For breast cancer patients, molecular tumor characterization (including protein expression levels and genetic alterations or amplifications) identifies features that predict drug responsiveness. This information can be used to design personalized therapeutic strategies. Approximately 75% of breast tumors express hormonal receptors for Estrogen (ER) and/or Progesterone (PR) [1,2]; these patients typically respond well to endocrine therapy with or without CD4/6 kinase inhibitors . Another major tumor growth driver of breast cancer is HER2 gene amplification, which is seen in ~20% of breast cancers. These patients can be treated successfully with monoclonal antibodies that block HER2 function [4,5]. Despite benefits to patient survival, molecular data is often difficult to obtain in metastatic settings when patients’ health or refusals preclude biopsy, or the tumor metastasizes to a difficult-to-sample region of the body. Additionally, tissue molecular analyses may be inconclusive due to insufficient tissue amounts from biopsies or interference of bone tissue decalcification procedures with immunohistochemical (IHC) stains. Without understanding the molecular drivers of patients’ disease, treatment regimens based on tumor characteristics can neither be appropriately prescribed nor adjusted to tackle evolving properties of progressive disease.
The recent development of “liquid biopsy” technologies that analyze circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) allows physicians to obtain molecular data for guiding individual patient’s treatment approaches . CTCs are cells that have shed into the bloodstream from a primary or metastatic tumor, representing an alternative source of tumor material for non-invasive disease assessment [7,8]. Importantly, liquid biopsies provide a systemic representation of existing tumor clones, giving insight into tumor heterogeneity, emergence of new drivers, and the divergence between primary and metastatic tumors [6,9]. Here we describe a patient with recurrent breast cancer, who at one point declined an additional bone biopsy.
Biocept’s Target Selector™ liquid biopsy  (Figure 1) revealed ER expression and HER2 gene amplification in CTCs (Figure 2). Based on these data, the patient was able to receive anti-HER2 therapy earlier, providing a clinical example of the utility of liquid biopsy testing to gather molecular data that was unsuccessful by standard image-guided biopsy. In this case, CTC results of newly found HER2 amplification were paramount towards altering treatment strategies and inclusion of HER2 targeted therapies, which ultimately extended patient survival and quality of life.
The patient is a 51 year-old female, first diagnosed with stage 2 (T2N1M0) invasive ductal carcinoma in April 2003 at age 37. The primary tumor was strongly ER and PR positive by IHC staining, and the HER2 fluorescence in situ hybridization (FISH) analysis was negative. Subsequently, she completed 4 cycles of dose-dense doxorubicin/cyclophosphamide (AC) and 4 cycles of paclitaxel, followed by luprolide/exemestane.
In late 2006, imaging revealed multiple osseous metastatic lesions. Her T11 spine bone biopsy confirmed metastatic adenocarcinoma, and the IHC analysis was negative for ER, PR, and HER2. It was noted that the samples were decalcified per standard protocol. She received external beam radiation to her spine followed by docetaxel/ capecitabine, continuing afterwards on endocrine therapy and zoledronic acid.
PET/CT scans in August 2009 revealed a new lesion in the right scapula, and the needle biopsy was positive for metastatic carcinoma; ER, PR, and HER2 IHC stains were technically inconclusive. Subsequently, she started fulvestrant therapy.
The patient’s osseous disease progressed in 2013 and she received further chemotherapy. Her cancer progressed further in May 2014, and the decision was made to re-evaluate her hormone receptor and HER2 status. A right iliac bone biopsy attempted in August was negative for tumor cells, and the patient refused a repeat biopsy.
Target Selector™ liquid biopsy on her blood specimen from July 10, 2014, showed HER2 amplification by FISH and ER expression. Based on the HER2 positivity in CTCs, she began HER2 directed therapy with chemotherapy. Restaging imaging in January 2015 showed interval decrease of abnormal osseous activity in the numerous osseous metastases since May 2014 and no evidence of visceral involvement.
Restaging scans in October 2015 revealed new lesions in the skull, bilateral femurs, and ribs. Another Biocept liquid biopsy on October 14, 2015 revealed positive ER expression and negative HER2; it is important to note that this was while she was on HER2 therapy. She underwent surgical nail placement of the bilateral femurs, during which bone biopsies were obtained in November 2015, confirming metastatic carcinoma with positive ER and HER2 (3+) expression by IHC. For the first time, metastases were also noted in the lungs and liver, and she was started on trastuzumab emtansine in December.
Imaging in April 2016 showed increased adenopathy in the right supraclavicular region, and aright supraclavicular lymph node biopsy in May showed increased ER and PR expression. The HER2 analyses were equivocal, and therefore a tertiary HERmark test was performed, resulting in a positive quantitative result of 25.11. The patient’s treatment was switched to vinorelbine, and anti-HER2 therapy was continued.
The patient had metastatic progression into the dura and right lower lung lobe in January 2017, while her osseous metastatic sites remained stable. Her restaging scans in June 2017 demonstrated an overall stability of her metastatic disease except for slight increase in left retroperitoneal lymphadenopathy. Her most recent Biocept liquid biopsy from July 2017 revealed one cytokeratin positive cell with negative HER2 expression. As of December 2017, our patient continues on anti-HER2 therapy with chemotherapy. Table 1 and Figure 3 summarize key events.
Successful treatment of advanced breast cancer is driven by the knowledge of ER/PR and HER2 expression, and biopsy of metastatic sites is recommended to reconfirm the expression of these markers. In one study, discordance in expression measurements for ER, PR and HER2 between primary and recurrent breast tumors occurred in 18.4%, 40.3%, and 13.6% of cases respectively and had a negative effect on prognosis and survival likely due to misuse of targeted therapies . In this study, the discordant biomarker expression was thought to be due to tumor heterogeneity, change in clinical phenotype, or suboptimal reproducibility of measurement methods, particularly IHC.
Here we describe clinical difficulties with obtaining adequate tumor tissue for reconfirmation of ER/PR and HER2 expression. Our patient with a long history of metastatic breast cancer with bone only involvement underwent 4 tumor biopsy procedures to reassess expression of ER/PR and HER2. Her primary tumor initially tested HER2-negative, but after several years of treatment her metastatic tumor was found to be HER2 overexpressed. This finding led to therapy with anti-HER2 agents and almost certainly extended her life expectancy. The change in the breast cancer tumor expression of the HER2 is well-described in the literature  and occurs in up to 20% of cases . Difficulties in assessing the patient’s bone-only metastases are described, especially when bone biopsy IHC analyses were inconclusive or negative for hormone receptors and HER2, despite initial hormone receptor positivity of the primary tumor. It is well established that pathology processing and decalcification of tumor tissue from bone biopsies can interfere with IHC stains, leading to inconclusive or negative ER, PR, and HER2 stains. This could explain the patient’s negative or inconclusive tumor bone biopsy IHC staining results.
A meta-analysis of 49 studies (6,825 patients) showed that presence of CTCs was significantly associated with shorter survival; the prognostic value of CTCs was significant in both early (DFS: HR, 2.86; OS: HR, 2.78) and metastatic breast cancer (PFS: HR, 1.78; OS: HR, 2.33) . Furthermore, declining numbers of CTCs while on systemic treatments for advanced breast cancer are predictive of response to therapy and improved survival [15-18]. Assessment of CTC tumor markers is available commercially, but the clinical relevance of expression of tumor markers such as HER2 is not well understood [19-21]. A prospective study in metastatic patients with HER2-negative primary tumors showed that 24%of patients had HER2positive CTCs later; and this was associated with significantly shorter progression-free (P = 0.001) and overall survival (P = 0.013) compared with patients without HER2-positive CTCs . Further prospective studies are needed to determine the potential role of HER2-targeted therapies for patients with HER2-positive CTCs and HER2-negative primary tumors.
The first HER2 positive test in this patient’s case was obtained by CTC FISH testing. We eventually were able to confirm HER2 overexpression in tumor tissue approximately 16 months later with a surgical tumor sample from her orthopedic surgery, while prior less invasive needle bone biopsies were inconclusive. Based on CTC HER2 positivity, our patient received anti-HER2 therapy sooner than otherwise would have been the case based on tumor tissue testing.
In summary, HER2 is an important target for breast cancer therapy, and anti-HER2 therapy improves survival of advanced breast cancer patients [23,24]. In the metastatic setting, challenges exist with obtaining tumor tissue for re-testing markers. Our case illustrates the clinical applicability of a CTC HER2 test, which provided critical molecular information concerning HER2 tumor status 16 months before the tissue test confirmed HER2 overexpression. We conclude that ultimately our patient derived a clinically meaningful benefit from blood CTC HER2 testing, and the earlier initiation of antiHER2 therapy extended her life-expectancy and improved her quality of life.
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6. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol. 2017;14(9):531-548.
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A 44-year old male Hispanic male non-smoker was diagnosed with lung adenocarcinoma in a right pleural effusion. Molecular testing of collected fluid failed to reveal a genetic aberration. Palliative chemotherapy was initiated, consisting of carboplatin/pemetrexed/bevacizumab for six cycles, followed by maintenance chemotherapy with pemetrexed/bevacizumab for 23 cycles. At the time of disease progression, tissue from the patient’s tumor was insufficient for further
A Liquid Biopsy Provided Actionable Results
As a result of a liquid biopsy test, the patient began second-line erlotinib therapy, which was continued for 22 months until disease progression with peritoneal carcinomatosis. At this point, a ROS1 gene translocation was identified from biopsied tissue and confirmed in the analysis of blood CTCs via a Biocept liquid biopsy test. Plasma-based NGS failed to reveal ROS1 or any actionable gene aberrations. The patient began crizotinib therapy with disease stabilization. Brain metastases were detected 21 and 34 months later, and both were treated with stereotactic brain radiation. Because of the emergence of resistance, the patient was switched to ceritinib and has been stable for 6 months.
In April 2014, an 85-year-old non-smoker female reported night sweats during an office visit. She had a history of stage IV-B follicular B-cell non-Hodgkin lymphoma, which was in remission following single-agent rituximab treatment in 2010. An initial PET-CT (positron emission tomography-computed tomography) scan revealed increased FDG (fluorodeoxyglucose) uptake in a left upper lobe nodule with both mediastinal and celiac axis adenopathy. An EUS (endoscopic ultrasound)-guided fine needle aspiration was used to biopsy the celiac axis node. This procedure was non-diagnostic, and she was treated empirically with rituximab for four cycles.
In July 2014, a PET-CT scan following rituximab treatment revealed a 30% increase in size of the left upper lobe nodule with a SUV (standardized uptake value) of 6.8, now with new hilar and higher mediastinal uptake. Three more tumor biopsies were conducted, with the final biopsy showing the tumor as CK-7 (cytokeratin-7), Napsin, and thyroid transcription factor (TTF)-1 positive. Molecular studies came back negative for ALK translocation via FISH, there was insufficient tissue to test for EGFR mutations, and the patient refused another biopsy.
A Liquid Biopsy Expanded Treatment Options
To make sure the most appropriate course of action was being taken for treatment, a blood sample was collected and sent to Biocept in November 2014. Biocept’s Target Selector™ detected ALK-rearrangement in CTCs by FISH and EGFR abnormalities were not detected. The patient was prescribed crizotinib in December 2014 and in 8 weeks a PET-CT scan revealed a dramatically reduced tumor load and almost complete interval reduction in radiotracer uptake and size of her left upper lobe nodule, mediastinal, hilar, and upper abdominal adenopathy.
Targeted Therapy Gave Patient More Time
The patient remained asymptomatic on therapy with crizotinib for 13 months. A thoracentesis was performed in late February 2016, showing adenocarcinoma, and in March 2016 she was started on second line therapy with the ALK inhibitor ceritinib, but unfortunately developed excessive gastrointestinal toxicity. After 2 weeks, treatment was stopped and resumed ALK inhibitor alectinib after toxcitiy was resolved. Within 4 weeks of treatment with alectinib, her effusion resolved completely.
In July 2016 she presented to the emergency room with angioedema secondary to an ACE (angiotensin-converting-enzyme) inhibitor and required emergent tracheotomy with endotracheal intubation. Patient did not pursue additional therapy and passed away.
Female patient with a 19-year history of Breast Cancer and NSCLC Stage 1a diagnosed 5 years prior presented in January 2014 with PET confirmed left jugular lymph node enlargement and a RUL nodule. Fine needle aspiration confirmed metastatic NSCLC, but the tissue material was insufficient for biomarker testing. Patient received three cycles of Carboplatinum + Taxol® (paclitaxel) while undergoing concurrent CyberKnife therapy to the left neck and supraclavicular lymph nodes concluding in May 2014. Follow-up PET scan showed improved supraclavicular lymph node and stable RUL nodule. At that time, the treating physician tested the patient to predict the therapeutic benefit of using an EGFR TKI. Based on these test results, the patient was started on Tarceva® (erlotinib), an EGFR TKI therapy, which was subsequently discontinued as the patient did not tolerate the drug due to mucositis.
On November 17, 2014, NSCLC disease progression in the RUL was confirmed to be adenocarcinoma by CT guided needle biopsy. Biomarker testing was ordered, but the biopsy tissue material was insufficient.
Liquid Biopsy In Addition To Tissue
The patient’s physician was familiar with ordering liquid biopsy testing from Biocept. Blood was drawn from the patient on November 21, 2014 and EGFR and ALK biomarker tests were ordered. Biocept’s liquid biopsy detected an ALK translocation in the blood and the results were reported to the patient’s physician on December 1, 2014.
Biocept’s Liquid Biopsy Enables Selection of Targeted Therapy – The “Gift of Time”
Physician referred the patient for evaluation to participate in an immunotherapy clinical trial. However, it was determined that the best course of therapy was to proceed with an ALK-targeted therapy. As a result, the patient was treated with Xalkori® (crizotinib), from December 2014 – July 2016.
The identification of a clinically actionable biomarker and subsequent targeted therapy resulted in 18 months of progression-free survival (PFS) for the patient. In June 2016, the patient progressed with brain lesions, a new 3×1 cm lesion in the thyroid, mediastinal lymphadenopathy, and multiple RLL nodules. Patient was referred for stereotactic RT for the two brain lesions, (each receiving 2100 cGy dose in 1 fraction).
The patient was re-tested with Biocept’s liquid biopsy tests for EGFR, ALK and ROS1. The absence of clinically actionable biomarkers facilitated selection of immunotherapy over the alternative of a second-line ALK inhibitor. The patient was started on Opdivo® (nivolumab) in July 2016, and as of this publication has stable disease with limited toxicities.
Sarah is a 52-year-old woman diagnosed with breast cancer. A core biopsy of her primary tumor showed that the tumor was HER2-negative and ER-positive. Based on her molecular profile, she was appropriately treated for ER+ breast cancer. Sarah had been disease-free for about 2 years after treatment for her initial cancer when she visited her oncologist, Dr. Thomas as a follow-up due to some shortness of breath. Through her work-up and imaging studies, it was discovered that the cancer had recurred, this time with lesions in Sarah’s lung.
“Can we do another biopsy?”
Dr. Thomas was concerned about whether they would be able to get enough tissue from a surgical biopsy of the lung lesion. There were additional concerns about the location of the lesion, and any adverse effects that may occur from the re-biopsy procedure. Sarah had already been scheduled for a biopsy when her doctor considered an alternative approach. Dr. Thomas ordered a liquid biopsy from Biocept.
Establishing biomarker status with a simple blood test
Sarah’s doctor explained that a “liquid biopsy” was really just a simple blood test and only required a sample of blood from Sarah. It would spare her from undergoing another biopsy and the potential adverse effects that may have resulted due to the location of her lesion. The blood sample was sent to Biocept’s laboratory to be tested for established, clinically actionable biomarkers, including HER2 and ER. Dr. Thomas received Sarah’s results 5 days later and her results were both HER2positive and ER-positive.
A liquid biopsy reveals tumor heterogeneity
After considering all therapy options, Dr. Thomas decided to treat Sarah based on the results of the liquid biopsy with a combination therapy including anti-HER2 therapy and chemotherapy. Sarah responded well to this regimen and is in remission.
More effective treatment thanks to a liquid biopsy
Sarah was able to receive the appropriate standard of care therapy without an invasive lung biopsy. Through Biocept’s test, Sarah doctors were able to determine the true biology of her tumor and concluded that she would benefit from the anti-HER2 therapy regimen.
Michelle is a 79-year-old grandmother and is retired in Florida. She was diagnosed with breast cancer approximately 6 years ago. A core biopsy of her primary tumor showed that the tumor was HER2-positive and ER-positive. Based on her molecular profile, she was appropriately treated for her breast cancer. Michelle had been disease-free for many years until she felt some bone pain that lead her to the doctor. After a thorough work up and imaging study, it was determined that Michelle’s cancer had likely recurred and metastasized to her bones.
“Can we do another biopsy?”
For patients with lesions to the bone, a biopsy is routine and possible. However in Michelle’s case, the HER2 result that was positive on her primary tumor showed up negative on the bone lesion. There were some concerns that the laboratory process using decalcification for the biopsy sample may have made it difficult to obtain the HER2 results. Michelle’s oncologist recommended ordering a liquid biopsy from Biocept.
Establishing biomarker status with a simple blood test
Michelle was told that through a “liquid biopsy,” a simple test that only required a sample of blood from Michelle, they might be able to retest the HER2 status without obtaining another biopsy of her bone lesions. It would spare her from the pain and discomfort of undergoing another biopsy. The blood sample was sent to Biocept’s laboratory to be tested for established, clinically actionable biomarkers, including HER2 and ER. Michelle’s oncologist received her results 5 days later and her results were positive for HER2, as they were on her primary tumor.
More effective treatment thanks to a liquid biopsy
Michelle was able to receive the appropriate standard of care therapy without the physician needing to go back into her bone to obtain aa additional biopsy. Through Biocept’s test, Michelle’s doctors were able to determine the true biology of her tumor and concluded that she would benefit from the anti-HER therapy that she needed to fight her breast cancer.
Lisa is a 58-year-old and recently diagnosed with early stage breast cancer. A core biopsy of her primary tumor showed that the tumor was HER2-negative. Based on the stage and the presentation of her disease, she was a good candidate for neo-adjuvant therapy.
“Could we have missed this patient’s HER2 status?”
For patients with small core biopsies, there exists the possibility that due to the heterogeneity of disease, a small biopsy can miss areas of tumor that are positive for HER2 (and other critical biomarkers). Lisa’s doctor was familiar with ordering a liquid biopsy test from Biocept.
Establishing biomarker status with a simple blood test
Lisa was told that through a “liquid biopsy,” a simple test that only required a sample of blood, they could double check to confirm that they had not missed any HER2 positive cells. Finding HER2 positivity in early stage breast cancer patients who are candidates for neo-adjuvant therapy can drastically change the treatment options. The blood sample was sent to Biocept’s laboratory to be tested for established, clinically actionable biomarkers, including HER2 and ER. Doctors received Lisa’s results 5 days later and her results were positive for HER2 tumor cells circulating in the blood.
More effective treatment thanks to a liquid biopsy
Lisa still received neo-adjuvant therapy, but now it was with an anti-HER2 agent regimen. Through Biocept’s test, Lisa’s doctors were able to determine the true biology of her tumor and concluded that she would benefit from the anti-HER2 therapy that she needed to fight her breast cancer. This change to targeted therapy generally increases her likelihood of disease free survival while reducing side effects.