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Liquid biopsy-based analysis by ddPCR and CAPP-Seq in melanoma patients

      Highlights

      • Cell-free DNA (cfDNA) from melanoma patients was analyzed by liquid biopsy.
      • CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) was performed.
      • CAPP-Seq and droplet digital PCR methods revealed gene mutations in cfDNA.
      • Gene mutations, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in cfDNA.
      • The MET copy numbers were consistent with the disease status in melanoma patients.

      Abstract

      Background

      The development of BRAF/MEK inhibitors in patients with metastatic melanoma harboring BRAF mutations has garnered attention for liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) or next-generation sequencing methods.

      Objective

      To investigate gene mutations in tumor DNA and cfDNA collected from 43 melanoma patients and evaluate their potential as biomarkers.

      Methods

      ddPCR and CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) techniques were performed to detect gene mutations in plasma cfDNA obtained from patients with metastatic melanoma.

      Results

      Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in the plasma cfDNA, and the results were partially consistent with the results of those identified in the tissues. Among the variants examined, copy numbers of MET mutations were consistent with the disease status in two melanoma patients.

      Conclusion

      Liquid biopsy using CAPP-Seq and ddPCR has the potential to detect tumor presence and mutations, especially when tissue biopsies are unavailable. MET mutations in cfDNA may be a potential biomarker in patients with metastatic melanoma.

      Abbreviations:

      cfDNA (cell-free DNA), ddPCR (droplet digital PCR), CAPP-Seq (CAncer Personalized Profiling by deep Sequencing), ctDNA (circulating-tumor DNA), NGS (next-generation sequencing)

      Keywords

      1. Introduction

      Melanoma is associated with high mortality, especially in the metastatic stage. However, BRAF/MEK inhibitors have recently been reported to improve the overall survival in melanoma patients with BRAF mutations [
      • Robert C.
      • Karaszewska B.
      • Schachter J.
      • Rutkowski P.
      • Mackiewicz A.
      • Stroiakovski D.
      • Lichinitser M.
      • Dummer R.
      • Grange F.
      • Mortier L.
      • Chiarion-Sileni V.
      Improved overall survival in melanoma with combined dabrafenib and trametinib.
      ]. The sensitivity and specificity of the real-time PCR-based test for detecting BRAF mutations are high for clinical use [
      • Franczak C.
      • Salleron J.
      • Dubois C.
      • Filhine-Trésarrieu P.
      • Leroux A.
      • Merlin J.L.
      • Harlé A.
      Comparison of five Different Assays for the detection of BRAF mutations in formalin-fixed paraffin embedded tissues of patients with metastatic melanoma.
      ], but it has limitations, such as the need for tissue collection from metastatic lesions. Additionally, tumor biopsy is invasive, and some tumor lesions may not be accessible [
      • Boyer M.
      • Cayrefourcq L.
      • Dereure O.
      • Meunier L.
      • Becquart O.
      • Alix-Panabières C.
      Clinical relevance of liquid biopsy in melanoma and merkel cell carcinoma.
      ]. Furthermore, mutation-positive results may be overlooked because mutational heterogeneity exists between primary and metastatic lesions [
      • Kaji T.
      • Yamasaki O.
      • Takata M.
      • Otsuka M.
      • Hamada T.
      • Morizane S.
      • Asagoe K.
      • Yanai H.
      • Hirai Y.
      • Umemura H.
      • Iwatsuki K.
      Comparative study on driver mutations in primary and metastatic melanomas at a single Japanese institute: a clue for intra- and inter-tumor heterogeneity.
      ]. Thus, real-time PCR is inferior to droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to evaluate BRAF mutations. Therefore, a less invasive and more sensitive method is required to overcome these challenges.
      Recently, attention has been focused on liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using ddPCR [
      • Santiago-Walker A.
      • Gagnon R.
      • Mazumdar J.
      • Casey M.
      • Long G.V.
      • Schadendorf D.
      • Flaherty K.
      • Kefford R.
      • Hauschild A.
      • Hwu P.
      • Haney P.
      Correlation of BRAF mutation status in circulating-free DNA and tumor and association with clinical outcome across four BRAFi and MEKi clinical trials.
      ]. cfDNA is fragmented DNA in the blood derived from tumor cells that have undergone apoptosis or necrosis, containing the same genetic mutations as the source tumor [
      • Calapre L.
      • Warburton L.
      • Millward M.
      • Ziman M.
      • Gray E.S.
      Circulating tumour DNA (ctDNA) as a liquid biopsy for melanoma.
      ]. It has been reported that cfDNA in many cancer types can be used to detect minimal cancer presence [
      • Dawson S.J.
      • Tsui D.W.
      • Murtaza M.
      • Biggs H.
      • Rueda O.M.
      • Chin S.F.
      • Dunning M.J.
      • Gale D.
      • Forshew T.
      • Mahler-Araujo B.
      • Rajan S.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      ] and monitor disease status [
      • Crowley E.
      • Di Nicolantonio F.
      • Loupakis F.
      • Bardelli A.
      Liquid biopsy: monitoring cancer-genetics in the blood.
      ,
      • Gray E.S.
      • Rizos H.
      • Reid A.L.
      • Boyd S.C.
      • Pereira M.R.
      • Lo J.
      • Tembe V.
      • Freeman J.
      • Lee J.H.
      • Scolyer R.A.
      • Siew K.
      Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma.
      ].
      Moreover, to improve circulating tumor DNA (ctDNA) detection, an ultrasensitive NGS-based method for ctDNA detection, named CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), was recently established [
      • Newman A.M.
      • Bratman S.V.
      • To J.
      • Wynne J.F.
      • Eclov N.C.
      • Modlin L.A.
      • Liu C.L.
      • Neal J.W.
      • Wakelee H.A.
      • Merritt R.E.
      • Shrager J.B.
      An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.
      ]. CAPP-Seq is the first NGS-based ctDNA analysis method that enables coverage of various malignancies, and it has an ultralow detection limit at low ctDNA input levels at a reasonable cost, thereby facilitating quantitation of ctDNA from early-stage tumors. The CAPP-Seq method shows similar sensitivity to digital PCR or amplicon-based approaches at hotspot alleles and enables simultaneous examination of multiple genomic positions without affecting sensitivity or specificity. This ultrasensitive method can detect ctDNA in patients with early and advanced stages of various malignancies [
      • Iwahashi N.
      • Sakai K.
      • Noguchi T.
      • Yahata T.
      • Matsukawa H.
      • Toujima S.
      • Nishio K.
      • Ino K.
      Liquid biopsy-based comprehensive gene mutation profiling for gynecological cancer using CAncer Personalized Profiling by deep Sequencing.
      ].
      In this study, we investigated gene mutations in both tumor DNA and cfDNA collected from melanoma patients to evaluate their potential as biomarkers, using ddPCR and CAPP-Seq methods. In addition to the analysis of BRAF mutations, we also investigated other gene mutations in tumor DNA and cfDNA, which might be used as biomarkers in melanomas with BRAF mutations.

      2. Materials and methods

      2.1 Ethical approval and informed consent

      The study design was approved by the Ethics Review Committee of Kumamoto University (authorization number: 1452). All methods were performed in accordance with the Declaration of Helsinki, as specified by the Kumamoto University Faculty of Medicine. All participating patients provided signed informed consent before enrollment.

      2.2 Patients and samples

      We obtained 15 plasma samples for CAPP-Seq analysis. Moreover, for ddPCR analysis, we collected an additional 28 plasma samples and corresponding tumor tissues from patients with melanoma (Table 1a). Eight normal tissue and plasma samples were used as controls.
      Table 1aThe profiles of 43 Japanese patients with melanoma.
      CaseAgeSexPathologic Staging (T, N, M)StageTypeTT (mm)Biopsy or resection siteMetastatic siteBRAF
      155M1a, 1a, 0IIIAnon-CSD/SSM0.92backLNV600E
      283F3b, 2a, 0IIIBnon-CSD/SSM3waistLNV600E
      357M4b, 1b, 0IIICAcral/ALM4.5soleLNV600E
      459M2b, 3, 1cIVnon-CSD/SSM1.9upper armlung, gallbladder, adrenal, skin, muscle, LNV600E
      574M4a, 1b, 1cIVnon-CSD/SSM8abdomenliver, spleen, bone, skin, LNV600E
      659F1a, 3, 1cIVnon-CSD/SSM0.9femurlung, liver, bone, peritoneum, LNV600E
      773M4b, 1b, 1cIVnon-CSD/SSM23backupper pharynx, lung, liver, skin, LNV600K
      879M2a, 1a, 1cIVnon-CSD/SSM1.8backbrain, liver, bone, LNV600K
      939Mx, 3, 1cIVNMNAchestbrain, lung, liver, peritoneum, pleural, bone, LNV600E
      1072M4b, 2b, 1bIVAcral/ALM16.5toelung, LNV600E
      1178Fx, x, 1cIVNANANAliver, pancreasV600E
      1282Mx, 3, 1cIVNANANAbrain, lung, liver, adrenal, peritoneum, skin, bone, LNV600K
      1322Mx, 0, 0NAMucosalNAconjunctivaV600E
      1457M4b, 1a, 1cIVnon-CSD/SSM14shoulderlung, LN
      1558F4b, x, 1cIVNM4.3headlung, bone
      1667F3b, 1a, 1cIVAcral/ALM2.3solelung, liver, LN
      1751F4b, 2a, 1aIVAcral/ALM4.5toeLN
      1857Mx, 2b, 1cIVAcral/ALMNAtoeLN
      1974F3b, 2c, 1cIVAcral/ALM2.7footlung, liver, skin, muscle, bone, LN
      2083Mx, x, 1bIVNANANAlung
      2171Fx, 1b, 1cIVMucosalNAesophagusliver, LN
      2250M2b, 1a, 1cIVMucosal1.8liplung, skin, LN
      2381Fx, 0, 0NAMucosalNAvagina
      2474Mx, 1b, 0NAMucosalNAesophagusLN
      2574Mx, 1b, 0NANANAlungLN
      2675M4b, 2b, 0IIICnon-CSD/SSM14abdomenLN
      2771F4a, 3, 0IIICnon-CSD/SSM10kneeLN
      2883F4b, 2b, 0IIICAcral/ALM8toeLNNA
      2965M4b, 2a, 0IIIBAcral/ALM4.5heelLN
      3063F3a, 3, 1cIVnon-CSD/SSM2.8headlung, liver, skin, LN
      3182M4b, 0, 1bIVAcral/ALM6palmlung
      3261Fx, 2b, 1aIVMucosalNAnasal cavityLN
      3366F4b, 0, 1bIVNM17femurlungV600E
      3472M4a, 1a, 0IIIAAcral/ALM4.2soleLN
      3575F4a, 2a, 0IIIANM4.5legLN
      3679F4a, 2a, 0IIIAAcral/ALM7toeLN
      3751F4b, 2a, 0IIIBAcral/ALM8fingerLN
      3874F4b, 1a, 0IIIBAcral/ALM6.6heelLN
      3970M2a, 3, 0IIICnon-CSD/SSM1.9legLNNA
      4071M4b, 3, 0IIICAcral/ALM7.5soleLN
      4176M4b, 2b, 0IIICAcral/ALM7fingerLN
      4282M3a, 3, 0IIICLMM2.3cheekLN
      4371Mx, 2c, 1cIVAcral/ALMNAheellung, peritoneum, small intestine, LN
      Tumors were classified according to the AJCC 7th edition staging system. CSD, melanoma on skin with chronic sun-induced damage; SSM, superficial spreading melanoma; ALM, acral lentiginous melanoma; LMM, lentigo maligna melanoma; TT, tumor thickness; LN, lymph node; NA, not available or not assessed.

      2.3 CAPP-Seq of cfDNA from melanoma patients

      The cfDNA from the plasma samples (2 mL) of pretreated patients was subjected to CAPP-Seq using a gene-sequencing panel as per previously reported methods [
      • Newman A.M.
      • Bratman S.V.
      • To J.
      • Wynne J.F.
      • Eclov N.C.
      • Modlin L.A.
      • Liu C.L.
      • Neal J.W.
      • Wakelee H.A.
      • Merritt R.E.
      • Shrager J.B.
      An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.
      ]. CAPP-Seq for 77 genes was performed using the AVENIO ctDNA expanded kit (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer’s instructions. The purified libraries were sequenced using an Illumina NextSeq System (Illumina, San Diego, CA, USA). Variants were called with the AVENIO Oncology Analysis Software (version 2.0.0; Roche Diagnostics). Germline mutations were excluded based on the following databases: ExAC 1.0, dbSNP 150, and 1000 Genomes phase_3_v5b (Rikengenesis, Kanagawa, Japan). Somatic mutations were determined based on the following databases: COSMIC v83 and TCGA 9.0 (Rikengenesis).

      2.4 cfDNA extraction from plasma samples

      cfDNA was isolated from plasma (1 mL) using the QIAamp circulating nucleic acid kit (Qiagen, Hilden, Germany). All plasma samples were stored at −20 °C before use. cfDNA concentration was measured using Qubit Fluorometric Quantitation (Thermo Fisher Scientific, Waltham, MA, USA).

      2.5 Genomic DNA (gDNA) extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples

      FFPE Sections (4 μm thick) of primary or metastatic tumor tissues from patients with no history of systemic treatment, including immune checkpoint inhibitors, were used for ddPCR analyses. gDNA was extracted and purified using the QIAamp DNA FFPE Tissue Kit (Qiagen). The gDNA concentration was measured using a NanoDrop Lite (Thermo Fisher Scientific).

      2.6 ddPCR analysis

      Copy numbers of BRAF, NRAS, TP53, GNAS, and MET DNA were analyzed in tissue samples, and cfDNA was derived from plasma samples and quantified by droplet digital PCR (ddPCR; QX200 Droplet Digital PCR System, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The probes for ddPCR were purchased from Bio-Rad with the following assay ID numbers: BRAF p.V(Val)600E(Glu); dHsaMDV2010027, BRAF p.V(Val) 600 K (Lys), dHsaMDV2010035, NRAS p.Q(Gln)61R(Arg) c.182A(Ala)>G(Gly); dHsaMDV2010071, NRAS p.Q(Gln) 61 K (Lys) c.181C(Cys)>A(Ala); dHsaMDV2010067, TP53 p. R (Arg)267 P(Pro), dHsaMDS2512068, GNAS p. R (Arg)201C(Cys), dHsaMDV2510562, and MET p.V(Val)1088 M(Met); dHsaMDS694595282. The reaction was carried out in a total volume of 20 μL, including the DNA sample, 1 μL of each probe, 1 μL of the restriction enzyme, and 10 μL of primers. The PCR amplifications were performed under the following conditions: 1 cycle at 95 °C for 10 min; 40 cycles at 94 °C for 30 s, at 55 °C (BRAF, NRAS, TP53, and GNAS) or 51 °C (MET) for 1 min, 1 cycle at 98 °C for 10 min, and 4 °C as the holding temperature. Data were processed using QuantaSoft Version 1.7 (Bio-Rad).

      3. Results

      3.1 Analysis of BRAF mutations using ddPCR

      Since the detection of BRAF mutations is used in clinical practice, we investigated the BRAF mutation to evaluate its potential as a biomarker using ddPCR (Fig. 1 a–d). According to the results of conventional analyses, patients (n = 13) positive for BRAF mutations were also found to be positive by ddPCR analysis (Fig. 1d and e). Furthermore, the results of the conventional tests revealed 12 patients negative for BRAF mutations, and all of these BRAF mutation-negative patients were also found to be negative for BRAF mutations on ddPCR analysis (Fig. 1d and f). Next, we studied BRAF mutations in cfDNA. Six of the 11 positive patients detected by conventional tests also harbored the same BRAF mutations in cfDNA (Fig. 1e). None of the negative patients identified using conventional tests harbored BRAF mutations in their cfDNAs, as detected by ddPCR (Fig. 1f). The sensitivity and specificity for BRAF mutation detection using ddPCR in cfDNA were 54.5 % and 100 %, respectively.
      Fig. 1
      Fig. 1Analysis of BRAF gene mutations using ddPCR.
      (a–c) Representative ddPCR results of BRAF mutations in Case No. 6 (a), Case No. 12 (b), and Case No. 18 (c). Blue dots (enclosed by red circles) indicate mutations of BRAF, green dots indicate wild-type BRAF, and grey dots indicate droplets without DNA of interest. cfDNA: cell-free DNA.
      (d) Summary of ddPCR results for BRAF mutations in tissues and cfDNA in 25 patients with malignant melanoma. THxID-BRAF (conventional real-time PCR) was used for the companion diagnostics of dabrafenib. CSD, melanoma on skin with chronic sun-induced damage; NM, nodular melanoma; SSM, superficial spreading melanoma; ALM, acral lentiginous melanoma; LMM, lentigo maligna melanoma. n.a.: not available. ddPCR: droplet digital PCR.
      (e, f) The overall ratio of patients with BRAF mutations in FFPE tissues or cfDNA detected by ddPCR to that of conventional real-time PCR.

      3.2 CAPP-Seq of cfDNA from patients with melanoma

      The results mentioned above using ddPCR for the detection of BRAF mutations in cfDNA indicated the potential of liquid biopsy, thereby prompting us to investigate gene mutations other than BRAF mutations in cfDNA that could be used as biomarkers. Using the CAPP-Seq method, we screened gene mutations in cfDNA from 15 patients and identified the variants (Fig. 2). While one in five patients (20 %) harbored some mutations in stage III, we could detect mutations in six of the ten patients (60 %) in stage IV. NRAS and MET mutations were detected in patients with BRAF mutations (Case Nos. 6 and 11, respectively). Moreover, GNAS and TP53 mutations were detected in Case No. 10.
      Fig. 2
      Fig. 2Summary of cfDNA gene alterations identified by CAPP-Seq from patients with melanoma.
      Unfiltered missense variants (left) and filtered missense variants (right) were detected by CAPP-Seq. Variants were identified using AVENIO Oncology Analysis Software. For filtered missense variants, germline mutations were excluded based on the following databases: ExAC 1.0, dbSNP 150, and 1000 Genomes phase_3_v5b; somatic mutations were determined based on the following databases: COSMIC v83 and TCGA 9.0. The profiles of 15 patients, including the stage, presence of BRAF mutation, sex, and tumor type, are shown as indicated. CNV: Copy number variation.

      3.3 Analysis of NRAS, TP53, GNAS, and MET gene mutations using ddPCR

      Among the mutations detected in the CAPP-Seq analysis, we focused on NRAS (p.Gln61Lys), NRAS (p.Gln61Arg), TP53 (p.Arg267Pro), GNAS (p.Arg201Cys), and MET (p.Val1088Met) because the variant allele fractions (VAFs) were high (> 5 %) among these mutations (Fig. 3). Based on these findings, we performed ddPCR analyses of the five mutations using additional tumor tissues and plasma cfDNA samples obtained from patients with melanoma (Fig. 4a, b, and Table 1b). Of the 19 patients, three patients analyzed using primary tumor tissues were positive for the NRAS (p.Gln61Lys) mutation (15.8 %), while one patient analyzed using plasma cfDNA was positive (5.3 %) (Fig. 4b and c). For the NRAS (p.Gln61Arg) mutation, six patients analyzed using primary tumor tissues (31.6 %) were positive, while two patients analyzed using cfDNA were positive (10.5 %). Similarly, two patients harbored a TP53 (p.Arg267Pro) mutation in the primary tissues (10.5 %), while one patient was positive for the mutation analyzed using cfDNA (5.3 %). Although the GNAS (p.Arg201Cys) mutation was the most frequently detected mutation in tumor tissues (47.4 %) among the five mutations, the positive ratio of this mutation assessed using liquid biopsy was relatively small (5.3 %). Interestingly, the MET (p.Val1088Met) mutation was the most frequently detected mutation among these five patients (15.8 %), and the positive ratio of this mutation assessed using the primary tissues was 26.3 %. All mutations detected in cfDNA were also positive in the tissues of the corresponding patients. None of the five mutations were detected in tissues or plasma samples from healthy controls (Fig. 4b).
      Fig. 3
      Fig. 3The list of genes and their variants detected in CAPP-Seq from 15 melanoma patients.
      Genes and variants boxed in yellow indicate those with high allele fractions (> 5 %) utilized for ddPCR analysis.
      Fig. 4
      Fig. 4Analysis of NRAS, TP53, GNAS, and MET gene mutations using ddPCR.
      (a) Representative ddPCR results of NRAS (p.Gln61Lys), NRAS (p.Gln61Arg), TP53 (p.Arg267Pro), GNAS (p.Arg201Cys), and MET (p.Val1088Met) mutations. Blue dots (enclosed by red circles) indicate mutations of each mutation, green dots indicate wild type, and grey dots indicate droplets without DNA of interest.
      (b) The profiles of 19 patients with melanoma and summary of their ddPCR results of each mutation in tissue and cfDNA (upper). Copy numbers (×103/mL) are shown. The summary of ddPCR results of each mutation in normal tissue and cfDNA in eight healthy subjects (bottom). Ctrls: normal controls.
      (c) The ratio of patients with each mutation in FFPE tumor tissues or cfDNA detected by ddPCR among the 19 patients with melanoma.
      Table 1bThe comparison of the results among three methods of real-time PCR, cfDNA-ddPCR, and cfDNA-CAPP-Seq.
      CaseGeneReal time PCRddPCRCAPP-Seq
      TissuePlasma
      1BRAF (p.Val600Glu)
      4
      6
      9
      10NA
      11
      19NA
      26NANA
      27NANA
      29NANA
      30NANA
      31NANA
      32NANA
      33NANA
      19GNAS (p.Arg201Cys)NA
      26NA
      27NA
      30NA
      11MET (p.Val1088Met)NANA
      27NA
      30NA
      19NRAS (p.Gln61Arg)NA
      29NA
      30NA
      29NRAS (p.Gln61Lys)NA
      10TP53 (p.Arg267Pro)NA
      30NA
      cfDNA: cell-free DNA; ddPCR: droplet digital PCR; CAPP-Seq: CAncer Personalized Profiling by deep Sequencing; NA: not available or not assessed.

      3.4 Analysis of MET gene mutations as a monitoring marker of melanoma

      As our results showed that the MET gene had the highest positive mutation ratio in cfDNA among the five gene mutations (Fig. 4c), we focused on the MET gene mutation and performed a longitudinal study of the relationship between MET copies and clinical course (evaluated by RECIST [
      • Eisenhauer E.A.
      • Therasse P.
      • Bogaerts J.
      • Schwartz L.H.
      • Sargent D.
      • Ford R.
      • Dancey J.
      • Arbuck S.
      • Gwyther S.
      • Mooney M.
      • Rubinstein L.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      ]) in Case Nos. 11 and 40. In Case No. 11, a double positive mutation of BRAF and MET was detected in cfDNAs, which could not be detected by regression of liver metastasis after treatment with the BRAF/MEK inhibitor (Fig. 5a). The patient was diagnosed with progressive disease nine months after treatment. Our results revealed that MET mutation copy numbers increased to 7388 copies/mL, while BRAF was not detected at the time of disease progression. In Case No. 40, while the MET mutation was detected before surgical resection, it was undetectable after resection. Positron emission tomography-computed tomography revealed no recurrence seven months after surgery (Fig. 5b). These cases indicated that the kinetics of the copy numbers of MET mutations in cfDNA was almost coincident with disease status. The kinetics of the VAF of MET mutation was similar to that of copy numbers; however, the kinetics of copy numbers was more clearly coincident with the disease status in these patients (data not shown).
      Fig. 5
      Fig. 5Analysis of MET gene mutations as a monitoring marker of melanoma on treatment with BRAF/MEK inhibitor (a) and surgical resection (b).
      (a) The longitudinal study of BRAF copies in cfDNAs and clinical course of Case No. 11 treated with BRAF/MEK inhibitors (dabrafenib and trametinib) (upper). The kinetics of MET copy numbers and LDH value are shown. Computed tomography showed disease regression of liver metastasis 1.5 months after the treatment and the recurrence in the pancreas ten months after treatment (bottom). The metastatic lesions are indicated (red circles). PR: partial response. PD: progressive disease.
      (b) The longitudinal study of MET in cfDNAs and clinical course of Case No. 40. The kinetics of BRAF copy numbers and LDH value are shown. Positron emission tomography-computed tomography revealed tumor presence on the right sole (red circle) before surgical resection and no recurrence seven months after resection.

      4. Discussion

      Our results indicate that liquid biopsy can detect tumor presence and mutations, especially when tissue biopsies are unavailable. Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in plasma cfDNA, and the results were partially consistent with the results obtained from tissues. Some cases in which the mutation could be detected only from the tissue may be because the amount of tumor-derived DNA in plasma cfDNA is smaller than that of tumor tissue [
      • Dawson S.J.
      • Tsui D.W.
      • Murtaza M.
      • Biggs H.
      • Rueda O.M.
      • Chin S.F.
      • Dunning M.J.
      • Gale D.
      • Forshew T.
      • Mahler-Araujo B.
      • Rajan S.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      ,
      • Schwarzenbach H.
      • Hoon D.S.B.
      • Pantel K.
      Cell-free nucleic acids as biomarkers in cancer patients.
      ]. These results suggest that cfDNAs are derived from the source tumor, and when tissue biopsy is unavailable, liquid biopsy may be a valuable method for detecting the presence of a source tumor. In addition to detecting tumors, tracking cfDNA content may be helpful for the prediction of mutations in tumors and follow-up of melanoma patients because cfDNA using ddPCR can detect minute residual tumors and metastatic tumors that are not accessible or cannot be detected by image inspection [
      • Abbosh C.
      • Birkbak N.J.
      • Wilson G.A.
      • Jamal-Hanjani M.
      • Constantin T.
      • Salari R.
      • Le Quesne J.
      • Moore D.A.
      • Veeriah S.
      • Rosenthal R.
      • Marafioti T.
      Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.
      ,
      • Takahashi H.
      • Kagawa N.
      • Tanei T.
      • Naoi Y.
      • Shimoda M.
      • Shimomura A.
      • Shimazu K.
      • Kim S.J.
      • Noguchi S.
      Correlation of methylated circulating tumor dna with response to neoadjuvant chemotherapy in breast cancer patients.
      ,
      • Olsson E.
      • Winter C.
      • George A.
      • Saal L.H.
      • et al.
      Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.
      ].
      In melanoma patients, LDH is the most available blood-based biomarker for monitoring disease status [
      • Huang S.K.
      • Hoon D.S.
      Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients.
      ]; however, its accuracy is limited. Therefore, a more accurate method is necessary, and liquid biopsy can overcome this limitation. cfDNA has been used as a predictive and prognostic tool for melanoma [
      • Váraljai R.
      • Elouali S.
      • Lueong S.S.
      • Hamprecht K.W.
      • Seremet T.
      • Siveke J.T.
      • Becker J.C.
      • Sucker A.
      • Paschen A.
      • Horn P.A.
      • Neyns B.
      • Weide B.
      • Schadendorf D.
      • Roesch A.
      The predictive and prognostic significance of cell‐free DNA concentration in melanoma.
      ]. BRAF mutation testing of cfDNA has the potential to monitor the response to BRAF inhibitors or combinations of BRAF and MEK inhibitors in patients with BRAF-mutant melanoma [
      • Sacco A.
      • Forgione L.
      • Carotenuto M.
      • Carotenuto M.
      • Ascierto P.A.
      • Botti G.
      • Normanno N.
      Circulating tumor DNA testing opens new perspectives in melanoma management.
      ,
      • Schreuer M.
      • Meersseman G.
      • Herrewegen S.V.D.
      • Jansen Y.
      • Chevolet I.
      • Bott A.
      • Wilgenhof S.
      • Seremet T.
      • Jacobs B.
      • Buyl R.
      • Maertens G.
      • Neyns B.
      Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors.
      ]. Interestingly, our results showed that MET variant content could be a new biomarker for melanoma disease status when treated with the BRAF/MEK inhibitor. Case Nos. 11 and 40 revealed parallel disease statuses with copy numbers of MET mutations (Fig. 5). Because VAF is reported to be affected by the detectability of the wild-type gene, some papers have reported that copy number, which represents the absolute amount of gene mutations, would be suitable for detecting mutations [
      • Calapre L.
      • Warburton L.
      • Millward M.
      • Ziman M.
      • Gray E.S.
      Circulating tumour DNA (ctDNA) as a liquid biopsy for melanoma.
      ,
      • Dawson S.J.
      • Tsui D.W.
      • Murtaza M.
      • Biggs H.
      • Rueda O.M.
      • Chin S.F.
      • Dunning M.J.
      • Gale D.
      • Forshew T.
      • Mahler-Araujo B.
      • Rajan S.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      ,
      • Vendrell J.A.
      • Quantin X.
      • Serre I.
      • Solassol J.
      Combination of Tissue and Liquid Biopsy Molecular Profiling to Detect Transformation to Small Cell Lung Carcinoma During Osimertinib Treatment.
      ,
      • Bos M.K.
      • Nasserinejad K.
      • Jansen M.P.H.M.
      • Angus L.
      • Atmodimedjo P.N.
      • de Jonge E.
      • Dinjens W.N.M.
      • van Schaik R.H.N.
      • Re M.D.
      • Dubbink H.J.
      • Sleijfer S.
      • Martens J.W.M.
      Comparison of variant allele frequency and number of mutant molecules as units of measurement for circulating tumor DNA.
      ]. The kinetics of the VAF of MET mutation were similar to that of copy number; however, the kinetics of copy number were more clearly coincident with our patients' disease status, consistent with previous reports.
      MET encodes a receptor tyrosine kinase c-MET for a hepatocyte growth factor (HGF), predominantly of epithelial origin [
      • Maroun C.R.
      • Rowlands T.
      The Met receptor tyrosine kinase: a key player in oncogenesis and drug resistance.
      ]. Activated HGF/c-MET signaling leads to melanoma progression. As melanoma patients harbor BRAF mutations that activate the RAF-MEK-ERK pathway, BRAF V600E/K is considered a therapeutic target. Although two therapies (vemurafenib and dabrafenib) targeting BRAF V600E/K are available, their treatment results are not consistently satisfactory, even in combination with downstream kinase MEK1/2 inhibitors (trametinib and cobimetinib) [
      • Volpe V.O.
      • Klufas D.M.
      • Hegde U.
      • Grant-Kels J.M.
      The new paradigm of systemic therapies for metastatic melanoma.
      ]. Most melanoma patients acquire resistance to these treatments after several months [
      • Welsh S.J.
      • Rizos H.
      • Scolyer R.A.
      • Long G.V.
      Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next?.
      ], thereby necessitating a novel target for melanoma treatment. In view of this, HGF/c-MET signaling inhibitors in melanoma cells have been investigated in clinical studies [
      • Daud A.
      • Kluger H.M.
      • Kurzrock R.
      • Schimmoller F.
      • Weitzman A.L.
      • Samuel T.A.
      • Moussa A.H.
      • Gordon M.S.
      • Shapiro G.I.
      Phase II randomised discontinuation trial of the MET/VEGF receptor inhibitor cabozantinib in metastatic melanoma.
      ,
      • Schöffski P.
      • Gordon M.
      • Smith D.C.
      • Kurzrock R.
      • Daud A.
      • Vogelzang N.J.
      • Lee Y.
      • Scheffold C.
      • Shapiro G.I.
      Phase II randomised discontinuation trial of cabozantinib in patients with advanced solid tumours.
      ]; therefore, detection of MET mutations may be necessary in HGF/c-MET inhibitor treatment. The MET proto-oncogene located on chromosome 7 (7q21−31) is widely expressed in epithelial cells [
      • Zhang Y.
      • Du Z.
      • Zhang M.
      Biomarker development in MET-targeted therapy.
      ]. MET protein mutations are frequently found in different cancers, such as gastric cancer, esophageal cancer, colorectal cancer, non-small cell lung cancer, brain tumors, and melanoma [
      • Maroun C.R.
      • Rowlands T.
      The Met receptor tyrosine kinase: a key player in oncogenesis and drug resistance.
      ,
      • Daud A.
      • Kluger H.M.
      • Kurzrock R.
      • Schimmoller F.
      • Weitzman A.L.
      • Samuel T.A.
      • Moussa A.H.
      • Gordon M.S.
      • Shapiro G.I.
      Phase II randomised discontinuation trial of the MET/VEGF receptor inhibitor cabozantinib in metastatic melanoma.
      ,
      • Mondelo-Macía P.
      • Rodríguez-López C.
      • Valiña L.
      • Aguín S.
      • León-Mateos L.
      • García-González J.
      • Abalo A.
      • Rapado-González O.
      • Suárez-Cunqueiro M.
      • Díaz-Lagares A.
      • Curiel T.
      Detection of MET alterations using cell free DNA and circulating tumor cells from cancer patients.
      ,
      • Czyz M.
      HGF/c-MET signaling in melanocytes and melanoma.
      ]. Although MET mutations have been detected in melanoma cells [
      • Saitoh K.
      • Takahashi H.
      • Sawada N.
      • Parsons P.G.
      Detection of the c-met proto-oncogene product in normal skin and tumours of melanocytic origin.
      ,
      • Natali P.G.
      • Nicotra M.R.
      • Di Renzo M.F.
      • Prat M.
      • Bigotti A.
      • Cavaliere R.
      • Comoglio P.M.
      Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.
      ], to the best of our knowledge, this is the first study to detect MET alterations in plasma cfDNA ddPCR.
      In conclusion, our results suggest that gene mutations, including BRAF, NRAS, TP53, GNAS, and MET, are detectable in cfDNA from melanoma patients using CAPP-Seq and ddPCR. Among the variants examined, the MET copy number was consistent with the disease status in a melanoma patient treated with the BRAF/MEK inhibitor; therefore, it may be helpful as a biomarker for patients undergoing BRAF/MEK inhibitor treatment. However, our study has a limitation, which may be attributed to the small number of cases, with longitudinal studies in patients with MET mutations being the only case study. Further studies with larger sample sizes are required to confirm its efficiency and accuracy.

      Declaration of Competing Interest

      The authors have no conflict of interest to declare.

      Funding

      This work was supported by the Japan Society for the Promotion of Science (JSPS, Grant Numbers 19K21328 and 20K17353) and a grant from the Kumamoto University Hospital.

      References

        • Robert C.
        • Karaszewska B.
        • Schachter J.
        • Rutkowski P.
        • Mackiewicz A.
        • Stroiakovski D.
        • Lichinitser M.
        • Dummer R.
        • Grange F.
        • Mortier L.
        • Chiarion-Sileni V.
        Improved overall survival in melanoma with combined dabrafenib and trametinib.
        N. Engl. J. Med. 2015; 372: 30-39
        • Franczak C.
        • Salleron J.
        • Dubois C.
        • Filhine-Trésarrieu P.
        • Leroux A.
        • Merlin J.L.
        • Harlé A.
        Comparison of five Different Assays for the detection of BRAF mutations in formalin-fixed paraffin embedded tissues of patients with metastatic melanoma.
        Mol. Diagn. Ther. 2017; 21: 209-216
        • Boyer M.
        • Cayrefourcq L.
        • Dereure O.
        • Meunier L.
        • Becquart O.
        • Alix-Panabières C.
        Clinical relevance of liquid biopsy in melanoma and merkel cell carcinoma.
        Cancers. (Basel). 2020; 12: 960
        • Kaji T.
        • Yamasaki O.
        • Takata M.
        • Otsuka M.
        • Hamada T.
        • Morizane S.
        • Asagoe K.
        • Yanai H.
        • Hirai Y.
        • Umemura H.
        • Iwatsuki K.
        Comparative study on driver mutations in primary and metastatic melanomas at a single Japanese institute: a clue for intra- and inter-tumor heterogeneity.
        J. Dermatol. Sci. 2017; 85: 51-57
        • Santiago-Walker A.
        • Gagnon R.
        • Mazumdar J.
        • Casey M.
        • Long G.V.
        • Schadendorf D.
        • Flaherty K.
        • Kefford R.
        • Hauschild A.
        • Hwu P.
        • Haney P.
        Correlation of BRAF mutation status in circulating-free DNA and tumor and association with clinical outcome across four BRAFi and MEKi clinical trials.
        Clin. Cancer Res. 2016; 22: 567-574
        • Calapre L.
        • Warburton L.
        • Millward M.
        • Ziman M.
        • Gray E.S.
        Circulating tumour DNA (ctDNA) as a liquid biopsy for melanoma.
        Cancer Lett. 2017; 404: 62-69
        • Dawson S.J.
        • Tsui D.W.
        • Murtaza M.
        • Biggs H.
        • Rueda O.M.
        • Chin S.F.
        • Dunning M.J.
        • Gale D.
        • Forshew T.
        • Mahler-Araujo B.
        • Rajan S.
        Analysis of circulating tumor DNA to monitor metastatic breast cancer.
        N. Engl. J. Med. 2013; 368: 1199-1209
        • Crowley E.
        • Di Nicolantonio F.
        • Loupakis F.
        • Bardelli A.
        Liquid biopsy: monitoring cancer-genetics in the blood.
        Nat. Rev. Clin. Oncol. 2013; 10: 472-484
        • Gray E.S.
        • Rizos H.
        • Reid A.L.
        • Boyd S.C.
        • Pereira M.R.
        • Lo J.
        • Tembe V.
        • Freeman J.
        • Lee J.H.
        • Scolyer R.A.
        • Siew K.
        Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma.
        Oncotarget. 2015; 6: 42008-42018
        • Newman A.M.
        • Bratman S.V.
        • To J.
        • Wynne J.F.
        • Eclov N.C.
        • Modlin L.A.
        • Liu C.L.
        • Neal J.W.
        • Wakelee H.A.
        • Merritt R.E.
        • Shrager J.B.
        An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.
        Nat. Med. 2014; 20: 548-554
        • Iwahashi N.
        • Sakai K.
        • Noguchi T.
        • Yahata T.
        • Matsukawa H.
        • Toujima S.
        • Nishio K.
        • Ino K.
        Liquid biopsy-based comprehensive gene mutation profiling for gynecological cancer using CAncer Personalized Profiling by deep Sequencing.
        Sci. Rep. 2019; 9: 10426
        • Eisenhauer E.A.
        • Therasse P.
        • Bogaerts J.
        • Schwartz L.H.
        • Sargent D.
        • Ford R.
        • Dancey J.
        • Arbuck S.
        • Gwyther S.
        • Mooney M.
        • Rubinstein L.
        New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
        Eur. J. Cancer. 2009; 45: 228-247
        • Schwarzenbach H.
        • Hoon D.S.B.
        • Pantel K.
        Cell-free nucleic acids as biomarkers in cancer patients.
        Nat. Rev. Cancer. 2011; : 426-437
        • Abbosh C.
        • Birkbak N.J.
        • Wilson G.A.
        • Jamal-Hanjani M.
        • Constantin T.
        • Salari R.
        • Le Quesne J.
        • Moore D.A.
        • Veeriah S.
        • Rosenthal R.
        • Marafioti T.
        Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.
        Nature. 2017; 545: 446-451
        • Takahashi H.
        • Kagawa N.
        • Tanei T.
        • Naoi Y.
        • Shimoda M.
        • Shimomura A.
        • Shimazu K.
        • Kim S.J.
        • Noguchi S.
        Correlation of methylated circulating tumor dna with response to neoadjuvant chemotherapy in breast cancer patients.
        Clin. Breast Cancer. 2017; 17: 61-69
        • Olsson E.
        • Winter C.
        • George A.
        • Saal L.H.
        • et al.
        Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.
        EMBO Mol. Med. 2015; 7: 1034-1047
        • Huang S.K.
        • Hoon D.S.
        Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients.
        Mol. Oncol. 2016; 10: 450-463
        • Váraljai R.
        • Elouali S.
        • Lueong S.S.
        • Hamprecht K.W.
        • Seremet T.
        • Siveke J.T.
        • Becker J.C.
        • Sucker A.
        • Paschen A.
        • Horn P.A.
        • Neyns B.
        • Weide B.
        • Schadendorf D.
        • Roesch A.
        The predictive and prognostic significance of cell‐free DNA concentration in melanoma.
        J. Eur. Acad. Dermatol. Venereol. 2021; 35: 387-395
        • Sacco A.
        • Forgione L.
        • Carotenuto M.
        • Carotenuto M.
        • Ascierto P.A.
        • Botti G.
        • Normanno N.
        Circulating tumor DNA testing opens new perspectives in melanoma management.
        Cancers. 2020; 12: 2914
        • Schreuer M.
        • Meersseman G.
        • Herrewegen S.V.D.
        • Jansen Y.
        • Chevolet I.
        • Bott A.
        • Wilgenhof S.
        • Seremet T.
        • Jacobs B.
        • Buyl R.
        • Maertens G.
        • Neyns B.
        Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors.
        J. Transl. Med. 2016; 14: 15
        • Vendrell J.A.
        • Quantin X.
        • Serre I.
        • Solassol J.
        Combination of Tissue and Liquid Biopsy Molecular Profiling to Detect Transformation to Small Cell Lung Carcinoma During Osimertinib Treatment.
        Published online, 2020
        • Bos M.K.
        • Nasserinejad K.
        • Jansen M.P.H.M.
        • Angus L.
        • Atmodimedjo P.N.
        • de Jonge E.
        • Dinjens W.N.M.
        • van Schaik R.H.N.
        • Re M.D.
        • Dubbink H.J.
        • Sleijfer S.
        • Martens J.W.M.
        Comparison of variant allele frequency and number of mutant molecules as units of measurement for circulating tumor DNA.
        Mol. Oncol. 2021; 15: 57-66
        • Maroun C.R.
        • Rowlands T.
        The Met receptor tyrosine kinase: a key player in oncogenesis and drug resistance.
        Pharmacol. Ther. 2014; 142: 316-338
        • Volpe V.O.
        • Klufas D.M.
        • Hegde U.
        • Grant-Kels J.M.
        The new paradigm of systemic therapies for metastatic melanoma.
        J. Am. Acad. Dermatol. 2017; 77: 356-368
        • Welsh S.J.
        • Rizos H.
        • Scolyer R.A.
        • Long G.V.
        Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next?.
        Eur. J. Cancer. 2016; 62: 76-85
        • Daud A.
        • Kluger H.M.
        • Kurzrock R.
        • Schimmoller F.
        • Weitzman A.L.
        • Samuel T.A.
        • Moussa A.H.
        • Gordon M.S.
        • Shapiro G.I.
        Phase II randomised discontinuation trial of the MET/VEGF receptor inhibitor cabozantinib in metastatic melanoma.
        Br. J. Cancer. 2017; 116: 432-440
        • Schöffski P.
        • Gordon M.
        • Smith D.C.
        • Kurzrock R.
        • Daud A.
        • Vogelzang N.J.
        • Lee Y.
        • Scheffold C.
        • Shapiro G.I.
        Phase II randomised discontinuation trial of cabozantinib in patients with advanced solid tumours.
        Eur. J. Cancer. 2017; 86: 296-304
        • Zhang Y.
        • Du Z.
        • Zhang M.
        Biomarker development in MET-targeted therapy.
        Oncotarget. 2016; 7: 37370-37389
        • Mondelo-Macía P.
        • Rodríguez-López C.
        • Valiña L.
        • Aguín S.
        • León-Mateos L.
        • García-González J.
        • Abalo A.
        • Rapado-González O.
        • Suárez-Cunqueiro M.
        • Díaz-Lagares A.
        • Curiel T.
        Detection of MET alterations using cell free DNA and circulating tumor cells from cancer patients.
        Cells. 2020; 9: 522
        • Czyz M.
        HGF/c-MET signaling in melanocytes and melanoma.
        Int. J. Mol. Sci. 2018; 19: 3844
        • Saitoh K.
        • Takahashi H.
        • Sawada N.
        • Parsons P.G.
        Detection of the c-met proto-oncogene product in normal skin and tumours of melanocytic origin.
        J. Pathol. 1994; 174: 191-199
        • Natali P.G.
        • Nicotra M.R.
        • Di Renzo M.F.
        • Prat M.
        • Bigotti A.
        • Cavaliere R.
        • Comoglio P.M.
        Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.
        Br. J. Cancer. 1993; 68: 746-750