Authors: Kaidi Mikhitarian (corresponding author) [1,5]; Renee Hebert Martin [2]; Megan Baker Ruppel [1]; William E Gillanders [1,6]; Rana Hoda [3]; Del H Schutte [4]; Kathi Callahan [1]; Michael Mitas [1]; David J Cole [1]
Background
There is a significant amount of ongoing work aimed at defining the role of circulating tumor cells (CTC) in peripheral blood (PBL) and disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients. However, due to a variety of available tumor cell detection methods and use of different gene-markers, recently published studies show a wide range of results that are often contradictory and difficult to compare to one another. The main tumor cell detection methods have been immunocytochemistry (ICC) with cytokeratin-specific antibodies [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] and RT-PCR analysis based on overexpression of cancer-associated gene-markers [4, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29]. PCR methodology for detection of breast cancer has most frequently employed mammaglobin (
Our laboratory has extensive experience in detection of cancer cells using multi-marker real-time RT-PCR methodology [31, 32, 33, 34, 35]. To address the clinical relevance of molecular detection of occult breast cancer, we initiated a multi-institutional prospective cohort study. The primary objective of the study was to determine whether the molecular detection of occult breast cancer by multi-marker real-time RT-PCR in patients with pathology-negative axillary lymph nodes (ALN) is a clinically relevant predictor of disease recurrence. An interim analysis of 489 patients enrolled in the study showed a statistically significant association between molecular detection of occult breast cancer in the ALN and traditional predictors of poor prognosis in subjects with pathology-negative ALN [33]. In addition, in a separate publication we show that the sensitivity of sentinel lymph node (SLN) analysis to predict pathologic status of ALN was significantly increased by the addition of molecular analysis [34].
There are several cancer-associated gene markers used in the detection of breast cancer cells. Based on the heterogenous nature of the breast cancer, the multi-marker panel approach has shown to increase the sensitivity of molecular assay to detect the presence of disseminated cancer cells. However, the prognostic value of each individual marker is not known and therefore the ultimate goal would be to identify genes that are capable of differentiating patients with poor prognosis from the patients with a more favorable prognosis. Having a tool to recognize the subset of patients with unfavorable molecular characteristics could potentially translate into a better clinical outcome. In this interim analysis we examine the detection rate of cancer cells in PBL and in BM using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with the traditional predictors of prognosis.
Methods
MIMS Trial Study Design
A prospective cohort study design was adopted where, upon recruitment, eligible participants with Stage I, IIa, or IIb breast cancer were requested to consent to tissue sampling from axillary lymph nodes (ALN), sentinel nodes (SLN), bone marrow (BM), and peripheral blood (PBL). Tissue sampling was accomplished at the time of surgical intervention. The study was carried out in compliance with the Helsinki Declaration ethical principles in medical research involving human subjects. All specimens were collected through the Medical University of South Carolina Institutional Review Board for Human Research approved protocols (HR 9551, HR 8374, HR 8903, HR 8432). Informed consent was obtained in accordance with each participating center's Institutional Review Board guidelines. The design, enrollment criteria, tissue acquisition protocols, and determination of gene expression values for patients enrolled in the MIMS trial are described in more detail in a separate publication [33]. The current study focuses on the subset of 215 patients with PBL samples and the subset of 177 patients with BM samples. Real-time RT-PCR analyses for cancer-associated genes were performed on all specimens at the Central Molecular Diagnostics Laboratory at the Medical University of South Carolina (MUSC). The Clinical Innovation Group (TCIG, Charleston, SC) (later known as the Data Coordination Unit (DCU) in the Department of Biostatistics, Bioinformatics and Epidemiology at MUSC) served as the coordinating center, and all study data were collected, processed and analyzed at this central facility.
Blood and bone marrow samples from breast cancer subjects
Bone marrow aspirates were obtained from patient's left and or right anterior or posterior iliac crests under anesthesia at the time of operation. A 10 or 20 cc syringe with a 16-18 gauge bone marrow aspirate needle was used to aspirate 3-6 ml of bone marrow into a syringe and then immediately transferred to a sterile EDTA vacutainer. Peripheral blood samples were obtained before surgery or following the induction of anesthesia. A total of 5-10 ml of blood was drawn from a peripheral vein into a sterile EDTA vacutainer. Blood and bone marrow samples were then shipped at room temperature to the Central Molecular Diagnostics Laboratory at the MUSC for immediate processing by Ficoll density gradient centrifugation (Ficoll-Paque Plus; Amersham Biosciences). All the specimens inside US arrived in 24 hours and international shipments arrived in 48 hours. One mL of bone marrow was used for Cytospin preparation and stained for ICC analysis. These bone marrow samples were evaluated by a cytopathologist for the presence of micrometastases using cytokeratin AE1/AE3. Please note that the specimen acquisition protocol was amended after the initiation of the MIMS trial and for that reason only a subset of patients was included in this analysis.
Blood and bone marrow samples from control subjects without evidence of malignancy
In order to define baseline expression levels for the molecular markers used in this study, PBL and BM samples from control subjects were procured. Informed consent was obtained for BM aspiration from 49 patients undergoing orthopedic surgery at MUSC and for PBL drawn from 49 healthy volunteers. None of the control subjects had any history or clinical evidence of malignancy. Four to six ml of BM aspirate or 5-10 ml of PBL was transferred to an EDTA vacutainer and sent to the Central Molecular Diagnostics Laboratory to be processed by Ficoll density gradient centrifugation and analyzed by real-time RT-PCR.
RNA isolation and cDNA synthesis
Buffy coats were obtained by Ficoll density gradient centrifugation, and total cellular RNA was isolated using a guanidinium thiocyanate-phenol-chloroform solution (RNA STAT-60[TM]; TEL-TEST, Friendswood, TX). Briefly, cells were re-suspended in 1 ml of RNA STAT-60[TM]. Total RNA was isolated as per the manufacturer's instructions with the exception that 1 [mu]L of a 50 mg/mL solution of glycogen (Sigma, St. Louis, MO) was added to the aqueous phase prior to addition of isopropanol. Glycogen was used as a nucleic acid carrier to enhance RNA precipitation. The RNA pellet was dissolved in 50 [mu]l of 1x RNA secure buffer (Ambion, Austin, TX). RNA was quantified by spectrophotometry at 260 nm. cDNA was made from 5 [mu]g of total RNA using 200 U of M-MLV reverse transcriptase (Promega, Madison, WI) and 0.5 [mu]g Oligo …
Комментариев нет:
Отправить комментарий