- Email: [email protected]
Commentary: Molecular detection of breast cancer markers
Molecular Diagnosis Vol. 6 No. 2 2001
Commentary
Molecular Detection of Breast Cancer Markers KATHRYN M. VERBANAC, PhD Greenville, North Carolina
PCR technology has brought the ability to exponentially amplify a previously undetectable amount of nucleic acid to a detectable level. The advent of RT-PCR led to a concerted search for genes that were preferentially expressed in cancer cells, and it brought the ability to detect a very small number of cells (one tumor cell in the midst of ⱖ1 million normal cells). Real-time PCR allows the quantitation of RNA expression and has been combined with laser-assisted microdissection to detect as few as 50 cells from archival tissue sections. Differential display, chromosomal gene array, and, most recently, complementary DNA microarrays allow a rapid screening of hundreds of genes or complementary DNAs from different normal and cancer tissues. Laser-capture microdissection of tumor cells from a given tissue block allows one to enrich the sample for more sensitive analysis and also yields information about intratumor heterogeneity. Although the onus initially was to identify all differentially expressed genes, scientists are now frequently urged to look for patterns of expression and not be concerned about understanding the identity or function of specific markers. The large variety of methods presently available to identify markers for cancer cell detection brings with it the challenge to process and evaluate the clinical usefulness of this information. The new fields of genomics, bioinformatics, and proteomics are emerging to help scientists cope with the barrage of data that results from these analyses. All these techniques seek to establish a molecular profile for cancer cells and associate certain markers or patterns of gene expression with specific subsets of patients with cancer. Identified markers may help in the understanding of oncogenesis and metastasis and serve as targets for intervention. Distinct molecular profiles may be associated with specific stages and prognosis patterns. Ultimately, it is
For decades, scientists have been in search of cancer markers that can distinguish cancer cells from normal cells to accurately diagnose and stage and effectively treat and monitor the disease. The major tissues of interest for breast cancer are breast tissue, bone marrow, axillary lymph nodes, and blood. Candidate targets include oncogenes, tissuespecific and tumor-associated markers, and markers associated with angiogenesis and metastasis. Gene products were initially sought as cancer markers. Cytokeratins (eg, K19, K20) and proliferation markers (Ki-67) remain the standard protein markers for breast cancer detected in tissue by immunohistochemistry. Promising circulating protein markers secreted or shed from the surface of breast tumor cells include carcinoembryonic antigen, CA15-3, and HER-2/neu oncoprotein, although American oncologists consider the clinical data not sufficient to recommend their routine use for breast cancer diagnosis or postoperative surveillance. At the chromosomal DNA level, loss of heterozygosity, gene rearrangements, translocations, mutations (BRCA-1 and -2) and amplifications (her-2/neu) have been found in specific groups of patients with breast cancer. Genomic comparative hybridization has been used to investigate gene copy number aberrations. Although extremely valuable in subsets of patients, such cancer-associated changes at the genomic DNA level do not occur in the majority of patients with cancer. From the Department of Surgery, The Brody School of Medicine at East Carolina University, Greenville, NC. Reprint requests: Kathryn M. Verbanac, PhD, Department of Surgery, The Brody School of Medicine at East Carolina University, 4S16 Brody Medical Sciences Bldg, Greenville, NC 27858. Email: [email protected] Copyright © 2001 by Churchill Livingstone威 1084-8592/01/0602-0001$35.00/0 doi: 10.1084/modi.2001.24167
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hoped that certain markers or expression patterns can detect disease at an earlier stage, as well as predict responsiveness to treatment, and thus lead to the selection of surgical and medical treatment tailored to each patient.
Marker Expression in Primary and Metastatic Breast Tumors In this issue of Molecular Diagnosis, Houghton et al. use a combination of molecular techniques, including genetic subtraction, DNA microarray analysis, and real-time PCR, to generate a transcriptional profile of breast cancer. This study documents marker expression in primary and metastatic breast tumors and in blood from patients with breast cancer. Three promising new markers are reported in addition to the previously reported mammaglobin. The investigators suggest that these genes may serve as diagnostic targets and candidates for T-cell vaccine development. Houghton et al. have recognized, as have others in the field, that a single marker is unlikely to be expressed in every patient with breast cancer and absent from all relevant normal cells. This report details their search for a combination of markers most likely to be useful clinically. Houghton et al. have also appropriately focused on mammaglobin as the most promising breast tissue–specific marker. Mammaglobin is a breast-specific marker first described in 1996 that is expressed in normal breast and frequently overexpressed in breast tumors [1,2]. Our laboratory first reported the use of mammaglobin for the detection of sentinel axillary lymph node metastases as we sought to develop a panel of markers for RT-PCR detection of breast cancer micrometastases to improve staging accuracy [3]. Mammaglobin, in marked contrast to the majority of markers tested, was not detected in normal lymph nodes from patients without cancer. We found that RT-PCR for mammaglobin increases the detection of breast cancer sentinel lymph node micrometastases, upstaging 27% of patients with histologically negative lymph nodes [4,5]. Combined with another promising marker, carcinoembryonic antigen, up to 44% of histologically negative patients would be upstaged. Other investigators have since reported mammaglobin messenger RNA (mRNA) expression in nodes of patients with breast cancer by in situ hybridization or RT-PCR [2,6–8]. RT-PCR analysis for mammaglobin is clearly a spe-
cific and sensitive method to detect breast cancer metastases to lymph nodes. Clinical recurrence will determine the significance of molecular staging, and long-term follow-up of patients enrolled into our multicenter trial is ongoing. Although Houghton et al. correctly state that mammaglobin expression is not a universal feature of breast cancer, data from our laboratory and others indicate that mammaglobin expression at the molecular level approaches universal. Using RTPCR, our laboratory has detected mammaglobin in 100% (7 of 7) of human breast cancer cell lines tested, 100% (35 of 35 specimens) of primary tumors tested, and 94% (41 of 45 specimens) of patients with histologically positive lymph nodes [3–5]. Other groups reported the molecular detection of mammaglobin expression in 98% to 100% of primary tumors [6–8] and 71%, 99%, 100%, and 100% of histologically positive lymph nodes [2,6– 8]. These data indicate a much greater expression level than the report of Houghton et al.; 70% of tumors in a panel which consisted of 46 primary and metastatic breast cancers. It would be helpful to have a full description of patient specimens to best interpret the extensive molecular analysis that Houghton et al. present. For example, it is surprising to find that lymph nodes containing breast tumor metastases appear to express more copies of the markers (normalized to actin) than the breast tumors themselves. It would be useful to know whether these nodes were positive for disease by hematoxylin and eosin staining or only positive by immunohistochemical analysis. More information on the patient sources would also be of general value to aid in understanding the molecular analysis. This could include, e.g., the number of patients from which the metastatic lymph nodes were derived and whether primary tumors and metastatic lymph nodes were derived from the same patient(s). More detailed specimen information would also affect conclusions about complementation of marker expression with other genes. These types of data will be critical for the future evaluation of the practical significance of molecular analyses. As discussed, our laboratory and others have established clearly that mammaglobin expression is absent from normal lymph nodes of patients without cancer. It is anticipated that the investigators will similarly analyze normal lymph nodes for expression of their newly discovered markers.
Commentary
Clearly, this is the critical specificity control tissue for nodal metastases. In our hands, we have found this to be the single most important criterion for marker selection. Although most marker candidates tested in our laboratory were expressed in all breast cancers tested, they were also expressed in 30% to 100% of normal lymph nodes from patients without cancer and thus lacked the necessary tumor specificity to provide accurate analysis of clinical specimens [3].
Detection of Circulating Neoplastic Cells Houghton et al. are astute in recognizing the potential prognostic value of RT-PCR for the detection of circulating tumor cells in peripheral blood of patients with breast cancer, especially with the use of selective markers. The detection of circulating tumor cells or markers may offer potential advantages to patients with breast cancer at several stages: (1) presurgery levels may reflect tumor burden and have prognostic significance; (2) postsurgery levels may allow the detection of subclinical disease, earlier detection of relapse, and thus earlier implementation of treatment; and (3) postsurgery levels may be indicative of response to chemotherapy and other forms of adjuvant therapy. More than a dozen potential markers have been investigated for detecting circulating breast cancer cells or soluble proteins; however, few have been found to be widely useful clinically. Watson et al. were the first to report the use of mammaglobin as a marker for circulating breast tumor cells [2]. Peripheral-blood stem cells from patients undergoing high-dose chemotherapy and autologous stem cell transplantation for metastatic breast cancer were obtained and subjected to RTPCR and probe hybridization to detect mammaglobin mRNA. Although mammaglobin mRNA was never detected in peripheral-blood stem cells from healthy donors, in 9 of 15 cases (60%) of metastatic breast cancer, peripheral-blood stem cells contained detectable mammaglobin mRNA, indicating the presence of circulating breast tumor cells. This number is similar to the detection level of mammaglobin in blood samples from the patients with metastatic cancer reported by Houghton et al. in this issue, using immunobead capture and RT-PCR. The validity of this approach and the utility of mammaglobin have been confirmed by two other
•
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European research groups. Zach et al. reported the use of a nested RT-PCR assay for mammaglobin RNA to detect circulating tumor cells [9]. Although not detected in healthy volunteers, mammaglobin transcripts were detected in the peripheral blood of 25% of 114 patients with breast cancer tested, 49% of those with known metastatic disease. A recent study by Gru¨newald et al. [10] also used nested RT-PCR to detect mammaglobin in blood samples of 8% of patients with invasive breast cancer. Notably, mammaglobin expression in the blood correlated with nodal status, metastasis, and CA-15-3 serum levels in this study. Houghton et al. have done an excellent job of showing the exquisite sensitivity of the blood RTPCR assay using immunobead capture. However, conclusions about marker specificity and complementation based on blood data should await analysis of additional normal blood samples since only 11 normal blood samples were examined for each marker. The finding that one of six normal blood samples (17%) expressed ␥-aminobutyrate type A receptor subunit suggests limited utility of this marker. Conclusions about marker specificity and complementation are complex in the absence of clinical data. It is important to study marker expression in relation to the time that the blood samples were procured relative to diagnosis, surgery, and treatment. Because it is well documented that tumor cells are released into the blood as a result of surgery, the detection of tumor cells is expected in most patients in the first weeks after surgery, but not necessarily in every patient months or years after surgery. (That is, 100% of all patients with metastatic breast cancer are unlikely to have circulating tumor cells. This should not be the endpoint for marker complementation studies.) Many other clinicopathological characteristics, including tumor size, nodal status, her-2/neu, and estrogen receptor/ progesterone receptor status would also be expected to correlate with the detection of circulating tumor cells. Finally, the type of therapy and timing of the blood procurement within the treatment course are important. Without more complete normal blood analysis and clinical correlates, it is difficult to predict whether marker detection and complementation are appropriate in all patient samples or may be inappropriate and even reflect a false-positive result. Without clinical follow-up, it is not known whether the lack of marker detection represents the
76
Molecular Diagnosis Vol. 6 No. 2 June 2001
lack of tumor cells that express the particular marker, a true absence of tumor cells, or assay insensitivity. Clearly, the clinical relevance of marker detection in the peripheral blood of patients with breast cancer should be evaluated in prospective studies. The merit of mammaglobin as a candidate for vaccine development warrants discussion. Mammaglobin is a secretory protein. Because there are no reports of mammaglobin localization on cell membranes, mammaglobin would thus have no utility as a target for antibodies, which recognize conformational determinants on cell-surface proteins. Mammaglobin-derived peptides are undoubtedly presented by cell surface major histocompatibility complex molecules and could theoretically serve as T-cell targets. However, T cells specific for selfantigens (such as mammaglobin) are normally deleted in the thymus during development, and those that might escape to the periphery are rendered functionally tolerant by a variety of mechanisms. Even if mammaglobin-reactive T-cell clones were present in the periphery and activated by a mammaglobin cancer vaccine such that immune selftolerance was broken, normal breast tissue would also be a target for these T-cell effectors. Most normal breast tissue expresses significant levels of mammaglobin and would be expected to express major histocompatibility complex–mammaglobin peptide targets; thus, a high therapeutic index (tumor to normal tissue ratio) would seem difficult to achieve. To conclude, the study reported by Houghton et al. uses a variety of complementary techniques to identify and characterize promising markers for the detection of occult breast cancer cells and should serve to further the field of molecular detection. Without a doubt, the molecular diagnosis of malignancy will become a routine part of many clinical evaluations. HER-2/neu is perhaps the best example of a marker that has made the bench-to-bedside transition and has had a positive impact on the lives of many patients with breast cancer. Follow-up studies of patients with melanoma and colon carcinoma indicate that molecular detection of micrometastases in lymph nodes [11–14] and blood [11,15] is prognostic and has clinical significance. We may also predict that the molecular detection of occult tumor cells will have a significant impact on future oncologic diagnosis and treatment. It has been suggested that micrometastatic tumor cells are biologi-
cally distinct from solid metastatic tumors and thus are likely to be responsive to different treatments [16]. The detection of micrometastases thus poses a challenge to research scientists and physicians to develop a better understanding of both the occult tumor cell and the clinical implications of its detection. Received February 2, 2001. Accepted February 7, 2001.
References 1. Watson MA, Fleming TP: Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 1996;56:860–865 2. Watson MA, Dintzis S, Darrow CM, et al.: Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res 1999;59:3028–3031 3. Min CJ, Tafra L, Verbanac KM: Identification of superior markers for PCR detection of breast cancer metastases in sentinel lymph nodes. Cancer Res 1998;58:4581–4584 4. Verbanac KM, Flemin T, Min CJ, Purser S, Tafra L: RT-PCR increases detection of breast cancer sentinel lymph node micrometastases. Breast Cancer Res Treatment 1999;57:41 (abstr) 5. Verbanac KM, Min CJ, Purser SM, et al.: RT-PCR analysis for mammaglobin and carcinoembryonic antigen detects metastases in histology-negative lymph nodes. Breast Cancer Res Treat 2000;64:37 (abstr) 6. Leygue E, Snell L, Dotzlaw H, et al.: Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol 1999;189:28–33 7. Kataoka A, Mori M, Sadanaga N, et al.: RT-PCR detection of breast cancer cells in sentinel lymph nodes. Int J Oncol 2000;16:1147–1152 8. Ooka M, Sakita I, Fujiwaa Y, et al.: Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients. Oncol Rep 2000;7:561–566 9. Zach O, Kasparu H, Krieger O, Hehenwarter W, Girschikofsky M, Lutz D: Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA. J Clin Oncol 1999;17:2015–2019 10. Gru¨newald K, Haun M, Urbanek M, et al.: Mammaglobin gene expression: A superior marker for breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin19. Lab Invest 2000;80:1071–1077 11. Mori M, Mimori K, Ueo H, et al.: Clinical signifi-
Commentary
cance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcriptase-polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J Clin Oncol 1998;16:128–132 12. Liefers GJ, Cleton-Jansen A-M, Van de Velde CJH, et al.: Micrometastases and survival in stage II colorectal cancer. N Engl J Med 1998;339: 223–228 13. Li W, Stall A, Shivers SC, et al.: Clinical relevance of molecular staging for melanoma. Ann Surg 2000; 231:795–803
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14. Bostick PJ, Mortin DL, Turner RR, et al.: Prognostic significance of occult metastases detected by sentinel lymphadenectomy and reverse transcriptase— polymerase chain reaction in early-stage melanoma patients. J Clin Oncol 1999;17:3238–3244 15. Hoon DSB, Bostick P, Kuo C, et al.: Molecular markers in blood as surrogate prognostic indicators of melanoma recurrence. Cancer Res 2000;60:2253– 2257 16. Pantel K, Cote RJ, Fodstad O: Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999;91:1113–1124
Commentary
Molecular Detection of Breast Cancer Markers KATHRYN M. VERBANAC, PhD Greenville, North Carolina
PCR technology has brought the ability to exponentially amplify a previously undetectable amount of nucleic acid to a detectable level. The advent of RT-PCR led to a concerted search for genes that were preferentially expressed in cancer cells, and it brought the ability to detect a very small number of cells (one tumor cell in the midst of ⱖ1 million normal cells). Real-time PCR allows the quantitation of RNA expression and has been combined with laser-assisted microdissection to detect as few as 50 cells from archival tissue sections. Differential display, chromosomal gene array, and, most recently, complementary DNA microarrays allow a rapid screening of hundreds of genes or complementary DNAs from different normal and cancer tissues. Laser-capture microdissection of tumor cells from a given tissue block allows one to enrich the sample for more sensitive analysis and also yields information about intratumor heterogeneity. Although the onus initially was to identify all differentially expressed genes, scientists are now frequently urged to look for patterns of expression and not be concerned about understanding the identity or function of specific markers. The large variety of methods presently available to identify markers for cancer cell detection brings with it the challenge to process and evaluate the clinical usefulness of this information. The new fields of genomics, bioinformatics, and proteomics are emerging to help scientists cope with the barrage of data that results from these analyses. All these techniques seek to establish a molecular profile for cancer cells and associate certain markers or patterns of gene expression with specific subsets of patients with cancer. Identified markers may help in the understanding of oncogenesis and metastasis and serve as targets for intervention. Distinct molecular profiles may be associated with specific stages and prognosis patterns. Ultimately, it is
For decades, scientists have been in search of cancer markers that can distinguish cancer cells from normal cells to accurately diagnose and stage and effectively treat and monitor the disease. The major tissues of interest for breast cancer are breast tissue, bone marrow, axillary lymph nodes, and blood. Candidate targets include oncogenes, tissuespecific and tumor-associated markers, and markers associated with angiogenesis and metastasis. Gene products were initially sought as cancer markers. Cytokeratins (eg, K19, K20) and proliferation markers (Ki-67) remain the standard protein markers for breast cancer detected in tissue by immunohistochemistry. Promising circulating protein markers secreted or shed from the surface of breast tumor cells include carcinoembryonic antigen, CA15-3, and HER-2/neu oncoprotein, although American oncologists consider the clinical data not sufficient to recommend their routine use for breast cancer diagnosis or postoperative surveillance. At the chromosomal DNA level, loss of heterozygosity, gene rearrangements, translocations, mutations (BRCA-1 and -2) and amplifications (her-2/neu) have been found in specific groups of patients with breast cancer. Genomic comparative hybridization has been used to investigate gene copy number aberrations. Although extremely valuable in subsets of patients, such cancer-associated changes at the genomic DNA level do not occur in the majority of patients with cancer. From the Department of Surgery, The Brody School of Medicine at East Carolina University, Greenville, NC. Reprint requests: Kathryn M. Verbanac, PhD, Department of Surgery, The Brody School of Medicine at East Carolina University, 4S16 Brody Medical Sciences Bldg, Greenville, NC 27858. Email: [email protected] Copyright © 2001 by Churchill Livingstone威 1084-8592/01/0602-0001$35.00/0 doi: 10.1084/modi.2001.24167
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74 Molecular Diagnosis Vol. 6 No. 2 June 2001
hoped that certain markers or expression patterns can detect disease at an earlier stage, as well as predict responsiveness to treatment, and thus lead to the selection of surgical and medical treatment tailored to each patient.
Marker Expression in Primary and Metastatic Breast Tumors In this issue of Molecular Diagnosis, Houghton et al. use a combination of molecular techniques, including genetic subtraction, DNA microarray analysis, and real-time PCR, to generate a transcriptional profile of breast cancer. This study documents marker expression in primary and metastatic breast tumors and in blood from patients with breast cancer. Three promising new markers are reported in addition to the previously reported mammaglobin. The investigators suggest that these genes may serve as diagnostic targets and candidates for T-cell vaccine development. Houghton et al. have recognized, as have others in the field, that a single marker is unlikely to be expressed in every patient with breast cancer and absent from all relevant normal cells. This report details their search for a combination of markers most likely to be useful clinically. Houghton et al. have also appropriately focused on mammaglobin as the most promising breast tissue–specific marker. Mammaglobin is a breast-specific marker first described in 1996 that is expressed in normal breast and frequently overexpressed in breast tumors [1,2]. Our laboratory first reported the use of mammaglobin for the detection of sentinel axillary lymph node metastases as we sought to develop a panel of markers for RT-PCR detection of breast cancer micrometastases to improve staging accuracy [3]. Mammaglobin, in marked contrast to the majority of markers tested, was not detected in normal lymph nodes from patients without cancer. We found that RT-PCR for mammaglobin increases the detection of breast cancer sentinel lymph node micrometastases, upstaging 27% of patients with histologically negative lymph nodes [4,5]. Combined with another promising marker, carcinoembryonic antigen, up to 44% of histologically negative patients would be upstaged. Other investigators have since reported mammaglobin messenger RNA (mRNA) expression in nodes of patients with breast cancer by in situ hybridization or RT-PCR [2,6–8]. RT-PCR analysis for mammaglobin is clearly a spe-
cific and sensitive method to detect breast cancer metastases to lymph nodes. Clinical recurrence will determine the significance of molecular staging, and long-term follow-up of patients enrolled into our multicenter trial is ongoing. Although Houghton et al. correctly state that mammaglobin expression is not a universal feature of breast cancer, data from our laboratory and others indicate that mammaglobin expression at the molecular level approaches universal. Using RTPCR, our laboratory has detected mammaglobin in 100% (7 of 7) of human breast cancer cell lines tested, 100% (35 of 35 specimens) of primary tumors tested, and 94% (41 of 45 specimens) of patients with histologically positive lymph nodes [3–5]. Other groups reported the molecular detection of mammaglobin expression in 98% to 100% of primary tumors [6–8] and 71%, 99%, 100%, and 100% of histologically positive lymph nodes [2,6– 8]. These data indicate a much greater expression level than the report of Houghton et al.; 70% of tumors in a panel which consisted of 46 primary and metastatic breast cancers. It would be helpful to have a full description of patient specimens to best interpret the extensive molecular analysis that Houghton et al. present. For example, it is surprising to find that lymph nodes containing breast tumor metastases appear to express more copies of the markers (normalized to actin) than the breast tumors themselves. It would be useful to know whether these nodes were positive for disease by hematoxylin and eosin staining or only positive by immunohistochemical analysis. More information on the patient sources would also be of general value to aid in understanding the molecular analysis. This could include, e.g., the number of patients from which the metastatic lymph nodes were derived and whether primary tumors and metastatic lymph nodes were derived from the same patient(s). More detailed specimen information would also affect conclusions about complementation of marker expression with other genes. These types of data will be critical for the future evaluation of the practical significance of molecular analyses. As discussed, our laboratory and others have established clearly that mammaglobin expression is absent from normal lymph nodes of patients without cancer. It is anticipated that the investigators will similarly analyze normal lymph nodes for expression of their newly discovered markers.
Commentary
Clearly, this is the critical specificity control tissue for nodal metastases. In our hands, we have found this to be the single most important criterion for marker selection. Although most marker candidates tested in our laboratory were expressed in all breast cancers tested, they were also expressed in 30% to 100% of normal lymph nodes from patients without cancer and thus lacked the necessary tumor specificity to provide accurate analysis of clinical specimens [3].
Detection of Circulating Neoplastic Cells Houghton et al. are astute in recognizing the potential prognostic value of RT-PCR for the detection of circulating tumor cells in peripheral blood of patients with breast cancer, especially with the use of selective markers. The detection of circulating tumor cells or markers may offer potential advantages to patients with breast cancer at several stages: (1) presurgery levels may reflect tumor burden and have prognostic significance; (2) postsurgery levels may allow the detection of subclinical disease, earlier detection of relapse, and thus earlier implementation of treatment; and (3) postsurgery levels may be indicative of response to chemotherapy and other forms of adjuvant therapy. More than a dozen potential markers have been investigated for detecting circulating breast cancer cells or soluble proteins; however, few have been found to be widely useful clinically. Watson et al. were the first to report the use of mammaglobin as a marker for circulating breast tumor cells [2]. Peripheral-blood stem cells from patients undergoing high-dose chemotherapy and autologous stem cell transplantation for metastatic breast cancer were obtained and subjected to RTPCR and probe hybridization to detect mammaglobin mRNA. Although mammaglobin mRNA was never detected in peripheral-blood stem cells from healthy donors, in 9 of 15 cases (60%) of metastatic breast cancer, peripheral-blood stem cells contained detectable mammaglobin mRNA, indicating the presence of circulating breast tumor cells. This number is similar to the detection level of mammaglobin in blood samples from the patients with metastatic cancer reported by Houghton et al. in this issue, using immunobead capture and RT-PCR. The validity of this approach and the utility of mammaglobin have been confirmed by two other
•
Verbanac 75
European research groups. Zach et al. reported the use of a nested RT-PCR assay for mammaglobin RNA to detect circulating tumor cells [9]. Although not detected in healthy volunteers, mammaglobin transcripts were detected in the peripheral blood of 25% of 114 patients with breast cancer tested, 49% of those with known metastatic disease. A recent study by Gru¨newald et al. [10] also used nested RT-PCR to detect mammaglobin in blood samples of 8% of patients with invasive breast cancer. Notably, mammaglobin expression in the blood correlated with nodal status, metastasis, and CA-15-3 serum levels in this study. Houghton et al. have done an excellent job of showing the exquisite sensitivity of the blood RTPCR assay using immunobead capture. However, conclusions about marker specificity and complementation based on blood data should await analysis of additional normal blood samples since only 11 normal blood samples were examined for each marker. The finding that one of six normal blood samples (17%) expressed ␥-aminobutyrate type A receptor subunit suggests limited utility of this marker. Conclusions about marker specificity and complementation are complex in the absence of clinical data. It is important to study marker expression in relation to the time that the blood samples were procured relative to diagnosis, surgery, and treatment. Because it is well documented that tumor cells are released into the blood as a result of surgery, the detection of tumor cells is expected in most patients in the first weeks after surgery, but not necessarily in every patient months or years after surgery. (That is, 100% of all patients with metastatic breast cancer are unlikely to have circulating tumor cells. This should not be the endpoint for marker complementation studies.) Many other clinicopathological characteristics, including tumor size, nodal status, her-2/neu, and estrogen receptor/ progesterone receptor status would also be expected to correlate with the detection of circulating tumor cells. Finally, the type of therapy and timing of the blood procurement within the treatment course are important. Without more complete normal blood analysis and clinical correlates, it is difficult to predict whether marker detection and complementation are appropriate in all patient samples or may be inappropriate and even reflect a false-positive result. Without clinical follow-up, it is not known whether the lack of marker detection represents the
76
Molecular Diagnosis Vol. 6 No. 2 June 2001
lack of tumor cells that express the particular marker, a true absence of tumor cells, or assay insensitivity. Clearly, the clinical relevance of marker detection in the peripheral blood of patients with breast cancer should be evaluated in prospective studies. The merit of mammaglobin as a candidate for vaccine development warrants discussion. Mammaglobin is a secretory protein. Because there are no reports of mammaglobin localization on cell membranes, mammaglobin would thus have no utility as a target for antibodies, which recognize conformational determinants on cell-surface proteins. Mammaglobin-derived peptides are undoubtedly presented by cell surface major histocompatibility complex molecules and could theoretically serve as T-cell targets. However, T cells specific for selfantigens (such as mammaglobin) are normally deleted in the thymus during development, and those that might escape to the periphery are rendered functionally tolerant by a variety of mechanisms. Even if mammaglobin-reactive T-cell clones were present in the periphery and activated by a mammaglobin cancer vaccine such that immune selftolerance was broken, normal breast tissue would also be a target for these T-cell effectors. Most normal breast tissue expresses significant levels of mammaglobin and would be expected to express major histocompatibility complex–mammaglobin peptide targets; thus, a high therapeutic index (tumor to normal tissue ratio) would seem difficult to achieve. To conclude, the study reported by Houghton et al. uses a variety of complementary techniques to identify and characterize promising markers for the detection of occult breast cancer cells and should serve to further the field of molecular detection. Without a doubt, the molecular diagnosis of malignancy will become a routine part of many clinical evaluations. HER-2/neu is perhaps the best example of a marker that has made the bench-to-bedside transition and has had a positive impact on the lives of many patients with breast cancer. Follow-up studies of patients with melanoma and colon carcinoma indicate that molecular detection of micrometastases in lymph nodes [11–14] and blood [11,15] is prognostic and has clinical significance. We may also predict that the molecular detection of occult tumor cells will have a significant impact on future oncologic diagnosis and treatment. It has been suggested that micrometastatic tumor cells are biologi-
cally distinct from solid metastatic tumors and thus are likely to be responsive to different treatments [16]. The detection of micrometastases thus poses a challenge to research scientists and physicians to develop a better understanding of both the occult tumor cell and the clinical implications of its detection. Received February 2, 2001. Accepted February 7, 2001.
References 1. Watson MA, Fleming TP: Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 1996;56:860–865 2. Watson MA, Dintzis S, Darrow CM, et al.: Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res 1999;59:3028–3031 3. Min CJ, Tafra L, Verbanac KM: Identification of superior markers for PCR detection of breast cancer metastases in sentinel lymph nodes. Cancer Res 1998;58:4581–4584 4. Verbanac KM, Flemin T, Min CJ, Purser S, Tafra L: RT-PCR increases detection of breast cancer sentinel lymph node micrometastases. Breast Cancer Res Treatment 1999;57:41 (abstr) 5. Verbanac KM, Min CJ, Purser SM, et al.: RT-PCR analysis for mammaglobin and carcinoembryonic antigen detects metastases in histology-negative lymph nodes. Breast Cancer Res Treat 2000;64:37 (abstr) 6. Leygue E, Snell L, Dotzlaw H, et al.: Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol 1999;189:28–33 7. Kataoka A, Mori M, Sadanaga N, et al.: RT-PCR detection of breast cancer cells in sentinel lymph nodes. Int J Oncol 2000;16:1147–1152 8. Ooka M, Sakita I, Fujiwaa Y, et al.: Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients. Oncol Rep 2000;7:561–566 9. Zach O, Kasparu H, Krieger O, Hehenwarter W, Girschikofsky M, Lutz D: Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA. J Clin Oncol 1999;17:2015–2019 10. Gru¨newald K, Haun M, Urbanek M, et al.: Mammaglobin gene expression: A superior marker for breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin19. Lab Invest 2000;80:1071–1077 11. Mori M, Mimori K, Ueo H, et al.: Clinical signifi-
Commentary
cance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcriptase-polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J Clin Oncol 1998;16:128–132 12. Liefers GJ, Cleton-Jansen A-M, Van de Velde CJH, et al.: Micrometastases and survival in stage II colorectal cancer. N Engl J Med 1998;339: 223–228 13. Li W, Stall A, Shivers SC, et al.: Clinical relevance of molecular staging for melanoma. Ann Surg 2000; 231:795–803
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