RT-PCR for Mammaglobin Genes, MGB1 and MGB2, Identifies Breast Cancer Micrometastases in Sentinel Lymph Nodes

Transcription

1 Anatomic Pathology / MAMMAGLOBIN GENE DETECTION IN BREAST CANCER SENTINEL LYMPH NODES RT-PCR for Mammaglobin Genes, MGB1 and MGB2, Identifies Breast Cancer Micrometastases in Sentinel Lymph Nodes Rodney J. Ouellette, MD, PhD, 1,2 Dominique Richard, PhD, 1 and Emmanuel Maïcas, MD, PhD 3 Key Words: MGB1; MGB2; Reverse transcription polymerase chain reaction; RT-PCR; Sentinel lymph node; SLN; Breast carcinoma DOI: /MMACTXT55L8QTKC1 Abstract In the present study, we examined the expression of the mammaglobin genes, MGB1 and MGB2, in the sentinel lymph nodes (SLNs) of patients with breast cancer and compared our results with the histologic status of the same SLNs. Compared with immunohistochemical staining for cytokeratin 8, which detected metastases in 17 of 42 patients, reverse transcription polymerase chain reaction (RT-PCR) for MGB1 or MGB2 genes was positive in 22 patients. The concordance between the expression of any mammaglobin and histologic status was 79% (33/42), with a sensitivity of 88% and specificity of 72%. The detection of patients with metastases was more sensitive when testing for both MGB1 and MGB2 (P <.0001) rather than MGB2 (P <.0005) or MGB1 (P <.05) alone. The increased detection rate relative to histologic examination suggests that using RT-PCR for the mammaglobin genes might identify patients at higher risk compared with patients with negative RT- PCR results. The sentinel lymph node (SLN) biopsy is becoming an alternative to complete axillary lymph node (ALN) dissection in the staging of breast cancer. Initially applied to patients with melanoma 1,2 and, subsequently, to patients with breast cancer, 3-7 the SLN concept is based on the rationale that SLNs should be the first nodes to receive primary lymphatic flow from the tumor and, therefore, should be more likely to contain disseminated tumor cells than would non-slns. Intraoperative identification of SLNs is achieved by the injection of vital dyes, radiolabeled colloids, or both in the peritumoral area before lymph node dissection. 3-7 One important advantage of the SLN biopsy is that it spares patients without metastasis from complete ALN dissection and its associated morbidity Consistent with the hypothesis that SLNs should not harbor metastases when the remaining nodes are free of metastases, previous studies have demonstrated that the histologic status of SLNs using routine histologic examination reflects that of ALNs with 96% to 100% accuracy. 4-7 Other studies involving more extensive and focused pathologic analysis of SLNs have revealed that the diagnostic accuracy was improved significantly. Indeed, serial sectioning, 12,13 immunohistochemical analysis, and a combination of both have been found to increase the detection rate of SLN micrometastases that had been missed by using standard H&E staining. However, the number of patients with positive nodes with tumors up-staged by the combination of serial sectioning and immunohistochemical analysis does not account for all patients with negative nodes who experience relapse and eventually die of the disease Even if all metastatic SLNs could be identified, application of these techniques to all SLNs is too labor-intensive and costly to be Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1 637

2 Ouellette et al / MAMMAGLOBIN GENE DETECTION IN BREAST CANCER SENTINEL LYMPH NODES used for routine examination. Clearly, a technique that is at once more sensitive and simple is needed for accurate staging of breast cancer. The reverse transcription polymerase chain reaction (RT-PCR) has proven to be a highly sensitive diagnostic tool in the detection of lymph node metastases in patients with breast cancer. Genetic markers such as MUC1, CK19, and CEA (carcinoembryonic antigen) have been shown to increase the number of positive lymph nodes compared with immunohistochemical staining The specificity and diagnostic value of these markers, however, remain controversial because they have been reported to be present and absent 23,24,28 in lymph nodes from patients without cancer. Mammaglobin (MGB1) and mammaglobin B (MGB2) are 2 related genes of the uteroglobin gene family that are overexpressed in breast tissue and metastatic lymph nodes from patients with breast cancer While MGB1 is expressed specifically in breast tissue, MGB2 expression also is found in the uterus and salivary gland. 31 Recent studies using RT-PCR assays have revealed high levels of expression of MGB1, MGB2, or both in human breast cancer cell lines and metastatic lymph nodes, 28,36-39 with an absence in noncancerous tissues. Compared with histologic results, markers for MGB1, MGB2, or both increase the number of positive lymph nodes detected. 28,36,37 Because of their high specificity and sensitivity, MGB1 and MGB2 markers might be ideal candidates for screening patients with breast cancer. To our knowledge, no single study has studied the dual use of MGB1 and MGB2 markers in SLNs. In the present study, patients with breast cancer underwent surgical removal of SLNs, and the expression of MGB1 and MGB2 was examined using RT-PCR. The RT-PCR results were compared with those obtained by immunohistochemical analysis and serial sectioning of the same SLNs. Materials and Methods Patients Fifty-three consecutive patients with breast carcinoma underwent SLN biopsy and complete ALN dissection at the Dr Georges-L. Dumont Hospital (Moncton, Canada) from May 1999 to October Informed consent for both surgical procedures and SLN analysis was obtained from all patients. Data for 42 patients were retained for analysis; data for 11 patients were excluded for the following reasons: diagnosis of primary breast oat cell cancer (n = 1), diagnosis of a T1 micrometastatic cancer (n = 3), diagnosis of a Tis cancer (n = 5), diagnosis of a cancer in both breasts (n = 1), and because SLN analysis was not done according to protocol (n = 1). Tumors were staged according to the latest classification by the American Joint Committee on Cancer 40 Table 1. Because characterization of HER-2/neu expression was not done routinely until the beginning of the year 2000, the HER-2/neu expression status was unknown for the 10 patients who entered the present study in 1999 (Table 1). ALN Dissection and SLN Biopsy Labeling of SLNs was done as previously described. 6 Briefly, technetium 99 sulfur colloid was injected around the tumor site 8 to 20 hours before surgery, and scintigraphy images were taken at 30-minute intervals. The next day, 5 ml of patent blue dye was injected intraoperatively a few minutes before the harvesting of SLNs. Each of the 42 patients underwent routine ALN dissection during the same surgery. A node was considered sentinel if it was bluestained or emitted a radioactive signal (using a γ probe). ALNs and SLNs were obtained immediately after surgery. In 63 of the 72 SLNs identified, an end portion of the lymph node, representing approximately 10% of the node, was removed by the pathologist and kept at 70 C for RT-PCR analysis. Histologic and Immunohistochemical Examination of Lymph Nodes Approximately 90% of each of the 72 SLNs was sliced into 2-mm sections and embedded in toto in paraffin. Three 5-µm sections were taken at 3 levels of the block and stained with H&E. An additional 5-µm section from level 1 was Table 1 Clinical Characteristics of 42 Patients Characteristic Results * Mean age (range) (y) (57) Mean tumor size (cm) (2.45) Tumor stage T1a 3 T1b 10 T1c 23 T2 6 Histologic diagnosis Invasive ductal carcinoma 38 Invasive lobular carcinoma 4 Estrogen receptor status Positive 36 Negative 6 Progesterone receptor status Positive 24 Negative 18 HER-2/neu status Positive 11 Negative 21 Unknown 10 Nodal staging pn0 (without isolated cells) 23 pn0 (with isolated cells) 3 pn1mi 3 pn1a 13 * Data are given as number of cases unless otherwise indicated. 638 Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1

3 Anatomic Pathology / ORIGINAL ARTICLE subjected to immunohistochemical staining with a monoclonal antibody against cytokeratin 8 (Cam 5.2, BD Bioscience, Franklin Lakes, NJ). The presence of 1 or more clearly identifiable atypical epithelial cells on H&E-stained or cytokeratin 8 immunohistochemically stained slides was scored as a micrometastasis. In each of 532 nonsentinel ALNs, node tissue was submitted in toto, and 1 section from 1 level was stained with H&E and examined. RNA Extraction Of the 72 SLNs, 9 were excluded from RT-PCR analysis because the node was too small and histologic examination would have been compromised or because the delay between node removal and reception was too long to ensure preservation of messenger RNA (mrna). Sixty-three frozen SLN portions were thawed and homogenized in Tri-zol reagent (Invitrogen Life Technologies, Huntsville, AL). Total RNA extraction was performed according to the manufacturer s instructions. Briefly, approximately 50 µg of lymph node specimen was added to 1 ml of reagent and homogenized. After incubation for 5 minutes at room temperature, 0.2 ml of chloroform was added. The sample was homogenized and centrifuged (12,000g) for 15 minutes at 4 C. The upper aqueous phase was removed, and RNA was precipitated by adding 0.5 ml of isopropyl alcohol. The sample again was centrifuged (12,000g) for 10 minutes at 4 C, and the pellet was rinsed twice with 1 ml of ethanol (75%). The RNA was resuspended in RNase-free water to a concentration of 0.5 µg/µl. RT-PCR for MGB1 and MGB2 Genes We added 0.5 µg of Oligo(dT) and 16 µl of RNasefree water to 5 µg of total RNA and incubated at 65 C for 10 minutes. Samples were permitted to renature at 4 C for 5 minutes. The following were then added to the mixture: 8 µl of 5 reverse transcriptase buffer (Invitrogen Life Technologies), 2 µl of a 0.1-mol/L concentration of dithiothreitol, 1 µl of a 25-mmol/L concentration of deoxynucleoside triphosphate (dntp), 1 µl of an RNase inhibitor buffer (Invitrogen Life Technologies), and 1 µl of Maloney murine leukemia virus. The reaction was carried out at 42 C for 1 hour and terminated by heating at 70 C for 10 minutes. All pre-pcr and post-pcr procedures were done in separate physical laboratory stations. Primers used for the reactions were those described by others for MGB1 32 : forward, 5'-AGCACTGCTACGCAGGCTCT-3' and reverse, 5'-ATAAGAAAGAGAAGGTGTGG-3'; and for MGB2 36 : forward, 5'-ACTCCTGGAGGACATGGTTGA-3' and reverse 5'-TCTGAGCCAAACGCCTTGGGT-3'. Reaction conditions were as follows: 10% or 4 µl of each lymph node complementary DNA (cdna) reaction was added to obtain a final concentration of 1 Taq DNA polymerase buffer (Applied Biosystems, Foster City, CA), a 200-µmol/L concentration of dntp, a 2.0-mmol/L concentration of magnesium chloride, a 0.64-µmol/L concentration of forward and reverse primers (MGB1 and MGB2), and 2.5 U of Taq DNA polymerase (Applied Biosystems). RNase-free water was added to give a final volume of 50 µl. Reactions were done using a touchdown PCR technique as follows: 2 cycles at 94 C for 1 minute, 60 C for 1 minute, and 72 C for 1 minute; then 2 cycles at 94 C for 1 minute, 58 C for 1 minute, and 72 C for 1 minute; and, finally, 37 cycles at 94 C for 1 minute, 56 C for 1 minute, and 72 C for 1 minute. Fifteen microliters of each sample was loaded and separated by gel electrophoresis on 2% agarose gels. Bands were visualized by transillumination UV light following staining with ethidium bromide. Twenty-five SLNs from patients with melanoma were used as negative control samples for MGB1 and MGB2 reactions. DNA fragments corresponding to MGB2 were observed in 2 of the 25 nodes (data not shown). The integrity of mrna for each sample was verified by RT-PCR using glyceraldehyde-3-phosphate dehydrogenase forward and reverse primers: forward, 5'-GTCAACG- GATTTGGTCTGTATT-3'; reverse, 5'-AGTCTTCTGGGTG- GCAGTGAT-3'. Statistical Analysis The diagnostic accuracy of cdna primers for MGB1, MGB2, or combination of both was evaluated by sensitivity (probability that the primers are expressed given that an SLN is histologically positive), specificity (probability that the primers are not expressed given that an SLN is histologically negative), and concordance (the number of times that the assay result was similar to the histologic result, divided by the total number of cases). The Fisher exact test (χ 2 ) was used to assess significance between cdna primers and the histologic detection of metastases. A P value of less than.05 was assumed to be statistically significant. Results Pathologic Characterization of SLNs and ALNs Fifty-three consecutive women with primary breast cancer and clinically negative axillary nodes underwent SLN biopsy followed by complete ALN dissection. After 11 patients were excluded for previously mentioned Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1 639

4 Ouellette et al / MAMMAGLOBIN GENE DETECTION IN BREAST CANCER SENTINEL LYMPH NODES reasons (see the Materials and Methods section), 42 patients remained in the study population (Table 1). Four surgeons performed the 42 cases. A mean of 1.5 SLNs (range, 1-3 SLNs) and 12.6 nonsentinel lymph nodes (ALNs; range, 4-25 nodes) were removed surgically from each of the 42 patients and analyzed for the presence of metastases. Serial sectioning and immunohistochemical analysis for cytokeratin 8 Image 1 revealed metastases in 17 patients (40%). The SLN was the only site of metastases in 11 (65%) of 17 cases with metastases. Of the 42 patients, 2 had positive ALNs and negative SLNs. The remaining 23 patients had no evidence of nodal metastases. Nodal staging according to the most recent American Joint Committee on Cancer classification 40 is shown in Table 1. RT-PCR Analysis in SLNs RT-PCR assays were used to detect the expression of MGB1 and MGB2 in SLNs, yielding products at 331 and 245 base pairs, respectively Image 2. In 10 of 12 cases in which a multiplex RT-PCR was used to examine the expression of MGB1 and MGB2, single RT-PCR assays yielded products with similar base pairs, thereby confirming the validity of the multiplex assay. The expression of either MGB1 or MGB2 was observed in 22 of 42 cases. Seven of the 42 cases were histologically negative. RT-PCR for either mammaglobin therefore increased the number of cancer-positive patients to a total of 52% (22/42) Figure 1. The 2 patients with positive ALNs and negative SLNs also had negative RT-PCR results. In control SLNs, MGB1 was absent in all 25 samples, while MGB2 was found in 2 cases (data not shown). A Image 2 Gel electrophoresis of single (lanes 3, 4, 6, 7) and multiplex (lanes 5 and 8) reverse transcription polymerase chain reaction for MGB1 and MGB2 from sentinel lymph nodes of 2 patients. bp, base pairs; M, marker; MGB, mammaglobin. The concordance between the expression of any mammaglobin and the histologic status of patients was 79%, with a sensitivity of 88% and a specificity of 72% Table 2. When the expression of individual mammaglobins was correlated with histologic data, MGB2 was more sensitive (76%) than MGB1 (59%), whereas MGB1 and MGB2 had identical specificity (80%; Table 2). The detection of patients with metastases was more sensitive when using assays for both mammaglobins (P <.0001) than either MGB2 (P <.0005) or MGB1 (P <.05) alone. Compared with pathologic staging, the expression of either mammaglobin was observed in 7 of 23 pn0 (without tumor cells), 3 of 3 pn0 (with isolated tumor cells), 1 of 2 pn1mi, and 11 of 12 pn1a tumors Table 3. B Image 1 Consecutive serial sections (5 µm) of a blue-labeled, radioactive sentinel lymph node. Arrows indicate areas of micrometastasis. A, Histologic staining (H&E, 20). B, Immunohistochemical staining (cytokeratin 8, 20). 640 Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1

5 Anatomic Pathology / ORIGINAL ARTICLE Discussion In the present study, the SLNs of patients with breast cancer were subjected to an RT-PCR assay to evaluate the expression of MGB1 and MGB2, 2 genes that have been shown to be expressed in high levels in breast cancer tumors and metastatic lymph nodes. 28,36-39 The RT-PCR results then were compared with those obtained by serial sectioning and immunohistochemical staining for cytokeratin 8. A meta-analysis revealed that the percentage of cases in which an SLN is histologically negative when, in fact, there are metastases in the ALN is approximately 5%. 41 Our results are consistent with those found in the literature, as we report a 5% failure rate (2 false-negative results in 42 cases). The SLNs from our 2 false-negative cases were also negative for MGB1 and MGB2 expression. RT-PCR assays have been developed to improve the sensitivity of detecting breast cancer. Because it is unlikely that a single gene is expressed consistently in all patients with breast cancer, it has been suggested that RT-PCR assays for several markers be used to increase the likelihood of identifying all patients with metastases. 38,39 In our cohort, one or both mammaglobins were expressed in 22 of 42 cases. MGB1 expression was detected in 15 cases, and expression of MGB2 was found in 18. That the simultaneous expression of both mammaglobins was detected in only 13 of 22 positive cases suggests that testing for the expression of both MGB1 and MGB2 identifies potential node-positive patients who otherwise would have been missed had a single gene been examined. In 12 cases, we subjected SLNs to a multiplex RT-PCR assay with both MGB1 and MGB2 markers. In 10 cases, the results obtained by the multiplex assay reflected those obtained by the combination of individual assays, thereby confirming the validity of our multiplex RT-PCR assay. While a number of genes are expressed in tumors and lymph nodes from patients with breast cancer, their diagnostic significance remains questionable because of their lack of specificity Consistent with the reported absence of MGB1 and MGB2 expression in lymph nodes from patients without breast cancer, 28,36-39 the specificity of MGB1 was confirmed by its lack of expression in SLNs obtained Figure 1 Correlation between reverse transcription polymerase chain reaction results for MGB1 and MGB2 and histologic status of sentinel lymph nodes from 42 women. histo, histologically; MGB, mammaglobin. from patients with melanoma. MGB2 expression, however, was detected in 8% of control SLNs (2/25). In the present study, the expression of either mammaglobin was detected in 7 cases that were immunohistochemically negative, thereby increasing by 28% the number of patients with metastases. A 58% increase has previously been reported when comparing the expression of both mammaglobins with histologic status. 28 In that study, however, the lymph nodes were examined by standard histologic examination only, which most likely accounts for the larger increase. The detection of MGB1, MGB2, or both in histologically uninvolved lymph nodes also has been reported. 36,37,42 The results presented herein, consistent with those reported by others, 28,36-39 suggest that MGB1 and MGB2 markers are highly sensitive tools for the detection of lymph node metastases. Historically, approximately two thirds of patients with breast cancer diagnosed as histologically node-negative (pn0) have long-term disease-free survival after surgical therapy However, one third of these node-negative Table 2 Correlation Between RT-PCR Assay Results and Histologic Status of Patients No. Histologically Negative No. Histologically Positive RT-PCR+ RT-PCR RT-PCR+ RT-PCR Concordance (%) Sensitivity (%) Specificity (%) Any mammaglobin MGB MGB RT-PCR, reverse transcription polymerase chain reaction. Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1 641

6 Ouellette et al / MAMMAGLOBIN GENE DETECTION IN BREAST CANCER SENTINEL LYMPH NODES Table 3 Correlation Between Nodal Staging of Patients and RT-PCR Assay Results * RT-PCR+ RT-PCR pn0 (without isolated cells) 7 16 pn0 (with isolated cells) 3 0 pn1mi 1 1 pn1a 11 1 RT-PCR, reverse transcription polymerase chain reaction. * The sentinel lymph nodes of 2 of the 42 patients were both histologically negative and RT-PCR negative (axillary lymph nodes were histologically positive) and were omitted. patients eventually experience relapse, 5,7,8 suggesting that a substantial proportion of patients are given an inaccurate diagnosis of pn0. Therefore, it is likely that a number of patients with false-negative diagnoses have ALN or other micrometastases that are not detected with routine pathologic examination. To examine the correlation between pathologic staging and RT-PCR results, we divided patients with stage pn0 into 2 categories according to immunohistochemical analysis results: pn0 with isolated tumor cells and pn0 without tumor cells. As shown in Table 3, all cases of pn0 with isolated tumor cells were RT-PCR positive for mammaglobins. On the other hand, 70% of patients with pn0 disease without tumor cells (16/23) had negative RT-PCR results, and 30% (7/23) had positive results for either mammaglobin. Follow-up studies will be required to determine whether the patients with mammaglobin-positive, immunohistochemically negative tumors reported herein have poorer outcomes and should be considered a higher risk group. Recent studies suggest that mrna for circulating MGB1 might be a useful diagnostic and monitoring tool Our results reveal that MGB1 and MGB2 markers have a high sensitivity (88%), suggesting that they might be useful for identifying micrometastases in the SLNs of patients with breast cancer. While we currently are unable to measure the impact of micrometastases on survival, future studies should reveal the clinical implications of these findings. From the 1 Beauséjour Medical Research Institute; 2 Department of Chemistry and Biochemistry, University of Moncton; and 3 Department of Pathology, Dr Georges-L. Dumont Hospital, Moncton, Canada. Supported by the Atlantic Canada Opportunity Agency (ACOA) and Dr Georges-L. Dumont Hospital Foundation, Moncton, Canada. Address reprint requests to Dr Ouellette: Beauséjour Medical Research Institute, 35 Providence St, Moncton, New Brunswick, Canada, E1C 8X3. Acknowledgment: We thank Marie-Reine Beaulieu for technical assistance. References 1. Morton DL, Wen D-R, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg. 1992;17: Krag DN, Meijer SJ, Weaver DL, et al. Minimal-access surgery for staging of malignant melanoma. Arch Surg. 1995;130: Krag DN, Weaver DL, Alex JC, et al. Surgical resection and radiolocalization of the sentinel lymph node in breast cancer using a gamma probe. Surg Oncol. 1993;2: Giuliano AE, Kirgan DM, Guenther JM, et al. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Arch Surg. 1994;220: Giuliano AE, Dale PS, Turner RR, et al. Improved axillary staging of breast cancer with sentinel lymphadenectomy. Ann Surg. 1995;222: Albertini JJ, Lyman GH, Cox C, et al. Lymphatic mapping and sentinel node biopsy in the patient with breast cancer. JAMA. 1996;276: Nwariaku FE, Euhus DM, Beitsch PD, et al. Sentinel lymph node biopsy, an alternative to elective axillary dissection for breast cancer. Am J Surg. 1998;176: Kissin MW, Querci della Rovere G, Easton D, et al. Risk of lymphoedema following the treatment of breast cancer. Br J Surg. 1986;73: Bryant M, Baum M. Postoperative seroma following mastectomy and axillary dissection. Br J Surg. 1987;74: Ivens D, Hoe AL, Podd TJ, et al. Assessment of morbidity from complete axillary dissection. Br J Cancer. 1992;66: Keramopoulos A, Tsionou C, Minaretzis D, et al. Arm morbidity following treatment of breast cancer with total axillary dissection: a multivariated approach. Oncology. 1993;50: Friedman S, Bertin F, Mouriesse H, et al. Importance of tumor cells in axillary node sinus margins ( clandestine metastasis) discovered by serial sectioning in operable breast carcinoma. Acta Oncol. 1988;27: Bettelheim R, Price KN, Gelber RD, et al. The International Breast Cancer Study Group (Ludwig Trial): prognostic importance of occult axillary lymph node micrometastases from breast cancers. Lancet. 1990;335: Trojani M, de Mascarel I, Bonichon F, et al. Micrometastases to axillary lymph nodes from carcinoma of breast: detection by immunohistochemistry and prognostic significance. Br J Cancer. 1987;55: Hainsworth PJ, Tjandra JJ, Stillwell RG, et al. Detection and significance of occult metastases in node-negative breast cancer. Br J Surg. 1993;80: Coté RJ, Peterson HF, Chaiwun B, et al. The International Breast Cancer Study Group: role of immunohistochemical detection of lymph-node metastases in management of breast cancer. Lancet. 1999;354: Schreiber RH, Pendas S, Ku NN, et al. Microstaging of breast cancer patients using cytokeratin staining of the sentinel lymph node. Ann Surg Oncol. 1999;6: Jakub JW, Diaz NM, Ebert MD, et al. Completion axillary lymph node dissection minimizes the likelihood of false negatives for patients with invasive breast carcinoma and cytokeratin positive only sentinel lymph nodes. Am J Surg. 2002;184: Motomura K, Komoike Y, Inaji H, et al. Multiple sectioning and immunohistochemical staining of sentinel nodes in patients with breast cancer. Br J Surg. 2002;89: Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1

7 Anatomic Pathology / ORIGINAL ARTICLE 20. Fisher ER, Swonidoss S, Lee CH, et al. Detection and significance of occult axillary node metastases in patients with invasive breast cancer. Cancer. 1978;45: Gardner B, Feldman J. Are positive axillary nodes in breast cancer markers for incurable disease? Ann Surg. 1993;218: Hellman S. Natural history of small breast cancers. J Clin Oncol. 1994;12: Noguchi S, Aihara T, Nakamori S, et al. The detection of breast carcinoma micrometastases in axillary lymph nodes by means of reverse transcriptase polymerase chain reaction. Cancer. 1994;74: Schoenfeld A, Luqmani Y, Smith D, et al. Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction. Cancer Res. 1994;54: Bostick PJ, Chatterjee S, Chi DD, et al. Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients. J Clin Oncol. 1998;16: Burchill SA, Bradbury MF, Pittman K, et al. Detection of epithelial cancer cells in peripheral blood by reverse transcriptase polymerase chain reaction. Br J Cancer. 1995;71: Trudeau WL, Fields KK, Sullivan DM, et al. Detection of cytokeratin 19 (CK19) transcript in the lymph nodes (LN) of patients with breast cancer by RT-PCR [abstract]. Proc Am Assoc Cancer Res. 1998;39: Ooka M, Sakita I, Fujiwara Y, et al. Selection of mrna markers for detection of lymph node micrometastases in breast cancer patients. Oncol Rep. 2000;7: Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res. 1996;56: Watson MA, Darrow C, Zimonjic DB, et al. Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13. Oncogene. 1998;16: Becker RM, Darrow C, Zimonjic DB, et al. Identification of mammaglobin B, a novel member of the uteroglobin gene family. Genomics. 1998;54: Watson MA, Dintzis S, Darrow CM, et al. Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res. 1999;59: Min CJ, Tafra L, Verbanac KM. Identification of superior markers for polymerase chain reaction detection of breast cancer metastases in sentinel lymph nodes. Cancer Res. 1998;58: Leygue E, Snell L, Dotzlaw H, et al. Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol. 1999;189: Corradini P, Voena C, Astolfi M, et al. Maspin and mammaglobin genes are specific markers for RT-PCR detection of minimal residual disease in patients with breast cancer. Ann Oncol. 2001;12: Aihara T, Fujiwara Y, Ooka M, et al. Mammaglobin B as a novel marker for detection of breast cancer micrometastases in axillary lymph nodes by reverse transcription polymerase chain reaction. Breast Cancer Res Treat. 1999;58: Kataoka A, Mori M, Sadanaga N, et al. RT-PCR detection of breast cancer cells in sentinel lymph modes. Int J Oncol. 2000;16: Manzotti M, Dell Oro S, Maisonneuve P, et al. Reverse transcription polymerase chain reaction assay for multiple mrna markers in the detection of breast cancer metastases in sentinel lymph nodes. Int J Cancer. 2001;95: Zehentner BK, Dillon DC, Jiang Y, et al. Application of a multigene reverse transcription PCR assay for detection of mammaglobin and complementary transcribed genes in breast cancer lymph nodes. Clin Chem. 2002;48: Singletary SE, Allred C, Ashley P, et al. Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol. 2002;20: Miltenburg DM, Miller C, Karamlou TB, et al. Meta-analysis of sentinel lymph node biopsy in breast cancer. J Surg Res. 1999;84: Branagan G, Hughes D, Jeffrey M, et al. Detection of micrometastases in lymph nodes from patients with breast cancer. Br J Surg. 2002;89: Fleming TP, Watson MA. Mammaglobin, a breast-specific gene, and its utility as a marker for breast cancer. Ann N Y Acad Sci. 2000;923: Gal S, Fidler C, Lo YM, et al. Detection of mammaglobin mrna in the plasma of breast cancer patients. Ann N Y Acad Sci. 2001;945: Zach O, Wagner H, Kasparu H, et al. Statistical validation of the mammaglobin-nested RT-PCR assay for tumor cell detection in blood of breast cancer patients. Biotechniques. 2001;31: Am J Clin Pathol 2004;121: DOI: /MMACTXT55L8QTKC1 643