Breast cancer is prevalent malignancy among women in worldwide (1). molecular diagnosis is recently proposed as a sensitive method than histological methods (2). detection of breast cancer is routinely performed by histological examination, but 30-35% of node negative patients are not diagnosed that may develop metastasis in next 5 years after local treatment (3). the sensitivity of histological method for micro metastasis detection is already low and is about 20% (4).
sentinel lymph node (sln) is a good predictive of axillary lymph node metastasis in breast cancer and sln analysis during the first surgery, can reduce the cost and low survival time of a second operation (5). using of reliable molecular diagnostic method may help and fill the gap between these subjects. assessing of some genes that are involved in breast cancer metastasis, may help for fast and accurate diagnosis, and with combining of them it is possible to develop the most complete method for breast cancer micro metastasis. to fulfill this need, selection of an ideal marker is very important that would have high specificity and sensitivity close to 100%. mammoglobin is a member of uteroglobin/clara cell protein family (secretoglobins) (6), that is ideal for detection of breast cancer metastasis evaluation in blood, lymph nodes, and bone marrow (7). mammoglobin is expressed in breast epithelial cells (8), and is over expressed in close to 70-80% of primary and metastatic breast cancers (9). in this study the expression status of mammoglobin protein is evaluated as a candidate molecular marker in sentinel lymph nodes using rt-pcr method that in histology examination, are reported as negative.
Methods
Sentinel lymph node preparation: all patients classified as coding groups and all private information was stored and saved secretly by responsible researcher of project. all patients staged according to the american joint committee on cancer (ajcc) standard criteria by the attending surgeon using relevant clinical data and histological analysis. sentinel node localization was determined for non palpable tumors by inserting a kopans needle into the mammographically imaged lesion using stereotactic coordinates, followed by injection of technetium-99m- labeled sulfur colloid.
30 sentinel lymph node rna samples are made available from the tumor bank of pathology department of medical multicenter cancer imam khomeini hospital, tehran medical university. also 10 bresat ductal carcinoma and 1 benign breast tissue were procured from stored personal archive of author. this study is designed to determine the utility of qualitative measurement of mammoglobin protein as marker in lymph nodes to reduction of breast cancer relapse possibility after or during the first surgery. in this order, the node negative (hematoxylin/eosin staining) lymph nodes are marked as case samples and node positive (metastatic samples or h/e positive) also breast cancer tissues are considered as control samples. rna quality was evaluated using eppendorf spectrophotometery and beta-actin was used as housekeeping gene in pcr analysis. rna of lymph node and breast tissue samples were considered to be of poor quality if either of the housekeeping gene gave signals that was at least 3.5 cycles above the mean of lymph node and other samples tested. in reverse transcription and quantitative polymerase chain reaction for samples, the rna was undergone to reverse transcription reaction to make a cdna before performing rt-pcr. the obtained rna was stored in 10μl aliqutes in -20 ͦ c until next processing.
Results
Based on findings, the highest single gene expression was found with 66.7% for
mammoglobin, followed by ck19 50% and mtdh, 26.7%.
Conclusion
Because of the presence of significant expression of at least three genes in 83.3% (40 of the 48) pathology negative patients, it seems that triple marker evaluation of most prevalent expressed gene instead of separate gene, will be most likely useful in metastasis prognosis. in evaluation of other tumor markers such as tumor’s stage, grade, er expression levels there was no significant variation with positive expression results of each genes.
Keywords
Breast cancer, micro metastases, sentinel lymph node, mammaglobin
