Clinical Utility of Breast Cancer DNA Markers in Serum

Institution: John Wayne Cancer Institute
Investigator(s): David Hoon, MSc, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7WB-0021 Award: $470,520
Award Type: STEP Award
Research Priorities
Detection, Prognosis and Treatment>Imaging, Biomarkers, and Molecular Pathology: improving detection and diagnosis

Initial Award Abstract (2001)
The incidence of early stage breast cancer is rapidly increasing due to advances in methods for its detection. Despite these improvements 20-30% of patients without evidence of metastatic disease at initial diagnosis will subsequently develop a recurrence. New methods to identify high-risk patients requiring aggressive adjuvant therapy and to those not requiring treatment are needed. Because cancer progression has been shown to result from the accumulation of genetic alterations within the cell, assays that identify these genetic events may serve as valuable prognostic indicators. However, studies evaluating tumors are limited as they assess only a single time-point in the disease course and require a biopsy, an invasive procedure that may be associated with potential morbidity. Also this method is not always practical for early stages of disease when detection may be critical. We have recently developed an assay that can identify tumor-associated genetic aberrations from a patient's blood sample. We have demonstrated that these circulating genetic marker alterations correlate with those occurring in the patient's tumors and thus may act as surrogate markers of disease.

Our hypothesis is that the sensitive genetic marker assay of serum will permit a minimally invasive approach to assess those genetic changes that may occur during tumor progression, and their assessment will have prognostic implications. We hypothesize that the serum genetic marker assay will allow earlier detection of breast cancer disease progression and identify disease-free patients who are likely to have disease recurrence within a short time.

We have developed a method to isolate "free" DNA from small amounts of blood and analyze the DNA for tumor genetic markers. The DNA markers are amplified and assessed using highly sensitive instrumentation. In addition to assessing blood, we will also analyze bone marrow. We will develop an array of genetic markers that are frequently found in breast tumors. These markers will then be used to assess blood (serum) isolated from patients taken both before and after surgery. Bone marrow will be assessed as blood is for DNA markers. We focus our efforts on early stage patients, particularly those undergoing sentinel Iymph node mapping, a technique used to identify early disease spreading to the tumor-draining Iymph nodes.

In this proposal one of the innovative approaches will be to use a more robust, rapid, high-throughput, and sensitive instrumentation to improve the assay efficacy.. The unique element of this proposal is that it permits a comprehensive approach for screening early stage breast cancer patients by using highly sensitive techniques for assessing critical sites of early disease spreading: sentinel lymph node(s), blood, and bone marrow. These findings may prove highly predictive of disease recurrence and help staging disease. Finally, more appropriate patient stratification into clinical treatment protocols may occur earlier in the disease course as a consequence of these genetic based tests. This addition of molecular oncology to current clinical and radiographic evaluations for disease screening and diagnosis may prove beneficial in the overall care of breast cancer patients in California.

Final Report (2005)
The development of breast tumors and spreading to distant sites involves the progressive accumulation of multiple numbers of genetic (DNA) aberrations in the breast cancer cells. One common type of DNA aberration is the loss of a DNA segment from a chromosome arm referred to as allelic imbalance (AI). This AI can be detected in all breast cancer tumors when the cancer tissues are assessed directly. The presence of AI continues to accumulate on multiple chromosomes as the tumor progresses and often causes loss of important regulatory genes.

The objective of the project was to determine if free circulating DNA could be detected in blood of breast cancer patients. If this was possible the analysis of patients' tumor would not be needed and most important a blood test could be developed to monitor patients with breast cancer. Our objective was to determine if this was a possible new approach to assess breast cancer. Furthermore, DNA-based tumor genetic testing of blood samples offers an easily accessible route by which to evaluate ongoing tumor genetic events which may have a role of predicting how the tumor will progress in the near future in patients. This approach provides for a minimally invasive method to monitor early breast disease progression.

The project consisted of three aims which were completed during the funding period. Aim 1 was to develop a multiplex capillary array electrophoresis assay to enhance detection of serum LOH markers. We have developed methodologies to allow for high-throughput and detecting multiple DNA markers at the same time from a single blood sample. In conjunction with this assay we developed analytical software programs. This is a unique and significant advance for serial tumor genetic analysis of bloods which has not been previously developed. This advancement will pave the way for new approaches of assessing circulating DNA in body fluids in cancer patients. The development of a robust assay allowed for assessment of patients of different disease status.

AIM 2 was to correlate clinicopathological factors to serum LOH markers. We demonstrated the detection frequencies of multiple AI markers with advancing breast tumor disease. These individual AI markers and combinations of markers were also correlated to known clinical and pathologic prognostic factors associated with breast cancer. Because breast cancer is often diagnosed early and is associated with a prolonged time to recurrence, we are in the process of following these patients with serial blood draws and marker analysis for long-term clinical correlations. These studies are ongoing and will be in the future assessed accordingly.

AIM 3 was to determine the clinicopathologic utility of AI markers in serum and bone marrow fluid of patients undergoing lymph node dissection. This study demonstrated that circulating DNA can be detected in bone marrow. Comparative studies on bone marrow and bloods from the same patients were performed. The studies were performed with different clinical stages of breast cancer. Further clinical follow-up to determine the prognostic and clinical implications of these findings. The significance of the development of an ultrasensitive DNA based assay for blood for early disease progression will advance and improve management of breast cancer. The future studies will involve assessment of circulating DNA in a multicenter treatment trial of breast cancer involving many institutions to valid the significance of the assay. We have initiated this trial which is ongoing accruing patients. Hopefully the assay in the future will be part of panel of test to better diagnose and predict outcome of breast cancer. The impact of the study is that breast cancer genetic, changes can be monitor in blood without the assessment of the actual tumor.

Symposium Abstract (2003)
Despite improvements in methods for early breast cancer detection it remains the leading site of new cancer diagnosis and the second most frequent cause of cancer mortality among women. Lymph node evaluation is an integral part in the assessment of breast cancer spread, however approximately 20% of lymph node negative patients will subsequently develop recurrent disease. Therefore additional methods are needed to enhance identification of those patients at increased risk for relapse. Breast cancer progression has been shown to result from an accumulation of genetic alterations, i.e.: loss of heterozygosity (LOH), within the cell and assays that identify these genetic events may serve as valuable prognostic indicators of disease progression. We have developed a method to isolate from a minimal amount of blood circulating free DNA which harbors genetic aberrations similar to those found in the primary tumor and therefore may serve as surrogate markers of disease.

In this study blood was collected from 71 breast cancer patients as follows: preoperatively from 30 AJCC stage I, 26 stage II and 7 stage III patients, and from 8 patients at the time of stage IV diagnosis. In addition, 24 patients paired bone marrow (BM) aspirates collected at the time of surgery were also available for evaluation. Serum and BM from patients and 35 healthy female donors (30 serum and 5 BM samples) were assessed for LOH using 8 microsatellite markers on 5 chromosomes (8, 10, 14, 16, 17) that frequently demonstrate LOH in primary breast cancers. LOH was identified in 20 (28%) of 71 patientsí serum samples. The incidence increased with advancing stage: 17% (5 of 30) stage I; 27% (7 of 26) stage II; 43% (3 of 7) stage III; and 63% (5 of 8) stage IV. LOH was most frequently identified on chromosome 17 occurring in 7 of 63 informative patients (11%). LOH was identified in 5 (21%) of 24 patientsí BM samples. In this group LOH was only present in 3 match-paired serum samples. No LOH was detected from any of the healthy female donorís serum or BM samples.

These findings demonstrate the successful identification of tumor-specific DNA in the blood and BM of breast cancer patients with correlation to stage of disease. This investigation provides an innovative approach for assessing breast cancer patientsí serum/BM for the presence of molecular surrogates that may identify subclinical disease progression and serve as significant predictors of patient outcome.

Microsatellite alterations detected in the serum of early stage breast cancer patients
Periodical:Annals of the New York Academy of Sciences
Index Medicus: Ann N Y Acad Sci
Authors: Taback B, Giuliano AE, Hansen NM, Hoon DSB
Yr: 2001 Vol: 945 Nbr: Abs: Pg:22-30

Identification of tumor-specific genetic alterations in the bone marrow of early stage breast cancer patients.
Periodical:Proceedings of the American Association for Cancer Research
Index Medicus: Proc Am Assoc Cancer Res
Authors: Taback B, Giuliano A, Hansen N, Singer R, Hoon D
Yr: 2003 Vol: 44 Nbr: Abs: Pg:563

Detection of tumor-specific genetic alterations in bone marrow from early-stage breast cancer patients
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Taback B, Giuliano AE, Hansen NM, Singer FR, Shu S, Hoon DS
Yr: 2003 Vol: 63 Nbr: 8 Abs: Pg:1884-7