Tumor Specific Plasma DNA

In 2011, there was an estimated 233,000 cases of invasive breast cancer, and 39,970 deaths
from breast cancer in the United States. The vast majority of patients are diagnosed with
Stage I-III resectable and potentially curable disease, and for these patients, the most
pressing questions are whether adjuvant endocrine or chemotherapy are indicated, and if so,
how to determine whether these treatments are working. Adjuvant systemic therapy reduces
relative recurrence rates by 30-50%, depending on the age of the patient and tumor
characteristics. However, patients with early stage disease often do not bear measurable
markers of disease such as an elevated cancer antigen 27-29 (CA27.29) or circulating tumor
cells. Patients with early stage breast cancer are typically treated with adjuvant therapy
based on historical evidence showing that such therapy prolongs survival in this population.

Lung cancer is the most common malignancy and the leading cause of cancer-related death in
the U.S. Approximately 220,000 new cases of lung cancer are diagnosed in the U.S. every year.
Unfortunately, lung cancers are often diagnosed at later stages than breast cancer, due in
part to little/no effective screening for lung cancer. As with breast cancer, patients are
commonly treated with chemotherapeutic agents, but treatment regimens can take several weeks
to months to elicit clinically detectable anti-tumor effects. A biomarker to assess early
tumor response to therapy would benefit this patient population.

The contents of dying tumor cells can be detected in the bloodstream, and this may be
enhanced by the leaky vasculature of solid tumors. Protein biomarkers of tumor cell death are
difficult to detect due to the complex nature of plasma and the lack of technical
sensitivity. In contrast, DNA is easier to detect through polymerase chain reaction (PCR)
amplification. Indeed, circulating tumor DNA has been detected in plasma from patients with
osteosarcoma, breast cancer, and colorectal cancer. Until recently, it was impractical to
develop an assay to routinely quantify circulating tumor DNA due to heterogeneity between
patients and tumors. Advances in genomic technology now permit sequencing a tumor genome to
identify patient-specific genomic aberrations. Major genomic alterations (i.e., insertions,
amplifications, deletions, inversions, translocations) can be readily detected using PCR
primers which will recognize tumor DNA but not normal DNA.

While this strategy may be generally applicable to diverse types of solid tumors, two issues
are apparent in breast cancer. Firstly, the incidence of chromosomal rearrangements varies
widely. Whole-genome sequencing of 15 breast tumors revealed a range of 1-231 major genomic
alterations (mean= 68), where 2 tumors had 1 alteration, and 9 tumors had > 20 alterations.
Single-base point mutations are more common but difficult to reliably detect using PCR.
Therefore, the investigators must consider that a small subset of patients may have a limited
number of genomic alterations available for this assay. Secondly, intratumoral heterogeneity
may mean that some genomic alterations are not present in every tumor cell. Such
heterogeneity has been inferred from FISH and immunohistochemistry (IHC) studies for many
years, and is now being verified at the genomic level. The investigators must consider that
only a subpopulation of tumor cells may be sensitive to cytotoxic therapy, so changes in the
levels of circulating tumor DNA may only be reflected with analysis of genomic alterations
specific to the sensitive cells.