Circulating Tumor Cells and DNA in Precision Oncology

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1		sridevi addanki	--	4819	2022-07-26 22:36:51	\|
2	format	Jason Zhu	-7 word(s)	4812	2022-07-27 08:15:18	\|

This entry is adapted from the peer-reviewed paper 10.3390/ijms23147843

Liquid biopsies allow for the detection of cancer biomarkers such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Elevated levels of these biomarkers during cancer treatment could potentially serve as indicators of cancer progression and shed light on the mechanisms of metastasis and therapy resistance. Thus, liquid biopsies serve as tools for cancer detection and monitoring through a simple, non-invasive blood draw, allowing multiple longitudinal sampling. These circulating markers have significant prospects for use in assessing patients’ prognosis, monitoring response to therapy, and developing precision medicine. In addition, single-cell omics of these liquid biopsy markers can be potential tools for identifying tumor heterogeneity and plasticity as well as novel therapeutic targets.

liquid biopsy early stage breast cancer late-stage breast cancer CTC ctDNA NGS single-cell sequencing Precision Oncology

1. Introduction

Breast cancer is the most prevalent malignancy among women in the United States, with an estimated 287,850 newly diagnosed cases in 2022 ^[1]. It is projected that around 43,250 women will die of the disease this year, making it the second biggest cause of cancer-related fatalities in women. Breast cancer is a complex disease, in which incidence increases with age as a result of the accumulation of somatic mutations in the mammary glands. Malignancy in the breast tissue is heterogeneous and broadly divided into different subtypes based on the molecular aberrations present in the tumor ^[2].

In the luminal A subtype, which accounts for 50–60% of breast cancer cases, the tumors are positive for estrogen receptor (ER) and/or progesterone receptor (PR) ^[3]. Because of lower levels of Ki67, a protein associated with the growth of the cancer cells, the luminal A subtype is low grade, less aggressive, and carries a good prognosis. Another subtype of breast cancer is characterized by the presence and overexpression of human epidermal growth factor receptor 2 (HER2). HER2-enriched tumors tend to grow faster and could have a worse prognosis if not for the effectiveness of HER2-targeted therapies such as trastuzumab and pertuzumab. The luminal B breast cancer subtype is also hormone receptor-positive, with the presence of ER and/or PR, but has higher levels of Ki67 and may also be HER2-positive ^[4]. Luminal B patients’ prognosis is slightly worse than that of luminal A patients.

The most ominous subtype is triple-negative breast cancer (TNBC), characterized by the lack of ER and PR expression and HER2 amplification ^[5]. TNBCs account for 15–20% of invasive breast cancers and are predominantly high-grade, high-risk tumors that occur more frequently in young women, especially Black women ^[6]. Inter- and intratumoral heterogeneity contribute to the aggressive nature of this subtype. Women presenting with TNBC have a higher rate of distant recurrence and poorer prognosis than do women with other subtypes of breast cancer ^[7].

Breast cancer is thus a heterogeneous and complex disease with a wide range of histologic characteristics, treatment responses, metastatic activity, and patient outcomes. Apart from age and genetic, hormonal, and reproductive components of breast cancer risk, there are modifiable factors such as excess body weight, physical inactivity, alcohol use, and receipt of hormone replacement therapy. Breast cancer incidence in women in the United States increased by an average of 0.3% per year between 2004 and 2018 ^[8]. Incidentally, the incidence of breast cancer increased among women 20–49 years old while it decreased in women older than 50 years. The US Preventive Services Task Force recommends mammography screening every 2 years for women 50–74 years old. However, a screening study including 993,000 people found that screening did not affect the incidence of stage IV illness ^[9]. In addition, although early stage cancer detection facilitates curative surgical resection in many solid tumors, including breast cancers, mammography is limited by its poor sensitivity, overdiagnosis, false-positive rates, and the discomfort and anxiety it causes patients. Thus, a better prognosis of breast cancer is impeded by a lack of early screening programs in young women and effective diagnostic tools in general ^[10]^[11]. These limitations highlight the importance of developing new technologies and strategies for the early identification and treatment of breast cancer.

Liquid biopsies are emerging as a minimally invasive method for early detection and risk management of breast cancer. The purpose is to examine the utility of liquid biopsy indicators of breast cancer, circulating tumor cells (CTCs), and circulating tumor DNA (ctDNA), as well as current improvements and technological advancements in the field.

2. CTCs in Early Stage Breast Cancer

Early stage breast cancer is more commonly diagnosed than more advanced breast cancer, but around 20% of these patients have recurrence ^[12]. CTCs in the early stages of breast cancer can generate micrometastases and thus are the seeds of the metastatic cascade. CTCs may also serve as surrogate markers for minimal residual disease (MRD).

Many researchers have looked at the detection and characterization of CTCs in early stage breast cancer peripheral blood. For HER2-positive patients with primary breast cancer, the GeparQuattro clinical study examined neoadjuvant chemotherapy (NACT) that included trastuzumab ^[13]. The researchers concluded that CTC numbers are low in early stage disease using the FDA-approved CellSearch system for CTC detection. Despite a drop in CTC levels following NACT, there was no correlation between persisting CTC levels and treatment response. They designed HER2 immunoscoring of CTCs to direct patients whose CTCs overexpress HER2 to receive HER2-targeted therapies.

Another group evaluated the prognostic significance of cytokeratin (CK-19)-positive CTCs in early stage breast cancer after NACT and identified it to be an independent risk factor ^[14]. Sandri et al. examined the possible role of CTCs in operable breast cancer and determined that 30% of patients have CTCs before and after surgery ^[15] Another study looked into the prognostic value of CTCs in early stage breast cancer and found that the persistence of CTCs before and after NACT identifies a patient subpopulation linked with a higher risk of recurrence ^[16]. Pierga and colleagues determined that CTCs enable the prediction of early metastatic relapse following NACT in large operable and locally advanced breast cancer ^[17]. The same group examined the clinical outcomes of CTC detection in non-metastatic breast cancer patients and reported that detecting ≥1 CTC/7.5 mL before NACT accurately predicts overall survival (OS) ^[18]. This was followed up by performing CTC counts on 118 patients before and after chemotherapy and examining survival. It was concluded that CTC detection is independently associated with significantly worse outcomes, especially 3–4 years after surgery ^[18]. The research group looked at CTC data from chemotherapy-naïve patients with stage I-III breast cancer at definitive surgery and discovered that having ≥1 CTC/7.5 mL predicts early recurrence and a shorter OS ^[19]. Researchers then investigated the presence of CTCs after NACT in stage I-III TNBC and concluded that ≥1 CTC is predictive of relapse and survival ^[20].

A large prospective trial of primary breast cancer patients revealed the independent prognostic value of CTCs before and after NACT ^[21]. The BEVERLY-2 trial evaluated the safety and efficacy of NACT with bevacizumab and trastuzumab to treat patients with HER2-positive inflammatory breast cancer (IBC) ^[22]. In the prospective survival analysis at 3 years of follow-up, CTC analysis predicted 81% vs. 43% percent disease-free survival (DFS) for patients with ≥1 CTC/7.5 mL of blood at baseline. CTC detection was also found to be a strong and independent predictor of survival in patients with nonmetastatic IBC in the BEVERLY-1 and BEVERLY-2 trials ^[23]. When the group of patients with pathologic complete response to NACT was merged with the group that had no CTC detection at baseline, a subgroup of IBC patients with a 3-year OS of 94% was discovered. As a result of the BEVERLY study, the role of CTCs in tumor spread and their potential application for IBC patient stratification were established.

3. CtDNA in Early Stage Breast Cancer

Many approaches have been used in the past decade to detect and measure ctDNA in patients with early stage breast cancer. For example, Beaver et al. detected ctDNA in the plasma of early stage breast cancer patients using primary breast tumors with matched pre- and post-surgery samples ^[24]. PIK3CA mutations identified using Sanger sequencing in tumor tissue were accurately detected in plasma samples using ddPCR.

Researchers have assessed ctDNA amounts in patients with early stage breast cancer for the purpose of reducing mortality by early detection and therapy modification. Riva et al. investigated the presence of ctDNA in a cohort of patients with nonmetastatic TNBC to examine whether ctDNA was associated with response to NACT and measure MRD after surgery ^[25]. Using ddPCR, they assessed ctDNA presence at baseline with a detection rate of 75%. Furthermore, there was a rapid decline in ctDNA levels during NACT as well as undetectable MRD. The researchers also found that a slow reduction in ctDNA levels during NACT was substantially linked to a shorter survival time. Phallen and colleagues sought the detection of early stage cancers using ctDNA ^[26]. They developed targeted error correction sequencing (TEC-seq), which allows direct examination of sequence changes in cfDNA using massive parallel sequencing. Fifty-eight cancer-related genes were investigated by this method, and somatic mutations were discovered in the plasma of 71% of the early stage breast cancer patients. There was a great degree of concordance between mutations found in the tumor samples and ctDNA. CancerSEEK is a blood test aimed at detecting eight different cancer types, including nonmetastatic breast cancer, by assessing the levels of circulating proteins and tumor-specific mutations in the circulating DNA ^[27].

A principal caveat of tissue biopsies is their inability to track changing genomic profiles over time, which liquid biopsies can overcome through serial sampling. Longitudinal fluid biopsy sampling allows precise monitoring of therapeutic efficacy and tracks the development of treatment resistance. Many recent studies have attempted sequencing ctDNA to generate mutation profiles to identify gene alterations in the resistant clones. Such mutation tracking showed ctDNA to be associated with relapse in early stage breast cancer ^[28]^[29]^[30]. ddPCR was used to track mutations discovered in the primary tumor for their presence in ctDNA in post-surgery and follow-up samples. Using massive parallel sequencing, the mutational profile of ctDNA was used to identify the genetic features of therapy-resistant tumor clones. The group also monitored early stage breast cancer with a lead time of ctDNA detection of 10.7 months following disease relapse ^[30]. These investigations elucidated the relevance of ctDNA in monitoring tumor burden and tracking the emergence of resistant clones to enable appropriate therapy selection.

Rothe and colleagues looked at the relationship between ctDNA and response to anti-HER2 therapy and discovered that HER-2-enriched tumors with no ctDNA had the greatest pathologic complete response rates at baseline, indicating that ctDNA can be used as a biomarker for NACT response in HER-2-amplified breast cancer ^[31]. Along these lines, Zhang et al. investigated the genomic variants of ctDNA for their potential use as actionable biomarkers in early stage breast cancer treatment ^[32]. Deep sequencing of plasma and matching tissue samples revealed that the intratumoral heterogeneity found in tumor tissues was reflected in ctDNA values. Furthermore, post-surgery ctDNA positivity was linked to a higher percentage of lymph node metastasis, indicating the possibility of recurrence and distant metastasis.

Researchers also looked into the use of ctDNA analysis for diagnosing early stage breast cancer after mammography results ^[33]. They analyzed primary breast tissue with the Illumina NGS TruSeq Custom Amplicon Low Input Panel and plasma with SafeSEQ (Sysmex Inostics). Additional ctDNA mutations in the TP53 and PIK3CA genes were discovered in the sequencing data that were not identified in the tissue specimens. Furthermore, age, tumor grade and size, immunohistochemistry subtype, Breast Imaging Reporting and Data System classification (BI-RADS) category, and lymph node positivity were all linked to ctDNA mutations.

Another study examined the efficacy of ctDNA to predict relapse in TNBC patients with residual disease after NACT ^[34]. Using the Oncomine NGS panel for ctDNA sequencing, the researchers demonstrated that recurrence in such patients may be predicted with high specificity but modest sensitivity. Moreover, recurrence was quick in the event of ctDNA detection. Diagnostic methods to correctly predict residual disease following NACT are needed in localized breast tumors. NACT can help guide treatment decisions such as the extent of surgical resection and the need for radiation treatment. Because present diagnostic techniques lack sensitivity, a therapy monitoring biomarker that can accurately discriminate residual disease from disease elimination would allow patients to obtain tailored therapy ^[35]^[36]. McDonald and colleagues created the targeted digital sequencing (TARDIS) method for multiplexed analysis of patient-specific cancer mutations ^[37]. This approach proved successful in detecting minute amounts of residual DNA in patients’ plasma. The researchers discovered that patients who obtained pathologic complete response had lower ctDNA concentrations than those with residual disease. In addition, during NACT, the drop in ctDNA levels was more pronounced in the group with pathologic complete response.

4. CTCs in Metastatic Breast Cancer

Although significant strides have been made toward improving breast cancer survival rates, resistance to treatment develops in many patients and eventually leads to death from metastatic breast cancer. CTCs are released into the bloodstream of patients with solid tumors, functioning as seeds for subsequent metastasis. Elevated levels of CTCs during cancer treatment are an indicator of cancer progression and therefore can reveal the mechanisms of metastasis. CTCs in metastatic breast cancer provide more information than those in early stage breast cancer because they reflect the dominant clones at metastatic homing sites and aid in quantifying the remaining tumor burden. Thus, CTCs are invaluable tools for cancer detection and monitoring through a simple, non-invasive blood draw, allowing multiple longitudinal sampling.

In early efforts to evaluate the predictive values of CTCs in metastatic breast cancer, CTCs were indeed revealed to be a strong independent prognostic marker for the disease ^[38]. Martin et al. sought to analyze the relationship between OS and CTC counts after the first round of chemotherapy ^[39]. They acquired CTC counts at the baseline, before starting the first cycle of chemotherapy, and after the first cycle of chemotherapy to examine the prognostic relevance of CTC measures before giving the second round of chemotherapy. The CTCs were separated into low count (0–4 CTCs) and high count (≥5 CTCs). Patients with 0–4 CTCs after the first chemotherapy cycle had a significantly better OS (median OS: 38.5 months vs. 8.7 months), PFS (median 9.4 vs. 3.0 months), and clinical benefit rate (77% vs. 44%) than patients with ≥5 CTCs. In conclusion, the researchers determined that CTC measures following the first chemotherapy cycle were an early and robust predictor of treatment outcomes in metastatic breast cancer patients.

A pooled analysis of individual patient data was acquired from 17 European centers to evaluate the clinical validity of CTC quantification in the metastatic breast cancer prognosis ^[40]. Using a 1944 eligible patient database derived from 20 different studies, the researchers found that patients with ≥5 CTCs at baseline had decreased PFS (HR 1.92, 95%, CI 1.73–2.14, p < 0.0001) and OS (HR 2.78, 95%, CI 2.42–3.19, p < 0.0001) compared with <5 CTCs/7.5 mL plasma at baseline. Furthermore, increased CTC counts 3–5 weeks and 6–8 weeks after treatment were associated with shorter PFS and OS. These data shed light on the independent prognostic value of CTCs for PFS and OS of metastatic breast cancer patients.

Because high CTC levels have been linked to poor prognosis, the SWOGS0500 trial was designed to see if switching chemotherapy in metastatic breast cancer patients with persisting CTCs after the first cycle of first-line chemotherapy would improve OS ^[41]. After 21 days of chemotherapy, patients with continuously increasing CTCs were randomly assigned to either continue receiving the initial therapy or switch to an alternative chemotherapy regimen. The investigators discovered that switching to an alternate cytotoxic therapy early after the first cycle of chemotherapy did not result in longer OS in individuals with persistently elevated CTCs. Patients with increased or persistent CTCs after first-line chemotherapy may benefit from immunological, targeted, or other therapeutic modalities rather than moving to another type of cytotoxic therapy, according to the findings.

Cristofanilli et al. tested the predictive utility of CTCs for stratifying the patients with stage IV metastatic breast cancer ^[42]. In a retrospective, pooled analysis based on 18 cohorts, 2436 metastatic breast cancer patients were classified as either stage IV aggressive (≥5 CTCs) or stage IV indolent (<5 CTCs) based on molecular subtype, disease location, and prior treatments. The stage IV indolent group was found to have a longer OS than the stage IV aggressive group. These results demonstrated that CTC levels are a valuable technique for staging and stratifying advanced metastatic breast cancer.

The DETECT study program aimed to assess treatment interventions in metastatic breast cancer patients using CTC phenotypes ^[41]. The trial’s goal was to compare the safety and quality of life measured by the occurrence of adverse events in patients treated with dual HER2-targeted therapy (trastuzumab plus pertuzumab) plus either endocrine therapy or chemotherapy. It was the first study to categorize participants according to the HER2 phenotype of their CTCs. The HER2 status of the primary tumor was the main criterion for grouping the patients into different DETECT trails, and the clearance of CTCs and PFS eventually estimated the clinical efficacy. In the DETECT III and IV trials, HER2-negative subjects were included, and CTCs were a significant prognostic indicator in these patients. It was reported that the presence of ≥1 CTC with strong HER2 immunostaining was associated with shorter OS. Thus, the study elucidated the biological role of HER2 positivity in CTCs ^[43].

In HR-positive, HER2-negative metastatic breast cancer patients, the STIC study was designed to assess the efficacy of CTC-driven vs. clinician-driven first-line therapeutic choices ^[44]. In this randomized, open-label, noninferiority phase 3 trial, patients were grouped into two arms: the CTC-driven arm was given chemotherapy if the CTC counts were ≥5 CTCs/7.5 mL or endocrine therapy if the CTCs were <5 CTCs/7.5 mL, and the clinician decided treatment for the control arm. According to the findings of the STIC trial, a high CTC count (5 CTCs/7.5 mL) indicates a significant negative prognostic factor for OS and PFS. The study results revealed that CTC counts could be reliable biomarkers for selecting the first-line therapy for HR-positive, HER2-negative metastatic breast cancer patients.

Another clinical trial, CirCe01, looked into the efficacy of CTC-based monitoring of patients with metastatic breast cancer after they had completed their third line of treatment ^[45]. Patients with ≥5 CTCs were randomized to a CTC-driven arm or a standard arm; patients in the CTC arm were assessed after each cycle of therapy, and those whose CTC levels predicted tumor development would be given an alternate line of treatment. However, due to accrual and compliance issues, the trial could not demonstrate the clinical usefulness of CTC monitoring.

5. ctDNA in Metastatic Breast Cancer

In metastatic breast cancer, CTCs can give clues about the genomic landscape of the different tumor populations and the tumor burden. Many studies have recently looked into the prognostic significance of ctDNA. Dawson et al. performed a comparative analysis of conventional serum marker CA15-3, CTCs, and ctDNA in 30 women with metastatic breast cancer receiving systemic therapy. ctDNA was detected in 97% of the patients, and their levels had a greater dynamic range and correlation than CA15-3 and CTCs ^[46]. Bettegowda et al. used the ddPCR method to detect ctDNA in 640 patients with various cancer types, finding that ctDNA levels were >75% in patients with metastatic breast cancer and 50% in patients with localized breast adenocarcinoma ^[47]. They also discovered ctDNA in patient samples with no CTCs, indicating that these biomarkers are separate entities. A meta-analysis of 10 eligible studies with 1127 breast cancer patients was conducted to determine the relationship between cfDNA and survival outcomes ^[48]. The meta-analysis found a robust link between cfDNA and OS (HR 2.41, 95% CI 1.83–3.16) and disease- and recurrence-free survival (HR 2.73, 95% CI 2.04–3.67). These results revealed the predictive and prognostic power of cfDNA in breast cancer.

Shaw et al. examined whether the mutation profiles of cfDNA would capture the heterogeneity exhibited in numerous single CTC profiles ^[49]. In 112 individuals with metastatic breast cancer, CTCs were counted using CellSearch and compared with matching cfDNA, serum CA15-3, and alkaline phosphatase. Multiple single epithelial cell adhesion molecule (EpCAM)-positive CTCs were recovered by DEPArray in five patients with ≥100 CTCs and compared with matched cfDNA and primary tumor tissue using targeted NGS of about 2200 mutations in 50 cancer genes ^[49]. Mutational heterogeneity in the PIK3CA, TP53, ESR1, and KRAS genes was mirrored between single CTCs and accurately represented in the cfDNA molecular profiles, highlighting the importance of using cfDNA to monitor the metastatic burden and make treatment decisions. The researchers also compared the efficacy of circulating biomarkers, including cfDNA and CTCs, to traditional breast cancer biomarkers, CA15-3 and AP, in predicting metastatic breast cancer prognosis and treatment response ^[50]. They concluded that cfDNA levels are the best predictor of disease response and PFS; however, a paired test analyzing both cfDNA and CTC counts provides additional prognostic information and allows patients to be stratified further.

Murtaza et al. found that ctDNA can represent the clonal hierarchy of breast cancer, making it a valuable tool for detecting inter- and intra-metastatic heterogeneity ^[51]. They performed parallel sequencing of sequential tissue biopsies and plasma ctDNA samples in ER-positive/HER2-positive metastatic breast cancer patients. They discovered that most ctDNA mutations were present in all tumor samples, whereas some rare mutations were only found in one metastatic sample. The PALOMA-3 study combined palbociclib, a CDK4/6 inhibitor, with fulvestrant, a selective ER degrader, to treat women with HR-positive, HER2-negative advanced breast cancer ^[52]. The study shed light on the early dynamics of ctDNA and established its utility as a biomarker for the CDK4/6 inhibition ^[53]. Darrigues et al. wanted to see if early changes in ctDNA levels are related to the efficacy of the combination drugs used in the PALOMA-3 trial, which established palbociclib and fulvestrant at the standard of care for ER-positive, HER2-negative metastatic breast cancer ^[54]. Their findings revealed that serial ctDNA studies prior to radiological evaluation can indeed monitor the efficacy of palbociclib and fulvestrant and that early ctDNA variation is a predictive factor for PFS. A pooled ctDNA analysis was performed, which combined results of 1503 patients from the MONALEESA-2, -5, and -7 trials to identify biomarkers for CDK4/6 inhibition in the advanced breast cancer ^[55]. The MONALEESA trials looked at the efficacy and safety of ribociclib, a CDK4/6 inhibitor, with a choice of endocrine partners as a first- or second-line treatment for patients with HR-positive, HER2-negative advanced breast cancer. The researchers discovered biomarkers for the response, such as FRS2, MDM2, PRKCA, ERBB2, AKT1, and BRCA1/2, and biomarkers for resistance, such as CHD4, BC11B, ATM, and CDKN2A/2B/2C.

Early ctDNA dynamics were found to be a predictor for PFS in advanced breast cancer in the BEECH trial ^[56]. The BEECH trial investigated the efficacy of combining capivasertib, an AKT inhibitor, with the first-line chemotherapeutic paclitaxel in metastatic breast cancers that were HER2-positive and HER2-negative and harbored PIK3CA mutations. ctDNA dynamics were assessed as a surrogate for PFS and an early predictor of treatment efficacy. The findings revealed that ctDNA dynamics early during treatment could be used as a proxy for PFS. Additionally, dynamic ctDNA analysis can improve early drug development significantly. The utility of ctDNA analysis to direct therapy in advanced breast cancer was explored by an open-label, multicohort, phase 2a, platform trial of ctDNA testing in 18 UK hospitals ^[57]. For ctDNA analysis, digital PCR and targeted sequencing were used, with a concordance of 96–99% (n = 800, kappa 0.89–0.93). Their findings show that targeted therapies against uncommon HER2 and AKT1 mutations have clinically relevant activity, indicating that these mutations could be used to treat breast cancer. They concluded that with adequate clinical validity for introduction into standard clinical practice, ctDNA testing could provide accurate, quick genotyping that permits the selection of mutation-directed therapy for patients with breast cancer. The study’s results highlight the importance of ctDNA analysis in the development of mutation-directed medicines.

6. Use of Circulating Tumor Markers for Precision Medicine

Liquid biopsies make longitudinal sampling possible through a patient’s treatment period, provide information on the changing mutation profile of the disease in real time, and serve as predictive markers for precision medicine. NGS-based approaches can be used to build cancer mutation profiles, which can then be used to create patient-specific panels for customized treatment. Coombes and colleagues developed a tailored ctDNA profiling method for detecting breast cancer recurrence ^[58]. A patient-specific assay was made by employing whole-exome sequencing data to select 16 variations from the primary tumor, which were then evaluated against longitudinal plasma samples to detect ctDNA using ultradeep sequencing. They showed that a patient-specific ctDNA assay could be a specific and sensitive tool for disease surveillance in patients with breast cancer. ESR1 mutations have recently been discovered in the plasma of ER-positive metastatic breast cancer patients, and ESR1-mutated ctDNA has been identified as a predictive marker for response to aromatase inhibitor therapy ^[59]^[60]. In addition, mutations in the TP53/PIK3CA genes in ctDNA have been sensitive and specific circulating blood biomarkers, with increasing ctDNA copies related to therapeutic response ^[46].

Butler et al. conducted whole-exome sequencing on the cfDNA and primary tumor of two metastatic patients ^[61]. They discovered significant heterogeneity between primary and metastatic disease, and the cfDNA mirrored the metastases. They discovered that the PIK3CA p.H1047R activating mutation is present in primary tumors but not in plasma or metastatic sites. ESR1 mutations were found in the plasma and at the metastatic location but not in the primary tumor ^[61].

The PI3K/AKT/mTOR pathway is frequently dysregulated in cancer and was shown to play a critical role in tumorigenesis and treatment resistance ^[62]. The loss of PTEN and PIK3CA gene mutations are the most common genomic events seen in human malignancies, including breast cancer ^[63]. Genomic aberrations in the PI3K pathway are reported to be increased in metastatic TNBC and suggested as a mechanism of chemotherapy resistance ^[64]^[65]. Several preclinical studies underpinned that the presence of PIK3CA mutations are predictive markers of sensitivity to PI3K pathway inhibitors. However, patients with documented PI3K aberrations did not show targeted therapy responses. There is a shortage of validated predictive biomarkers for PI3K pathway inhibitors. ctDNA mutation profiling allows tracking the gain or loss of mutations in the PI3K pathway during cancer evolution and aids in patient-tailored therapy decisions.

PARP inhibitors are synthetically lethal to TNBC tumors harboring BRCA1/2 aberrations by impairing the DNA repair mechanisms ^[66]. Detecting the genomic alterations through longitudinal plasma sampling enables the identification of resistant genes to PARP inhibitors such as olaparib and veliparib. These studies show that ctDNA detection can be used to follow the molecular alterations for developing strategically targeted therapy.

7. Leveraging Next-Generation Sequencing Methods for the Application of Liquid Biopsy Markers toward Personalized Medicine of Breast Cancer

ctDNA sequencing is a non-invasive method for detecting cancer mutations that reduce biopsy-related costs and inconveniences in patients with breast cancer. For sensitive detection of known molecular alterations, methods such as ddPCR and BEAMing are more appropriate. However, ctDNA sequencing using NGS enables a greater detection rate of mutations per patient sample and copy number variations. In addition, NGS of ctDNA is convenient for investigating the presence of genetic aberrations, and this approach is an increasingly favorable method in precision oncology, where tissue biopsies are often inadequate for molecular characterization.

In the past decade, immense progress in the field of NGS has expedited the development of liquid biopsy markers for diagnostic assays. Recent studies indicate that CTCs can be used to predict late recurrence and hence guide treatment selection. However, a major challenge in the field has been the conflicting reports involving CTCs and ctDNA analysis in metastatic breast cancer ^[67]. These discrepancies are because of the low yields of intact CTCs and bulk sequencing strategies, resulting in the missing of cellular heterogeneity. Nevertheless, efforts have been made to improve CTC yields by developing multiple markers for selection and improved sequencing methods ^[68]. CTCs provide a snapshot of the genomic abnormalities found in metastatic sites and can be queried to assess mutational changes occurring during the metastatic process and the development of drug resistance ^[69]^[70]. Single-cell investigations of patient-derived CTCs revealed genomic changes such as single nucleotide variants, copy number variants, microsatellite instability, and inter- and intrachromosomal rearrangements. Furthermore, single-cell transcriptomics aids in the identification of clinically differentiated tumor subgroups and prognostic biomarkers for the therapy response ^[71]. As a result, the approach may overcome intratumoral heterogeneity issues and produce distinct lineage patterns for the biomarker development ^[69].

Emerging technologies such as the single-cell sequencing of CTCs can be a potential tool to identify spatiotemporal tumor heterogeneity, plasticity, and novel pharmacological targets for predicting clinical outcomes and treatment response ^[69]^[72]. Single-cell sequencing of CTCs can also reveal tumor evolution through the treatment regimen for the early detection of therapy resistance ^[73]. Digital measurement of intracellular ER signaling in single CTCs has been found to predict residual disease in patients with localized breast cancer treated with NACT ^[71]. Furthermore, the 17-gene CTC score suggested that endocrine therapy is insufficient to block ER signaling in functional ESR1-mutant populations, resulting in early metastatic breast cancer progression. A study conducted on circulating breast cancer cells demonstrated that the expression of PD-L1 is highly increased in CTCs in HR-positive, HER2-negative breast cancer patients ^[74]. These findings emphasize the importance of identifying CTC subpopulations that cause metastasis in order to select patients for therapies such as immune checkpoint inhibition. In addition, single-cell sequencing technology can contribute to a better understanding of the immune system’s complexity and offer new cancer treatment targets ^[75].

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