Detection of Occult Micrometastases in Non-Small Cell Lung Carcinoma by Reverse Transcriptase-Polymerase Chain Reaction

doi:10.1378/chest.113.6.1526

Chest

Volume 113, Issue 6, June 1998, Pages 1526-1532

https://doi.org/10.1378/chest.113.6.1526 Get rights and content

Background

The 5-year survival rate following surgical resection of Stage I or Stage II non-small cell lung carcinoma (NSCLC) is 30% to 50%, probably because of undetected occult micrometastases (OMs) at the time of surgery. Other investigators have detected OMs in bone marrow and histologically negative lymph nodes from patients with NSCLC using immunohistochemical staining to cytokeratins and cell surface glycoproteins.

Study objective

To develop and evaluate an assay based on the reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of OMs in NSCLC.

Patients

Twenty-eight patients with benign or malignant thoracic pathology. Samples of primary tumors and lymph nodes were collected at the time of surgical resection or mediastinoscopic lymph-node biopsy.

Results

Using RT-PCR to detect messenger RNA (mRNA) transcripts for MUC1 (a cell surface glycoprotein present in lung tissue but absent from normal lymph nodes), OMs were identified in 33 of 88 lymph nodes determined to be free of tumor by hematoxylin and eosin staining. Eleven of 11 control mediastinal lymph nodes from patients without malignancy failed to express detectable MUC1 transcripts. Dilutional experiments demonstrate that the assay can detect one MUC1-positive NSCLC cell in 1×10⁷ MUC1-negative cells. A comparison of our RT-PCR assay to immunohistochemistry specific for the MUC1 glycoprotein suggests that RT-PCR may be more sensitive than immunohistochemistry for the detection of NSCLC OMs.

Conclusions

This study demonstrates that RT-PCR for MUC1 mRNA can detect the presence of MUC1 mRNA in histologically negative lymph nodes from patients with NSCLC. The prognostic significance of these findings is currently unknown.

Section snippets

Tissue Samples and Cell Lines

Institutional review board approval was obtained prior to initiating the study. Twenty-three patients with suspected or proven NSCLC who were undergoing either surgical resection or staging via mediastinoscopic lymph node biopsy participated in this study. Informed consent was obtained from all patients prior to tissue collection. From these patients, 18 primary tumors, 60 mediastinal lymph nodes, and 43 peribronchial lymph nodes were obtained. In five patients who underwent only

Assay Sensitivity

The DNA-based assay was determined to be sensitive enough to detect MUC1 mRNA at a concentration of as low as 1 cancer cell (NCI-H2009) per 10⁷ mouse fibroblast cells (NIH 3T3). The degree of amplification was not quantitative in the cell line tested (Fig 1).

Assay Specificity

The H2009 NSCLC cell line was included as a positive control in each analysis. This cell line demonstrated expression of MUC1 mRNA every time it was tested. As expected, normal lung tissue also demonstrated expression of MUC1 mRNA.⁴⁰

DISCUSSION

This study has demonstrated that RT-PCR with primers specific for MUC1 mRNA can amplify a 248-bp fragment from RNA isolated from normal lung tissue, NSCLC cell lines, primary NSCLC tumors, and mediastinal lymph nodes collected from patients with NSCLC. The RT-PCR assay described has the ability to detect a single NSCLC cell in 10 million control cells. Furthermore, the RT-PCR assay may be more sensitive than immunohistochemical staining for the MUC1 glycoprotein.

In this study, MUC1 mRNA was

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The basic anatomic lymph node drainage patterns from lung cancers have remained relatively constant since their early descriptions. Sentinel lymph node mapping and other reviews of anatomic resections have provided additional information regarding drainage patterns of lymphatic metastases. In light of these efforts, topics related to lymphatic metastases, such as skip metastasis and micrometastasis, still remain the subject of investigation. A review of the recent literature shows that the incidence of macroscopic skip metastasis is approximately 25%. Despite the occurrence of skip metastases, a generalized lymphatic drainage pattern is observed and is consistent with the drainage patterns that are observed among nonskip metastases to the mediastinum. Direct mediastinal drainage or the inability to detect micrometastatic disease due to observational errors or technical inadequacies may explain the presence of skip metastasis. This is supported by the fact that a review of the recent literature also shows that the incidence of micrometastasis is approximately 19%. The similarities between these 2 values suggest that an improved ability to detect micrometastatic disease is needed. This manuscript reviews the patterns of lymph node drainage from a historical and current perspective to facilitate an understanding of the existing knowledge with respect to skip metastasis and micrometastasis.
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Various authors demonstrated the presence of these metastases in patients with breast [21], colon [7], prostate [22], and gastric cancers [23]. Other investigators have demonstrated that the immunohistochemical detection of micrometastases in NSCLC is possible and correlate with a higher recurrence rate and poorer survival rate [24]. More recently, efforts have been made to detect micrometastases in LNs at molecular levels.
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Lymph node involvement is the most important factor affecting the prognosis and treatment of patients with potentially resectable NSCLC. Radiographic imaging is inadequate to ascertain lymph node involvement. Currently, lymph nodes are assessed pathologically using conventional histologic techniques; however, lymph node micrometastases may be missed, leading to inaccurate staging and suboptimal treatment. Assessment of occult involvement using antibody expression improves the sensitivity of lymph node analysis, and more advanced techniques, using molecular biologic methods, may further improve lymph node staging. Optimizing outcomes of patients with lung cancer depends on accurate lymph node staging, and the development of the strategies that improve the assessment of lymph node micrometastases will be beneficial.
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Active tumor immunotherapy may provide hope for patients with non–small-cell lung cancer (NSCLC) because, in more than 20 years, current therapies have yet to change mortality statistics. Creating an efficacious vaccine involves selection of important tumor antigens and formulation of their immunogenic epitopes into a construct for delivery to antigen-presenting cells. The method of immunization will confer significant properties to the potency of the vaccine and might require augmentation with certain adjuvant agents like interleukin- 12 and granulocyte-macrophage colony-stimulating factor. So far, clinical trials in NSCLC immunotherapy have shown promise with the induction of immune responses and the presence of clinical responses compared with historical controls treated with standard therapy. Immunotherapy could merge seamlessly into the current standard of care for NSCLC with the emergence of data supporting a beneficial role of chemotherapy and radiation in the production of antitumor immune responses. With continued work in this field, active immunotherapy may provide the necessary therapy for the successful treatment of this common disease.
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: Funded by the Department of Surgery, University of Minnesota and by a grant from the Minnesota Medical Foundation.

: Reprint requests: Christopher T. Salerno, MD, Department of Surgery, University of Minnesota Hospital and Clinics, 420 Delaware St SE, UMHC Box 207, Minneapolis, MN 55455

View full text

Chest

Clinical Investigations: Lung CancerDetection of Occult Micrometastases in Non-Small Cell Lung Carcinoma by Reverse Transcriptase-Polymerase Chain Reaction

Background

Study objective

Patients

Results

Conclusions

Section snippets

Tissue Samples and Cell Lines

Assay Sensitivity

Assay Specificity

DISCUSSION

Chest

Lancet

Ann Thorac Surg

Curr Opin Immunol

Urology

Eur J Cancer

Gene

Biochem Biophys Res Commun

Rb and pl6INK4a expression in resected non-small cell lung tumors.

Cancer Res

Immunocytological detection of bone marrow micrometastases in operable non-small cell lung cancer.

Cancer Res

Immunohistochemical assessment of individual tumor cells in lymph nodes of patients with non-small cell lung cancer.

J Clin Oncol

Frequency and distribution of occult micrometastases in lymph nodes of patients with non-small cell lung carcinoma.

J Natl Cancer Inst

Micrometastases from squamous cell carcinoma in neck dissection specimens.

Eur Arch Otorhinolaryngol

Epithelial tumor cells in bone marrow of patients with colorectal cancer: immunohistochemical detection, phenotypic characterization, and prognostic significance.

J Clin Oncol

The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma.

J Pathol

Identification of occult micrometastases in pericolic lymph nodes of Dukes' B colorectal cancer patients using monoclonal antibodies against cytokeratin and CC49.

Cancer

Identification of submicroscopic lymph node metastases in patients with malignant melanoma.

Semin Surg Oncol

Detection of submicroscopic lymph node metastases in patients with melanoma.

Arch Surg

The immunohistochemical detection of lymph node metastases from infiltrating lobular carcinoma of the breast.

Br J Cancer

Demonstration of mucinous-like carcinoma-associated antigen in bone marrow and lymph node biopsies from patients with breast carcinoma.

Int J Biol Markers

Detection of occult metastatic lobular carcinoma in axillary lymph nodes using anticytokeratin monoclonal antibodies.

Conn Med

Detection of micrometastases from primary breast cancer.

Can J Surg

Detection of micrometastatic tumor cells in bone marrow of breast carcinoma patients.

J Surg Oncol

Immunohistochemical detection and significance of axillary lymph nodes in breast carcinoma.

Anal Quant Cytol Histol

A preliminary report on the usefulness of monoclonal antibodies to CA-153 and MCA in the detection of micrometastases in axillary lymph nodes draining primary breast carcinoma.

Eur J Cancer

Prediction of early relapse with operable breast cancer by detection of occult bone marrow micrometastases.

J Clin Oncol

Clinical Investigations: Lung Cancer
Detection of Occult Micrometastases in Non-Small Cell Lung Carcinoma by Reverse Transcriptase-Polymerase Chain Reaction

Rb and pl6^INK4a expression in resected non-small cell lung tumors.