Detection of somatic mutations in circulating cell-free DNA from patients with esophageal squamous cell carcinoma


 Background: The aim of this study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in circulating cell-free DNA (cfDNA) from patients with esophageal squamous cell carcinoma (ESCC). Methods: Paired multi-regional tumor tissues, cfDNA and white blood cells (WBCs) collected from five ESCC patients before treatment from a prospective study (NCT02395705). Of them, samples from Cohort 1 (E102 and E110) were sequenced by whole-exome sequencing (WES) and those from Cohort 2 (E104, E111 and E121) were sequenced by targeted captured sequencing with a panel of 560 cancer-related genes respectively. To call somatic single nucleotide variations (SNVs) by comparing the solid tumor or cfDNA with matched WBCs, the minimal variant allele frequency (VAFmin) as 0.1% and P value <0.05 were allowed. Results: Genomic DNA (gDNA) and plasma-derived cfDNA from 26 samples were successfully sequenced. In Cohort 1, 596 (596/712, 83%) and 562 (562/796, 71%) were heterogeneous SNVs in E102 and E110 respectively. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R2 = 0.78, P <0.0001). In Cohort 2, 296 (296/323, 92%), 384 (384/423, 91%) and 331 (331/357, 93%) were heterogeneous SNVs in E104, E111 and E121respectively. cfDNA could recover an average of 60.7% (31/51; range, 35.7%-76.2%) of somatic mutations present in matched solid tumors. The correlation of VAFs between cfDNA and matched solid tumor was significantly positive (r2 =0.92, P <0.0001).Conclusions: Both sequencing approaches revealed the highly intratumoral heterogeneity in ESCC and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA. Further validation in cfDNA is required to define its potential utility for ESCC in clinical practice. Trial registrationAll patients selected in this study were from the registered clinical trial from ClinicalTrials.gov (NCT02395705). Date of registration: March 24, 2015.

are still unclear, although both environmental and genetic factors are suspected to play roles [5][6][7]. The management of ESCC is dependent on the characteristics of the patient and those of the tumor, mainly the TNM stage. Very early stage tumors may be suitable for endoscopic resection, whereas locally advanced cancers are treated with chemotherapy, chemoradiotherapy, surgical resection or combinations of these. Patients with ESCC that are not suitable for surgical management are treated with systemic chemotherapy [8].
In 1948, Mandel and Metais discovered the presence of circulating cell-free DNA (cfDNA) in human blood samples [9]. Thirty years later, it was demonstrated that serum and plasma from cancer patients contain higher concentrations of cfDNA than those from healthy individuals [10]. Later, it was found that the former harbor tumor-speci c molecular alterations, suggesting that tumor-derived cfDNA, that is, circulating tumor DNA (ctDNA), can appear in the circulation [11,12]. Despite these ndings and the increased attention during the last decade, the exact origin and molecular release mechanism of cfDNA is still not fully understood. It is assumed that release of cfDNA occurs though apoptosis and necrosis of normal as well as malignant cells [13]. Moreover, some studies provided evidence for an active release via secretion of extracellular vesicles such as exosomes [14][15][16]. It is likely that mechanisms of compaction and release of cfDNA into circulation, which may differ depending on its origin, will be re ected by different fragment sizes [17]. The cell of origin and the mechanism of cfDNA release into blood can mark cfDNA with speci c fragmentation signatures, potentially providing precise information about cell type, gene expression, cell physiology or pathology, or action of treatment [18][19][20]. cfDNA is typically found as double-stranded fragments of approximately 150 to 200 base pairs (bp) in length, corresponding to nucleosome-associated DNA [21]. The fraction of cfDNA that is released from primary tumors or metastases (i.e. ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers [11,12].
Recent studies have demonstrated technical feasibility and clinical applications of cfDNA including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. The potential to assess the genetic pro le of a patient's tumor from a simple blood draw, without the need for an invasive biopsy, makes cfDNA analysis an attractive tool [22]. However, a key initial question is whether the mutational pro le established through cfDNA testing reliably reproduces the mutational pro le derived from a direct tumor biopsy, which remains the standard of care [23]. To date, the related studies on cfDNA for ESCC are highly limited. To determine the potential to be surrogate of tissue biopsy, we did this study to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cfDNA from patients with ESCC.

Patients and sample collection
All ve ESCC patients (E102, E104, E110, E111, E121) from the ongoing clinical trial (ClinicalTrials.gov NCT02395705) in Cancer Hospital, Chinese Academy of Medical Sciences. All patients selected received no treatments before surgery and gave informed consent. Blood samples were collected in K 2 EDTA tubes (BD, USA) before surgery. Matched fresh tumor tissues were obtained during operation at the same day and stored at −80°C within 30 minutes after collection. Three sub-regional tumor tissues were analyzed in each patient, except patient E102 in whom four regions were analyzed. The study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki and approved by the Independent Ethics Committee of the Cancer Hospital Chinese Academy of Medical Sciences (Approval No. NCC2020C-207).
DNA Extraction and quanti cation.
Peripheral blood samples were processed within 2 hours after collection. Plasma was separated from blood by centrifugation at 1,600 g for 10 min at 4°C followed by the second centrifugation at 16,000 g for 10 min at 4°C to remove debris. After the rst centrifugation, the white blood cells (WBCs) were collected. Afterwards, Plasma and WBCs were stored at −80°C until DNA extraction. Germline DNA (gDNA) of tumor tissues and WBCs was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany). cfDNA was extracted from 4 mL plasma using the QIAamp Circulating Nucleic Acid Kit (QIAGEN, Germany) and eluted in 50 μL of AVE buffer. The quality of gDNA about degradation and contamination was monitored on 1% agarose gel, while the concentration was measured by Qubit DNA Assay Kit in Qubit 2.0 Flurometer (Invitrogen, USA). The quality and quantity of cfDNA were assessed with Agilent Bioanalyzer 2100 (Agilent Technologies, USA) and Qubit dsDNA HS Assay Kit (Invitrogen, USA).
Library construction and sequencing of DNA.
Indexed Illumina NGS libraries were prepared from cfDNA and gDNA. Brie y, fragmentation was carried out on gDNA by hydrodynamic shearing system (Covaris, Massachusetts, USA) to generate 180-280 bp fragments. cfDNA was used for library construction without additional fragmentation. The NGS libraries were constructed using the Agilent SureSelect XT Custom Kit (Agilent Technologies, USA) following the manufacture's instruments. Every sequencing library was prepared using a combination of the KAPA Hyper Prep Kit (Kapa Biosystems, USA) and the SureSelect Target Enrichment System (Agilent Technologies, USA). End repair and A-tailing reactions were performed in reaction of 60μL volumes. The mixtures were incubated at 20°C for 30 minutes and 65°C for 30 minutes respectively. Adapter ligation was performed using 110 μL and samples were incubated at 16°C overnight using Agilent SureSelect Adapter. After post-ligation clean-up, the ligated fragments were ampli ed in 50uL reaction containing KAPA HiFi HotStart ReadyMix and KAPA library Ampli cation Primer Mix. The following PCR ampli cation protocol was used: initial denaturation 98°C for 45 s; 14-16 cycles (minimum number required for optimal ampli cation depending on the input DNA amount) of denaturation 98°C for15s, annealing 60°C for 30s, and extension 72°C for 30s; nal extension 72°C for 1 min; and hold in 4°C. The quality and quantity of library were determined by Qubit 2.0 DNA Assays (Invitrogen, USA) and Agilent Bioanalyzer 2100 (Agilent, USA).
At rst, the whole-exome sequencing (WES) was carried out on both cfDNA and gDNA in Cohort 1. The captured DNA was sequenced on the NovaSeq 6000 platform (Illumina, USA) with a mean sequencing depth of 300×. Afterwards, the targeted deep sequencing was performed on captured DNA of three patients in Cohort 2 by an established panel of 560 tumor-related genes (All-in-one Cancer Panel, Novogene, China) (a list of 560 genes was in Additional les 1) on the same sequencing platform with 150bp reads at a mean sequencing depth of 1000×.

Data processing and somatic mutation detection
The original uorescence image les obtained from NovaSeq platform were transformed to short reads (raw data) by base calling and recorded in FASTQ format, which contained sequence information and corresponding sequencing quality information. After excluding reads containing adapter contamination and low-quality or unrecognizable nucleotides, clean data were applied for downstream bioinformatical analyses. At the same time, the total reads number, sequencing error rate, percentage of reads with average quality >20 and with average quality >30. Valid sequencing data was mapped the 150 bp pairedend reads to the reference hg38 by the BWA-MEM in default mode to get the original mapping results stored in BAM format [24], with the mapping e ciency achieved > 99%. The GATK was used to remove the duplicates for solid samples. We used SAMtools [25] to Picard MarkDuplicates [26] to sort BAM les and do duplicate marking, local realignment, and base quality recalibration to generate nal les of solid tumor or cfDNA and matched WBCs for computing the sequence coverage and depth. The MuTect and Strelka [27,28]were respectively applied to call somatic single nucleotide variations (SNVs) and small insertions and deletions (InDels) by comparing the solid tumor or cfDNA with matched WBCs sample, allowing the minimal variant allele frequency (VAF min ) as 0.1% and the P value <0.05. Then we ltered out those variants: (1) the number of supporting tumor reads in WBCs sample >2 or the VAF in WBCs > 1%; or (2) the read depth of solid tumor or cfDNA < 100; or (3) the supporting tumor reads in solid tumor or cfDNA appeared in only one strand; or (4) the number of supporting tumor reads in solid tumor or cfDNA ≤10 * the number of supporting tumor reads in WBCs samples. Subsequently, the ltered variants were annotated by ANNOVAR [29]. All quali ed mutations including silent and non-silent SNVs were used to construct the phylogenetic trees. At rst, we used the appearance of all the SNVs in each sample to generate a binary table. Then Camin-Sokal parsimony method using PHYLIP [30]based on the binary table was used to construct the phylogenetic tree. The relationship of VAFs between cfDNA and matched solid tumor was analyzed by the Pearson's correlation analysis and the P-value < 0.05 was considered signi cant.

Results
Overall, all ve patients (E102, E104, E110, E111, E121) with ESCC were analyzed in this study. The baseline characteristics of ve patients in our study were showed in Table 1. The mean concentration of cfDNA from 4mL plasma was 0.74ng/uL (range, 0.35-1.28ng/ul). The predominant type of cfDNA extracted had a fragment size centered around 160 bp exhibited by Agilent Bioanalyzer 2100 (supplementary Figure S1A and B) (Additional les 3). The gDNA extracted was not degraded and not contaminated with RNA or protein exhibited by agarose gel electrophoresis (supplementary Figure S1C) (Additional les 3). The WES was successfully performed in those two patients of 9 samples. The quality of WES data with mapping ratio of >99% and exome enrichment of >99% was acceptable (supplementary Table S1) (Additional les 2). Overall, the most common type of mutation was C>T transition and the major type of mutation was missense mutation (supplementary Figure S2) (Additional les 3), which had been observed in previous study of ESCC [31]. The total mutations (silent and non-silent) for solid tumor ranged from 290 to 483, with a median value 342 (221 for silent ones and 123 for non-silent ones). We found 915 and 863 mutations (376 silent and 340 non-silent ones) for two cfDNA samples respectively. Tumor mutation burden (TMB) for solid tumor samples ranged from 4.92 to 8.19 mutations /M, whereas cfDNA samples had higher TMB from 14.63 to 15.51 mutations /M. A total of 694 non-silent mutations were discovered in 9 samples. In E102, a total of 712 mutations (silent and non-silent mutations) were identi ed in tumor tissues, 119 of which were ubiquitous in T1, T2, T3 and T4 tumor regions. Meanwhile, 796 mutations (silent and non-silent mutations) were identi ed in E110, 234 of which were ubiquitous in T1, T2 and T4 tumor regions. To explore intratumoral heterogeneity and the genomic evolution of ESCC, phylogenetic trees were constructed on the basis of somatic mutations (both silent and non-silent mutations) identi ed in each region. The phylogenetic trees varied extensively in two cases, which showed evidence of spatial intratumoral heterogeneity, with an average of 76.6% (E102 of 83.2% and E110 of 70.6%) of somatic variants having spatial heterogeneity ( Figure 1A).
13 genes previously reported in ESCC were con rmed in this cohort, including TP53, MUC16, CTNND2, DNAH9, EP300, NOTCH3, SYNE1, RP1, TTN, ATM, KMT2D, NOTCH1 and USH2A [31,32]. However, only MUC16, CTNND2 and NOTCH3 were detected in matched cfDNA ( Figure 1B). In addition, 12 of 20 (60.0%) tumor-associated mutations identi ed were found in cfDNA, which were the important driver genes in four oncogenic pathways, including NOTCH (NOTCH3, NCOR2, SPEN), RTK-RAS (ARHGAP35, KSR1, IRS1), WNT (DVL3, FRAT1) and Hippo (TAOK2, LLGL2, TEAD2) signaling pathways ( Figure 2). In addition, we found two novel cancer-related genes (MUC4 and MUC17) in both of the two patients which were not previously reported in ESCC. The details of somatic mutations and prevalence in two ESCC patients were showed in supplementary Table S2 and S3 (Additional les 2). The Pearson's correlation analysis of VAFs between cfDNA and matched solid tumor showed the positive linear relationship (R 2 =0.78, P <0.0001) ( Figure 3). The VAFs of ubiquitous and heterogeneous somatic mutations in cfDNA and solid tumor were listed in supplementary  Table S5) (Additional les 2). The most common type of mutation was C>T transition and the majority of variant type was missense mutation, which were the same as Cohort 1 via WES (supplementary Figure S3) (Additional les 3). The total mutations (silent and non-silent) for solid tumor ranged from 107 to 194, with a median value of 142. We found 494, 769 and 262 mutations (245, 407 and 121 non-silent ones respectively) for three cfDNA samples respectively. A total of 333 genes were mutated (non-silent mutations) in 12 samples (supplementary Table S6 and S7) (Additional les 2). The phylogenetic trees were constructed on the basis of somatic mutations (both silent and non-silent mutations) identi ed in each region of those three cases. The phylogenetic trees varied extensively as well, which showed evidence of spatial intratumoral heterogeneity, with an average of 91.7% (1011/1103; range, 90.5%-92.7%) of heterogeneous somatic mutations ( Figure 4A).
32 mutated genes previously reported in ESCC were con rmed in this cohort [31][32][33]. In patient E104, 323 variants were detected in three sub-regions, 27 out of which were ubiquitous variants. KMT2D, TP53, UBR5 from the identi ed variants were previously con rmed as frequent mutations in ESCC [31,32]. In patient E111, 423 mutations were detected totally, 39 out of which were ubiquitous variants in three regions. Among them, CSMD3, PTCH1, TP53, PIK3CA were important and associated with ESCC. In Patient E121, 357 mutations were detected totally, 26 of which were ubiquitous variants. Among them, FBXW7, LRP1B, TP53, MTOR and EP300 were con rmed as frequent mutations in ESCC ( Figure 4B). To determine the VAFs of tumor mutations in cfDNA. The VAF min we setup in algorithm for detection of mutations was 0.1%. The rst quantile and third quantile of VAFs in cfDNA were 0.49-0.88%, 0.44-0.77% and 0.41-0.875% for patient E104, E111 and E121 respectively (supplementary Figure S4) (Additional les 3). In total, there were 14 mutations with VAFs of >5% detected in cfDNA. The results showed ESCC had low ctDNA in the blood, which was similar to one previous study on EAC [34]. Moreover, the variants called from cfDNA were much more than the ones called from solid tumor tissues. Some true positive calls were missed in our solid tumor samples due to the intratumor heterogeneity and sampling biases. Some were potential false positive calls possibly due to clonal hematopoiesis (CH). Therefore, we focused on the ESCC recurrent genes to answer the following questions: how many mutated genes in solid tumor were identi ed in cfDNA? How many important mutations were found in cfDNA only? For patient E104, 10 out of 16 (62.5%) ESCC-associated mutations detected in solid tumor were recovered in paired cfDNA, including CSMD3, KMT2D, LRP1B, SYNE1, ATR, EP300, PRDM1, UBR5, FSTL5, LIFR. In E111, SYNE1, BRCA2, ATR, EP300, ASXL1, MTOR, PKHD1, PTCH1, UBR5, BRIP1, KMT2A, NUP214, NOTCH2, CREBBP. In E121, 35.7% (5/14) ESCC-associated mutations were found in both solid tumor and cfDNA, including KMT2D, LRP1B, EP300, PKHD1, PRDM1. Moreover, KMT2D, SYNE1 and UBR5 were shared by cfDNA and all regions of tumor in E104. PTCH1 was the shared one in E111 and LRP1B was shared by three sub-regional tumors and cfDNA in E121 ( Figure 5). Mutations in TP53 were often detected in solid tumor only, whereas mutations in CBL, POLE, PTCH1 and NFE2L2 were only detected in cfDNA.
Furthermore, the correlation of VAFs between cfDNA and matched solid tumor was strong (R 2 =0.92) with a signi cant P-value of <0.0001 ( Figure 6). The VAFs of somatic mutations in cfDNA and solid tumors were showed in supplementary Table S8 (Additional les 2).

Discussion
Analysis of somatic alterations in tumor tissue has become routine practice in clinical oncology. Although these alterations are highly informative, sampling tumor tissue has limitations as tissue biopsies are often di cult to obtain and are subjected to sampling bias resulting from temporal and spatial tumor heterogeneity [33]. Therefore, alternative strategies, such as liquid biopsies, are currently evaluated for applicability in different clinical settings. Liquid biopsies have a few advantages compared to tissue biopsies; they are less invasive, safe and may overcome di culties of intratumoral heterogeneity [35].
Together with the possibility of multiple assessments over time, the use of cfDNA may be able to predict response to treatment and patterns of therapeutic resistance earlier and more accurately than radiological imaging [34,36]. The ability to analyze tumor-derived DNA from a routine blood draw without the need for an invasive tumor biopsy represents a critical advance with potentially transformative clinical applications. In particular, the minimally invasive nature of cfDNA analysis provides a means of molecular pro ling for tumors that are di cult or unsafe to biopsy and allows a practical means for monitoring tumor DNA serially over time without the risk and potential complications of standard tumor biopsy. In addition, cfDNA analysis may better capture the molecular heterogeneity harbored by multiple distinct clonal populations in a patient's tumor, as compared with a needle biopsy of a single tumor lesion. Finally, cfDNA analysis offers the potential for tumor detection or monitoring in patients without clinically evident disease [35].
Recent developments in NGS technologies, enable the testing of cfDNA with high sensitivity and speci city [37]. However, the low amount of cfDNA in the blood presents challenges in constructing sequencing libraries with high quality and complexity, especially in ESCC. Notably, to minimize the likelihood of detecting mutations due to CH, some mutations involved in CH that were also observed in the matched WBCs samples should be excluded [38]. Thus, we simultaneously sequenced the leukocytes fraction of an individual to be able to properly attribute the mutations in their cfDNA to tumors [39]. In this pilot study, we respectively used the WES and targeted sequencing with a panel of 560 cancer-associated genes to detect mutations in paired multi-regional solid tumors and cfDNA. Some ESCC-associated mutations identi ed previously were detected in cfDNA in two cohorts, including MUC16, CTNND2, NOTCH3, CSMD3, KMT2D, LRP1B, SYNE1, BRCA2, NFE2L2, EP300, ATR, ATM, ASXL1, MTOR, PTCH1, PKHD1, PRDM1, UBR5, KMT2A, NOTCH2, ERCC3, URIP1, CBL, FSTL5, LIFR and POLE [31][32][33]. All the results showed that both platforms had the ability to detect ESCC-associated mutations in cfDNA. We found the targeted sequencing in cohort 2 identi ed more important genes than WES in cohort 1. Since ESCC has low tumor fraction in cfDNA, at a similar price, targeted sequencing with panel of higher sequencing depth is better for detecting variants of low VAFs than WES of compromised sequencing depth, which is why most groups used NGS panels encompassing a select set of genes commonly in studies on cfDNA. From our results, multi-regions of primary tumor and matched cfDNA WES was used to identify somatic mutations present in each of tumor regions and matched cfDNA, which demonstrated highly spatial intratumoral heterogeneity in all tumors. Moreover, 12 cancer-associated mutations identi ed were found in cfDNA, which were the important driver genes in four oncogenic signaling pathways, including NOTCH, RTK-RAS, WNT and Hippo signaling pathways which play important roles in the occurrence and development of tumor [40]. Although a few ubiquitous mutations were found in cfDNA due to limited number of cases, the VAFs of those common alterations between paired solid tumor and cfDNA were signi cantly correlated. The results suggested that genetic information from limited number of tumor tissues biopsied could not represent the whole genetic pro le of ESCC and cfDNA could be an adjuvant tool for revealing the genetic characteristics of ESCC.
Afterwards, we performed target captured sequencing with a panel of 560 cancer-related genes of higher depth in another cohort. For the previously con rmed variants in ESCC, the mutation density in cfDNA was signi cantly higher than solid tumor, which suggested that cfDNA could present more information of mutations than solid tumor. On the one hand, cfDNA could recover an average of 60.7% (31/51; range, 35.7%-76.2%) of mutations present in solid tumor samples, which was similar to the rates of previous studies on other cancers [38,41,42]. The Pearson's correlation of VAFs between cfDNA and matched solid tumor was signi cantly positive, which indicated genomic pro le of cfDNA could represent the information of ubiquitous and heterogeneous mutations in solid tumors. On the other hand, many somatic mutations which were appeared in three sub-regions were not identi ed in their matched cfDNA. For instance, we found that some important ESCC-related mutations identi ed in solid tumors of some patients, including TP53 R273H and TP53 R175H mutations, were not detected in matched cfDNA, and out of expectation those mutations had high VAFs in solid tumor. The majority of these variants of high VAFs in solid tumor had important functions in the development of ESCC. In addition, TP53 R273H mutation was shown in three sub-regions of patient E111 with VAF of 22.98%, 42.86% and 34.79% respectively, but only 0.93% in cfDNA. This observation agreed with a previous study that one ESCC patient had a TP53 N1311 mutation with VAF of 52.9 % in tumor and 1.3% in cfDNA from preoperative plasma [43]. One possible explanation could be that the colonies of tumor cells with driver mutations are resistant to apoptosis, and rarely release mutated DNA fragments to plasma.
Successful monitoring of response to treatment carries several important implications. First, it provides physicians with a rapid assessment of the success of the treatment choice and may allow for modi cation of therapeutic regimens if inadequate response is noted. Second, poor clinical response to neoadjuvant treatment may be identi ed early during therapy, thereby allowing changes in the treatment plan, including surgical management [34]. Therefore, as a repeat or serial testing tool, cfDNA testing for treatment selection and tracking therapeutic response is increasingly used as an alternative to repeat invasive biopsy and may reveal actionable mutations that guide treatment decisions in these patients [22]. Recently, data from one study with longitudinal EAC samples indicated that ctDNA levels correlate with and precede evidence of response to therapy, which demonstrated that the potential of ctDNA as a dynamic biomarker to monitor treatment response in patients with EAC. Moreover, the VAFs of some mutations were lower or equaled zero in postoperative plasma from ESCC patients [34]. Similarly, somatic mutations can be detected in preoperative cfDNA from patients with ESCC at stage IIA to IIIB , and at a lower frequency in postoperative cfDNA in another study [44]. Pectasides et al. recently explored cfDNA as a tool to identify therapeutic targets not detectable from standard tissue-based testing in gastric and esophageal adenocarcinomas (GEA) with metastatic lesions. cfDNA pro ling may ultimately provide a more accurate representation of disseminated disease in GEA [45]. These results demonstrated that ctDNA is a valuable biomarker for tracking tumor status and evaluating treatment effect. One such study in our medical center is ongoing (ClinicalTrials.gov NCT02395705), which involves the evaluation of the survival bene t of neoadjuvant chemotherapy (cisplatin plus paclitaxel) versus surgery alone in ESCC patients. This study will also determine the feasibility of using circulating biomarkers, including cfDNA, to reliably predict the sensitivity of neoadjuvant chemotherapy and early screen patients with insensitive response to chemotherapy before treatment so as to reduce unnecessary chemotherapy and change therapeutic plan for those ESCC patients. In addition, longitudinal sampling of cfDNA from diagnosis to relapse will be done to monitor minimal residual disease after surgery and detect the recurrence. The targeted sequencing approach with a panel of 560 cancer-related genes presented here will be applied to this clinical trial to analyze cfDNA. The results from our pilot study and previously related studies demonstrated that integration of real-time cfDNA analysis into clinical trials and eventually into standard clinical management has the potential to become a valuable tool for revealing tumor heterogeneity and monitoring therapeutic response.
Inevitably, there were some limitations in our study. First, our study cohort size was limited. Although the number of patients enrolled was small, we collected multi-regional samples of each tumor, which could exactly indicate the intratumoral heterogeneity in ESCC. As far as we know, compared to most studies which sampled single region of tumor, it is the rst time to sequence multi-regional tumors and matched cfDNA in ESCC that could exactly reveal the high intratumoral heterogeneity in ESCC and the ability of cfDNA to detect both ubiquitous and heterogeneous mutations and represent the genetic characteristics of solid tumor. Second, owing to lack of materials we could not do an independent validation of the mutations detected in cfDNA. To overcome this shortcoming, we carefully monitored sequencing error rates for all positions for which we identi ed mutations in cfDNA by our prede ned algorithm and criteria of data processing. In addition, we used two NGS approaches to sequence two cohorts independently and to some extent, they could validate each other mutually. Technically, we found the targeted sequencing of cancer-speci c gene panel with high depth is suitable for detecting variants of low VAFs in cfDNA in some tumors, such as ESCC. In the last decade, cfDNA has become a very hot topic in oncology and it is almost impossible to keep pace with the number of new papers published every day. However, there are quite a few fundamental problems for which we do not have an answer. First, our basic knowledge on cfDNA is far from being complete. We still do not know exactly all of the mechanisms leading to the release of cell-free DNA into circulation. Second, there are some technical and methodological issues that have to be solved. So far, there has not been consensus on a "gold standard" for the isolation of cfDNA. Finally, computational approaches should be improved to perform the genetic analysis of cfDNA sensitively deconvoluting the cancer-speci c signals from the mixture of cancer and normal signals in cfDNA.

Conclusions
Both WES and targeted sequencing approaches revealed the highly spatial intratumoral heterogeneity in ESCC and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA.

Declarations
Ethics approval and consent to participate The study was performed in accordance with the ethical guidelines of the Declaration of Helsinki and approved by the Independent Ethics Committee of the Cancer Hospital Chinese Academy of Medical Sciences (Approval No. NCC2020C-207).

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information les and are available from the corresponding author on reasonable request. The occurrence of known ESCC-related mutations in Cohort 1. Phylogenetic trees were constructed from all somatic mutations detected in multi-regional tumors, with ubiquitous mutations on the trunk of the tree, and heterogeneous mutations on the branches. Underneath each phylogenetic tree is the number (n) and percentage of heterogeneous mutations (A). 13 genes previously reported in ESCC were con rmed in this cohort, including TP53, MUC16, CTNND2, DNAH9, EP300, NOTCH3, SYNE1, RP1, TTN, ATM, KMT2D, NOTCH1 and USH2A. Only MUC16, CTNND2 and NOTCH3 are detected in cfDNA (B).

Figure 2
The occurrence of oncogenic pathway genes. 12 (12/20, 60.0%) cancer-associated mutations identi ed were found in cfDNA, which were the important driver genes in ve oncogenic pathways. Each pathway corresponds to related driver genes in the heat map. The occurrence of con rmed ESCC-associated mutations in Cohort 2. Phylogenetic trees were constructed from all somatic mutations detected in multi-regional tumors, with ubiquitous mutations on the trunk of the tree, and heterogeneous mutations on the branches. Underneath each phylogenetic tree is the number (n) and percentage of heterogeneous mutations (A). 32 genes previously reported in ESCC were con rmed in this cohort, 28 of which were detected in cfDNA (B). The distribution of the overlapped ESCC-related genes between multi-regional solid tumors and cfDNA. In PTCH1 was the shared one in E111 (B, right) and LRP1B was shared in E121 (C, right).