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Annals of Surgical Oncology 10:117-125 (2003)
© 2003 Society of Surgical Oncology

ORIGINAL ARTICLES

Clinical Relevance of Reverse Transcriptase-Polymerase Chain Reaction for the Detection of Axillary Lymph Node Metastases in Breast Cancer

Masahiro Sakaguchi, MD, Arvind Virmani, PhD, Marla W. Dudak, MD, George N. Peters, MD, A. Marilyn Leitch, MD, Hossein Saboorian, MD, Adi F. Gazdar, MD and David M. Euhus, MD

From the Departments of Surgery (MWD, GNP, AML, DME) and Pathology (MS, AV, HS, AFG), University of Texas Southwestern Medical Center, Dallas, Texas.

Correspondence: Address correspondence and reprint requests to: David M. Euhus, MD, Division of Surgical Oncology, E6.222, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9155; Fax: 214-648-7965; E-mail: david.euhus{at}utsouthwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The mammary sentinel lymph node procedure can increase the detection of axillary metastases by 45% compared with standard axillary dissection. Some investigators have reported that reverse transcriptase-polymerase chain reaction (RT-PCR) increases metastasis detection even more, but it is uncertain whether a positive RT-PCR test in the face of a negative histological evaluation is clinically meaningful.

Methods: RT-PCR for epithelial glycoprotein 2 and cytokeratin 19 was performed on sentinel and pooled nonsentinel axillary lymph nodes from 108 women with clinical stage I or II breast cancer who were followed up for a median of 40 months.

Results: Axillary metastases were detected on standard tissue sections in 26% and by RT-PCR in 30%. Results for the two tests were concordant for 80% of the cases. RT-PCR upstaged 16%. Tumors from women whose lymph nodes were positive only by RT-PCR were phenotypically similar to those from women with no metastases detected by any method. Moreover, 4-year actuarial distant disease-free survival was 100% for women with metastases detected by RT-PCR only, as compared with 74% for those with metastases detected by routine histology (P = .03) and 93% for those with no metastases detected by either method (P = .04).

Conclusions: Analysis of sentinel lymph nodes by RT-PCR for epithelial glycoprotein 2 and cytokeratin 19 is unlikely to provide clinically useful information.

Key Words: Breast cancer • Sentinel nodes • RT-PCR • Cytokeratin-19 • EGP-2 • Survival


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most important prognostic factor for patients with breast cancer is the presence of axillary lymph node metastases. However, 23% of stage I (T1N0M0) breast cancer patients who are treated with mastectomy will eventually die of the disease.1 Better identification of micrometastatic disease in axillary lymph nodes may allow for more accurate assessment of prognosis in women with breast cancer.

Special techniques, such as serial sectioning and immunohistochemical (IHC) staining, improve the detection of axillary micrometastases. Serial sectioning alone can upstage 7% to 33% of axillae,2–4 and single-section IHC upstages 20%.2 The sentinel lymph node procedure itself has improved the detection of micrometastases by allowing pathologists to focus on only one or two lymph nodes. In one series, application of the sentinel lymph node procedure to clinical stage I and II breast cancer patients increased the metastasis detection rate from 29% to 42%, largely by enhanced detection of micrometastases.5

As methods for detecting micrometastatic disease become more sensitive, it is appropriate to question whether clinically meaningful information is obtained by the identification of tiny foci of tumor cells in axillary nodes. Using serial sectioning, the Multicentre International (Ludwig) Breast Cancer Study Group identified axillary metastases in 83 (9%) of 921 cases initially designated as node negative by routine processing. The 5-year disease free survival was 58% for women with occult axillary metastases detected by serial sectioning, as compared with 74% for those with no occult metastases (P = .003).3 The addition of single-section IHC to the evaluation of axillary nodes from the same study more than doubled the detection rate of occult metastases. The 10-year disease-free survival for the cases with occult metastases detected by IHC was 55%, as compared with 63% for those with no occult metastases detected by IHC (P = .09).2 On the basis of these studies, detection of occult metastases by serial sectioning or IHC seems to provide useful prognostic information, although the case for IHC is not as strong.

A third, potentially more sensitive, method for detecting occult axillary metastases is the reverse transcriptase-polymerase chain reaction (RT-PCR). This is a molecular test designed to detect epithelial cell messenger RNA (mRNA), which should not normally be present in lymph nodes. Two epithelial cell markers were used in our study: cytokeratin 19 (CK-19) and epithelial glycoprotein 2 (EGP-2). CK-19 is expressed by most epithelial cells and many types of cancer, but not by lymph node lymphocytes, peripheral blood cells, or bone marrow cells.6 This suggests that CK-19 RT-PCR may be useful for the detection of micrometastases in breast cancer. EGP-2 is also known as Ber-Ep4. It is expressed on the surface of most epithelial cells and tumors,6 but not by mesothelial cells.7 Studies of bone marrow indicate that IHC can detect 1 breast cancer cell among 104 to 105 normal cells.8,9 In comparison, RT-PCR is 10 to 100 times more sensitive; it can detect 1 cancer cell among 106 normal cells.10

The purpose of this study was to assess the clinical relevance of RT-PCR for the detection of lymph node metastases in breast cancer. The criteria selected for estimation of clinical relevance were prediction of distant disease-free survival and correlation with the pattern of prognostic biomarker expression in the primary tumors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General Study Design
Sentinel and nonsentinel axillary lymph nodes were evaluated by routine histology and RT-PCR for a series of 108 women undergoing surgery for clinical stage I or II breast cancer between November 1996 and July 1998. The study was approved by the institutional review board, and informed consent was documented for each subject. Clinicopathologic features of the primary cancers and outcome measures were compared for cases with no axillary metastases detected by any method, those with axillary metastases detected by RT-PCR only, and those with histologically apparent metastases that were also positive by RT-PCR. In addition, immunostaining for CK-19 was performed for all sentinel and nonsentinel axillary nodes from patients whose nodes were positive by RT-PCR but negative by routine histology.

Characteristics of the Patients
Thirty-two patients underwent total mastectomy, and 76 underwent breast-conserving surgery (Table 1). The patients ranged in age from 34 to 84 years, with a mean of 57 years. The mean pathologic tumor size was 1.7 cm. All tumors were invasive breast carcinomas. Ninety-five were invasive ductal carcinoma, 5 of which had only microinvasive disease, and 13 were invasive lobular carcinoma. Five patients whose nodes were negative by both histology and RT-PCR were lost to follow-up. For the remaining 103 patients, follow-up ranged from 10 to 57 months, with a median of 40 months.


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  		TABLE 1. Clinicopathologic features of the 108 patients
 
Sentinel Lymph Node Technique
Our approach for the identification of sentinel lymph nodes has been previously described.11 Briefly, all patients underwent lymphoscintigraphy after injection of 500 µCi of 99mTc-labeled sulfur colloid near the tumor. In addition, 5 mL of isosulfan blue was injected around the primary tumor 5 to 15 minutes before skin incision. A handheld gamma counter was used in the operating room, and sentinel nodes were recognized by blue staining or by gamma counts that were significantly higher than the adjacent background. Completion level I and II axillary dissection was performed for 45 patients early in this series whether the sentinel lymph node was positive or not. For the subsequent patients, axillary dissection was performed only if the sentinel node was found to contain metastases by routine histological examination.

Lymph Node Processing
A small sliver (approximately 1/5 of the node) of each sentinel lymph node and each nonsentinel axillary lymph node was placed in growth factor medium (ACL4)12 supplemented with 5% fetal bovine serum immediately after the specimen was received in the cutting room (generally less than 1 hour after removal). For RT-PCR, samples from the sentinel nodes were processed separately, whereas those from nonsentinel axillary nodes were pooled. For routine histological evaluation, each sentinel node was bisected and then step-sectioned at three levels. This study was initiated before the introduction of IHC for the assessment of mammary sentinel lymph nodes in our institution, so IHC results are not available for comparison.

Lymph node tissue was prepared for RT-PCR as follows. Cells were collected by scraping node fragments in sterile phosphate-buffered saline (PBS), after which the sample was passed through a nylon filter to remove large fragments. The cell suspension was then centrifuged at 500 x g for 10 minutes at room temperature. Cell pellets were washed once and resuspended in 2 mL of PBS containing .01% bovine serum albumin. Epithelial cell enrichment was performed with Dynabeads (Dynal A. S., Oslo, Norway) coated with anti-epithelial cell antibodies at a cell:bead ratio of 5:1. The enrichment step was performed at 4°C for 1 hour with gentle rotation. The immunobeads were recovered by three cycles of washing with PBS and magnetic attraction.

RT-PCR
Total RNA was isolated from the magnetically selected cell pellets by using a Microscale RNA extraction kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. The RNA pellet was dissolved in diethylpyrocarbonate-treated water and used directly for reverse transcription without further dilution. RT-PCR was performed as previously described.12a Briefly, RNA was heat-denatured at 80°C with 1 µg of oligo-deoxythymidine primer and then cooled on ice. Complementary DNA was synthesized with 500 U of RT at 42°C for 60 minutes in a final volume of 50 µl. Five microliters of the RT product was used for PCR amplification. The primer sequences for CK-19 were sense: 5'-AGGTGGATTCCGCTCCGGGCA-3' and antisense: 5'-ATCTTCCTGTCCCTCGAGCA-3'; for EGP-2, they were sense: 5'-GAACAATGATGGGCTTTATGA-3' and antisense: 5'-TGAGAATTCAGGTGCTTTTT-3'. The PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide, and the amplicons were visualized under a UV transilluminator (Fig. 1).



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  		FIG. 1. Representative reverse transcriptase-polymerase chain reaction (RT-PCR) gels. (A) Improvement in specificity with immunobead enrichment. Six lymph nodes from patients without breast cancer were run before (-) and then after (+) immunobead enrichment. (B) Assay sensitivity: 0 or 10 cultured breast cancer cells (HCC1187) were mixed with 1 x 107 cells from 3 lymph nodes obtained from women without breast cancer. Immunobead enrichment was performed before RT-PCR. The assay detects 1 breast cancer cell among 106 lymph node cells. (C) Results for two cases with no axillary metastases detected by routine histology. SLN, sentinel lymph node; Ax, pooled axillary lymph nodes; CK, cytokeratin; bp, base pair.

 
Each assay included a positive control derived from the human breast cancer cell line HCC1187, which is known to express both CK-19 and EGP-2. Specificity of the assay was verified by using a panel of lymph nodes from six patients with benign disease. None of these samples was positive for CK-19 or EGP-2 by RT-PCR.

Statistical Analysis
In general, means were compared by using Student’s t-tests, and proportions were compared by using Fisher’s exact test. Disease-free survival was calculated with the method of Kaplan and Meier, and differences were compared by using the method of Greenwood.13 All statistical analyses were two tailed, and alpha was set at .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Twenty-eight (26%) of the 108 patients had lymph node metastases identified by routine histological processing. The mean number of positive lymph nodes per patient with metastases was 2.6, with a range of 1 to 14. Histology and RT-PCR were concordant in 80% of cases (Table 2). Of the 28 patients with histologically positive nodes, CK-19 mRNA was detected by RT-PCR in 19 (68%) and EGP-2 in 13 (46%). Six patients had CK-19 expression only, whereas none expressed EGP-2 only. Of the 80 patients with histologically negative nodes, CK-19 mRNA was detected by RT-PCR in 9 (11%) and EGP-2 in 11 (14%). Overall, RT-PCR was positive in 13 (16%) of the 80 patients with no metastases detected by histology. This suggests that RT-PCR may detect occult lymph node metastases in 16% of histologically node-negative patients.


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  		TABLE 2. RT-PCR status by histology status
 
To estimate the clinical relevance of a positive RT-PCR test, the cases were categorized into three groups (1) no metastases detected by either histology or RT-PCR (n = 67), (2) no metastases detected by histology, but RT-PCR positive (n = 13); and (3) metastases detected by both histology and RT-PCR (n = 19). The remaining nine patients, who were histologically positive, but RT-PCR negative, were not considered further. After a median follow-up of 40 months, there were three local recurrences: two in women who were node negative by all tests and one in a woman who was node positive by histology. During the same period, distant metastases were diagnosed in eight women: four who were node negative by all tests and four who were node positive by histology. There were no recurrences in any woman whose lymph nodes were RT-PCR positive but histology negative. Actuarial distant disease-free survival at 48 months was 93% for women who were node negative by both histology and RT-PCR, 100% for those who were RT-PCR positive only, and 74% for those with lymph node metastases detected by routine histology and RT-PCR (Fig. 2). The 48-month distant disease-free survival was significantly greater for women whose nodes were positive by RT-PCR only as compared with women whose nodes were negative by both RT-PCR and routine histology (P = .04). It should be noted that most of the patients in our series received adjuvant hormonal therapy consisting of tamoxifen or anastrozole regardless of the estrogen-receptor status. Thirty-seven percent of the patients whose nodes were negative by all tests received cytotoxic chemotherapy, as compared with 38% for those whose nodes were RT-PCR positive only (P = 1.00) and 100% for those whose nodes were positive by histology (P < .0001; Table 3).



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  		FIG. 2. Life table plots of distant disease-free survival by lymph node status. {image}, reverse transcriptase-polymerase chain reaction (RT-PCR) positive only; negative by both tests; {image}, positive by hematoxylin and eosin and RT-PCR.

 

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  		TABLE 3. Primary tumor characteristics, adjuvant therapy, and outcome by node status
 
We also evaluated the expression of prognostic biomarkers by the primary tumor as a surrogate end point for outcome (Table 3). As compared with tumors from women who were node negative by both tests, tumors from women with lymph node metastases detected by histology were larger (1.5 vs. 2.8 cm; P = .01) and more likely to be associated with lymphatic or vascular invasion (10% vs. 65%; P < .01). There was also a trend toward more nondiploid tumors and more Her-2/neu–overexpressing tumors, but these results did not reach statistical significance (P = .07 and P = .14, respectively). In contrast, the primary tumors from women with lymph nodes that were RT-PCR positive only were more like the tumors from women whose lymph nodes were negative by both tests than those from women with lymph nodes that were positive by both tests. Specifically, there was no trend for larger tumors or tumors associated with lymphatic or vascular invasion.

To further evaluate the possibility that RT-PCR was identifying microscopic disease missed by routine histology, IHC for CK-19 was performed on both sentinel and nonsentinel axillary lymph node tissue recut from the original paraffin blocks for the cases that were positive by RT-PCR but negative by routine histology (mean of six blocks per patient). The paraffin blocks could not be found for 2 of the 13 cases. One of the remaining 11 cases was positive for a micrometastasis by CK-19 immunostaining, but the rest were negative. This metastasis was confirmed on review of the hematoxylin and eosin sections. This patient had already undergone completion axillary dissection.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although several promising prognostic markers for blood,14 bone marrow,15,16 and primary tumors17,18 are currently under evaluation, detection of lymph node metastases remains the most thoroughly validated and generally accepted predictor of distant metastases in breast cancer. Introduction of the sentinel lymph node technique has reduced the morbidity of axillary staging and has improved the sensitivity.5,19 Because the sentinel node procedure retrieves a comparatively small volume of tissue, it is possible to use enhanced detection methods for the identification of minute metastatic foci within these nodes. Serial sectioning and IHC are two established methods that have increased the sensitivity for metastasis detection.3,2,20 This study evaluates what may be an even more sensitive tool: RT-PCR for the detection of epithelial cell mRNA.

The RT-PCR test amplifies unique or overexpressed genes in epithelial cells that should not, ordinarily, be present in lymphoid organs. We detected mRNA for CK-19 or EGP-2 in axillary lymph nodes from 16% of cases in which no metastases were identified by routine histology. This is in keeping with the 14% to 40% rate reported in the literature (Table 4). Because only a small portion of all available lymph node tissue is processed for RT-PCR and because the processing destroys the tissue, it was not possible to test identical sections by both RT-PCR and histology. This is the likely explanation for the 32% of cases with histologically confirmed metastases that were negative by RT-PCR. Another possible explanation is a delay in lymph node processing, which could have resulted in degradation of the mRNA. This is unlikely, because the pathologists personally retrieved the lymph nodes from the operating room as they became available and immediately processed them.


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  		TABLE 4. Clinical studies evaluating RT-PCR for the detection of lymph node metastases in breast cancer
 
RT-PCR is a very sensitive test; it detects as few as 1 breast cell among 106 lymphocytes, but there is considerable uncertainty as to the specificity of the test for detecting metastatic breast cancer. Of all the CKs evaluated to date, CK-19 is the least likely to be expressed in hematopoietic cells6 and, therefore, is the most specific CK for detection of epithelial cancer cells. Despite this, several features of CK-19 expression limit its clinical utility for metastasis detection. First, it is not expressed exclusively by epithelial cells. CK-19 expression has been detected in endothelial cells and fibroblasts,6 both components of intact lymph nodes. The epithelial cell enrichment step we included would have reduced contamination by cells that only weakly express CK-19. Second, a CK-19 pseudogene has been described. Pseudogenes are DNA segments arising from the integration of reverse transcripts of mRNA into the genome. Pseudogenes are thought to arise in germ cells21 and so are present in cells of every type and can be amplified by RT-PCR if the mRNA preparation is contaminated by DNA.22,23 Again, our epithelial cell enrichment step would have reduced the number of cells lacking CK-19 protein expression, but it would not have eliminated pseudogene amplification in these cells. The addition of deoxyribonuclease treatment could have further reduced positive signals from CK-19 pseudogenes, but this was not done. Of note, the 5' sense primer we selected for CK-19 mRNA amplification capitalizes on known sequence differences between CK-19 mRNA and CK-19 pseudogenes, making it unlikely that pseudogene amplification contributed significantly to our RT-PCR–positive cases. Finally, breast cancers demonstrate heterogeneous patterns of gene expression, and it is doubtful that evaluation of a few genes would be sufficient to detect metastases for every case. A recent evaluation of a 12-gene panel found a unique pattern of epithelial cell gene expression for every case evaluated; no single gene was expressed in every lymph node with breast cancer metastases.24 Of the genes evaluated, carcinoembryonic antigen (CEA), mammoglobin, mammoglobin B, prolactin-inducible protein, CK-19, erbB2, and muc1 seemed to hold the most promise for metastasis detection.

The extreme sensitivity of molecular tests for the detection of epithelial cells in lymph nodes makes it essential that the panel of markers selected is not merely breast cell specific, but metastatic breast cancer cell specific. Fragments of breast cancer and benign breast epithelium can be identified in axillary lymph nodes after needle biopsy of the breast in 28% to 93% of cases.25,26 This epithelial cell displacement is most commonly observed in sentinel lymph nodes.27 The artifactual nature of this phenomenon can be recognized microscopically because it is typically associated with hemosiderin-laden macrophages, foreign body giant cells, and physically altered red blood cells and lymphocytes. A molecular test could not make this distinction. In addition, cross-contamination of lymph node samples with breast cancer tissue can occur in the cutting room if the pathologist uses the same blade to cut both specimens. Because RT-PCR is so sensitive, this could produce false-positive results. Although protocols in our cutting room make cross-contamination unlikely, it is not possible to definitively exclude this as a factor in our study, and cross-contamination could seriously limit broad clinical application of RT-PCR for the detection of lymph node metastases.

Any useful molecular test for nodal metastases must predict relapse. Our results clearly demonstrate that detection of CK-19 and/or EGP-2 mRNA in axillary lymph nodes does not predict relapse. Indeed, distant disease-free survival was statistically significantly greater for cases that were positive by RT-PCR only than for cases that were negative by both RT-PCR and routine histology. Because our sample size was relatively small and our follow-up fairly short, it is possible that a larger study with longer follow-up could determine that RT-PCR–positive lymph nodes are correlated with outcome. One recent study that used a new technique for extracting and amplifying mRNA from archival formalin-fixed paraffin-embedded tissue found that detection of CEA mRNA in pooled axillary nodes was associated with a 10-year disease-free survival of 66.1%, as compared with 87.6% for cases that were negative by both RT-PCR and histology (P = .0008).28 This is an intriguing study that may point the way to successful application of molecular techniques for lymph node staging, but several issues remain unresolved. First, the primary tumors of patients with metastases detected by RT-PCR were phenotypically indistinguishable from those that were negative by both RT-PCR and histology. Second, in contrast to other investigators, who report that CEA mRNA is detected in only 63% of cases with metastases confirmed by histology,24 these investigators report a 100% detection rate; finally, although RT-PCR results were not available for any patient at the time of initial treatment, 88% of those with metastases subsequently detected by RT-PCR had received systemic adjuvant therapy, as compared with only 72% of those whose nodes were negative by RT-PCR (P = .05). This suggests that factors other than the RT-PCR result differentiate these two groups. Independent confirmation of these results is required.

No other studies have demonstrated decreased disease-free survival in patients with lymph node metastases detected by RT-PCR only, although some have correlated RT-PCR results with prognostic biomarker expression of primary tumors as a surrogate end point for outcome (Table 4). One study, which included 126 breast cancer patients, upstaged 15 (14%) of 106 histologically node-negative patients on the basis of CK-19 RT-PCR performed on pooled axillary nodes.29 As compared with tumors from women whose nodes were negative by both histology and RT-PCR, those from women who were positive by RT-PCR only were larger (2.5 vs. 1.9 cm; P < .05) and more frequently associated with lymphatic invasion (53% vs. 18%; P < .01). We did not observe a similar correlation in our series. This discrepancy is explained by differences in tissue processing. Whereas these investigators submitted one half of each axillary node for RT-PCR, we submitted only a small sliver from each node. Consequently, their metastasis detection rate by histology was only 16%, whereas ours was 26%. A more thorough histological assessment in this prior study would, no doubt, have reclassified more patients as histology positive and altered the distribution of prognostic factors between the groups. Others have identified correlations between RT-PCR–positive lymph nodes and primary tumors that are estrogen receptor negative or Her-2/neu positive30 or those associated with vascular invasion.31 In some studies that have used multiple markers, the number of positive markers was correlated with adverse prognostic features.30,32 None of these studies has reported outcome data.

Molecular assessment of axillary lymph nodes is potentially an ultrasensitive method for the detection of metastatic disease. Current markers, however, are not specific for metastatic breast cancer cells, and tumor heterogeneity is likely to limit the sensitivity of single-marker tests. As additional markers are described, it may be possible to develop panels with acceptable sensitivity and specificity. Clinical utility can be established only if these tests predict relapse.


   Footnotes
 
Presented at the 54th Annual Cancer Symposium of the Society of Surgical Oncology, Washington, DC, March 15–18, 2001.

Although evaluation of axillary lymph nodes with reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 19 and epithelial glycoprotein 2 apparently upstaged 16% of breast cancer patients, a positive RT-PCR test did not predict disease-free survival.

Received for publication January 17, 2002. Accepted for publication September 24, 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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