Bruce M. Elliott

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.

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.

Limitations of peroneal artery bypass grafting for limb salvage

PURPOSE The purpose of this study was to compare the results of peroneal bypass grafting for limb salvage with the results of other tibial and pedal bypass grafts performed concurrently. METHODS Thirty-four peroneal bypass grafts with autologous vein were performed for limb salvage between September 1986 and June 1992. These constituted 18% of an overall experience of 194 tibial or pedal bypasses performed during that time. Preoperative and intraoperative arteriograms were reviewed to identify anatomic characteristics associated with successful limb salvage. RESULTS Secondary patency rates for peroneal bypass grafts (70%) compared with the other tibial and pedal bypass grafts (65%) did not differ significantly at 48 months by life-table analysis. Limb salvage achieved by peroneal artery bypass grafting was significantly worse (55%) than that achieved by the remaining tibial and pedal bypasses (67%) at 48 months. Limb salvage was 33% at 7 months for those undergoing peroneal artery bypass grafting as opposed to 57% at 48 months for patients undergoing other tibial or pedal revascularizations with tissue necrosis. Four anatomic features were identified that were associated with failure after peroneal artery bypass grafting. These were peroneal length less than 10 cm (p = 0.012), peroneal artery diameter less than 2 mm (p = 0.035), absence of arteriographically demonstrated collaterals perfusing the foot (p = 0.0001), and little or no visualization of the pedal arch (p = 0.008). CONCLUSIONS Although successful grafts may avoid amputation in carefully selected cases, alternatives to peroneal artery bypass grafting should be considered when less than favorable anatomic conditions are encountered, particularly in the presence of forefoot tissue necrosis.

Racial differences in operation for peripheral vascular disease: results of a population-based study

Operation for non-coronary atherosclerotic peripheral vascular occlusive disease may vary among race and gender groups. Using a state-wide registry, the authors identified all operations performed for infrarenal peripheral vascular disease over a 12-month period in a single south-eastern state. Procedures performed included reconstruction for aortoiliac (n=641) and infrainguinal (n=1129) disease and major amputation (n=1077). The incidence for patients over age 50 was calculated using census data. Operation for aortoiliac disease was significantly more likely for white patients (relative risk 3.79, 95% C.I. 2.84-5.15), but less likely for infrainguinal peripheral vascular disease (relative risk 0.64, 95% C.I. 0.56-0.73) and amputation (relative risk 0.17, 95% C.I. 0.15-0.19). Trends toward lower operative mortality in blacks with aortoiliac disease (10.6% versus 12.0%), PVD (3.2% versus 3.5%), and amputation (5.5 versus 8.7%) failed to attain statistical significance. Patient race was associated with the type and location of operation performed for peripheral vascular disease.

Percutaneous endoluminal stent-graft repair of an old traumatic femoral arteriovenous fistula

A stent-graft was custom made to close a high-flow traumatic arteriovenous fistula of the left superficial femoral artery, present for 30 years, in a 60-year-old man with congestive heart failure and ischemic ulceration in the left foot. A balloon expandable Palmaz stent (P394; 2.5 mm × 3.9 cm) was covered with a polytetrafluoroethylene (PTFE) graft and was inserted percutaneously through an 11 Fr vascular sheath. Follow-up Doppler ultrasound at 6 months demonstrated occlusion of the arteriovenous fistula, patency of the artery, and luminal integrity of the artery and vein.

Distal wound complications following pedal bypass: analysis of risk factors.

Wound complications of the pedal incision continue to compromise successful limb salvage following aggressive revascularization. Significant distal wound disruption occurred in 14 of 142 (9;8%) patients undergoing pedal bypass with autogenous vein for limb salvage between 1986 and 1993. One hundred forty-two pedal bypass procedures were performed for rest pain in 66 patients and tissue necrosis in 76. Among the 86 men and 56 women, 76% were diabetic and 73% were black. All but eight patients had a history of diabetes and/or tobacco use. Eight wounds were successfully managed with maintenance of patent grafts from 5 to 57 months. Exposure of a patent graft precipitated amputation in three patients, as did graft occlusion in an additional patient. One graft was salvaged by revision to the peroneal artery and one was covered by a local bipedicled flap. Multiple regression analysis identified three factors associated with wound complications at the pedal incision site: diabetes mellitus (p=0.03), age >70 years (p=0.03), and rest pain (p=0.05). Ancillary techniques (“pie-crusting”) to reduce skin tension resulted in no distal wound problems among 15 patients considered to be at greatest risk for wound breakdown. Attention to technique of distal graft tunneling, a wound closure that reduces tension, and control of swelling by avoiding dependency on and use of gentle elastic compression assume crucial importance in minimizing pedal wound complications following pedal bypass.

Protamine use during peripheral vascular surgery: A prospective randomized trial

PURPOSE One hundred twenty patients undergoing aortic reconstruction (40), infrainguinal bypass (49), and carotid endarterectomy (31) were prospectively enrolled into a double-blind randomized trial to investigate the utility of routine heparin reversal with protamine. METHODS All patients underwent systemic heparinization with 90 U/kg body weight during operation and after revascularization were randomized to receive either protamine or saline solution for heparin reversal. Blood loss was measured throughout the surgical procedure, and indexes of coagulation and the requirement for blood and blood products were documented during operation and the first 24 hours after operation. RESULTS Plasma heparin concentration, partial thromboplastin time, and activated clotting time were significantly higher (p < 0.05) in those receiving saline solution at 20 minutes and 1 hour after administration. Total surgical blood loss was not significantly different between study groups. No significant differences were found in blood product requirement, intravenous fluid administered, hematocrit, or wound hematomas between groups at 24 hours. In addition, no difference was seen in the surgeons subjective intraoperative assessment of hemostasis after administration of either study drug. Furthermore, after study drug administration protamine was associated with a deleterious effect on subsequent intraoperative blood loss (318 +/- 33 ml vs 195 +/- 18 ml, p < 0.05). CONCLUSIONS Although protamine effectively reverses heparin anticoagulation, its routine use after elective peripheral vascular surgical reconstruction does not appear to provide any clinical benefit.

Diabetes mellitus is the major risk factor for African Americans who undergo peripheral bypass graft operation.

OBJECTIVE African Americans, especially African American women, have a greater risk of lower extremity ischemia that necessitates an infrainguinal bypass graft operation and amputation. Because the prevalence of diabetes mellitus is proportionally greater in this ethnic/racial group, the relative contribution of diabetes was compared with other potential risk factors. METHODS This study was designed as a retrospective case control study at the University and Veterans Hospitals. In a 5-year period, 764 consecutive patients who required infrainguinal revascularizations were compared with a statewide population that was described by the 1995 Behavior Risk Factor Surveillance System database. The main outcome measure was the requirement for infrainguinal revascularization. RESULTS Diabetes mellitus was more common among African American women who underwent bypass graft operation (70%; odds ratio [OR], 24.9; 95% confidence interval [CI], 20.3 to 30.4) than African American men (46%; OR, 11.6; 95% CI, 8.9 to 15.2), white women (49%; OR, 15.9; 95% CI, 13.0 to 19.5), or white men (42%; OR, 14.8; 95% CI, 12.5 to 17.4). Overall, bypass graft operation was associated more strongly with diabetes mellitus for all groups (OR, 15.7; 95% CI, 13.5 to 18. 3) than with smoking (OR, 4.5; 95% CI, 3.8 to 5.2) or hypertension (OR, 4.6; 95% CI, 4.0 to 5.3). Life-table analysis revealed limb salvage to be worse at 3 years among African American patients (64% vs 75%; P <.005) despite similar primary and cumulative secondary graft patency rates. CONCLUSION Diabetes mellitus is the dominant risk factor that contributes to the need for bypass graft operation, especially among African American women. A greater prevalence of diabetes mellitus may account for the higher incidence of tissue necrosis and the increased requirement for distal bypass grafting and may contribute to the reduction in long-term limb salvage that was observed with these women.

Limitations of Subfascial Ligation for Refractory Chronic Venous Stasis Ulceration

Eighteen subfascial ligations of deep venous perforators were performed on 17 patients with refractory venous stasis ulceration. Thirteen patients also required concomitant or subsequent split thickness skin grafting. Primary indications included: (1) recurrence of ulceration during adequate support therapy with failure to heal using conservative measures (10 cases–55%) and (2) failure to heal with support therapy alone (eight cases 45%). Five limbs had ulcers greater than 30 cm2 and two had giant ulcers (>50 cm2). Most extremities had evidence of venous reflux by photoplethysmography or Doppler ultrasound (10 of 11) or chronic deep venous thrombosis by venography (six of seven). Mean hospital stay was 23 days±17, range six to 68 days. Early complications, including incisional breakdown or partial skin graft loss, were common and occurred in 10 patients. With a mean follow-up interval of 28 months (range nine to 49 months), most limbs (N=10) were judged cured, including both with giant ulcers, and three significantly improved. By life table analysis, 63% were free from significant ulcer recurrence at 42 months. Four limbs were not significantly improved following surgery. Most patiens with refractory venous ulceration will benefit from subfascial ligation of deep venous perforators and skin grafting, although recurrent or persistent ulceration remains problematic for a significant number of patients.