Sandra M. Waldrop

Sentinel node staging of early breast cancer using lymphoscintigraphy and the intraoperative gamma detecting probe

Lymphoscintigraphy combined with intraoperative gamma-probe detection of sentinel lymph nodes in patients with inoperable early primary breast cancers is effective for staging the disease. The clinical alternative is axillary lymph node dissection, which is a far more invasive procedure and is accompanied by significant morbidity. Accuracy of staging is enhanced by immunohistochemical staining of micrometastases, which pathologists can easily perform for one to three sentinel lymph nodes, but not for 20 to 30 nodes, using axillary dissection procedure. Optimum methodology is presented for performing sentinel lymph node imaging and is important for accurate identification of sentinel node(s).

Rapid immunohistochemistry of sentinel lymph nodes for metastatic melanoma

Sentinel lymph node (SLN) biopsy is performed on patients with malignant melanoma (MM) to assess the need for selective complete lymphadenectomy. Melanoma metastasis to regional lymph nodes is an important prognostic indicator in patients with MM. This study assesses the sensitivity and specificity of rapid immunohistochemistry (RIHC) in intraoperative delineation of melanoma metastasis to SLN. RIHC for S-100 protein, HMB45, and a melanoma marker cocktail (melan A, HMB45, and tyrosinase) was performed on 71 SLNs obtained from 28 patients with MM. Frozen sections (6 micro thick) on plus slides were fixed for 2 to 3 minutes in cold acetone and then stored at -70 degrees C. The EnVision kit (Dako, Carpinteria, CA) for rapid immunohistochemistry (RIHC) on frozen tissue sections was used, and the staining technique took 19 minutes. Together with preparation of the frozen sections and fixation in acetone, immunostained slides were available in approximately 25 minutes. Of the 71 SNLs examined, 7 showed melanoma metastasis in permanent sections. RIHC of frozen sections detected metastatic melanoma in 6 SLNs, with a sensitivity of 86% for HMB45 and 71% for S-100 protein and the melanoma cocktail and a specificity of 97% for HMB45 and 100% for S-100 and the melanoma cocktail. We conclude that RIHC for HMB45, S-100 protein, and the melanoma cocktail may help detect melanoma metastasis in SLN intraoperatively, leading to total lymph node dissection and obviating the need for 2 surgical procedures. Section folds and background stain can make interpretation difficult. Intraoperative time constraints require a more rapid technique. A recent consensus group has discouraged frozen-section examination of SLN.

Automated in situ hybridization and immunohistochemistry for cytomegalovirus detection in paraffin-embedded tissue sections.

Cytomegalovirus (CMV) infections cause serious morbidity and mortality in immunocompromised individuals. CMV detection methods include serology, viral culture, polymerase chain reaction, and histologic examination [hematoxylin and eosin, immunohistochemistry (IHC), in situ hybridization (ISH)]. Until recently, ISH was performed manually. We compared automated ISH and automated IHC (Bond-max system, Leica Microsystems) in 72 cases (multiple organ systems) previously evaluated for CMV by conventional methods [hematoxylin and eosin, IHC (Dako Autostainer Plus), polymerase chain reaction, and/or viral culture]. By automated ISH, 27 cases were CMV-positive (25 positive, 2 equivocal on original diagnosis), 43 were negative (10 positive, 29 negative, 4 equivocal on original diagnosis), and 2 were equivocal (positive on original diagnosis). By automated IHC, 31 cases were CMV-positive (28 positive, 3 equivocal on original diagnosis), 39 were negative (7 positive, 29 negative, 3 equivocal on original diagnosis), and 2 were equivocal (positive on original diagnosis). Using original CMV diagnosis as gold standard, automated ISH and automated IHC had sensitivities of 67.6% and 75.7%, respectively (P value=0.25), and both had specificities of 100%. Combined automated ISH and IHC had sensitivity of 75.7% and specificity of 100%. Positive predictive values for automated ISH, automated IHC, and combination of ISH and IHC were 92.6%, 90.3%, and 90.3%, respectively. Negative predictive values were 67.4%, 74.4%, and 76.3%, respectively. We recommend either automated ISH or IHC for CMV detection in formalin-fixed, paraffin-embedded tissues. Although with moderate sensitivities, these methods have high specificities and positive predictive values, permit rapid turnover, require only set-up time, and result in strong stain intensity with minimal background.

Epstein-Barr Virus (EBV)-Encoded RNA: Automated In-Situ Hybridization (ISH) Compared with Manual ISH and Immunohistochemistry for Detection of EBV in Pediatric Lymphoproliferative Disorders:

Detection of Epstein-Barr virus (EBV) may be achieved by various methods, including EBV-encoded RNA (EBER) in-situ hybridization (ISH) and immunohistochemistry (IHC) for latent membrane protein (LMP-1). We compared novel automated ISH and IHC techniques in pediatric lymphoproliferative disorders with results obtained by manual ISH. Thirty-seven pediatric cases previously studied by manual EBER ISH (including 18 EBER-positive, 15 EBER-negative, and 4 EBER-equivocal cases) were used for the study. Automated EBER ISH and automated LMP-1 IHC were performed using the BondMax autostainer and prediluted EBER probe and EBV cell surface 1 to 4 at 1:50 dilution, respectively. Results of each of the automated techniques for EBV detection were compared with results by manual EBER ISH. Compared with manual EBER ISH as the gold standard, automated ISH had a sensitivity and specificity of 94% and 69%, respectively, accuracy of 83%, positive predictive value (PPV) of 79%, and negative predictive value (NPV) of 90%. Automated IHC had a sensitivity of 44%, specificity of 93%, accuracy of 67%, PPV of 88%, and NPV of 59%. Automated ISH and IHC correlated significantly (P < 0.045). Automated ISH is useful for diagnosis of EBV-related pediatric neoplasms, being easy to perform and interpret and requiring only the technologists time to set up and having a high sensitivity and NPV. The automated IHC protocol is of too low sensitivity for routine use, although results show high specificity and PPV.

Immunohistochemical evaluation of sentinel lymph nodes in breast carcinoma patients.

Sentinel lymph node sampling has become an alternative to axillary lymph node dissection to provide prognostic and treatment information in breast cancer patients. The role of immunohistochemistry has yet to be established. A total of 241 sentinel lymph nodes (in 270 slides) from 91 patients with invasive carcinoma (73 ductal, 9 lobular, 8 mixed lobular/ductal, 1 NOS) were studied for presence of macrometastases (> 0.2 cm), identified in hematoxylin and eosin sections, and occult metastases (micrometastases [≤ 0.2 cm], clusters of cells, isolated carcinoma cells), identified only by immunohistochemistry. Intraoperative touch preparations, frozen sections, seven hematoxylin and eosin levels (L1–L7), and two AE1–3 cytokeratin immunohistochemistries (L1, L4–5) of the entire bisected or trisected sentinel lymph node were examined. Thirty-one (34%) patients had 50 positive sentinel lymph nodes. Twenty-six (33%) sentinel lymph nodes had metastatic carcinoma (11 macrometastases, 11 micrometastases, 3 clusters of cells, 1 isolated carcinoma cells) by touch preparations, frozen sections, and one hematoxylin and eosin (L1). Thirty-eight (43%) were positive by AE1–3 immunohistochemistry (L1) (11 macrometastases, 8 micrometastases, 13 clusters of cells, 6 isolated carcinoma cells), significantly more than by touch preparations, frozen sections, hematoxylin and eosin L1, or hematoxylin and eosin L2–7. Cytokeratin immunostain on L4–5 demonstrated 31 (34%) positive sentinel lymph nodes, a similar frequency to cytokeratin immunostain on L1. Size of sentinel lymph node metastasis did not correlate with size, histologic grade, or type of primary breast carcinoma. AE1–3 (L1) immunohistochemistry is highly sensitive in delineating sentinel lymph node metastasis, especially clusters of cells and isolated carcinoma cells. The prognostic significance of clusters of cells and isolated carcinoma cells and the value of AE1–3 immunohistochemistry on frozen sections need to be determined.

Gd(ABE-DTTA)-enhanced cardiac MRI for the diagnosis of ischemic events in the heart†

To demonstrate that contrast‐enhanced MRI (ceMRI) with the aid of Gd(ABE‐DTTA) is able to detect ischemic events in the heart in a canine ischemia/reperfusion (30/40 minutes) model.