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Title: Whole Genome Sequencing of Triple Negative Breast Cancer      
Breast Cancer Subject Participant ID 700064 (Source Sample names: 6888 and 206). We used massively parallel DNA sequencing technologies to screen entire genomes, in an unbiased manner, for genetic changes associated with tumor growth and metastasis. We describe the complete genome sequence analysis of four DNA samples from a 44-year old African-American patient with basal-like breast cancer: peripheral blood, the primary tumor, a brain metastasis that developed within a year of initial therapy, and a first-passage xenograft derived from the primary tumor. A total of 50 validated mutations were discovered within coding, RNA, or splice site sequences. Of these, 20 mutations were abundantly present in all three tumors, including mutations in CSMD1 and JAK2. These two genes subsequently were found to be mutated in other breast tumors. The metastasis contained two de novo mutations not present in the primary tumor, and was significantly enriched for 20 shared mutations, suggesting that they may be involved in the metastatic process. The xenograft contained no unique coding, RNA, or splice site mutations and retained all primary tumor mutations, albeit at different frequencies. However, a significant increase in copy number alterations was observed in the xenograft as compared to the primary tumor. We validated 28 large deletions and six inversions, as well as seven translocations in at least one of the three tumor samples. Among them, a 26 kb deletion in MECR was solely identified, assembled, and validated in the brain metastasis and two overlapping large deletions on chromosome 5 encompassing CTNNA1, a potential tumor suppressor gene, were identified in all three tumors. The differential mutation frequencies and structural variation patterns between primary and metastatic tumors suggest that metastatic tumors may arise from minor subpopulations of cells within the primary. Namely, the metastatic and xenografting processes apparently select for cells harboring a distinct subset of the primary tumor mutation repertoire.
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Breast Neoplasms
Transplantation, Heterologous
Washington University, St. Louis, MO, USA
Richard Wilson, PhD
Principal Investigator
National Institutes of Health, Bethesda, MD, USA
Funding Source
TitleNameInstitute Principal InvestigatorRichard Wilson, PhDWashington University, St. Louis, MO, USA Funding SourceHG002079National Institutes of Health, Bethesda, MD, USA
Data Use Certificate
Cancer Research
A previously healthy, premenopausal 44 year old African American woman presented to the medical oncologist with newly diagnosed inflammatory left breast cancer, clinical stage IIIB, T4dN1M0. Patient's past medical history was insignificant; no prior cancer, hypertension, heart disease or history of any surgical interventions. The patient's family history was obtained by the treating medical oncologist, and was positive for cancer in maternal grandfather with lung cancer, one uncle with throat cancer and another with brain cancer. All three were deceased. Patient reported that she noted that left breast was larger than right. A mammogram performed 3 days later showed no significant masses, calcifications or other abnormalities. Patient subsequently presented to the surgeon following a 3 week history of an enlarging left breast mass with edema, erythema and increasing pain. A left breast excisional biopsy and skin biopsy were performed. Breast biopsy showed invasive ductal carcinoma, poorly differentiated, with extensive lymphatic invasion. The tumor was ER negative (0%), PR positive (11%) and HER2 negative (0) by immunohistochemistry. Skin biopsy showed invasive carcinoma involving deep dermal lymphatics. Staging work-up was obtained prior to initiating neoadjuvant chemotherapy. Bone scan and CT of chest, abdomen and pelvis were negative for distant metastatic disease, positive only for left axillary lymphadenopathy. After informed consent was obtained, the patient was enrolled in an institutional protocol studying the effect of neoadjuvant chemotherapy on breast cancers, bone marrow cancer cells, and circulating cancer cells. Specimens of breast tumor mass, bone marrow and peripheral blood were collected and preserved prior to initiation of neoadjuvant chemotherapy. The patient initiated neoadjuvant chemotherapy with four cycles of dose dense doxorubicin and cyclophosphomide followed by four cycles of paclitaxel. Left breast mastectomy with axillary node dissection performed 3 weeks following chemotherapy completion showed multifocal, poorly differentiated invasive mammary carcinoma with ductal and lobular features. Six of thirteen lymph nodes were positive. Pathologic stage was T2N2. Right breast simple mastectomy showed no evidence of malignancy. Post-treatment specimens of breast mass, bone marrow and peripheral blood were collected and preserved for the effect of neoadjuvant chemotherapy study at that time as well. Patient subsequently received four week course of radiation therapy to the left chest wall and regional lymphatics followed by tamoxifen 20 mg daily. Despite these therapies, patient returned in approximately 11 months from diagnosis complaining of severe right hip pain, nausea and vomiting. Patient was found to have relapsed to the right cerebellum. Also of note: while never pathologically diagnosed, right hip pain was suspected to be due to progressive metastatic disease. Neurosurgical consultation was obtained, and patient was scheduled for sub-occipital craniectomy for resection of right cerebellar lesion. Prior to the procedure, informed consent was obtained for an institutional protocol entitled Longitudinal Cancer Genome Profiling Using Sequential Tissue Biopsies in Patients with Breast Cancer. After ensuring adequate tumor was obtained for diagnostic purposes, specimens from the cerebellar mass were collected and preserved for protocol. Pathology from craniectomy showed metastatic carcinoma, consistent with breast primary. Patient tolerated the procedure well. However, after the procedure, patient's neurologic status continued to decline and the patient expired 11 months after her initial diagnosis.

This patient sample set represented a unique opportunity to investigate several interesting questions that directly impinge upon clinical understanding and basic science research into cancer biology. In the first area, clinical understanding, it is fairly rare to obtain a metastatic sample from a patient whose primary tumor and a matched normal were already banked. In this case, the metastatic sample was even more rare, as brain tumors are typically not removed, due to the traumatic impact of neurosurgery. The particular clinical understanding gained from studying a set of samples from the same patient that included normal, primary disease and metastatic disease is enormous due to the rarity of these sample sets, as indicated, and due to the exquisite detail obtained by whole genome sequencing. In the context of basic science research, this patient had a portion of her primary tumor, prior to any chemotherapy, placed into a NOD/SCID mouse for propagation and further study as an HIM (human-in-mouse) xenograft. While scientists have used such a system in the past for tumor studies, nobody has ever answered, at the highest level of resolution offered by DNA sequencing, a whole genome comparison between the tumor from the human and the tumor once implanted into the HIM. Our choice to sequence the resected primary tumor xenograft also afforded a unique ability to compare this genome sequence to the primary tumor and to the metastatic tumor genomes.

Other inclusion and exclusion criteria included the following:
Must have been newly diagnosed with clinical stage II or III breast cancer who underwent neoadjuvant chemotherapy (chemotherapy prior to surgery), over the age of 18, not pregnant, and willing and able to sign the consent form. Uncorrected coagulopathy, bleeding tendency or other conditions that might increase the risk of a biopsy procedure. Prior history of other invasive malignancies is not an exclusion criterion, unless the disease is active and progressing at the time of protocol screening.

National Human Genome Research Institute