Resection of Cavity Shave Margins in Stage 0-III Breast Cancer Patients Undergoing Breast Conserving Surgery: A Prospective Multicenter Randomized Controlled Trial.

OBJECTIVE: Single-center studies have demonstrated that resection of cavity shave margins (CSM) halves the rate of positive margins and re-excision in breast cancer patients undergoing partial mastectomy (PM). We sought to determine if these findings were externally generalizable across practice settings. METHODS: In this multicenter randomized controlled trial occurring in 9 centers across the United States, stage 0-III breast cancer patients undergoing PM were randomly assigned to either have resection of CSM ("shave" group) or not ("no shave" group). Randomization occurred intraoperatively, after the surgeon had completed their standard PM. Primary outcome measures were positive margin and re-excision rates. RESULTS: Between July 28, 2016 and April 13, 2018, 400 patients were enrolled in this trial. Four patients (2 in each arm) did not meet inclusion criteria after randomization, leaving 396 patients for analysis: 196 in the "shave" group and 200 to the "no shave" group. Median patient age was 65 years (range; 29-94). Groups were well matched at baseline for demographic and clinicopathologic factors. Prior to randomization, positive margin rates were similar in the "shave" and "no shave" groups (76/196 (38.8%) vs. 72/200 (36.0%), respectively, P = 0.604). After randomization, those in the "shave" group were significantly less likely than those in the "no shave" group to have positive margins (19/196 (9.7%) vs. 72/200 (36.0%), P < 0.001), and to require re-excision or mastectomy for margin clearance (17/196 (8.7%) vs. 47/200 (23.5%), P < 0.001). CONCLUSION: Resection of CSM significantly reduces positive margin and re-excision rates in patients undergoing PM.

Venous thromboembolic events in the rehabilitation setting.

Venous thromboembolism (VTE) is a disease entity that encompasses both deep venous thrombosis and pulmonary embolism. During the past decade there have been significant advances in the understanding of prophylaxis and treatment of VTE. There is an extensive research base from which conclusions can be drawn, but the heterogeneity within the rehabilitation patient population makes the development of rigid VTE protocols challenging and overwhelming for the busy clinician. Given the prevalence of this condition and its associated morbidity and mortality, we review the evidence for the prevention, identification, and optimal treatment of VTE in the rehabilitation population. Our goal is to highlight studies that have the most clinical applicability for the care of VTE patients from a physiatrist's perspective. At times, information about acute care protocols is included in our discussion because these situations are encountered during the consultation process that identifies patients for rehabilitation needs.

The expression of TRMT2A, a novel cell cycle regulated protein, identifies a subset of breast cancer patients with HER2 over-expression that are at an increased risk of recurrence.

BACKGROUND: Over-expression of HER2 in a subset of breast cancers (HER2+) is associated with high histological grade and aggressive clinical course. Despite these distinctive features, the differences in response of HER2+ patients to both adjuvant cytotoxic chemotherapy and targeted therapy (e.g. trastuzumab) suggests that unrecognized biologic and clinical diversity is confounding treatment strategies. Furthermore, the small but established risk of cardiac morbidity with trastuzumab therapy compels efforts towards the identification of biomarkers that might help stratify patients. METHODS: A single institution tissue array cohort assembled at the Clearview Cancer Institute of Huntsville (CCIH) was screened by immunohistochemistry staining using a large number of novel and commercially available antibodies to identify those with a univariate association with clinical outcome in HER2+ patients. Staining with antibody directed at TRMT2A was found to be strongly associated with outcome in HER2+ patients. This association with outcome was tested in two independent validation cohorts; an existing staining dataset derived from tissue assembled at the Cleveland Clinic Foundation (CCF), and in a new retrospective study performed by staining archived paraffin blocks available at the Roswell Park Cancer Institute (RPCI). RESULTS: TRMT2A staining showed a strong correlation with likelihood of recurrence at five years in 67 HER2+ patients from the CCIH discovery cohort (HR 7.0; 95% CI 2.4 to 20.1, p < 0.0004). This association with outcome was confirmed using 75 HER2+ patients from the CCF cohort (HR 3.6; 95% CI 1.3 to 10.2, p < 0.02) and 64 patients from the RPCI cohort (HR 3.4; 95% CI 1.3-8.9, p < 0.02). In bivariable analysis the association with outcome was independent of grade, tumor size, nodal status and the administration of conventional adjuvant chemotherapy in the CCIH and RPCI cohorts. CONCLUSIONS: Studies from three independent single institution cohorts support TRMT2A protein expression as a biomarker of increased risk of recurrence in HER2+ breast cancer patients. These results suggest that TRMT2A expression should be further studied in the clinical trial setting to explore its predictive power for response to adjuvant cytotoxic chemotherapy in combination with HER2 targeted therapy.

Primary cardiac sarcomas: a clinicopathologic analysis of a series with follow-up information in 17 patients and emphasis on long-term survival.

Although cardiac sarcomas are rare in comparison to their soft tissue counterparts, they are the second most common type of primary cardiac neoplasm. Of the few hundred cases reported, most has been based on autopsy series. A series of 27 cardiac sarcomas removed at surgery for curative and diagnostic intent were reviewed for clinicopathologic features with correlation to available postoperative follow-up data in 17 patients. There were 6 angiosarcomas, 6 myxofibrosarcomas, 3 malignant peripheral nerve sheath tumors, 3 leiomyosarcomas, 2 synovial sarcomas, 1 epithelioid hemangioendothelioma, 1 chondrosarcoma, 1 osteosarcoma, and 4 poorly differentiated sarcomas. There was a wide age and size range with slight female predilection. There were 20 cases that arose in the atria/pulmonary vessels, 4 in the ventricles, 1 in mitral valve, and 2 in epi/pericardium. There was a slight left predilection. The histologic grade was low in 4, moderate in 3, and high in 20 cases. Six high-grade and 1 low-grade tumors were also treated with adjuvant chemotherapy and/or radiation. In 17 patients with follow-up data, 6 of 12 patients with high-grade tumor died (4 within 5 days of the initial surgery, 1 in 21 months, and 1 in 131 months), and 1 patient with moderate-grade tumor and all 4 patients with low-grade tumor were alive without evidence of disease at the end of follow-up. Tumor grade appeared to be prognostically important in cardiac sarcoma. Long survival was achieved in patients who survived the initial surgery well.

Stage IA vulvar squamous cell carcinoma: an analysis of tumor invasive characteristics and risk.

Early invasive vulvar squamous cell carcinoma (SCC) with less than 1.0 mm of invasion (FIGO stage IA) has been shown to have a minimal risk of lymph node metastasis and is associated with an excellent prognosis. The prognostic significance of other histologic parameters other than depth of invasion, however, remains controversial. Seventy-eight consecutive cases of vulvar SCC having a depth of invasion of 5.0 mm or less were reviewed and the clinical outcome compared with the type of surgical excision, the presence of concurrent lymph node metastases, the depth of tumor invasion, the tumor thickness, the tumor horizontal spread, the estimated tumor volume, tumor histologic subtype, tumor histologic grade, tumor pattern of invasion, tumor multifocality, presence of perineural invasion, presence of angiolymphatic invasion and the presence of precursor lesions, including the type of vulvar intraepithelial neoplasia and presence of lichen sclerosus. The only histologic feature for predicting concurrent lymph node metastasis was tumor depth of invasion. The 3 most important features of stage IA tumors in predicting tumor recurrence were the depth of invasion, presence of SCC at the surgical margins, and the histologic grade.

Epidermal growth factor receptor is not amplified in schwannomas.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor of the ErbB family. This family of receptors plays an active role in cellular growth and mitogenesis. It is well established that the overexpression of ErbB receptors in human cancers, most commonly because of true genomic amplification, correlates with a more aggressive clinical course. There is limited data published on the expression and amplification of EGFR in schwannomas. Both neurofibromas and schwannomas are capable of progression to malignant peripheral nerve sheath tumors (MPNSTs). A subset of human MPNSTs, both NF-1-related and sporadic, overexpress EGFR via true genomic amplification of the short arm of chromosome 7 (7p12). The goal of this study is to assess whether EGFR is expressed and/or amplified in human schwannomas. Twenty schwannomas in 12 women and 8 men (mean age, 51 years) were analyzed for EGFR expression via immunohistochemistry and fluorescence in situ hybridization. None of the 20 cases were positive for EGFR expression via immunohistochemistry; 3 tumors showed focal nonspecific Golgi staining. None of the cases demonstrated true genomic amplification of the EGFR region via fluorescence in situ hybridization. The mitogenic signaling for schwannomas is unlikely to be related to overexpression or amplification of EGFR; however, acquiring this signaling pathway might contribute to the progression of a subset of benign peripheral nerve sheath tumors to MPNST.

Molecular and morphologic distinctions between infiltrating ductal and lobular carcinoma of the breast.

Histopathologic distinction between ductal and lobular carcinomas of the breast has been made since 1941. Together, these two subtypes account for >95% of all mammary carcinomas. With the recent advances in molecular techniques, our understanding of the biology behind these carcinomas has greatly expanded. The genomic aberrations in mammary carcinoma are highly complex and appear to be more associated with tumor grade rather than any histopathologic subtype. Protein and RNA expression profiling reveals a classification of mammary carcinoma that has some overlap with traditional histopathology and can at least partially explain clinical behavior. The goal of this review is to present what is currently known about the molecular profiles of infiltrating ductal and lobular carcinoma and how they relate to conventional histopathology and biologic behavior.

The expression of the cytoskeletal focal adhesion protein paxillin in breast cancer correlates with HER2 overexpression and may help predict response to chemotherapy: a retrospective immunohistochemical study.

Paxillin, a cytoskeletal focal adhesion adaptor protein, has been shown to be transcriptionally up-regulated and phosphorylated by human epidermal growth factor receptor-2 (HER2) signaling in vitro. Paxillin expression may also correlate with HER2 amplification in breast cancer patients. In the current study, we sought to explore the relationship further between paxillin expression and clinicopathologic features and clinical outcome in breast cancer. A total of 314 primary invasive breast carcinomas were assessed for paxillin expression via immunohistochemistry. Paxillin immunoreactivity was compared with estrogen receptor/progesterone receptor status, HER2 status by silver in situ hybridization, age, tumor size, stage, Bloom-Richardson grade, nodal status, disease-free survival (DFS), and overall survival (OS). Paxillin expression was identified in 27.7% of breast carcinomas as diffuse cytoplasmic staining and the expression correlated with HER2 overexpression (p < 0.001). The influence of paxillin on clinical outcome, in particular the response to chemotherapy, appeared to differ depending on the HER2 status of the tumor. For the subset of HER2 nonamplified cases treated with chemotherapy, patients whose tumor showed a loss of paxillin expression demonstrated a significantly lengthened DFS and OS. In contrast, loss of paxillin expression in the HER2 amplified subset of patients who received chemotherapy correlated with a significantly worse outcome. These data suggest that paxillin up-regulation may be a part of the HER2 pathway in some breast cancers and, furthermore, paxillin expression may also influence the clinical response to chemotherapy, depending upon the HER2 status of a given patient's tumor. Further study of a role for paxillin expression in predicting response to cytotoxic regimens or targeted treatments is warranted.

Incidence of simultaneous epidural and vascular injection during lumbosacral transforaminal epidural injections.

BACKGROUND CONTEXT: The incidence of vascular penetration during contrast confirmed fluoroscopically guided transforaminal lumbosacral epidural injections has been reported as 8.9% to 21.3% depending on the level of injection. Recently, intermittent fluoroscopy was shown to miss more than half of the vascular injections observed under live fluoroscopy. The number of misses increased when epidural and vascular contrast flow appeared simultaneously, even if the fluoroscopic image was taken during contrast injection. To date, no studies have documented the incidence of simultaneous epidural and vascular contrast injections. Also, most previous studies of vascular injections did not document use of live fluoroscopy during contrast injection, so the incidence of vascular injections may be higher than reported. PURPOSE: To determine the incidence of simultaneous epidural and vascular contrast injection during lumbosacral transforaminal epidural injections. STUDY DESIGN/SETTING: A prospective, observational, in vivo, study. PATIENT SAMPLE: Patients receiving lumbar transforaminal epidural injections at a university-based outpatient spine center. OUTCOME MEASURES: Incidence of epidural, vascular, and simultaneous epidural and vascular contrast patterns. METHODS: One interventional spine physician and three fellows under his direct supervision recorded contrast patterns observed during 191 fluoroscopically guided lumbosacral transforaminal epidural steroid injections with injection of contrast observed under live fluoroscopy. RESULTS: The incidence of simultaneous epidural and vascular injection during lumbosacral transforaminal epidural injections was 8.9%. The incidence of a vascular injection alone was 4.2%, for a total vascular injection incidence of 13.1%. Subjects with a simultaneous epidural and vascular injection who returned for repeat injection had a statistically significant chance of a repeat simultaneous epidural and vascular injection when the injection was done at the same location. Fluoroscopy time was significantly increased, an average of 8.8 seconds, when a vascular injection was identified. There was no statistically significant correlation between the incidence of simultaneous epidural and vascular injection with subjects' age, sex, level of injection, side of injection, needle gauge, or diagnosis. CONCLUSIONS: Simultaneous epidural and vascular injection is twice as likely to occur as vascular injection alone. Use of intermittent fluoroscopy can miss the transient appearance of the vascular component of these injections, giving the false impression of successful contrast placement. In light of these results, live fluoroscopy is recommended during contrast injection for confirmation of lumbosacral transforaminal epidural injections.

Clinicopathologic findings in mesial temporal sclerosis treated with gamma knife radiotherapy.

Mesial temporal sclerosis (MTS) is the most common cause of medically intractable temporal lobe epilepsy. Histologic findings include hippocampal atrophy with neuronal loss in the dentate, CA1, and CA3/CA4 regions with gliosis. The conventional treatment of patients with intractable epilepsy secondary to MTS has been surgical excision. Gamma knife radiotherapy (GKR) has recently been suggested as a less invasive alternative to surgery. To date, the histologic changes that occur in this setting after GKR have not been well described. The clinicopathologic features of 4 patients with MTS who received GKR and underwent subsequent surgical resection or autopsy were retrospectively reviewed. The study group is composed of 4 patients (3 women, 1 men) with ages 55, 48, 22, and 20 years, respectively, at the time of GKR. There were 2 patients who had a history of infantile febrile seizures, and 2 who had a central nervous infection during infancy. All 4 patients had a long-standing (13-36 years) history of temporal lobe seizures resistant to medical management. Imaging studies, electroencephalogram, and surgical specimens all confirmed the diagnosis of MTS. The oldest of the 4 patients died 1 month after receiving GKR, presumably because of post-gamma knife persistent seizure complications. The postmortem neuropathology on this patient was unremarkable for any radiation effect changes but showed evidence of MTS. The remaining 3 patients underwent surgical resection for persistent seizures at 18, 22, and 20 months, respectively, post-gamma knife. These 3 surgical specimens showed variable degrees of radiation effect changes in the temporal lobe, hippocampus, and amygdala, including chronic (lymphocytes and macrophages) perivascular inflammation (3/3), vascular sclerosis (3/3), foci of edema with necrosis (3/3; extensive in 2 patients), reactive astrocytosis (3/3), microglial proliferation (1/3), and microcalcifications (1/3). Patients with MTS who underwent GKR can develop typical radiation changes over time. Treatment of individuals with MTS via GKR may not always be adequate in controlling seizures. Radiation therapy effect may contribute to persistent seizures after GKR in some patients with MTS.

Reflex UroVysion testing of bladder cancer surveillance patients with equivocal or negative urine cytology: a prospective study with focus on the natural history of anticipatory positive findings.

A proportion of patients under surveillance for recurrent bladder carcinoma with no immediate evidence of bladder tumor recurrence have positive multitarget fluorescence in situ hybridization (FISH; UroVysion, Vysis, Downers Grove, IL) results. The course of these "anticipatory positive" cases and the time to bladder tumor recurrence remains unknown. We followed up 250 patients with urine cytologic results, concurrent multitarget FISH, and cystoscopic examination for recurrent urothelial carcinoma. Of 81 cases (32.4%) with FISH-positive results, tumor recurrence developed in 60 (74.0%). Of 169 (67.6%) FISH-negative cases, recurrent urothelial carcinoma developed in 22 (13.0%). Of 211 patients (84.4%) with negative cystoscopic examination results, 56 (26.5%) had positive FISH results, and in 35 (62.5%) of these patients, recurrent urothelial carcinoma developed. Approximately 27% of patients under bladder carcinoma surveillance without immediate evidence of tumor recurrence will have a positive FISH result, defining the anticipatory positive subset. In about 65% of this anticipatory positive group, recurrent bladder urothelial carcinoma developed within 29 months.

Loss of breast cancer metastasis suppressor 1 protein expression predicts reduced disease-free survival in subsets of breast cancer patients.

PURPOSE: This study aims to determine the effect of loss of breast cancer metastasis suppressor 1 (BRMS1) protein expression on disease-free survival in breast cancer patients stratified by estrogen receptor (ER), progesterone receptor (PR), or HER2 status, and to determine whether loss of BRMS1 protein expression correlated with genomic copy number changes. EXPERIMENTAL DESIGN: A tissue microarray immunohistochemical analysis was done on tumors of 238 newly diagnosed breast cancer patients who underwent surgery at the Cleveland Clinic between January 1, 1995 and December 31, 1996, and a comparison was made with 5-year clinical follow-up data. Genomic copy number changes were determined by array-based comparative genomic hybridization in 47 breast cancer cases from this population and compared with BRMS1 staining. RESULTS: BRMS1 protein expression was lost in nearly 25% of cases. Patients with tumors that were PR negative (P=0.006) or HER2 positive (P=0.039) and <50 years old at diagnosis (P=0.02) were more likely to be BRMS1 negative. No overall correlation between BRMS1 staining and disease-free survival was observed. A significant correlation, however, was seen between loss of BRMS1 protein expression and reduced disease-free survival when stratified by either loss of ER (P=0.008) or PR (P=0.029) or HER2 overexpression (P=0.026). Overall, there was poor correlation between BRMS1 protein staining and copy number status. CONCLUSIONS: These data suggest a mechanistic relationship between BRMS1 expression, hormone receptor status, and HER2 growth factor. BRMS1 staining could potentially be used in patient stratification in conjunction with other prognostic markers. Further, mechanisms other than genomic deletion account for loss of BRMS1 gene expression in breast tumors.

Perceptions of diagnostic terminology and cytopathologic reporting of fine-needle aspiration biopsies of thyroid nodules: a survey of clinicians and pathologists.

INTRODUCTION: Guidelines on thyroid fine-needle aspiration biopsy (FNABs) reporting calls for unambiguous diagnostic terminology in order to maximize treatment. This study evaluates how pathologists follow the guidelines and clinicians understand the diagnostic categories in terms of patient care. DESIGN: Survey 1 asked pathologists who perform/interpret FNABs which of "atypical," "indeterminate," "suspicious," and "nondiagnostic" they routinely use. Survey 2 asked clinicians who treat thyroid nodules to correlate these categories to the options of "negative FNAB/follow-up," "repeat FNAB," and "proceed to surgery." The anonymous, voluntary results were entered into a database and analyzed. MAIN OUTCOME: Pathologists' and clinicians' response rates were 70% and 35%, respectively. Survey 1: 27% of pathologists used three, 27% used one, and 44% used two categories. Survey 2: 98% clinicians would repeat the FNAB with a "nondiagnostic" and 96% opted for surgery with a "suspicious" diagnosis. "Indeterminate" prompted 58% to repeat the FNAB and 32% to send the patient to surgery. "Atypical" would lead 37% to repeat the FNAB and 52% to send the patient to surgery. CONCLUSIONS: Pathologists actively use the terminology "suspicious," "indeterminate," or "atypical," which cause confusion in some clinicians. These results support the need for a more standardized terminology for FNAB reporting and education of the clinicians on that terminology.

Validation of a five-tier cytodiagnostic system for thyroid fine needle aspiration biopsies using cytohistologic correlation.

OBJECTIVE: Approximately 70-80% of thyroid fine needle aspiration biopsies (FNAB) can distinguish benign from malignant thyroid nodules. However, much interpretive diagnostic difficulty arises with the remaining 20-30% of cases. These problematic thyroid aspirations have been placed in various diagnostic categories, which collectively have led to confusion and a negative impact on the clinical management of patients with thyroid nodules. We present our experience using a five-tier system, including the diagnostic terminology: benign, indeterminate, suspicious, malignant, and unsatisfactory. DESIGN: Thyroid FNABs were diagnosed using a five-tier system from 200 consecutive patients and the subsequent surgical excisions were correlated. MAIN OUTCOME: Overall, there was an excellent association between the five diagnostic categories and predicting benign versus neoplastic thyroid nodules (LR = 96.06, X(2) = 76.49, and phi = 0.618, df = 4, p < 0.0001). A negative cytologic diagnosis carries a negative predictive value of 92%, while an indeterminate, suspicious, and malignant cytologic diagnosis carries a positive predictive value of 50, 71, and 100% respectively. The estimated sensitivity for an indeterminate, suspicious, or malignant cytologic diagnosis varied from 70 to 89%, while the specificity increased from 57 to 92 to 100%, respectively. CONCLUSIONS: The data presented shows that the five diagnostic categories of thyroid FNAB are excellent at distinguishing benign from neoplastic thyroid nodules. Both the indeterminate and suspicious categories, while not statistically different from each other in predicting benign from neoplasia, are statistically different from obviously benign and obviously malignant categories. These results support the need for an indeterminate and/or suspicious category.

Breast cancers with brain metastases are more likely to be estrogen receptor negative, express the basal cytokeratin CK5/6, and overexpress HER2 or EGFR.

Brain metastases (BM) from breast cancer are associated with significant morbidity and mortality. In the current study, we have examined a cohort of breast cancer patients who went on to develop BM for clinical-pathologic features and predictive markers that identify this high-risk subgroup of patients at the time of diagnosis. The primary tumors from 55 patients who developed BM were used to construct a tissue microarray. The clinical and pathologic features were recorded and the tissue microarray was stained for estrogen receptor, human epidermal growth factor receptor 2, cytokeratin 5/6, and epidermal growth factor receptor by immunohistochemistry. This cohort of patients was compared against a group of 254 patients who remain free of metastases (67 mo mean follow-up), and another cohort of 40 patients who developed mixed visceral and bone metastatic disease without brain recurrence over a similar period of time. Breast cancer patients who went on to develop BM were more likely to be <50 years old (P<0.001), and the primary tumors were more likely to be estrogen receptor negative (P<0.001) and high grade (P=0.002). The primary tumors were also more likely to express cytokeratin 5/6 (P<0.001) and epidermal growth factor receptor (P=0.001), and to overexpress human epidermal growth factor receptor 2 (P=0.001). The data presented above suggest a profile for breast cancer patients at increased risk for developing BM. Predictive factors to help identify patients with metastatic breast cancer who are at an increased risk for developing central nervous system recurrence might allow for screening of this population for early detection and treatment or for the development of targeted strategies for prevention.

Novel prognostic immunohistochemical biomarker panel for estrogen receptor-positive breast cancer.

PURPOSE: Patients with breast cancer experience progression and respond to treatment in diverse ways, but prognostic and predictive tools for the oncologist are limited. We have used gene expression data to guide the production of hundreds of novel antibody reagents to discover novel diagnostic tools for stratifying carcinoma patients. PATIENTS AND METHODS: One hundred forty novel and 23 commercial antisera, selected on their ability to differentially stain tumor samples, were used to stain paraffin blocks from a retrospective breast cancer cohort. Cox proportional hazards and regression tree analysis identified minimal panels of reagents able to predict risk of recurrence. We tested the prognostic association of these prospectively defined algorithms in two independent cohorts. RESULTS: In both validation cohorts, the Kaplan-Meier estimates of recurrence confirmed that both the Cox model using five reagents (p53, NDRG1, CEACAM5, SLC7A5, and HTF9C) and the regression tree model using six reagents (p53, PR, Ki67, NAT1, SLC7A5, and HTF9C) distinguished estrogen receptor (ER)-positive patients with poor outcomes. The Cox model was superior and distinguished patients with poor outcomes from patients with good or moderate outcomes with a hazard ratio of 2.21 (P = .0008) in validation cohort 1 and 1.88 (P = .004) in cohort 2. In multivariable analysis, the calculated risk of recurrence was independent of stage, grade, and lymph node status. A model proposed for ER-negative patients failed validation in the independent cohorts. CONCLUSION: A panel of five antibodies can significantly improve on traditional prognosticators in predicting outcome for ER-positive breast cancer patients.

Analytical validation and interobserver reproducibility of EnzMet GenePro: a second-generation bright-field metallography assay for concomitant detection of HER2 gene status and protein expression in invasive carcinoma of the breast.

Fluorescence in situ hybridization (FISH) has both excellent sensitivity and specificity in detecting HER2 gene amplification in invasive breast carcinoma. FISH has not been widely implemented in clinical practice because of reagent costs and the special instrumentation and expertise required to perform and integrate the assay. Immunohistochemistry (IHC) for HER2 protein is widely used, but false-positive and false-negative results are problematic. We developed a bright-field assay to visualize HER2 gene amplification and concomitant HER2 protein expression (EnzMet GenePro). This assay detects HER2 gene amplification via deposition of metallic silver by enzyme metallographytrade mark (EnzMettrade mark, Nanoprobes, Yaphank, NY) combined with HER2 protein detection by IHC using alkaline phosphatase and fast red K substrate visualization (CB11;Ventana, Tucson, AZ). The assay was performed on 94 invasive breast carcinomas, for which FISH (PathVysiontrade mark, Vysis, Downer's Grove, IL), conventional IHC (CB11), and enzyme metallography (EnzMettrade mark) results were known. The EnzMettrade mark component of the assay was scored as either HER2 gene amplified, polysomic, or nonamplified. The IHC component was scored using the conventional FDA scale of 0 to 3+. Concordance of the EnzMet component of the assay versus FISH was assessed and showed an excellent correlation (Pearson coefficient of 0.95; P < 0.001). The combination of gene and protein detection (EnzMet GenePro) displayed a specificity of 100% and an accuracy of 92.6% (95% confidence interval 85.3-97.0), facilitated recognition of gene/protein discordances, and allowed for efficient interpretation of the slide by conventional light microscopy. The interobserver kappa for each component was excellent (IHC, kappa = 0.94; and EnzMettrade mark, kappa = 0.96). EnzMet is the first bright-field ISH assay in our experience that routinely and nonambiguously detects endogenous HER2 signals, essential for a reliable clinical HER2 assay, and in combination with HER2 protein enables improved diagnosis in borderline cases.

The influence of polysomy 17 on HER2 gene and protein expression in adenocarcinoma of the breast: a fluorescent in situ hybridization, immunohistochemical, and isotopic mRNA in situ hybridization study.

Breast carcinomas with amplification of HER2 on chromosome 17 are associated with HER2 protein overexpression, adversely affecting prognosis and predicting response to Herceptin therapy. Chromosome 17 polysomy is encountered in assessing HER2 gene status, and its impact on HER2 gene and protein expression remains unclear. This impact was investigated in breast carcinomas identified by fluorescence in situ hybridization (FISH) to have a gain of chromosome 17 (CEP17+; n = 56), using a dual probe assay, which detects HER2 gene copy number and enumerates chromosome 17 (HER2/CEP17; Vysis). Cases were immunostained for HER2 protein (CB-11, Ventana), and scored blinded to FISH. A subgroup was evaluated by isotopic in situ hybridization for HER2 mRNA expression. Controls included ten HER2 amplified and ten nonamplified tumors, eusomic for chromosome 17. Immunohistochemistry (IHC) for HER2 protein was negative (0 or 1+) in 69% (39 of 56), 2+ in 27% (15 of 56), and 3+ in 3% (2 of 56) of CEP17+ cases. The mean CEP17 copy number among the three groups was similar (3.1, 3.0, and 3.1 for IHC 0/1+, 2+, and 3+, respectively). Isotopic in situ hybridization for HER2 mRNA performed on 26 CEP17+ cases (16 IHC 0-1+, 10 IHC 2+ or 3+) showed no increased HER2 mRNA expression (normalized to beta-actin mRNA). The mRNA expression and the IHC staining of the HER2-amplified and nonamplified controls was concordant with their FISH status. These results suggest that chromosome 17 polysomy in the absence of HER2 amplification does not have a significant biologic influence on HER2 gene expression in breast carcinoma.

The incidence of topoisomerase II-alpha genomic alterations in adenocarcinoma of the breast and their relationship to human epidermal growth factor receptor-2 gene amplification: a fluorescence in situ hybridization study.

Clinical and in vitro evidence supports the concept that human epidermal growth factor receptor-2 ( HER2 ) gene amplification prediction of response to anthracycline-based chemotherapy in breast cancer is not a direct effect of HER2 overexpression, but the result of coamplification of topoisomerase II-alpha ( TOP2A ). We investigated the relationship of TOP2A to HER2 genomic alterations by fluorescence in situ hybridization (FISH) and the correlations with polysomic states for chromosome 17 (CEP17). One hundred thirty-eight cases of breast cancer HER2 gene amplified by 2-color FISH ( HER2 /CEP17) were reevaluated with a 3-color probe set ( HER2 /CEP17/ TOP2A ) to investigate the frequency of coamplification and deletion of TOP2A . TOP2A was never amplified in the absence of HER2 amplification and was coamplified with HER2 in 68 (50%) of 137 cases; HER2 gene copy number was higher than the TOP2A copy number ( P < .01). Of the 137 cases with HER2 amplification, 23 (16%) showed a monoallelic deletion of TOP2A . Of the 43 cases not amplified for HER2 , 27 (63%) were CEP17 eusomic, 13 (30%) polysomic, and 3 (7%) monosomic. Of the HER2 nonamplified cases, 2 (5%) showed monoallelic deletion of both the HER2 and TOP2A . The current study demonstrates the complex interrelationship between the HER2 and TOP2A genes in breast cancer. The clinical implications of TOP2A amplification and deletion in breast cancer need to be further defined. If TOP2A gene dosage can be confirmed to correlate with tumor responsiveness to anthracycline-based therapy in the clinical setting, FISH testing for TOP2A status may be warranted to aid in the selection of the most appropriate therapy.

Canine model of surgical stress response comparing standard laparoscopic, microlaparoscopic, and hand-assisted laparoscopic nephrectomy.

OBJECTIVES: To compare the postoperative stress induced by standard laparoscopic, microlaparoscopic, and hand-assisted laparoscopic (HALS) nephrectomy in an animal model. METHODS: A total of 39 dogs underwent standard laparoscopic (n = 19), microlaparoscopic (n = 11), or HALS (n = 9) left nephrectomy. The serum cortisol levels were measured preoperatively, at skin closure, and 2, 4, and 6 hours postoperatively. RESULTS: Compared with the preoperative level, a sharp rise was noted in the serum cortisol taken at skin closure that correlated with both operative time (P = 0.003) and method (P = 0.009 for HALS versus microlaparoscopy and P = 0.02 for HALS versus standard). HALS had the lowest cortisol increase and shortest operative time, and microlaparoscopy had the greatest cortisol increase and longest operative time (P = 0.03 for cortisol increase, HALS versus microlaparoscopy). Two hours postoperatively, HALS was the only method associated with a continuing increase in cortisol levels (P = 0.01 and P = 0.02 compared with the other methods) and had the greatest cortisol level relative to baseline. The cortisol level decreased at all postoperative points in the other groups. By 4 hours, all methods were associated with similar and falling cortisol levels. CONCLUSIONS: HALS nephrectomy was associated with a greater operative stress response in the first 2 postoperative hours in dogs compared with standard laparoscopic and microlaparoscopic nephrectomy that was partially ameliorated by the lower intraoperative cortisol production in the more rapid HALS procedure. Microlaparoscopy provided no benefit in terms of reduced surgical stress. The stress differences among the techniques were insignificant by 4 hours postoperatively.