John S. Metcalf

Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel

Real‐time RT‐PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer‐associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c‐myc, p97, vim and Ki67) in 51 negative‐control normal lymph nodes and in 17 histopathology‐positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22‐ to 2.8 × 105‐fold above normal mean expression, whereas PIP was overexpressed from 30‐ to 2.2 × 106‐fold above normal in 13 lymph nodes. Real‐time RT‐PCR analysis of pathology‐negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.

Molecular detection of micrometastatic breast cancer in histopathology-negative axillary lymph nodes correlates with traditional predictors of prognosis: An interim analysis of a prospective multi-institutional cohort study

Objective:We sought to establish the clinical relevance of micrometastatic disease detected by reverse transcription polymerase chain reaction (RT-PCR) in axillary lymph nodes (ALN) of breast cancer patients. Background:The presence of ALN metastases remains one of the most valuable prognostic indicators in women with breast cancer. However, the clinical relevance of molecular detection of micrometastatic breast cancer in sentinel lymph nodes (SLN) and nonsentinel ALN has not been established. Methods:Four hundred eighty-nine patients with T1–T3 primary breast cancers were analyzed in a prospective, multi-institutional cohort study. ALN were analyzed by standard histopathology (H&E staining) and by multimarker, real-time RT-PCR analysis (mam, mamB, muc1, CEA, PSE, CK19, and PIP) designed to detect breast cancer micrometastases. Results:A positive marker signal was observed in 126 (87%) of 145 subjects with pathology-positive ALN, and in 112 (33%) of 344 subjects with pathology-negative ALN. In subjects with pathology-negative ALN, a positive marker signal was significantly associated with traditional indicators of prognosis, such as histologic grade (P = 0.0255) and St. Gallen risk category (P = 0.022). Mammaglobin was the most informative marker in the panel. Conclusion:This is the first report to show that overexpression of breast cancer–associated genes in breast cancer subjects with pathology-negative ALN correlates with traditional indicators of disease prognosis. These interim results provide strong evidence that molecular markers could serve as valid surrogates for the detection of occult micrometastases in ALN. Correlation of real-time RT-PCR analyses with disease-free survival in this patient cohort will help to define the clinical relevance of micrometastatic disease in this patient population.

A pilot study evaluating the efficacy of a fully acetylated poly-N-acetyl glucosamine membrane formulation as a topical hemostatic agent ☆ ☆☆

BACKGROUND Topical hemostatic agents are frequently needed for control of intraoperative bleeding. Currently available topical products each have potential drawbacks, making a more effective topical hemostatic agent desirable. This study was performed to evaluate the effectiveness of a particular formulation of a newly available polysaccharide polymer, poly-N-acetyl glucosamine (p-GlcNAc), as a topical hemostatic agent for use in the operating room. Swine splenic incision and splenic capsular stripping hemorrhage models were initially used, with a subsequent pilot human study then performed. METHODS For the swine splenic incision model, anesthetized immature female Yorkshire white swine had a 3 x 8 mm incision created on the spleen. One of 3 agents (p-GlcNAc membrane, oxidized cellulose, or absorbable collagen) was sequentially applied to individual wounds and digitally compressed for 20 seconds. The wound was observed without pressure for 2 minutes. Up to 8 wounds per animal were created in 7 animals. For the swine splenic capsular stripping model a 2 x 2 cm area of capsular stripping on the surface of the spleen to a depth of 3 mm was created. Either p-GlcNAc membrane or oxidized cellulose was applied and digitally compressed for 60 seconds, followed by observation without pressure for 2 minutes. Six wounds per animal were created in 2 animals. If bleeding persisted in either model, a new cycle of compression was applied. These steps were repeated until hemostasis was achieved. No change in hemodynamics or coagulation factors was observed in either model. Subsequently, 10 consecutive patients undergoing elective small-bowel surgery were enrolled on pilot study. A 5 x 3 x 3 mm cruciate incision was created midway between the mesenteric and antimesenteric borders of the small bowel. Either p-GlcNAc membrane formulation or oxidized cellulose was applied (the sequence alternated per patient) with a 400-mg weight used for even, direct pressure. A second cruciate incision was then created on the contralateral side of the bowel to evaluate the second material. The number of applications required for hemostasis was assessed. Hemodynamics, small-bowel pathologic condition, and hematologic parameters were evaluated. RESULTS The p-GlcNAc membrane required fewer cycles of compression in the swine splenic incision model to achieve hemostasis than either absorbable collagen or oxidized cellulose (1.25 vs 2.58 and 3.41, respectively; P < .01) and caused more effective immediate cessation of bleeding (79% for p-GlcNAc vs 17% for both absorbable collagen and oxidized cellulose). With the more traumatic splenic capsular stripping model, p-GlcNAc required fewer cycles of compression to achieve hemostasis than oxidized cellulose (average, 2.5 versus 6.8 respectively; P < .01) and was able to achieve hemostasis with greater efficacy (50%) in 2 applications than did oxidized cellulose (0%; P < .01). When used in the human pilot study, p-GlcNAc membranes required fewer cycles of compression than oxidized cellulose (2.5 vs 5.4, respectively; P < .002), was able to stop bleeding with greater efficacy in 1 cycle of compression (50% vs 0%, respectively; P < .01), and ultimately accomplished hemostasis in 80% of the cases as opposed to 20%. CONCLUSIONS On the basis of its greater hemostatic efficacy as compared with collagen or oxidized cellulose-based products, p-GlcNAc holds promise as an effective topical hemostatic agent and deserves further evaluation.

Merkel cell carcinoma in situ

Background:  Merkel cell carcinoma (MCC) is a dermal small blue‐cell tumor that occurs in the elderly on the sun damaged head and neck. Epidermal involvement is unusual and MCC limited to the epidermis is very rare.

Melanoma thickness and histology predict sentinel lymph node status

BACKGROUND It remains unclear which patients with melanoma will benefit most from lymphatic mapping and sentinel lymphadenectomy. The purpose of this study is to determine whether primary melanoma histopathologic features could be applied to predict sentinel node status. METHODS One hundred twelve patients underwent sentinel node biopsy between May 1995 and August 1999. Reported histologic features were assessed for predictive value by univariate and multivariate logistic regression. RESULTS The sentinel node was located successfully in 105 of the 112 patients (94%). Twenty-one of these 105 patients (20%) had sentinel nodes that were positive for metastatic disease. Multivariate analyses revealed that tumor thickness greater than 1.5 mm (P = 0.01), ulceration (P <0.01), and lymphovascular invasion (P = 0.05) predicted the presence of micrometastases. CONCLUSIONS The presence of unfavorable histopathology such as ulceration and lymphovascular invasion may identify a group of patients with thin melanomas who would benefit from sentinel lymphadenectomy.

Detection of occult breast cancer micrometastases in axillary lymph nodes using a multimarker reverse transcriptase-polymerase chain reaction panel ☆

BACKGROUND Axillary lymph node status in breast cancer patients remains the single most important predictor of outcomes. Current methods of histopathologic analysis may be inadequate because 30% of node-negative patients recur. The purpose of this study was to test the hypothesis that a multigene reverse transcriptase-polymerase chain reaction (RT-PCR) panel provides a more sensitive method to detect axillary lymph node metastases than routine pathologic examination. STUDY DESIGN Sixty-one consecutive breast cancer patients were evaluated, with nine normal control patients. Nodes > 1 cm were bisected for histopathologic and RT-PCR analysis. Nodal tissue was homogenized, and total RNA was converted into cDNA with reverse transcriptase. Reverse transcriptase-polymerase chain reaction analysis was performed with primers specific for keratin-19, c-myc, prolactin inducible protein (PIP), and beta-actin using ethidium bromide gel electrophoresis. Reverse transcriptase-polymerase chain reaction positive/ pathology negative axillary lymph nodes were reevaluated using step sectioning and immunohistochemical staining. RESULTS Thirty-seven patients had pathologically negative axillary lymph nodes, of which 15 (40%) were positive by RT-PCR analysis. Two RT-PCR negative results (one probably from tissue processing error and the other secondary to sampling error) among the 24 histologically positive specimens were detected (8%). The number of patients in each pathologic stage was 26 patients in stage I; 18, stage IIA; 7, stage IIB; 7, stage IIIA; 3, stage IIIB; and 0 patients in stage IV. By RT-PCR staging, 8 of 26 patients went from stage I to IIA (30%), and 7 of 18 from stage IIA to IIB (39%). Of the RT-PCR positive individuals who were stage I by pathologic analysis, 100% were found to be c-myc positive, 0% keratin-19 positive, and 0% PIP positive; for stage IIIB patients these markers were 50%, 100%, and 100% respectively. Additionally, an increasing number of positive markers per specimen appeared to correlate with larger primary tumor size (p < 0.01) and decreased predicted 5-year survival (r = 0.950, p < 0.002). CONCLUSIONS Multimarker RT-PCR analysis appears to be a readily available and highly sensitive method for the detection of axillary lymph node micrometastases. Longterm followup of RT-PCR positive patients will be required to determine its clinical relevance. If validated as a predictor of disease recurrence, this method would provide a powerful complement to routine histopathologic analysis of axillary lymph nodes.

Circumscribed storiform collagenoma (sclerosing fibroma).

In the past several years we have examined eight dermal nodules that have morphologic features identical to the nodules described in patients with Cowdens disease. The patients in this series had no other clinical manifestations of Cowdens disease. In an attempt to better define this distinctive entity, we subjected tissue sections to a battery of histochemical and immunohistochemical stains and examined tisssue from one of the nodules ultrastruc-turally. Although we found similarities between these nodules and other common dermal fibrotic lesions, we believe that they are distinctive architecturally (they are sharply circumscribed and have a strikingly uniform slo-riform pattern) and immunohistochemically (with uniformly scattered factor 13a-positive cells). Because of the unique histologic features, we propose that the term “circumscribed storiform collagenoma” be applied to these nodules.

Squamous syringometaplasia in lobular panniculitis and pyoderma gangrenosum.

Squamous metaplasia of eccrine sweat glands has been most frequently described in chronic cutaneous ulcerations with associated epidermal hyperplasia. We found examples of the process in skin biopsy specimens from five patients: three had associated lobular panniculitis and two had lesions of pyoderma gangrenosum. The metaplasia was located in the mid-to-deep reticular dermis in all five patients and extended into the superficial subcutis in one. Immunohistochemical stains for CEA and S-100 protein were used to accentuate the relationship of the metaplastic islands with eccrine ducts. It is postulated that necrosis of a portion of the eccrine duct is the stimulus for this process.

Bronchial mucoepidermoid carcinoma metastatic to skin. Report of a case and review of the literature.

A case of bronchial mucoepidermoid carcinoma is reported, the presentation of which was as cutaneous metastases. Histologic, histochemical, and ultrastructural features of the neoplasm are described, and the literature pertaining to bronchial mucoepidermoid carcinoma is reviewed. This case illustrates the potential for aggressive behavior in a mucoepidermoid neoplasm, the histologic features of which are considered low grade by some authors. Because such metastatic lesions may be morphologically identical to tumors that have been described as primary cutaneous mucoepidermoid carcinomas, this differential must be considered by the histopathologist confronted by such a neoplasm.

Tumors composed of malignant epithelial and melanocytic populations: a case series and review of the literature

Over the last 30 years, there have been approximately 49 case reports of tumors composed of both malignant epithelial and malignant melanocytic populations. Herein, we present four additional cases of combined tumors that consist of two phenotypically distinct but intermingled populations of malignant cells. Each tumor was composed of an invasive melanoma closely associated with either a basal cell carcinoma or a squamous cell carcinoma. We review all previous case reports in the English literature and attempt to clarify the terminology and summarize what is currently known about these unique tumors.