Abstract

   Metastatic breast cancer is one of the leading causes of cancer-related
   death in women. Limited studies have been done on the genomic evolution
   between primary and metastatic breast cancer. We reconstructed the
   genomic evolution through the 16-yr history of an ER^+ HER2^− breast
   cancer patient to investigate molecular mechanisms of disease relapse
   and treatment resistance after long-term exposure to hormonal therapy.
   Genomic and transcriptome profiling was performed on primary breast
   tumor (2002), initial recurrence (2012), and liver metastasis (2015)
   samples. Cell-free DNA analysis was performed at 11 time points
   (2015–2017). Mutational analysis revealed a low mutational burden in
   the primary tumor that doubled at the time of progression, with driver
   mutations in PI3K–Akt and RAS–RAF signaling pathways. Phylogenetic
   analysis showed an early branching off between primary tumor and
   metastasis. Liquid biopsies, although initially negative, started to
   detect an ESR1 E380Q mutation in 2016 with increasing allele frequency
   until the end of 2017. Transcriptome analysis revealed 721 (193 up, 528
   down) genes to be differentially expressed between primary tumor and
   first relapse. The most significantly down-regulated genes were TFF1
   and PGR, indicating resistance to aromatase inhibitor (AI) therapy. The
   most up-regulated genes included PTHLH, S100P, and SOX2, promoting
   tumor growth and metastasis. This phylogenetic reconstruction of the
   life history of a single patient's cancer as well as monitoring tumor
   progression through liquid biopsies allowed for uncovering the
   molecular mechanisms leading to initial relapse, metastatic spread, and
   treatment resistance.

   Keywords: multifocal breast carcinoma

INTRODUCTION

   Breast cancer is the most common malignancy affecting women in the
   United States, causing the most cancer-related deaths ([49]Howlader et
   al. n.d.; [50]Breast Cancer Statistics and Resource 2017). However,
   patients normally do not die from the primary disease, but as a
   consequence of metastasis ([51]Fouad et al. 2015). Hormone
   receptor–positive disease accounts for >60% of all breast cancer
   ([52]American Cancer Society n.d.). The 20-yr recurrence risk of
   distant metastasis in estrogen receptor (ER)-positive breast cancer is
   between 10% and 40% and depends on the size of the primary tumor and
   number of positive lymph nodes ([53]Pan et al. 2017). More than
   one-half of the recurrence happens after 5 yr of initial diagnosis
   ([54]Dowling et al. 2019). Little is known about the triggers of
   metastasis or how the metastatic disease is different from the primary
   tumor. Developing sequencing technology made it possible to identify
   the cancer drivers and closely track disease evolution on the genomic
   and transcriptomic level.

   Here we present a patient who was diagnosed with stage III ER-positive
   breast cancer and developed distant recurrences after 10 years of
   remission. The patient survived late-stage disease for another 6 years
   and passed away as a result of extensive disease burden and
   cancer-related complications. Tissue samples were collected from the
   patient's breast tumor at initial diagnosis, first recurrence in
   supraclavicular lymph node, and distant metastasis in the liver. Eleven
   liquid biopsies were collected after distant metastasis. DNA and RNA
   from the samples were extracted, sequenced, and analyzed. From the
   genomic and transcriptomic analysis, we were able to identify potential
   disease drivers and resistance mechanisms at each stage of the disease,
   reconstruct the cancer evolutionary tree, and investigate the
   relationship between the metastases and primary tumor.

RESULTS

Clinical Presentation

   A 46-yr-old premenopausal Caucasian female presented with stage III
   invasive ductal carcinoma of the breast in January 2002. The patient‘s
   treatment history is summarized in [55]Figure 1A. Solid and liquid
   samples collected from 2002 to 2017 are gathered in [56]Table 1.

Figure 1.

   [57]Figure 1.
   [58]Open in a new tab

   Patient disease history with treatment and tumor molecular profiles.
   (A) Disease and treatment history, with red explosions indicating
   disease progression and an × mark on the time axis indicating treatment
   on hold because of adverse events. T1, M1, and M2 on the time line
   indicate when solid tumor samples were collected. Treatment duration is
   plotted according to the time axis on top. Treatment strip color is
   assigned per drug class: blue for chemotherapies; red for estrogen
   receptor antagonists; pink for aromatase inhibitors; purple for mTOR
   inhibitor; yellow for tyrosine kinase inhibitors; lilac for MEK
   inhibitor; and green for immunotherapy. (B) Driver mutations from solid
   tissue samples and liquid biopsies (cfDNA). The table is shaded with a
   stream graph representing the mutation load during disease progression.
   Driver mutations are shown with colored bands with the band widths
   representing mutation allele frequency.

Table 1.

   Sample information
   Tumor sample Test Average coverage
   Sample Specimen site Collection year ER PR HER2
   T1 Right breast 2002 + + – WES 190×
   RNA-seq 46M
   M1 Right scalene lymph node 2012 + – – WES 181×
   RNA-seq 75M
   M2 Liver 2015 + + – Targeted Sequencing (FoundationOne 315 gene) 462×
   Liquid biopsies Blood 2015–2016 NA NA NA Targeted Gene Sequencing
   (Guardant 70 gene) Min 15,000×
   Liquid biopsies Blood 2017 NA NA NA Targeted Gene Sequencing
   (FoundationAct 67 gene) 10,000×
   [59]Open in a new tab

   Solid and liquid samples collected from 2002 to 2017.

   At initial diagnosis, the patient's tumor was characterized as
   ER-positive, progesterone receptor (PR)-positive, and human epidermal
   growth factor receptor 2 (HER2)-negative by immunohistochemistry (IHC).
   Right breast mastectomy and axillary lymph node dissection were
   performed. The patient was treated adjuvantly with doxorubicin and
   cyclophosphamide followed by paclitaxel (AC-T). She was then maintained
   on tamoxifen for 2 years, followed by 8 years of aromatase inhibitor
   therapy (anastrozole).

   In May 2012, the patient returned to the clinic with recurrent disease
   at the right supraclavicular lymph node and possible metastatic nodules
   in the right upper lobe of the lung and anterior mediastinum. The lymph
   node metastasis was ER-positive, PR-negative, and HER2-negative. She
   started on single-agent exemestane. Everolimus was added in July 2012.
   Radiation therapy to the regional lymphatics and right chest wall was
   completed in December 2012. In January 2013, restaging positron
   emission tomography (PET) demonstrated a complete metabolic response.
   The disease was considered stable for 24 mo under this regimen.

   In May 2014, progressive liver lesions were shown on computerized
   tomography (CT) scan. In response to disease progression, the patient
   was moved to the next line of treatment with fulvestrant and
   exemestane.

   In September 2014, evidence of progression in the liver, pleura, and
   bones was found through CT scan. The treatment was switched to
   single-agent capecitabine. The patient was stable for 6 mo.

   In March 2015, new lesions were found in the patient's liver. The
   previous disease in her mediastinal lymph nodes increased in size. The
   liver lesion tested ER-positive, PR-positive, and HER2-negative by IHC.
   In April 2015, treatment was changed to paclitaxel plus everolimus and
   sorafenib.

   To guide treatment and track tumor evolution, genomic testing was
   performed first through targeted DNA sequencing of a liver biopsy
   (through FoundationOne test) and subsequently liquid biopsies (through
   Guardant360 and FoundationAct tests) from August 2015 to November 2017.
   Mutational findings are summarized in [60]Figure 1B.

   In August 2015, all treatments were held for 2 wk because of severe
   neutropenia. The regimen was then changed to everolimus plus
   vinorelbine and crizotinib.

   In February 2016, because of progression in the liver, the patient
   started a new line of treatment with trametinib, eribulin, and
   everolimus. A CT scan in April 2016 suggested a 20% reduction in liver
   tumor volume. In July 2016, crizotinib was added to the regimen as a
   result of progression in the liver and the right pleura.

   In early September 2016, the patient finished 11 cycles of eribulin.
   Her CT scan showed slight progression on the chest wall. She was
   switched to carboplatin plus exemestane and cabozantinib. Everolimus
   was added to the regimen at the end of December 2016.

   In late February 2017, new lesions were detected on the patient's bone
   scan. The patient started a new regimen of atezolizumab, fulvestrant,
   everolimus, and cabozantinib. Protein-bound paclitaxel was added in
   early May. In June 2017, everolimus was held because of
   thrombocytopenia and neutropenia. Later in June, slight progression was
   seen on a CT scan, and trametinib was added.

   In October 2017, all therapy was held because of her implant that had
   eroded through the chest wall, which was then surgically removed. The
   patient continued to have an open area at the right breast but wished
   to continue with aggressive treatment after surgery. Treatment was
   restarted at the beginning of November 2017. At the end of November, CT
   showed progression in the lungs and retroperitoneal lymph nodes. In
   mid-December 2017, the patient's therapy was changed to protein-bound
   paclitaxel, cabozantinib, atezolizumab, abemaciclib, and fulvestrant.

   At the end of January 2018, the patient struggled with wound healing
   and a large pneumothorax developed on the right with communication at
   the wound site. There were no good options to treat the pneumothorax
   based on her status. Because of the significant tumor burden in the
   liver, the patient was not capable of tolerating further treatment. The
   patient was referred to hospice and passed away a week later.

   The patient was enrolled into the multicenter clinical trial
   “Identifying Molecular Drivers of Cancer (CCD)” ([61]NCT02470715).
   Therapies were selected by incorporating recommendations of a molecular
   tumor board consisting of oncologists, pharmacists, nurses, genetic
   counselors, bioinformaticians, patient advocates, and molecular
   biologists. The therapies ultimately given were furthermore based on
   the treating physician's consideration of patient preferences, drug