The concept of collecting serum or plasma in the clinic is straightforward, and in general, standard operating procedures are well established. This ease of collection and general acceptance of individuals to provide blood specimens, makes this fluid an ideal source for developing diagnostic assays. However, variability exists for each patient sample depending on the facility or clinic setting where the blood is drawn with respect to processing and transporting of the specimens. Except for specialized centers, blood samples additionally could be coming from clinic visits to general practitioners, surgeons or oncologists, reflecting multiple points along a diagnostic and treatment course. Standardizing this collection to reflect the many potential phases of breast cancer diagnostics remains a challenge. However, the overall breadth of collection remains attractive for proteomic-based studies, as samples can easily be stratified to reflect breast cancer-related clinical parameters such as menopause/age, body mass index (BMI)/obesity, race, pathology stage, cell origin (ductal vs. lobular), ER/PR/Her2 status, and genetic subtypes.
One model serum collection cohort that could be used to design protein-based diagnostic assays is as follows. For the past 5 years, we have collected over 1,000 serum samples at two sites in a study design focused on women given a BIRADS 4 mammogram report. The Breast Imaging-Reporting and Data System (BIRADS) is used by radiologists to make treatment or follow-up recommendations by categorizing mammographic findings based on the likelihood of malignancy (Obenauer et al., 2005
) across seven categories ranging from 0 (“incomplete assessment”) to 6 (“confirmed malignant”). A BIRADS 4 lesion is categorized as a “suspicious abnormality,” even though the majority (80%) will be benign conditions. Because a malignancy cannot be ruled out, a biopsy will be performed, even for those lesions further stratified into BIRADS 4a, 4b, and 4c (Lazarus et al., 2006
), with a 4a lesion designation as a “low level of suspicion.” Thus, each year, a large number of women undergo a biopsy that will yield a benign diagnosis. Beyond pathological cytology, biomarkers of benign disease from any sample type, genetic, metabolite or protein, are lacking.
Our study was designed to collect prebiopsy serum from women with a BIRADS 4 mammogram who were about to have a breast biopsy procedure. The ultimate goal is to develop a proteomic-based blood test that would be used prior to biopsy that could better discriminate those BIRADS 4 designated women that are more likely to have benign disease, or confirm cancer suspicions, and therefore better justify a tissue biopsy decision. The specifics regarding sample collection have been reported (Shin et al., 2006
), and are summarized as follows. Patients underwent either stereotactic or ultrasound-guided core biopsy, or needle localized excisional breast biopsy. After informed consent, which included a short questionnaire for assessment of each subject's risk factors for breast cancer, serum was collected immediately prior to the biopsy procedure. The questionnaire data included age, race, BMI, age at menarche, parity, age at first live birth, and age at menopause. Each serum specimen was assigned a diagnosis retrospectively after final pathology review. As would be expected, final pathologies for the benign cohort included fibroadenomas, fibrocystic disease, papillomas, atypical ductal hyperplasia without associated carcinoma or carcinoma in situ
, lobular carcinoma in situ
, and benign breast tissue (Shin et al., 2006
). The cancer cohort included ductal carcinoma in situ
and primarily early stage invasive ductal and lobular carcinomas (Shin et al., 2006
). An initial cohort was used for extensive MALDI-TOF serum protein profiling following weak cation exchange affinity bead enrichment. Expression of a panel of 12 low molecular weight peptides could be used with a genetic algorithm to correctly classify 88% of the cancer samples and 86% of the benign disease samples (Shin et al., 2006
). Despite evidence of protein differences between the two disease classifications, use of this single affinity method alone has proven to be not sensitive enough to routinely distinguish cancer from benign disease classifications.
The advantages of using this type of serum cohort center on the study design, as several known biases related to sample collection are minimized. All of the samples are collected in the same way in a similar clinic environment, from patients with the same BIRADS 4 designation. Because it is done just prior to biopsy, there is no diagnosis at the time of collection, so psychological and stress issues for each subject are similar. All associated risk factor information for breast cancer is collected, and can readily be combined with subsequent pathology and genetic properties associated with each sample. The ease and breadth of collection also facilitate their use in any developed high throughput detection platform. The clinical decisions made that brought each subject to the point of biopsy are also very similar. Because the BIRADS 4 diagnosis is based on a radiological test, samples are collected from all possible types of malignant and benign breast diseases. The collection protocol itself is potentially transportable to any breast cancer-related surgical facility that routinely performs breast biopsy procedures. Overall, collection protocols can be standardized and readily integrated into current clinical decision-making workflow associated with a benign or malignant diagnosis.
The disadvantages to use of this cohort are multifaceted. The primary disadvantage is related to all of the challenges of developing any blood-based protein biomarker diagnostic for cancer. A core problem is the enormous dynamic protein concentration range of blood proteins, with 20 proteins accounting for over 98% of the total protein amounts. Detecting a cancer specific protein in blood derived from the tumor is thus hampered by the very low concentrations of these proteins that would be present in blood. Previous studies that have used proteomic profiling approaches for detecting low molecular weight serum proteins (reviewed in Laronga and Drake, 2007
) including our own BIRADS 4 study (Shin et al., 2006
), are weakened by lack of protein identification and the dynamic concentration range issues (Anderson and Anderson, 2002
; Diamandis, 2004
), as well as study design bias, overgeneralization of results, and sample processing issues (Hu et al., 2005
; McLerran et al., 2008a
; Ransohoff, 2005
). Although our BIRADS 4 collection strategy addresses the bias and processing issues, the dynamic concentration range and tumor specificity remains a large barrier to assay development. There is also a sample collection time line issue in our approach, in that we have to collect mostly benign disease samples (4 to 1) to obtain enough cancers to have statistically relevant numbers for even the most basic breast cancer subtype stratifications based on BMI, age/menopause status, tumor pathology, and tumor genetic subtypes. Addressing these cumulative weaknesses form the basis for the proposed glycoprotein targeting strategy described in the next section.