Clinicopathological features and BRCA1 and BRCA2 mutation status in a prospective cohort of young women with breast cancer.

BACKGROUND: Breast cancer in young women is more likely to have higher risk features and be associated with germline BRCA1/BRCA2 mutations. We present the clinicopathologic features of breast cancers in a prospective cohort of young women, and associations between surrogate molecular subtype and BRCA1/BRCA2 mutation status. METHODS: Histopathological features, biomarker status, tumour stage and BRCA status were collected. Invasive tumours were categorised as luminal A-like (ER + and/or PR + , HER2-, grade 1/2), luminal B-like (ER + and/or PR + , HER2 + , or ER + and/or PR + , HER2-, and grade 3), HER2-enriched (ER/PR-, HER2 + ) or triple-negative. RESULTS: In all, 57.3% (654/1143) of invasive tumours were high grade. In total, 32.9% were luminal A-like, 42.4% luminal B-like, 8.3% HER2-enriched, and 16.4% triple-negative. Among different age groups, there were no differences in molecular phenotype, stage, grade or histopathology. 11% (131) of tumours were from BRCA mutation carriers; 64.1% BRCA1 (63.1% triple-negative), and 35.9% BRCA2 (55.3% luminal B-like). DISCUSSION: The opportunity to provide comparisons across young age groups, BRCA mutation status, surrogate molecular phenotype, and the identification of more aggressive hormone receptor-positive phenotypes in this population provides direction for future work to further understand and improve disparate outcomes for young women with luminal B-like cancers, particularly BRCA2-associated cancers, with potential implications for tailored prevention and treatment.

Encapsulated papillary carcinoma with and without frank invasion: Comparison of clinicopathologic features and role of axillary staging.

To investigate clinical and pathologic features of encapsulated papillary carcinomas (EPCs) that may be associated with invasive disease and characterize the axillary staging practices for EPCs at our institution. A pathology database search for cases containing "papillary carcinoma" was performed. Slides were reviewed by two pathologists. Clinicopathological features and axillary staging practices of EPCs with and without invasion were compared. Twenty-five cases of EPCs were identified. Fifteen cases contained a frank invasive tumor (60%), which were all pT1 (0.7 ± 0.56 cm), and the majority were ER-positive, HER2-negative, low-grade IDC-NST. Seventeen patients underwent sentinel lymph node biopsies (SLNB). No nodal metastases were identified. Follow-up was available for 24 patients (mean = 39 ± 29 months); 23 had no NED. Patients that presented with a self-palpated mass (versus screening) were more likely to have an invasive component; however, no pathologic or radiologic features differentiated EPCs with and without frank invasion. Pathologic and radiologic characteristics did not differentiate EPCs with and without frank invasion. EPCs have an excellent prognosis supported by the notable disease-free survival and negative nodal status in our cohort, which supports the notion that patients with EPCs may forgo axillary staging.

Plasma DNA as a "liquid biopsy" incompletely complements tumor biopsy for identification of mutations in a case series of four patients with oligometastatic breast cancer.

PURPOSE: Circulating tumor DNA in plasma may present a minimally invasive opportunity to identify tumor-derived mutations to inform selection of targeted therapies for individual patients, particularly in cases of oligometastatic disease where biopsy of multiple tumors is impractical. To assess the utility of plasma DNA as a "liquid biopsy" for precision oncology, we tested whether sequencing of plasma DNA is a reliable surrogate for sequencing of tumor DNA to identify targetable genetic alterations. METHODS: Blood and biopsies of 1-3 tumors were obtained from 4 evaluable patients with advanced breast cancer. One patient provided samples from an additional 7 tumors post-mortem. DNA extracted from plasma, tumor tissues, and buffy coat of blood were used for probe-directed capture of all exons in 149 cancer-related genes and massively parallel sequencing. Somatic mutations in DNA from plasma and tumors were identified by comparison to buffy coat DNA. RESULTS: Sequencing of plasma DNA identified 27.94 ± 11.81% (mean ± SD) of mutations detected in a tumor(s) from the same patient; such mutations tended to be present at high allelic frequency. The majority of mutations found in plasma DNA were not found in tumor samples. Mutations were also found in plasma that matched clinically undetectable tumors found post-mortem. CONCLUSIONS: The incomplete overlap of genetic alteration profiles of plasma and tumors warrants caution in the sole reliance of plasma DNA to identify therapeutically targetable alterations in patients and indicates that analysis of plasma DNA complements, but does not replace, tumor DNA profiling. TRIAL REGISTRATION: Subjects were prospectively enrolled in trial NCT01836640 (registered April 22, 2013).

Genotype-phenotype associations in breast pathology: Achievements of the past quarter century.

The first genotype-phenotype relationship in breast pathology developed in 1994 with the discovery of the CDH1 gene. This finding eventually provided biological insight into the characteristic morphology of invasive lobular carcinoma. Subsequent investigative efforts have uncovered additional molecular alterations largely responsible for the histology of several breast neoplasms including secretory carcinoma, adenoid cystic carcinoma, tall cell carcinoma with reversed polarity, fibroepithelial lesions, and most recently, adenomyoepithelioma. Evaluation of the genomic landscape of other special types of breast cancer with distinctive growth patterns, such as invasive mucinous carcinoma, have yet to uncover recurring cytogenetic and/or molecular alterations. Despite the lack of a hallmark alteration in mucinous carcinoma, it is important to note the relative decrease in PIK3CA mutations compared with invasive carcinoma of no special type. In this review, we describe the clinical and pathologic features of breast tumors with recognized genotype-phenotype correlations and summarize the molecular alterations of mucinous carcinoma.

Molecular and epigenetic profiles of BRCA1-like hormone-receptor-positive breast tumors identified with development and application of a copy-number-based classifier.

BACKGROUND: BRCA1-mutated cancers exhibit deficient homologous recombination (HR) DNA repair, resulting in extensive copy number alterations and genome instability. HR deficiency can also arise in tumors without a BRCA1 mutation. Compared with other breast tumors, HR-deficient, BRCA1-like tumors exhibit worse prognosis but selective chemotherapeutic sensitivity. Presently, patients with triple negative breast cancer (TNBC) who do not respond to hormone endocrine-targeting therapy are given cytotoxic chemotherapy. However, more recent evidence showed a similar genomic profile between BRCA1-deficient TNBCs and hormone-receptor-positive tumors. Characterization of the somatic alterations of BRCA1-like hormone-receptor-positive breast tumors as a group, which is currently lacking, can potentially help develop biomarkers for identifying additional patients who might respond to chemotherapy. METHODS: We retrained and validated a copy-number-based support vector machine (SVM) classifier to identify HR-deficient, BRCA1-like breast tumors. We applied this classifier to The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast tumors. We assessed mutational profiles and proliferative capacity by covariate-adjusted linear models and identified differentially methylated regions using DMRcate in BRCA1-like hormone-receptor-positive tumors. RESULTS: Of the breast tumors in TCGA and METABRIC, 22% (651/2925) were BRCA1-like. Stratifying on hormone-receptor status, 13% (302/2405) receptor-positive and 69% (288/417) triple-negative tumors were BRCA1-like. Among the hormone-receptor-positive subgroup, BRCA1-like tumors showed significantly increased mutational burden and proliferative capacity (both P < 0.05). Genome-scale DNA methylation analysis of BRCA1-like tumors identified 202 differentially methylated gene regions, including hypermethylated BRCA1. Individually significant CpGs were enriched for enhancer regions (P < 0.05). The hypermethylated gene sets were enriched for DNA and chromatin conformation (all Bonferroni P < 0.05). CONCLUSIONS: To provide insights into alternative classification and potential therapeutic targeting strategies of BRCA1-like hormone-receptor-positive tumors we developed and applied a novel copy number classifier to identify BRCA1-like hormone-receptor-positive tumors and their characteristic somatic alteration profiles.

Papillary and sclerosing lesions of the breast detected and biopsied by MRI: Clinical management, upgrade rate, and association with apocrine metaplasia.

Benign papillary and sclerosing lesions of the breast (intraductal papillomas, complex sclerosing lesions, radial scars) are considered high-risk lesions due to the potential for upgrade to carcinoma on subsequent surgical excision. Optimal clinical management of such lesions remains unclear due to variable reported upgrade rates. Apocrine metaplasia is a common finding in breast tissue and its role in MRI enhancing lesions is increasingly being recognized. The purpose of this study was to investigate the MRI features of papillary and sclerosing lesions of the breast, evaluate the clinical management and upgrade rate of such lesions, and examine the contribution of apocrine metaplasia to the imaging findings. A 13-year retrospective review of MRI-guided biopsies identified 70 MRI-detected and -biopsied papillary and sclerosing lesions. Sixteen lesions without atypia underwent surgical excision; only one case (6%) was upgraded to pleomorphic lobular carcinoma in situ. The majority (64%) of biopsies contained apocrine metaplasia either within or adjacent to the targeted lesion. We found that half of MRI-detected lesions had T2 hyperintense foci (2-5 mm) or masses (>5 mm) adjacent to the lesion. Histologic correlation showed apocrine cysts were likely responsible for this imaging finding in 56% of these cases.

Personal history of proliferative breast disease with atypia and risk of multifocal breast cancer.

BACKGROUND: A history of proliferative breast disease with atypia (PBDA) may be indicative of an increased risk not just of breast cancer but also of a more aggressive form of breast cancer. METHODS: Multifocal breast cancer (MFBC), defined as 2 or more tumors in the same breast upon a diagnosis of cancer, is associated with a poorer prognosis than unifocal (single-tumor) breast cancer. PBDA, including atypical ductal hyperplasia and atypical lobular hyperplasia, is a known risk factor for breast cancer. Using New Hampshire Mammography Network data collected for 3567 women diagnosed with incident breast cancer from 2004 to 2014, this study assessed the risk of MFBC associated with a previous diagnosis of PBDA. RESULTS: Women with a history of PBDA were found to be twice as likely to be subsequently diagnosed with MFBC as women with no history of benign breast disease (BBD; odds ratio [OR], 2.23; 95% confidence interval [CI], 1.08-4.61). Ductal carcinoma in situ on initial biopsy was associated with a 2-fold increased risk of MFBC in comparison with invasive cancer (OR, 2.13; 95% CI, 1.58-2.88). BBD and proliferative BBD without atypia were not associated with MFBC. CONCLUSIONS: Women with a history of previous PBDA may be at increased risk for MFBC. Women with a history of PBDA may benefit from additional presurgical clinical workup. Cancer 2018;124:1350-7. © 2017 American Cancer Society.

Triple-Negative Breast Cancer: Next-Generation Sequencing for Target Identification.

Presently, the ability to study disease at the most fundamental molecular level has led to a reclassification of human cancers into numerous subtypes that vary in disease progression and response to therapy. Similar to most solid tumors, breast cancer is a heterogeneous disease with considerable variation in histologic and biological features. Triple-negative breast cancer (TNBC) is a subtype of breast cancer in which the estrogen receptor and progesterone receptor are not expressed, and human epidermal growth factor-receptor 2 is not amplified or overexpressed. Data derived from highly complex molecular technologies, such as microarrays and next-generation sequencing, have identified gene expression and somatic mutation profile subsets of TNBC that reflect biological behavior more accurately and may lead to further effective therapeutic targets, better prognosis, and improved outcomes. Herein, we review the genomic findings of TNBC and discuss current efforts in precision medicine as they relate to TNBC.

A Patient-Specific 3D-Printed Form Accurately Transfers Supine MRI-Derived Tumor Localization Information to Guide Breast-Conserving Surgery.

BACKGROUND: Wire-localized excision of nonpalpable breast cancer is imprecise, resulting in positive margins 25-30% of the time. METHODS: Patients underwent preoperative supine magnetic resonance imaging (MRI). A radiologist outlined the tumor edges on consecutive images, creating a three-dimensional (3D) view of its location. Using 3D printing, a bra-like plastic form (the Breast Cancer Locator [BCL]) was fabricated, with features that allowed a surgeon to (1) mark the edges of the tumor on the breast surface; (2) inject blue dye into the breast 1 cm from the tumor edges; and (3) place a wire in the tumor at the time of surgery. RESULTS: Nineteen patients with palpable cancers underwent partial mastectomy after placement of surgical cues using patient-specific BCLs. The cues were in place in <5 min and no adverse events occurred. The BCL accurately localized 18/19 cancers. In the 18 accurately localized cases, all 68 blue-dye injections were outside of the tumor edges. Median distance from the blue-dye center to the pathologic tumor edge was 1.4 cm, while distance from the blue dye to the tumor edge was <5 mm in 4% of injections, 0.5-2.0 cm in 72% of injections, and >2 cm in 24% of injections. Median distance from the tumor center to the BCL-localized wire and to the clip placed at the time of diagnosis was similar (0.49 vs. 0.73 cm) on specimen mammograms. CONCLUSIONS: Information on breast cancer location and shape derived from a supine MRI can be transferred safely and accurately to patients in the operating room using a 3D-printed form.

Concordance of DNA methylation profiles between breast core biopsy and surgical excision specimens containing ductal carcinoma in situ (DCIS).

The utility and reliability of assessing molecular biomarkers for translational applications on pre-operative core biopsy specimens assume consistency of molecular profiles with larger surgical specimens. Whether DNA methylation in ductal carcinoma in situ (DCIS), measured in core biopsy and surgical specimens are similar, remains unclear. Here, we compared genome-scale DNA methylation measured in matched core biopsy and surgical specimens from DCIS, including specific DNA methylation biomarkers of subsequent invasive cancer. DNA was extracted from guided 2mm cores of formalin fixed paraffin embedded (FFPE) specimens, bisulfite-modified, and measured on the Illumina HumanMethylation450 BeadChip. DNA methylation profiles of core biopsies exhibited high concordance with matched surgical specimens. Within-subject variability in DNA methylation was significantly lower than between-subject variability (all P<2.20E-16). In 641 CpGs whose methylation was related with increased hazard of invasive breast cancer, lower within-subject than between-subject variability was observed in 92.3% of the study participants (P<0.05). Between patient-matched core biopsy and surgical specimens, <0.6% of CpGs measured had changes in median DNA methylation >15%, and a pathway analysis of these CpGs indicated enrichment for genes related with wound healing. Our results indicate that DNA methylation measured in core biopsies are representative of the matched surgical specimens and suggest that DCIS biomarkers measured in core biopsies can inform clinical decision-making.

Targeted next-generation sequencing detects a high frequency of potentially actionable mutations in metastatic breast cancers.

BACKGROUND: Metastatic breast cancer is a genetically heterogeneous disease and effective therapies for advanced stage disease are limited. METHODS: In this study, distant metastases of 22 formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were sequenced using the Ion Torrent PGM and the 50 gene AmpliSeq Cancer Hotspot Panel v2 from 10ng of extracted DNA using 318 chips. Data analysis was performed with the Ion Torrent Variant Caller Plugin (hg19) and Golden Helix's SVS software for annotation and prediction of the significance of the variants. RESULTS: All patients were female with a median age of 61years (range 37-85years). Metastatic sites included liver (n=6, 27%), skin (n=5, 23%), brain (n=4, 18%), lymph node (n=3, 14%), lung (n=2, 9%), retroperitoneum (n=1, 4.5%), and colon (n=1, 4.5%). Overall, 28 variants in 11 genes were observed. Five (23%) samples showed no alterations and 17 (77%) showed at least one potentially biologically significant variant (BSV) defined as having FDA-approved drugs or clinical trials evaluating their significance. BSVs included mutations in the following genes: TP53 (n=8), APC (n=4), PIK3CA (n=5), MET (n=2), ERBB2 (n=2), AKT1 (n=1), CDKN2A (n=1), KRAS (n=1), and FGFR3 (n=1). CONCLUSIONS: Potentially actionable mutations were identified in the majority of breast cancer metastases. Evaluating metastatic breast tumors using a NGS approach provides a better understanding of the mechanisms behind tumor progression and evolution and also identifies additional potentially beneficial therapeutic targets for patient management or eligibility for clinical trials.

Proliferative lesion of anogenital mammary-like glands in the setting of Cowden syndrome: case report and review of the literature.

Mammary-like glands are normal appendages of anogenital skin and can give rise to epithelial and stromal tumors that closely resemble breast tumors. Cowden syndrome is an autosomal-dominant cancer-predisposition syndrome that is associated with increased risk of various benign and malignant tumors including breast cancers. Here, we report the first case of a proliferative lesion of mammary-like glands in the setting of Cowden syndrome. A 27-year-old female with Cowden syndrome (R130Q-PTEN mutation) presented with a 1-cm tender, polypoid perianal lesion. An excisional biopsy revealed a circumscribed, lobulated lesion with fibromyxoid stroma and epithelial hyperplasia with apocrine and columnar cell changes that was arranged in papillary, micropapillary and focal cribriform architecture. The features strikingly resembled proliferative changes commonly seen in the breast. Interestingly, the patient subsequently developed an atypical complex sclerosing lesion of the breast. Given the increased risk of breast neoplasia in Cowden syndrome, and the morphologic relationship between breast glands and mammary-like glands, this case raises the possibility of an increased risk of neoplasia arising in mammary-like glands in the setting of Cowden syndrome.

Molecular Phenotype of Breast Cancer According to Time Since Last Pregnancy in a Large Cohort of Young Women.

BACKGROUND: The increase in breast cancer risk during pregnancy and postpartum is well known; however, the molecular phenotype of breast cancers occurring shortly after pregnancy has not been well studied. Given this, we investigated whether nulliparity and the time interval since pregnancy among parous women affects the breast cancer phenotype in young women. MATERIALS AND METHODS: We examined molecular phenotype in relation to time since pregnancy in a prospective cohort of 707 young women (aged ≤40 years) with breast cancer. Parity was ascertained from study questionnaires. Using tumor histologic grade on central review and biomarker expression, cancers were categorized as luminal A- or B-like, HER2 enriched, and triple negative. RESULTS: Overall, 32% were luminal A-like, 41% were luminal B-like, 9% were HER2 enriched, and 18% were triple negative. Although, numerically, patients diagnosed >5 years after pregnancy had more luminal A-like subtypes than women with shorter intervals since pregnancy, there was no evidence of a relationship between these intervals and molecular subtypes once family history of breast cancer and age at diagnosis were considered. CONCLUSION: Distribution of breast cancer molecular phenotype did not differ significantly among young women by parity or time interval since parturition when important predictors of tumor phenotype such as age and family history were considered. IMPLICATIONS FOR PRACTICE: Distribution of breast cancer molecular phenotype did not differ among parous young women by time interval since pregnancy. The implication of these findings for clinical practice suggests that pregnancy-associated breast cancers may be seen up to 5 years beyond parturition.

Implementation of a Molecular Tumor Board: The Impact on Treatment Decisions for 35 Patients Evaluated at Dartmouth-Hitchcock Medical Center.

BACKGROUND: Although genetic profiling of tumors is a potentially powerful tool to predict drug sensitivity and resistance, its routine use has been limited because clinicians are often unfamiliar with interpretation and incorporation of the information into practice. We established a Molecular Tumor Board (MTB) to interpret individual patients' tumor genetic profiles and provide treatment recommendations. PATIENTS AND METHODS: DNA from tumor specimens was sequenced in a Clinical Laboratory Improvement Amendments-certified laboratory to identify coding mutations in a 50-gene panel (n = 34) or a 255-gene panel (n = 1). Cases were evaluated by a multidisciplinary MTB that included pathologists, oncologists, hematologists, basic scientists, and genetic counselors. RESULTS: During the first year, 35 cases were evaluated by the MTB, with 32 presented for recommendations on targeted therapies, and 3 referred for potential germline mutations. In 56.3% of cases, MTB recommended treatment with a targeted agent based on evaluation of tumor genetic profile and treatment history. Four patients (12.5%) were subsequently treated with a MTB-recommended targeted therapy; 3 of the 4 patients remain on therapy, 2 of whom experienced clinical benefit lasting >10 months. CONCLUSION: For the majority of cases evaluated, the MTB was able to provide treatment recommendations based on targetable genetic alterations. The most common reasons that MTB-recommended therapy was not administered stemmed from patient preferences and genetic profiling at either very early or very late stages of disease; lack of drug access was rarely encountered. Increasing awareness of molecular profiling and targeted therapies by both clinicians and patients will improve acceptance and adherence to treatments that could significantly improve outcomes. IMPLICATIONS FOR PRACTICE: Case evaluation by a multidisciplinary Molecular Tumor Board (MTB) is critical to benefit from individualized genetic data and maximize clinical impact. MTB recommendations shaped treatment options for the majority of cases evaluated. In the few patients treated with MTB-recommended therapy, disease outcomes were positive and support genetically informed treatment.

The PI3K/mTOR dual inhibitor P7170 demonstrates potent activity against endocrine-sensitive and endocrine-resistant ER+ breast cancer.

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway has been implicated in anti-estrogen resistance in breast cancer. We tested the therapeutic potential of the novel PI3K/mTOR dual inhibitor P7170 in a panel of anti-estrogen-sensitive and anti-estrogen-resistant models of ER+ breast cancer. Estrogen receptor-positive (ER+) breast cancer cells were treated ±P7170. Fresh cores from primary ER+/HER2- tumors from two patients were treated ±P7170 ex vivo. Mice bearing breast cancer xenografts were randomized to treatment with vehicle, fulvestrant, P7170, or combinations, and tumor volumes were measured. Tissues and cells were analyzed for markers of pathway activity, cell viability, and apoptosis. In cell lines, P7170 exhibited IC50 values in the range of 0.9-7 nM and induced apoptosis. P7170 potently inhibited mTOR activity (≤ 25 nM) and inhibited PI3K at higher concentrations (≥ 200 nM). P7170 completely inhibited MCF-7 tumor growth, significantly inhibited growth of fulvestrant-resistant T47D tumors, and suppressed tumor cell proliferation but did not induce apoptosis. While P7170 inhibits PI3K and mTOR in ER+/HER2- human breast cancer cells and tumors ex vivo, in vivo data indicate that the primary mechanism of P7170 anti-tumor action is inhibition of mTOR and cell proliferation. P7170 is a novel agent worthy of further investigation for the treatment of ER+ breast cancer.

Breast MRI-detected cystic apocrine metaplasia: imaging features with microvessel analysis and histologic correlation.

OBJECTIVE: The purpose of this article is to characterize the histologic vascular features and distinguishing MRI features of cystic apocrine metaplasia to better understand imaging-pathology concordance. MATERIALS AND METHODS: Retrospective review of 261 consecutive MRI-guided biopsy cases was performed. Pathology results were reviewed for all biopsies; cystic apocrine metaplasia was identified as the predominant finding in 19 cases (7%). CD31 immunohistochemistry was subsequently performed on the most representative block of cystic apocrine metaplasia, and microvasculature was evaluated using computer-assisted image analysis. The contrast-enhanced MRI examinations correlating with the cystic apocrine metaplasia cases were independently reviewed by two radiologists specializing in breast imaging; lesions were analyzed for morphologic, kinetic, and T2 characteristics. RESULTS: On MRI review, 17 of 19 (89%) lesions were 10 mm or smaller. Washout kinetics were present in 11 of 19 (58%) lesions, and 14 of 19 (74%) lesions were at least partially hyperintense on T2-weighted sequences relative to adjacent glandular tissue. Cystic apocrine metaplasia had a higher percentage area (mean, 4.1%) of CD31-immunostained microvessels compared with background fibroglandular tissue (mean, 1.2%). CONCLUSION: Cystic apocrine metaplasia should be considered in the differential diagnosis of a T2-hyperintense enhancing focus or subcentimeter smoothly marginated mass, even if associated with washout kinetics. Cystic apocrine metaplasia contains a statistically significant increase in microvessel area compared with background fibroglandular tissue and fat and, therefore, may be considered a concordant result for this set of imaging findings.

Automated processing of fluorescence in-situ hybridization slides for HER2 testing in breast and gastro-esophageal carcinomas.

BACKGROUND: HER2 fluorescence in-situ hybridization (FISH) is used in breast and gastro-esophageal carcinoma for determining HER2 gene amplification and patients' eligibility for HER2 targeted therapeutics. Traditional manual processing of the FISH slides is labor intensive because of multiple steps that require hands on manipulation of the slides and specifically timed intervals between steps. This highly manual processing also introduces inter-run and inter-operator variability that may affect the quality of the FISH result. Therefore, we sought to incorporate an automated processing instrument into our FISH workflow. METHODS: Twenty-six cases including breast (20) and gastro-esophageal (6) cancer comprising 23 biopsies and three excision specimens were tested for HER2 FISH (Pathvysion, Abbott) using the Thermobrite Elite (TBE) system (Leica). Up to 12 slides can be run simultaneously. All cases were previously tested by the Pathvysion HER2 FISH assay with manual preparation. Twenty cells were counted by two observers for each case; five cases were tested on three separate runs by different operators to evaluate the precision and inter-operator variability. RESULTS: There was 100% concordance in the scoring between the manual and TBE methods as well as among the five cases that were tested on three runs. Only one case failed due to poor probe hybridization. In total, seven cases were positive for HER2 amplification (HER2:CEP17 ratio >2.2) and the remaining 19 were negative (HER2:CEP17 ratio <1.8) utilizing the 2007 ASCO/CAP scoring criteria. Due to the automated denaturation and hybridization, for each run, there was a reduction in labor of 3.5h which could then be dedicated to other lab functions. CONCLUSION: The TBE is a walk away pre- and post-hybridization system that automates FISH slide processing, improves work flow and consistency and saves approximately 3.5h of technologist time. The instrument has a small footprint thus occupying minimal counter space. TBE processed slides performed exceptionally well in comparison to the manual technique with no disagreement in HER2 amplification status.