Selecting Patients for Oncotype DX Testing Using Standard Clinicopathologic Information.

INTRODUCTION: Indiscriminate ordering of Oncotype DX (ODX) is expensive and of poor value to patients, physicians, and health care providers. The 3 Magee equations, Gage Algorithm, and University of Tennessee predictive algorithm all use standard clinicopathologic data to provide surrogate ODX scores. In this hypothesis-generating study, we evaluated whether these prognostic scores could be used to identify patients unlikely to benefit from additional ODX testing. PATIENTS AND METHODS: Retrospective data was collected from 302 patients with invasive ductal breast cancer and available ODX scores. Additional data was available for: Magee equations 1 (212 patients), 2 (299 patients), 3 (212 patients), Gage Algorithm (299 patients), and University of Tennessee predictive algorithm (286 patients). ODX scores were banded according to the TAILORx results. RESULTS: Correlation with ODX scores was between 0.7 and 0.8 (Gage), 0.8 and 0.9 (Magee 2, University of Tennessee predictive algorithm), and > 0.9 (Magee 1 and 3). Magee 3 was the most robust and is proposed as a screening tool: for patients aged ≤ 50 years, ODX testing would be not required if the Magee 3 score was < 14 or ≥ 20; for those aged > 50 years, ODX would not be required if the Magee 3 score was < 18 or ≥ 26. Using these cut-offs, 110 (51.9%) of 212 patients would be deemed as not requiring ODX testing, and 109 (99.1%) of110 patients would be appropriately managed. CONCLUSIONS: Use of all formulae, and the Magee 3 equation in particular, are proposed as possible screening tools for ODX testing, resulting in significantly reduced frequency of ODX testing. This requires validation in other populations.

The assessment of 8th edition AJCC prognostic staging system and a simplified staging system for breast cancer: The analytic results from the SEER database.

The prognostic value of the prognostic staging system that incorporated estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor-2 (Her-2), and histological grade has been validated in breast cancer (BC) patients, but the staging system seems to be somewhat complex. Recently, an updated bioscore system based on these tumor biological factors was proposed. The purpose of this study was to compare the prognostic stratification between prognostic staging system of American Joint Commission on Cancer (AJCC) and a simplified staging system based on the bioscore system and anatomic TNM staging for BC patients. A total of 44 593 patients with invasive ductal carcinoma who underwent radical resection between 2010 and 2011 were reviewed using the SEER database. The patients were reclassified into different groups according to the anatomic staging system, prognostic staging system, risk bioscore system, and simplified staging system, respectively. The prognostic differences between different groups were compared and clinicopathologic features were analyzed. The anatomic TNM staging failed to clearly distinguish the prognostic difference between stage IIIB and stage IIIC. Therefore, we proposed an adjusted anatomic staging, in which T1N3 and T2N3 were downstaged from stage IIIC to stage IIIB, and T4N2 was upstaged from stage IIIB to stage IIIC. Histological grade III, ER(-), PR(-), and Her-2(-) were identified as independent prognostic factors in the multivariate analysis, and these factors were separately marked as 1 point. There were significant survival differences among different risk points except for the comparison between 0 and 1 point. The higher the risk points, the poorer the prognosis of BC patients. In addition, the curve distance between stage IIA and stage IIB was not significantly broaden according to the prognostic staging system. However, the prognostic stratification for BC patients could be significantly improved by the simplified staging system incorporated the bioscore system and adjusted anatomic staging. Several drawbacks may still exist in the prognostic staging system of AJCC. A simplified staging system that incorporated risk score system and the anatomic staging could provide more accurate prognostic information for BC patients.

Treatment patterns, clinical outcomes, health resource utilization, and cost in patients with BRCA-mutated metastatic breast cancer treated in community oncology settings.

PURPOSE: This retrospective study of community oncology patients with breast cancer gene (BRCA)-mutated metastatic breast cancer (MBC) examined treatment outcomes and health resource utilization (HRU) and costs for a sample of patients with human epidermal growth factor receptor 2 (HER2)-negative disease who were either hormone receptor positive (HR+) or triple negative breast cancer (TNBC). METHODS: Evidence from the Vector Oncology Data Warehouse, a repository of electronic medical records/billing data and provider notes, was analyzed. Treatment outcomes were progression-free survival (PFS) and overall survival (OS) from start of first-line therapy in the metastatic setting. HRU and cost measures were collected from the time of MBC diagnosis to end of the record. HRU included hospitalizations, emergency room visits, infused/parenteral supportive care drugs, and outpatient visits. Costs were computed both as total and monthly costs. RESULTS: 57 HR+ and 57 TNBC patients (2013-2015) met inclusion criteria. Eight TNBC patients did not get treatment. HR+ patients had median first line PFS of 12.1 months and TNBC patients had 6.1 months. HR+ patients had median OS from start of first line of 38.4 months, and TNBC patients had 23.4 months. Rate of use of infused/parenteral supportive care drugs was 25.5% overall and 36.7% among TNBC patients with 15.8% among HR+ patients. CONCLUSION: There is an unmet need in BRCA-mutated patients with MBC, including those with HR+ and TNBC disease. The unmet need among TNBC patients was most evident in that 12% were not treated and TNBC patients appeared to have poor treatment outcomes. MICRO ABSTRACT: Reviewed medical records for outcomes, resource utilization, and costs in 114 community patients with BRCA mutated metastatic breast cancer. 57 hormone positive (HP); 57 triple negative (TN). RESULTS: median PFS: 12.1 months HP; 6.1 TN. HP OS was 38.4; TN 23.4. Rate of infused supportive care drugs: 25.5% HP; 36.7% TN. Patients with TN disease need better therapeutic options.

DSCAM-AS1 up-regulation in invasive ductal carcinoma of breast and assessment of its potential as a diagnostic biomarker.

BACKGROUND: The long non-coding RNA (lncRNA) DSCAM-AS1 has been demonstrated to participate in the pathogenesis of breast cancer and tamoxifen resistance. OBJECTIVE: To evaluate expression profile of DSCAM-AS1 in invasive ductal carcinoma of breast and its suitability as a biomarker for diagnosis of breast cancer. METHODS: We evaluated expression of DSCAM-AS1 in 108 breast tissues including tumoral and adjacent non-cancerous tissues (ANCTs) by means of quantitative real time PCR. RESULTS: DSCAM-AS1 was up-regulated in tumoral tissues compared with ANCTs (Fold change = 2.86, P = 0.011). Its expression was significantly higher in patients aged less than 55 compared with older patients (P = 0.02). However, its expression levels had not a good performance as a diagnostic biomarker for breast cancer. CONCLUSIONS: The significant up-regulation of DSCAM-AS1 in tumoral tissues compared with ANCTs provides further evidences for participation of this lncRNA in the pathogenesis of breast cancer.

Assessment of expression of interferon γ (IFN-G) gene and its antisense (IFNG-AS1) in breast cancer.

BACKGROUND: The role of long non-coding RNAs has been extensively appreciated in the contexts of cancer. Interferon γ-antisense RNA1 (IFNG-AS1) is an lncRNA located near to IFN-γ-encoding (IFNG) gene and regulates expression of IFNG in Th1 cells. METHODS: In the present study, we evaluated expression of IFNG and IFNG-AS1 in 108 breast samples including tumoral tissues and their adjacent non-cancerous tissues (ANCTs) using real-time PCR. IFNG-AS1 was significantly upregulated in tumoral tissues compared with ANCTs (expression ratio = 2.23, P = 0.03). RESULTS: Although the expression of IFNG was higher in tumoral tissues compared with ANCTs (relative expression = 1.89), it did not reach the level of significance (P = 0.07). IFNG expression was significantly higher in HER2-negative tumoral tissues compared with HER2-positive ones (P = 0.01) and in grade 1 samples compared with grade 2 ones (P = 0.03). No other significant difference was found in expressions of genes between other groups. CONCLUSION: Significant strong correlations were detected between expression of IFNG and IFNG-AS1 in both tumoral tissues and ANCTs. The present study provides evidences for participation of IFNG and IFNG-AS1 in the pathogenesis of breast cancer and warrants future studies to elaborate the underlying mechanism.

The impact and indications for Oncotype DX on adjuvant treatment recommendations when third-party funding is unavailable.

OBJECTIVES: Industry-supported decision impact studies demonstrate that Oncotype Dx (ODX) changes treatment recommendations (TR) in 24-40% of hormone receptor+/HER2- patients. ODX is not reimbursed by third-party payers in Australia, potentially resulting in more selective use. We sought to evaluate the impact of self-funded ODX on TRs. METHODS: Data collected included demographics, tumor characteristics, indication for ODX and pre- and post-recurrence score (RS) TR. Primary endpoint was frequency of TR change and associations with TR change were sought. RESULTS: Eighteen physicians contributed 382 patients (median age 54). A total of 232 (61%) of tumors were T1 and were grade 1, 2 and 3 in 49 (13%), 252 (66%) and 79 (21%). A total of 257 (67%) were node negative. Assay indications were: confirm need for chemotherapy (CT) (36%), confirm omission of CT (40%) and genuine equipoise (24%). RS was low (≤17) in 55%, intermediate (18-31) in 36% and high (≥32) in 9%. Thirty-eight percent of patients had TR change post-ODX. Sixty-five percent of patients recommended CT pre-ODX changed to hormone therapy alone (HT)-more likely if lower grade and if ER and/or PR > 10%. Fourteen percent of patients with pre-ODX TR for HT added CT-more likely if ER and/or PR ≤10% and if Ki67 > 15% Overall, TR for CT decreased from 47% to 24%. CONCLUSION: Patient-funded ODX changed TRs in 38% of patients, de-escalating 65% from CT to HT and adding CT to 14% of those recommended HT. These changes were greater than an industry-funded study suggesting that physicians can identify situations where the assay may influence decisions.

The Assessment of HER2 Gene Status by Fluorescence In Situ Hybridization in Invasive Breast Carcinomas With Equivocal HER2 Immunostaining: Experience From a Single Institution.

BACKGROUND: A subset of breast carcinomas harbors overexpression of the human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) should be performed in breast carcinomas with equivocal HER2 immunostaining (immunohistochemistry [IHC] HER2 2+). The aim of our study is to investigate clinicopathologic factors associated with HER2 status in breast invasive carcinomas with IHC HER2 2+ through FISH analysis. METHODS: This is a retrospective study including the FISH analysis of 111 patients with invasive breast carcinomas with equivocal HER2 immunostaining. RESULTS: The mean age was 49.51 ± 10.48 years, and invasive breast carcinoma of no special type was the most histological type in our study (96.4%). Most patients had tumors positive for hormones receptors (88.2% positive for estrogen receptor and 81.4% for progesterone receptor). On FISH, the HER2 amplification rate was 22.5%. There was no significant association of HER2 status with any clinicopathologic factors ( P > .05). CONCLUSIONS: Our study shows that there are no reliable clinicopathologic factors to predict the HER2 status in breast tumors with equivocal HER2 immunostaining, supporting the necessary usage of FISH in such circumstances.

Mitotic index matter: how to improve the assessment of mitosis in order to better classify G2 breast cancer and luminal A category.

G2 ductal infiltrating carcinomas are a heterogeneous group of tumours with ambiguous clinical significance. This is because G2 carcinomas are almost always the largest category and poorly reproducible. Mitotic count (MC) is one of the causes of poor histological grading reproducibility. The phosphoistone H3 (PPH3) antibody improves identification of mitotic figures. The aim of our study is to demonstrate whether using a new histological grading system based on PPH3 immunostaining to assess MC can re-stratify G2 category. We selected 100 cases of G2 invasive carcinoma. The mitotic score was accurately re-evaluated performing MC on PPH3 immunostained sections. 21/100 G2 cases (21%) showed the same mitotic score both with hematoxilin and eosin (H&E) and PPH3 while 79 cases (79%) with PPH3 shifted to a higher mitotic score. After re-grading the 100 G2 cases based on the assessment of mitotic score with PPH3 only 53 cases (53%) were confirmed as G2, while 47 cases (47%) had shifted to G3. Finally we reclassified early tumours in the surrogate molecular subtype according to the 2013 St. Gallen Conference criteria and found that 13/40 cases (33%) classified as luminal A were G3 with the PPH3 mitotic score and could benefit from chemotherapy. In conclusion, PPH3 improving MC gives a better categorization by halving the G2 group. In particular, applied to the surrogate subtype luminal A breast cancer it identified cases that could benefit from adjuvant cytotoxic chemotherapy.

Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer.

BACKGROUND: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. METHODS: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test. RESULTS: Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman's r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131). CONCLUSION: Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific.

An Analysis of Oncotype DX Recurrence Scores and Clinicopathologic Characteristics in Invasive Lobular Breast Cancer.

The Oncotype DX breast cancer assay (Genomic Health, Redwood City, CA) is increasingly being used to guide treatment decisions for patients with early stage, hormone-positive, Her-2-negative breast cancer. The utility of the Oncotype DX in decision making for treatment of invasive lobular carcinoma (ILC) has not been investigated as the results reported by Genomic Health are largely in a population with invasive ductal carcinoma (IDC). The authors hypothesized that the Oncotype DX recurrence score (RS) distribution for ILC is different than that for IDC. We performed a retrospective analysis of early stage breast cancer patients treated at Penn State Cancer Institute from 2001 to 2011 and identified 102 patients with ILC. We also pulled RS data from our institution's prospective registry of consecutive patients with early stage IDC treated during the same time period. Median follow-up was 55 months. We found that the RS distribution for ILC differed significantly from that of IDC (p = 0.024). We also found a statistically significant difference in the RS distribution between the pure ILC and pleomorphic ILC subtypes (p = 0.027). The Oncotype DX RS distribution in ILC is unique, differing significantly from that in ductal carcinoma. Consequently, the clinical usefulness and cost-effectiveness of the Oncotype DX in guiding treatment for ILC should be further investigated.

Implementation of a risk assessment program in a breast-imaging community practice.

BACKGROUND: To review the initial results of the implementation of a risk assessment program in our outpatient community-based breast-imaging practice. METHODS: From May 1, 2011 through November 4, 2013 a total of 96,389 patients presented to our facility. Each patient was required to complete a health history form. 24,850 (25.7 %) were flagged through our radiology information system (RIS) as potentially being at increased risk for breast cancer per responses on the health history form. Patient flagging was based on a system we developed. Letters were generated informing flagged patients and her physician that the patient could be a candidate for further breast cancer risk assessment. Genetic testing performed was primarily for BRCA1/BRCA2; testing for other mutations was offered based on personal and family history of the patient. RESULTS: 1,088 genetic counseling appointments occurred during the time frame from the 24,850 patients flagged, leading to 887 genetic tests performed. Forty-three mutations were detected: 21 BRCA1, 19 BRCA2, 1 MLH1, 1 MSH6 and 1 CHEK2. There were 717 negative tests, 20 variants of unknown significance; the remaining cases were cancelled. 464 high-risk MRIs were performed after the patient was referred from the program, from which 52 biopsies were performed due to a finding on MRI revealing 14 malignancies. CONCLUSIONS: In our experience after implementation of a risk assessment program, 24,850 potential at-risk patients have been flagged, revealing 40 BRCA 1 or 2 mutations. Screening with breast MRI provided to at-risk patients revealed 14 cancers diagnosed that may have otherwise been undetected.

Triple assessment of sentinel lymph node metastasis in early breast cancer using preoperative CTLG, intraoperative fluorescence navigation and OSNA.

BACKGROUND: Sentinel lymph node biopsy (SLNB) became a standard surgical procedure for patients with early breast cancer; however, the optimal method of sentinel lymph node (SLN) identification remains controversial. The current study presents the protocol of our institution for preoperative and intraoperative SLN detection. METHODS: Fifty female patients with early breast cancer and clinically node-negative axilla were enrolled in this study. All patients underwent preoperative CT lymphography (CTLG), intraoperative SLNB using fluorescence navigation, intraoperative one-step nucleic acid amplification (OSNA) and postoperative hematoxylin and eosin histopathological analysis. Prediction of metastasis by CTLG and detection of metastasis by OSNA were compared to results of histopathology as standard reference. RESULTS: SLN were identified by preoperative CTLG and intraoperative SLNB with fluorescence navigation in all patients, the identification rate was 100 %. SLN metastases were detected as positive by OSNA in 9 patients (18 %), 4 were (++), 4 were (+) and 1 was (+I). SLN metastases were detected as positive by histopathology in 10 patients (20 %). The concordance rate between OSNA and permanent sections was 90 %. The negative predictive value of CTLG was 80 %. CONCLUSION: Use of CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that cannot use the radioisotope method. OSNA provided accurate intraoperative method, allowing for completion of axillary node dissection during surgery and avoidance of second surgical procedure in patients with positive SLNs, thereby reducing patient distress and, finally, saving hospital costs.

Assessment of changes in the BRCA2 and P53 genes in breast invasive ductal carcinoma in northeast Brazil.

BACKGROUND: BRCA protein interacts with at least 13 different proteins that have been implicated with cancer susceptibility and loss of BRCA function is correlated to sensitivity to DNA crosslinking agents in preclinical models. RESULTS: BRCA2 methylation frequency was 44%, p53 Pro22 allele frequency was 32% and heterozygous frequency of Arg/Pro72 genotype was 60% which could be associated as risk factor for metastasis (p = 0.046 OR = 4.190). Regarding to polymorphism of codon 249 the frequency of Arg249 allele presented 82% which was considered not statistically significant. CONCLUSIONS: There was not statistical significance to BRCA2 promoter methylation with any parameters chosen. However, our findings suggest that patients who present heterozygous genotype at codon 72 of p53 gene may have a major susceptibility to any type of metastasis and this could serve as potential auxiliary biomarker for poor prognosis.

[Assessment of HER2 gene amplification in breast cancer: a comparison of dual-color in-situ hybridization and fluorescence in-situ hybridization].

OBJECTIVE: To compare dual-color in-situ hybridization (DISH) with fluorescence in situ hybridization(FISH) in evaluating the human HER2 gene status in invasive breast cancer. METHODS: HER2 gene status in 110 cases of breast invasive ductal carcinomas with a 2+ score on immunohistochemistry (IHC) was investigated by FISH and DISH. The 2007 and 2013 ASCO (American Society of Clinical Oncology)/CAP (College of American Pathologists) HER2 guideline were used respectively to evaluate the agreement between these two techniques. RESULTS: (1) Using the 2007 ASCO/CAP guideline, the overall concordance between FISH and DISH was 97.3% (107/110). Fifty of 51 samples with amplification by FISH were also detected by DISH and the remaining one sample was equivocal.Eight of 10 equivocal samples by FISH were equivocal by DISH and the remaining two samples were negative. Forty-nine samples with no amplification by FISH were all negative by DISH. The concordance was 98.0%, 8/10 and 100.0% respectively for the FISH positive, equivocal and negative groups. (2) Using the 2013 ASCO/CAP guideline, the overall concordance between FISH and DISH was 90.0% (99/110). Fifty-three of 55 samples with amplification by FISH were also detected by DISH and the remaining two were equivocal and negative respectively. Two of 12 equivocal samples by FISH were equivocal by DISH and the remaining ten samples were negative in 7 cases and equivocal in 3 cases. Forty-three samples with no amplification by FISH were all negative by DISH. The concordance was 96.4%, 3/12 and 100.0% respectively for the FISH positive, equivocal and negative groups. Pearson correlation analysis indicated that the HER2 status detected by FISH and DISH were significantly correlated with each other (R=0.584, P<0.01). CONCLUSIONS: There is a high concordance between DISH and FISH for assessing the HER2 gene status in invasive breast cancer. DISH is a new option for assessing HER2 gene status of breast cancer in clinical practice. The clinical significance of the discordance between DISH and FISH in equivocal cases warrants further study.

Assessment of clinical outcomes in breast cancer patients treated with taxanes: multi-analytical approach.

Polymorphisms in genes encoding CYPs (Phase I) and ABCB1 (Phase III) enzymes may attribute to variability of efficacy of taxanes. The present study aims to find the influence of CYP and ABCB1 gene polymorphisms on taxanes based clinical outcomes. 132 breast cancer patients treated with taxanes based chemotherapy were genotyped for CYP3A4*1B, CYP3A5*3, CYP1B1*3, CYP2C8*3, ABCB1 1236C>T, 2677G>T/A and 3435C>T polymorphisms using PCR-RFLP. Associations of genetic variants with clinical outcomes in terms of response in 58 patients receiving neo-adjuvant chemotherapy (NACT), and chemo-toxicity in 132 patients were studied. Multifactor dimensionality reduction (MDR) analysis was performed to evaluate higher order gene-gene interactions with clinical outcomes. Pathological response to taxane based NACT was associated with GA genotype as well as A allele of CYP3A5*3 polymorphism (Pcorr=0.0465, Pcorr=0.0465). Similarly, association was found in dominant model of CYP3A5*3 polymorphism with responders (Pcorr=0.0465). Haplotype analysis further revealed ACYP3A4-ACYP3A5 haplotype to be significantly associated with responders (Pcorr=0.048). In assessing toxicity, significant association of variant (TT) genotype and T allele of ABCB1 2677G>T/A polymorphism, was found with 'grade 1 or no leucopenia' (Pcorr=0.0465, Pcorr=0.048). On evaluating higher order gene-gene interaction models by MDR analysis, CYP3A5*3; ABCB11236C>T and ABCB1 2677G>T/A; ABCB1 3435C>T and CYP1B1*3 showed significant association with treatment response, grade 2-4 anemia and dose delay/reduction due to neutropenia (P=0.024, P=0.004, P=0.026), respectively. Multi-analytical approaches may provide a better assessment of pharmacogenetic based treatment outcomes in breast cancer patients treated with taxanes.

Assessment of changes in the brca2 and p53 genes in breast invasive ductal carcinoma in northeast Brazil

BACKGROUND: BRCA protein interacts with at least 13 different proteins that have been implicated with cancer susceptibility and loss of BRCA function is correlated to sensitivity to DNA crosslinking agents in preclinical models. RESULTS: BRCA2 methylation frequency was 44%, p53 Pro22 allele frequency was 32% and heterozygous frequency of Arg/Pro72 genotype was 60% which could be associated as risk factor for metastasis (p = 0.046 OR = 4.190). Regarding to polymorphism of codon 249 the frequency of Arg249 allele presented 82% which was considered not statistically significant. CONCLUSIONS: There was not statistical significance to BRCA2 promoter methylation with any parameters chosen. However, our findings suggest that patients who present heterozygous genotype at codon 72 of p53 gene may have a major susceptibility to any type of metastasis and this could serve as potential auxiliary biomarker for poor prognosis.

Sentinel and nonsentinel lymph node assessment using a combination of one-step nucleic acid amplification and conventional histological examination.

BACKGROUND: Clinical significance of intraoperative sentinel lymph node (SLN) metastases detection using one-step nucleic acid amplification (OSNA) has not been thoroughly investigated. The aim of this study was to assess the usefulness of using a combination of OSNA and conventional histological examinations. MATERIALS AND METHODS: We included 772 consecutive patients with clinical node-negative cTis-cT3 primary breast cancer who underwent SLN biopsy with intraoperative OSNA and multi-section histological examination at our institution. We estimated the concordance rate and compared SLN metastases detection rates between the two methods. We also compared non-SLN metastasis detection rate between patients who tested positive in OSNA and those who tested positive in histology. RESULTS: Among 772 patients, SLN metastases were intraoperatively detected in 211 (26.4%) by either OSNA or histology, in 168 (21.8%) by OSNA, and in 150 (19.4%) by histology. The concordance rate between OSNA and histological examination was 89.2%, but only 123 (58.8%) patients tested positive in both OSNA and histology; 45 were positive in OSNA only and 43 were positive in histology only. SLN status as per both OSNA and histology was significantly correlated with the presence of non-SLN metastases and multivariate analysis-identified independent predictive factors of non-SLN metastases. CONCLUSIONS: Intraoperative SLN metastases detection may be more accurate with a combination of OSNA and histological examination than with OSNA or histological examination alone. By using both methods, we can reduce the risk of false negative rate in SLN biopsy, and may prevent physicians from overlooking patients with non-SLN metastases.

Assessment of DNA methylation status in early stages of breast cancer development.

BACKGROUND: Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development. METHODS: To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARß2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n=52), ductal hyperplasia (n=23), atypical ductal hyperplasia (n=31), ductal carcinoma in situ (DCIS, n=95) and AJCC stage I invasive ductal carcinoma (IDC, n=34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed. RESULTS: Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, <0.0001 and <0.0001 for MINT17, MINT31, RARß2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics. CONCLUSION: DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential.

Evaluation of reliability of FISH versus brightfield dual-probe in situ hybridization (BDISH) for frontline assessment of HER2 status in breast cancer samples in a community setting: influence of poor tissue preservation.

AIMS: To evaluate the reliability of novel brightfield microscopy-based dual in situ hybridization (BDISH) methods for frontline HER2 status analysis in selected suboptimally preserved breast cancer tissue samples reflecting of the worst scenario in a community. METHODS AND RESULTS: A total of 320 morphologically poorly preserved breast invasive ductal carcinomas from the archives of 2 tertiary institutions in Brazil were selected for a tissue microarray-based analysis. 4B5 antibody was used for immunohistochemistry. Fluorescence in situ hybridization (FISH), DuoCISH, ZytoDot CISH, and silver in situ hybridization (SISH) were performed and compared. The highest agreement was observed between SISH and FISH. In addition, SISH was easier to assess in both amplified and nonamplified cases when compared with the other chromogenic methods, due to the sharpness of its dots. DuoCISH produced false-positive results, associated with thicker ill-defined dots, causing poor distinction between nonamplification and low amplification. ZytoDot CISH showed lower sensitivity, with increased frequency of false-positive results. CONCLUSIONS: SISH is the most reliable of the BDISH methods, with sensitivity and specificity highly comparable with FISH. It is also less deleterious than other BDISH methods, producing signals that were more distinct and therefore more readily analyzable even in poorly preserved tissue.

Multimodel assessment of BRCA1 mutations in Taiwanese (ethnic Chinese) women with early-onset, bilateral or familial breast cancer.

Although evidence suggests an importance of genetic factors in the development of breast cancer in Taiwanese (ethnic Chinese) women, including a high incidence of early-onset and secondary contralateral breast cancer, a major breast cancer predisposition gene, BRCA1, has not been well studied in this population. In fact, the carcinogenic impacts of many genetic variants of BRCA1 are unknown and classified as variants of uncertain significance (VUS). It is therefore important to establish a method to characterize the BRCA1 VUSs and understand their role in Taiwanese breast cancer patients. Accordingly, we developed a multimodel assessment strategy consisting of a prescreening portion and a validated functional assay to study breast cancer patients with early-onset, bilateral or familial breast cancer. We found germ-line BRCA1 mutations in 11.1% of our cohort and identified one novel missense mutation, c.5191C>A. Two genetic variants were initially classified as VUSs (c.1155C>T and c.5191C>A). c.1155C>T is not predicted to be deleterious in the prescreening portion of our assessment strategy. c.5191C>A, on the other hand, causes p.T1691K, which is predicted to have high deleterious probability because of significant structural alteration, a high deleterious score in the predictive programs and, clinically, triple negative characteristics in breast tumors. This mutant is confirmed by transcription activation and yeast growth-inhibition assays. In conclusion, we show as high a prevalence of germ-line BRCA1 mutation in high-risk Taiwanese patients as in Caucasians and demonstrate a useful strategy for studying BRCA1 VUSs.