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

Abstract

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.

Figures

FIGURE 1. Expression of cancer-associated genes in normal control lymph nodes. Real-time RT-PCR analysis of lymph nodes from 51 negative control patients [32 males (+), 19 females (O)] was performed as described in Materials and Methods using primer pairs for the indicated genes. Ct values for each gene were determined from triplicate reactions. ΔCt values were obtained by subtracting the mean Ct value of β2-microglobin from the mean Ct value of each respective gene. Horizontal lines indicate ΔCt threshold values (3 standard deviations below the mean). Mean ΔCt and ΔCt threshold for each gene are as follows: CEA 22.23, 18.45; mam 21.5, 16.9; mamB 22.49, 16.63; PIP 19.18, 12.68; PDEF 17.64, 10.61; CK19 17.25, 9.04; muc1 9.35, 4.43.

FIGURE 2. Frequency of gene expression in breast cancer subjects with pathology-positive [Path (+)] and pathology-negative [Path (-)] ALN. Real-time RT-PCR analysis of ALN from 145 subjects with pathology-positive ALN and 344 subjects with pathology-negative ALN was performed as described in the Materials and Methods using primer pairs for the indicated genes. Marker positivity was determined based on ΔCt threshold values. y-axis shows the percent of subjects positive for each respective gene.

FIGURE 3. Sensitivity of detection of metastatic disease by multimarker RT-PCR. This analysis includes lymph nodes from 126 patients with pathology-positive/marker-positive ALN (filled triangles) and 112 pathology-negative/marker-positive ALN (filled circles). The percent of patients positive with gene overexpression detected by real-time RT-PCR is shown using the most frequently expressed gene (mam), and then adding other genes to the marker panel in order of their ability to complement mam, or the existing marker panel.