE tumor and stroma tissue from 10 breast carcinoma tumors

Abstract The observation that cancer cells can undergo aerobic glycolosis (the Warburg Effect) has been the target of a great deal of research in recent years, specifically directed to development of novel agents which can disrupt this pathway and potentially retard cancer growth. Recent studies, by Lisanti and colleagues, have suggested that the critical site of aerobic glycolysis may not be the tumor epithelial cells, but instead the ‘Warburg Effect’ is occurring in the tumor associated fibroblasts and other stromal cells. In order to better define the importance of ‘tumor’ vs. ‘stroma’ components of the Warburg effect, we have developed a quantitative multiplexed assay which can be performed directly in formalin fixed paraffin embedded (FFPE) patient tissue. This multiplex assay is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which renders dissected tissue to a completely solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). For mass spectrometric analysis, we developed 9 different quantitative assays for each of the Warburg Effect components (LDHA, PKM2, FBA(a), PGKI, LDHA, PMI, EnolaseA, and TPI) as well as Caveolin-1 which was identified as a marker of active aerobic glycolysis in tumor fibroblasts. We then quantitated all of these proteins in multiplex on matched tumor and stromal tissue laser microdissected from 10 different FFPE breast carcinoma tumor samples. This analysis demonstrated broadly divergent levels of glycolytic enzyme expression across the set of tumor samples, and in most cases, much higher enzyme expression in tumor cells as opposed to tumor associated stroma cells. However there were several cases where the stromal level of Warburg protein expression was elevated above matched tumor epithelial cell expression. These results demonstrate that by using the Liquid Tissue® technology, we can separately microdissect tumor and stroma in FFPE tumor sections and characterize the specific tumor/stroma components, be they pharmacodynamic analyses, drug target analysis, or seed vs. soil studies. Second, this technology reinforces the capability of SRM based mass spectrometry studies to function in massively multiplexed fashion, where 20 to 40 or more specific analytes can be quantitated by analysis of 0.5ug of protein lysate from a single FFPE tumor section. Finally, we are in the process of clinically validating this Warburg Effect multiplex assay and will then perform the assay on a larger, completely annotated tumor cohort in order to extend our knowledge of how tumor vs. stroma expression of Warburg effect components impacts tumor progression and therapeutic response. 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 1514. doi:10.1158/1538-7445.AM2011-1514

Concordant HER2 status between malignant CSF cells and primary breast carcinoma tissue

1104 Background: Leptomeningeal diseases (LMD) from breast cancer are detected in up to 19% of patients with brain metastasis and their prognosis is extremely poor. Diagnosis of leptomeningeal disease requires positive cerebrospinal fluid (CSF) cytology. However, it is not known whether HER2 status of malignant CSF cells coincides with that of original breast carcinoma cells. We intended to determine whether CSF cytology specimen is suitable for evaluating HER2 status by fluorescence in situ hybridization (FISH) in LMD to provide the basis for HER2 targeted intrathecal therapy. Methods: Both formalin-fixed paraffin-embedded (FFPE) breast carcinoma tissue and liquid based CSF cytology specimen were tested for HER2 status in 16 patients who developed LMD at National Cancer Center between Dec 2004 and Jul 2008. We evaluated HER2 gene amplification by FISH on destained CSF cytology slides which contained minimum 20 malignant cells per slide, and compared with HER2 status by immunohistochemistry (IHC) in FFPE breast carcinoma tissue. HER2 was determined positive when FISH ratio &gt; 2.2 or IHC 3+ according to ASCO/CAP guideline. Results: Concordance rate of HER2 status between CSF cytology by FISH and FFPE tissue by IHC was 100% as shown in the table. Conclusions: When CSF cytology specimen was appropriately prepared yielding adequate cellularity without dry artifact, CSF cytology was suitable for evaluating HER2 status by FISH in LMD. Intrathecal HER2 targeted therapy could be attempted when FFPE breast carcinoma tissue is HER2 positive in view of highly concordant HER2 status by our data. Supported by NCC Grant No 0610240- 3. [Table: see text] No significant financial relationships to disclose.

Evaluation of the prognostic and predictive value of HER family mRNA expression in high-risk early breast cancer: A Hellenic Cooperative Oncology Group (HeCOG) study

The aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as well as to investigate the predictive value of HER2 mRNA expression for adjuvant treatment with paclitaxel. RNA was extracted from 268 formalin-fixed paraffin-embedded (FFPE) tumour tissue samples of high-risk breast cancer patients enrolled in the randomised HE10/97 trial, evaluating the effect of dose-dense anthracycline-based sequential adjuvant chemotherapy with or without paclitaxel. The mRNA expression of all four HER family members was assessed by kinetic reverse transcription-polymerase chain reaction (kRT–PCR). The overall concordance between kRT–PCR and IHC/FISH for HER2 status determination was 74%. At a median follow-up of 8 years, multivariate analysis showed that EGFR and HER2 mRNA expression was associated with reduced overall survival (OS). HER3 and HER4 mRNA level had a favourable prognostic value in terms of OS and disease-free survival (DFS), respectively. Adjusting for HER2 mRNA expression, OS and DFS did not differ between treatment groups. These data indicate that EGFR as well as HER2 are prognostic factors of worse clinical outcomes, whereas HER3 and HER4 gene transcription is associated with better prognosis in high-risk early breast cancer. However, HER2 mRNA expression did not predict clinical benefit from paclitaxel. Kinetic RT–PCR represents an alternative method for evaluating the expression of HER family members in FFPE breast carcinomas.

Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial

Estrogen receptor (ER) and progesterone receptor (PgR) protein expression carry weak prognostic and moderate predictive utility for the outcome of early breast cancer patients on adjuvant chemohormonotherapy. We sought to study the predictive significance and correlations of transcriptional profiling of the ER, PgR and microtubule-associated protein Tau (MAP-Tau) genes in early breast cancer. Messenger RNA (mRNA) was extracted from 279 formalin-fixed paraffin-embedded breast carcinomas (T1-3N0-1M0) of patients enrolled in the Hellenic Cooperative Oncology Group (HeCOG) trial HE 10/97, evaluating epirubicin-alkylator based adjuvant chemotherapy with or without paclitaxel (E-T-CMF versus E-CMF). Kinetic reverse transcription polymerase chain reaction (kRT-PCR) was applied for assessment of the expression of estrogen receptor, progesterone receptor and MAP-Tau genes in 274 evaluable patients. Cohort-based cut-offs were defined at the 25th percentile mRNA value for ER and PgR and the median for MAP-Tau. Two hundred and ten patients (77%) were ER and/or PgR-positive by immunohistochemistry (IHC). Positive ER and MAP-Tau mRNA status was significantly associated with administration of hormonal therapy and low grade, while MAP-Tau mRNA status correlated with premenopausal patient status. MAP-Tau strongly correlated with ER and PgR mRNA status (Spearmann r = 0.52 and 0.64, P < 0.001). The observed chance corrected agreement between determination of hormonal receptor status by kRT-PCR and IHC was moderate (Kappa = 0.41) for ER and fair (Kappa = 0.33) for PgR. At a median follow-up of 8 years, univariate analysis adjusted for treatment showed positive ER mRNA status to be of borderline significance for reduced risk of relapse (HR = 0.65, 95% CI 0.41-1.01, P = 0.055) and death (HR = 0.62, 95% CI 0.36-1.05, P = 0.077), while positive MAP-Tau mRNA status was significantly associated with reduced risk of relapse (HR = 0.50, 95% CI 0.32-0.78, P = 0.002) and death (HR = 0.49, 95% CI 0.29-0.83, P = 0.008). In multivariate analysis, only axillary nodal metastases (HR = 2.33, 95% CI 1.05-5.16, P = 0.04) and MAP-Tau mRNA status (HR = 0.46, 95% CI 0.25-0.85, P = 0.01) independently predicted patient outcome. However, MAP-Tau mRNA levels did not predict enhanced benefit from inclusion of paclitaxel in the adjuvant chemotherapy regimen (test for interaction P = 0.99). No correlation was evident between increasing ER and PgR mRNA transcription and increasing benefit from endocrine therapy in 203 ER and/or PgR IHC-positive patients receiving adjuvant hormone therapy (Wald P = 0.54 for ER, 0.51 for PR). ER gene transcription carries weak predictive significance for benefit from endocrine therapy or for outcome, with no apparent dose-response association. The predictive significance is possibly exerted via MAP-Tau gene expression, an ER-inducible tubulin modulator with strong predictive significance for patient outcome. However, MAP-Tau mRNA did not predict benefit from the addition of a taxane to adjuvant chemotherapy. Further study of the biologic function and utility of MAP-Tau for individualising adjuvant therapy is warranted.

Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situhybridization and immunohistochemistry

IntroductionOne of the most common genetic aberrations associated with breast cancer is the amplification and overexpression of the ERBB2 proto-oncogene located at chromosome 17, bands q12-21. The amplification/overexpression occurs in 25 to 30% of all breast cancers. In breast cancer, aneusomy of chromosome 17, either monosomy or polysomy, is frequently observed by conventional cytogenetics and fluorescence in situ hybridization (FISH). The aim of this study was to discover whether or not numerical aberrations on chromosome 17 have a correlation to the amplification or overexpression of the ERBB2 gene and to analyze their clinical implications in subgroups showing 2+ or 3+ positive scores by immunohistochemistry (IHC).MethodsWe used FISH on a series of 175 formalin-fixed paraffin-embedded breast carcinomas to detect ERBB2 amplification, using a dual-probe system for the simultaneous enumeration of the ERBB2 gene and the centromeric region of chromosome 17, as well as using IHC to detect overexpression. We analyzed clinical and pathological variables in a subgroup of patients with 2+ and 3+ IHC scores (147 patients), to describe any differences in clinicopathological characteristics between polysomic and non-polysomic cases with the use of the χ2 test.ResultsWe found 13% of cases presenting polysomy, and three cases presented monosomy 17 (2%). According to the status of the ERBB2 gene, instances of polysomy 17 were more frequently observed in non-amplified cases than in FISH-amplified cases, suggesting that the mechanism for ERBB2 amplification is independent of polysomy 17. Polysomy 17 was detected in patients with 2+ and 3+ IHC scores. We found that nodal involvement was more frequent in polysomic than in non-polysomic cases (P = 0.046).ConclusionsThe determination of the copy number of chromosome 17 should be incorporated into the assesment of ERBB2 status. It might also be helpful to differentiate a subgroup of breast cancer patients with polysomy of chromosome 17 and overexpression of ERBB2 protein that probably have genetic and clinical differences.

Does the correlation between EBNA-1 and p63 expression in breast carcinomas provide a clue to tumorigenesis in Epstein-Barr virus-related breast malignancies?

Several investigators have identified Epstein-Barr virus (EBV) particles in breast carcinomas, a fact that supports a role for EBV in mammary tumorigenesis. The possible mechanism involved in this process is not clear. The present study was carried out in an attempt to determine whether there is a relationship between latent infection with EBV and p53 and p63 expression in breast carcinomas. Immunohistochemistry developed with 3.3-diaminobenzidine tetrahydrochloride was performed in 85 formalin-fixed paraffin-embedded breast carcinomas using anti-EBV EBNA-1, anti-p63, anti-p53, anti-estrogen receptor (ER) and anti-progesterone receptor (PR) antibodies. The cases were selected to represent each of the various histologic types: intraductal carcinoma (N=12), grade I invasive ductal carcinoma (N=15), grade II invasive ductal carcinoma (N=15), grade III invasive ductal carcinoma (N=15), tubular carcinoma (N=8), lobular carcinoma (N=10), and medullary carcinoma (N=10). The ductal breast carcinomas were graded I, II and III based on the Scarff-Bloom and Richardson grading system modified by Elston and Ellis. One slide containing at least 1000 neoplastic cells was examined in each case. ER, PR, p63, p53 and EBNA-1 were positive in 60, 40, 11.8, 21.2 and 37.6% of carcinomas, respectively. There was a correlation between EBNA-1 and p63 expression (P<0.001), but not between EBNA-1 and p53 (P=0.10). These data suggest a possible role for p63 in the mammary tumorigenesis associated with Epstein-Barr virus infection.