Targ Oncol DOI 10.1007/s11523-015-0361-1
DAY-TO-DAY PRACTICE
Detection of KIT and PDGFRA mutations in the plasma of patients with gastrointestinal stromal tumor Guhyun Kang & Byung Noe Bae & Byeong Seok Sohn & Jung-Soo Pyo & Gu Hyum Kang & Kyoung-Mee Kim
Received: 19 November 2014 / Accepted: 28 January 2015 # Springer International Publishing Switzerland 2015
Abstract In subsets of gastrointestinal stromal tumors (GISTs), mutations of the KIT and PDGFRA receptor tyrosine kinases correlate with tumor prognosis and response to tyrosine kinase inhibitors (TKIs). Determining genotypes in TKI-resistant GISTs is challenging due to the potential risks and limitations of repeated biopsies during the course of treatment. We prospectively collected plasma samples from three GIST patients harboring KIT mutations that were detected in tissue DNA. The plasma samples were then analyzed for mutations in KIT, PDGFRA, and BRAF via next-generation sequencing. We were able to identify primary KIT mutations in all plasma samples. Additional mutations, including KIT exon
17 S821F and PDGFRA exon 18 D842V, were detected in the patient-matched plasma samples during follow-up and appeared to result in decreased sensitivity to TKIs. Our results demonstrate an approach by which primary and secondary mutations are readily detected in blood-derived circulating tumor DNA from patients with GIST. These mutations can be used as biomarkers for prediction of treatment response. The identification of a resistance mutation in plasma DNA will allow early change to alternative TKIs or dose escalation of imatinib for optimal patient management.
Keywords Gastrointestinal stromal tumor . Next-generation sequencing . Resistance mutation . Circulating tumor DNA . Tyrosine kinase inhibitor G. Kang Department of Pathology, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, South Korea B. N. Bae Department of Surgery, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, South Korea B. S. Sohn Department of Medicine, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, South Korea J.5
Progressive disease
1.58/3043 1.52/2046 5.72/1312 4.45/3009 KIT exon 11 Del K558-E562 KIT exon 17 S821F 61.11/1954 (mesentery) KIT exon 9 Dup A502-Y503 54.46/2681 (lung) PDGFRA exon 18 D842V
KIT exon 11 Del K558-E562a 79.01/6695 1/10 25 Stomach 59/male 1
Mutation AF (%)/DP Size (cm) Mitosis/ Mutation HPF Location
Tissue
Clinicopathologic characteristics and mutation results for three patients with gastrointestinal stromal tumor
Case 1 A 59-year-old man with neurofibromatosis type 1 presented with episodes of lower abdominal pain. CT disclosed a
Case series
Case no. Age at diagnosis/gender Primary tumor
Patients and methods Patients with malignant or advanced GIST were recruited as part of a preliminary study to assess the diagnostic performance of a NGS-based mutation analysis of ctDNA. Written informed consent was obtained from the patients, and blood samples were collected during TKI treatment. This study was approved by the institutional review board of Inje University Sanggye Paik Hospital. Table 1 summarizes the clinicopathologic features of three cases, which were characterized by different clinical responses to TKI: partial response, stable disease, and progressive disease. The cancer panel used in this study (SeaSun Biomaterials, Daejeon, Korea) covers exons 9, 11, 13, 14, 17, and 18 of KIT; exon 18 of PDGFRA, and exon 15 of BRAF. DNA extraction from tissue and plasma samples and amplicon library preparation were carried out according to manufacturers’ protocols and as described previously [8]. Emulsion polymerase chain reaction (PCR), breaking, and bead enrichment were done using the GS Junior Titanium emPCR Kit Lib-L, emPCR Reagents Lib-L Kit, Oil and Breaking Kit, and the Bead Recovery Reagents Kit (Roche Diagnostics, Mannheim, Germany). Sequencing was performed to over 1300-fold coverage using the GS Junior system (Roche Diagnostics). The results were analyzed with the GS Amplicon Variant Analyzer (version 2.7; Roche Diagnostics). To detect variants, filters were set to display sequence variances occurring in more than 1 % of bidirectional reads per amplicon [9].
Table 1
Plasma
AF (%)/DP
switching to an alternative TKI, although the strategy that yields the optimal outcomes remains unclear [1]. Diagnostic imaging techniques are currently used to assess response to TKI in patients with GIST. However, these methods display limited sensitivity and specificity, and treatment changes are generally triggered by clinical and/or computerized tomography (CT)-morphologic progression [5]. Serial or repeated biopsies are invasive and often unattainable and thus are not widely accepted as means of predicting treatment response or detecting resistance mutations. Moreover, as tumors are heterogeneous and continuously evolving, biopsies of one or two accessible sites may not be representative of the entire tumor, and the mutations present in a minor subclone can be missed [6]. An alternative approach that may overcome these issues is the analysis of peripheral blood, in which cell-free DNA fragments from multiple lesions in a single patient are mixed together [7]. Here, we have used a next-generation sequencing (NGS) platform to perform mutation analyses of cell-free circulating tumor DNA (ctDNA) from the plasma of GIST patients on TKI therapy.
Partial response
Best response to TKI
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25-cm mass arising from the stomach. Under endoscopic ultrasound guidance, three tissue fragments were obtained using a 22-gauge needle via a transgastric approach. A histologic diagnosis of GIST was made based on positive immunostaining for KIT, and further mutation analysis of the biopsy specimen revealed a deletion of K558-E562 in KIT exon 11. The patient had a partial response after 3 months of treatment with imatinib, and the tumor remained stable as assessed by CT scans at regular visits every 3 months. A missense mutation in KIT exon 17 (S821F), as well as the primary mutation, was detected in the plasma sample collected 10 months after biopsy. The missense mutation in KIT has not been reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Six months later, the last follow-up CT demonstrated a new enhancing nodule in the pelvic cavity (Fig. 1a). Case 2 A 61-year-old man was diagnosed with small intestinal GIST and received adjuvant imatinib (400 mg/day) for a year. After withdrawal of imatinib treatment, the patient developed local recurrence in the small bowel mesentery at 24 months. A subsequent wedge resection was performed at 28 months to rule out primary lung cancer, which confirmed to be a metastatic GIST. Molecular analysis of the mesenteric and lung masses identified a duplication of A502-Y503 in KIT exon 9. As patients with this mutation respond less well to imatinib, the regimen was then changed to sunitinib 37.5 mg/day. A CT scan at 40 months showed residual tumors at the sites of previous operations with a suspicious metastatic lesion in the liver (Fig. 1b). A PDGFRA exon 18 D842V mutation was also detected in the plasma sample of this patient at the last follow-up. This mutation had not been identified in the tumor tissues. Case 3 A 82-year-old woman presented with palpable abdominal masses. She had a history of small bowel resection for GIST. The clinical and radiologic features of the masses were suggestive of hepatic metastases of GIST. She was started on imatinib 400 mg daily and achieved a partial response after 5 months of treatment. Even though the dose was reduced to 200 mg/day because of unacceptable side effects, the patient had a stable disease status over 30 months of follow-up. At 60 months, ctDNA only contained the primary tumor mutation in KIT exon 11 (P551A) without any detectable mutation in exons 9, 13, 14, 17, or 18 of KIT; exon 18 of PDGFRA; or exon 15 of BRAF (Fig. 1c).
Discussion There is an outstanding need for methods that can be used to detect mutations efficiently and effectively and thereby inform the selection of drugs that have validated roles against targeted
mutations. However, the mutations in primary tumor tissues may not reflect current molecular status in a patient with recurrent or metastatic disease [10]. Using BEAMing technology and allele-specific ligation PCR, two recent studies have recovered KIT/PDGFRA mutations from ctDNA in the plasma samples of patients with GIST [11, 5]. However, because they are designed to detect specific, commonly observed mutations in hot spot regions, negative test results cannot be interpreted as demonstrating the absence of mutations in the genes [10]. As compared with these two methods, NGS holds major advantages in terms of cost, turnaround time, and breadth of mutation coverage (i.e., the ability to simultaneously detect all mutation types for a large number of genes in a single assay) and might offer an opportunity for increasing sensitivity. In this report, we further validated the utility of ctDNA analysis for detecting mutations associated with acquired TKI resistance in patients with malignant or advanced GIST. It was possible to identify a primary tumor mutation present in ctDNA at an allele frequency of 1.58 %, and there was 100 % concordance between mutations found in the primary tumors and those found in the ctDNA. Of note, an additional KIT or PDGFRA mutation was detected in two of the plasma samples; the presence of which was associated with poor response to TKI. The identification of resistant mutations represents one of the most immediate clinical applications and would allow alternative therapies to be initiated early [12]. A highly significant example is the detection of KIT exon 17 substitution S821F in the plasma of case 1 during imatinib therapy, before relapse became detectable radiologically. To our knowledge, this is a novel mutation that has not been reported in GISTs. The present case suggests that it might be associated with decreased sensitivity to imatinib. In case 2, our patient’s tumors contained KIT exon 9 mutations, and he was found to have a secondary PDGFRA exon 18 D842V mutation. The D842V is the single most common PDGFRA mutation that confers resistance to both imatinib and sunitinib. Uncommonly, imatinib resistance also occurs through the mutation of a different kinase that is less sensitive to this drug. Previous reports have described a GIST with a primary KIT G565R mutation that acquired a secondary PDGFRA D842V mutation, as well as a tumor with a primary KIT mutation in exon 9 that developed an additional mutation in PDGFRA exon 14 [13, 14]. The ctDNA as a diagnostic, prognostic, and theranostic tool in cancer patients is an appealing approach, at least in theory, since it is noninvasive and easily repeated [15]. However, there are several hurdles to adopt ctDNA into clinical routine practice: (1) lack of standardization of techniques, (2) difficulty in controlling the pre-analytical phase to obtain robust and reproducible results, and (3) high cost restricting patients’ access [12, 15, 16].
Targ Oncol
Targ Oncol
Fig. 1
Clinical course of each patient and the mutations that were detected in tissue and plasma samples: a case 1, b case 2, and c case 3 (IM imatinib, SU sunitinib)
Overall, our results demonstrate that the analysis of ctDNA for drug-resistant mutations can complement current invasive biopsy procedures and provide promising biomarkers for TKI-resistant GISTs, thus allowing dose escalation of imatinib or switching to another TKI on a timely basis. However, our preliminary results need to be validated in independent cohorts of patients and further large prospective multicenter studies are needed to introduce ctDNA into the routine clinical practice.
7.
8.
9.
Conflict of interest The authors declare that they have no conflict of interest.
References 10. 1. Demetri GD, von Mehren M, Antonescu CR, DeMatteo RP, Ganjoo KN, Maki RG, Pisters PW, Raut CP, Riedel RF, Schuetze S, Sundar HM, Trent JC, Wayne JD (2010) NCCN task force report: update on the management of patients with gastrointestinal stromal tumors. J Natl Compr Cancer Netw 8(Suppl 2):S1–S41, quiz S42-44 2. Corless CL, Barnett CM, Heinrich MC (2011) Gastrointestinal stromal tumours: origin and molecular oncology. Nat Rev Cancer 11(12): 865–878. doi:10.1038/nrc3143 3. Dematteo RP, Ballman KV, Antonescu CR, Maki RG, Pisters PW, Demetri GD, Blackstein ME, Blanke CD, von Mehren M, Brennan MF, Patel S, McCarter MD, Polikoff JA, Tan BR, Owzar K (2009) Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumour: a randomised, double-blind, placebocontrolled trial. Lancet 373(9669):1097–1104. doi:10.1016/S01406736(09)60500-6 4. Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H, Roberts PJ, Eisenberg BL, von Mehren M, Fletcher CD, Sandau K, McDougall K, Ou WB, Chen CJ, Fletcher JA (2006) Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol 24(29):4764–4774 5. Maier J, Lange T, Kerle I, Specht K, Bruegel M, Wickenhauser C, Jost P, Niederwieser D, Peschel C, Duyster J, von Bubnoff N (2013) Detection of mutant free circulating tumor DNA in the plasma of patients with gastrointestinal stromal tumor harboring activating mutations of CKIT or PDGFRA. Clin Cancer Res 19(17):4854–4867. doi:10.1158/1078-0432.CCR-13-0765 6. Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF, Kingsbury Z, Wong AS, Marass F, Humphray S, Hadfield J, Bentley D, Chin TM, Brenton JD, Caldas C, Rosenfeld
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13.
14.
15.
16.
N (2013) Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497(7447):108–112. doi: 10.1038/nature12065 Chan KC, Jiang P, Zheng YW, Liao GJ, Sun H, Wong J, Siu SS, Chan WC, Chan SL, Chan AT, Lai PB, Chiu RW, Lo YM (2013) Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. Clin Chem 59(1):211–224. doi:10.1373/clinchem.2012.196014 Lee B, Han G, Kwon MJ, Han J, Choi YL (2014) KRAS mutation detection in Non-small cell lung cancer using a peptide nucleic acidmediated polymerase chain reaction clamping method and comparative validation with next-generation sequencing. Korean J Pathol 48(2):100–107. doi:10.4132/KoreanJPathol.2014.48.2.100 Kohlmann A, Klein HU, Weissmann S, Bresolin S, Chaplin T, Cuppens H, Haschke-Becher E, Garicochea B, Grossmann V, Hanczaruk B, Hebestreit K, Gabriel C, Iacobucci I, Jansen JH, te Kronnie G, van de Locht L, Martinelli G, McGowan K, Schweiger MR, Timmermann B, Vandenberghe P, Young BD, Dugas M, Haferlach T (2011) The Interlaboratory RObustness of Nextgeneration sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories. Leukemia 25(12):1840–1848. doi:10. 1038/leu.2011.155 Richardson AL, Iglehart JD (2012) BEAMing up personalized medicine: mutation detection in blood. Clin Cancer Res 18(12): 3209–3211. doi:10.1158/1078-0432.CCR-12-0871 Yoo C, Ryu MH, Na YS, Ryoo BY, Park SR, Kang YK (2014) Analysis of serum protein biomarkers, circulating tumor DNA, and dovitinib activity in patients with tyrosine kinase inhibitor-refractory gastrointestinal stromal tumors. Ann Oncol Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 10(8):472–484. doi:10.1038/nrclinonc.2013.110 Debiec-Rychter M, Cools J, Dumez H, Sciot R, Stul M, Mentens N, Vranckx H, Wasag B, Prenen H, Roesel J, Hagemeijer A, Van Oosterom A, Marynen P (2005) Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the P KC412 i nhibi tor against i m atinib-r esis tant muta nts . Gastroenterology 128(2):270–279 Lim KH, Huang MJ, Chen LT, Wang TE, Liu CL, Chang CS, Liu MC, Hsieh RK, Tzen CY (2008) Molecular analysis of secondary kinase mutations in imatinib-resistant gastrointestinal stromal tumors. Med Oncol 25(2):207–213. doi:10.1007/s12032-007-9014-2 Ilie M, Hofman V, Long E, Bordone O, Selva E, Washetine K, Marquette CH, Hofman P (2014) Current challenges for detection of circulating tumor cells and cell-free circulating nucleic acids, and their characterization in non-small cell lung carcinoma patients. What is the best blood substrate for personalized medicine? Ann Transl Med 2(11):107. doi:10.3978/j.issn.2305-5839.2014.08.11 Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV (2013) The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int J Mol Sci 14(9):18925–18958. doi:10. 3390/ijms140918925
DAY-TO-DAY PRACTICE
Detection of KIT and PDGFRA mutations in the plasma of patients with gastrointestinal stromal tumor Guhyun Kang & Byung Noe Bae & Byeong Seok Sohn & Jung-Soo Pyo & Gu Hyum Kang & Kyoung-Mee Kim
Received: 19 November 2014 / Accepted: 28 January 2015 # Springer International Publishing Switzerland 2015
Abstract In subsets of gastrointestinal stromal tumors (GISTs), mutations of the KIT and PDGFRA receptor tyrosine kinases correlate with tumor prognosis and response to tyrosine kinase inhibitors (TKIs). Determining genotypes in TKI-resistant GISTs is challenging due to the potential risks and limitations of repeated biopsies during the course of treatment. We prospectively collected plasma samples from three GIST patients harboring KIT mutations that were detected in tissue DNA. The plasma samples were then analyzed for mutations in KIT, PDGFRA, and BRAF via next-generation sequencing. We were able to identify primary KIT mutations in all plasma samples. Additional mutations, including KIT exon
17 S821F and PDGFRA exon 18 D842V, were detected in the patient-matched plasma samples during follow-up and appeared to result in decreased sensitivity to TKIs. Our results demonstrate an approach by which primary and secondary mutations are readily detected in blood-derived circulating tumor DNA from patients with GIST. These mutations can be used as biomarkers for prediction of treatment response. The identification of a resistance mutation in plasma DNA will allow early change to alternative TKIs or dose escalation of imatinib for optimal patient management.
Keywords Gastrointestinal stromal tumor . Next-generation sequencing . Resistance mutation . Circulating tumor DNA . Tyrosine kinase inhibitor G. Kang Department of Pathology, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, South Korea B. N. Bae Department of Surgery, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, South Korea B. S. Sohn Department of Medicine, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, South Korea J.5
Progressive disease
1.58/3043 1.52/2046 5.72/1312 4.45/3009 KIT exon 11 Del K558-E562 KIT exon 17 S821F 61.11/1954 (mesentery) KIT exon 9 Dup A502-Y503 54.46/2681 (lung) PDGFRA exon 18 D842V
KIT exon 11 Del K558-E562a 79.01/6695 1/10 25 Stomach 59/male 1
Mutation AF (%)/DP Size (cm) Mitosis/ Mutation HPF Location
Tissue
Clinicopathologic characteristics and mutation results for three patients with gastrointestinal stromal tumor
Case 1 A 59-year-old man with neurofibromatosis type 1 presented with episodes of lower abdominal pain. CT disclosed a
Case series
Case no. Age at diagnosis/gender Primary tumor
Patients and methods Patients with malignant or advanced GIST were recruited as part of a preliminary study to assess the diagnostic performance of a NGS-based mutation analysis of ctDNA. Written informed consent was obtained from the patients, and blood samples were collected during TKI treatment. This study was approved by the institutional review board of Inje University Sanggye Paik Hospital. Table 1 summarizes the clinicopathologic features of three cases, which were characterized by different clinical responses to TKI: partial response, stable disease, and progressive disease. The cancer panel used in this study (SeaSun Biomaterials, Daejeon, Korea) covers exons 9, 11, 13, 14, 17, and 18 of KIT; exon 18 of PDGFRA, and exon 15 of BRAF. DNA extraction from tissue and plasma samples and amplicon library preparation were carried out according to manufacturers’ protocols and as described previously [8]. Emulsion polymerase chain reaction (PCR), breaking, and bead enrichment were done using the GS Junior Titanium emPCR Kit Lib-L, emPCR Reagents Lib-L Kit, Oil and Breaking Kit, and the Bead Recovery Reagents Kit (Roche Diagnostics, Mannheim, Germany). Sequencing was performed to over 1300-fold coverage using the GS Junior system (Roche Diagnostics). The results were analyzed with the GS Amplicon Variant Analyzer (version 2.7; Roche Diagnostics). To detect variants, filters were set to display sequence variances occurring in more than 1 % of bidirectional reads per amplicon [9].
Table 1
Plasma
AF (%)/DP
switching to an alternative TKI, although the strategy that yields the optimal outcomes remains unclear [1]. Diagnostic imaging techniques are currently used to assess response to TKI in patients with GIST. However, these methods display limited sensitivity and specificity, and treatment changes are generally triggered by clinical and/or computerized tomography (CT)-morphologic progression [5]. Serial or repeated biopsies are invasive and often unattainable and thus are not widely accepted as means of predicting treatment response or detecting resistance mutations. Moreover, as tumors are heterogeneous and continuously evolving, biopsies of one or two accessible sites may not be representative of the entire tumor, and the mutations present in a minor subclone can be missed [6]. An alternative approach that may overcome these issues is the analysis of peripheral blood, in which cell-free DNA fragments from multiple lesions in a single patient are mixed together [7]. Here, we have used a next-generation sequencing (NGS) platform to perform mutation analyses of cell-free circulating tumor DNA (ctDNA) from the plasma of GIST patients on TKI therapy.
Partial response
Best response to TKI
Targ Oncol
Targ Oncol
25-cm mass arising from the stomach. Under endoscopic ultrasound guidance, three tissue fragments were obtained using a 22-gauge needle via a transgastric approach. A histologic diagnosis of GIST was made based on positive immunostaining for KIT, and further mutation analysis of the biopsy specimen revealed a deletion of K558-E562 in KIT exon 11. The patient had a partial response after 3 months of treatment with imatinib, and the tumor remained stable as assessed by CT scans at regular visits every 3 months. A missense mutation in KIT exon 17 (S821F), as well as the primary mutation, was detected in the plasma sample collected 10 months after biopsy. The missense mutation in KIT has not been reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Six months later, the last follow-up CT demonstrated a new enhancing nodule in the pelvic cavity (Fig. 1a). Case 2 A 61-year-old man was diagnosed with small intestinal GIST and received adjuvant imatinib (400 mg/day) for a year. After withdrawal of imatinib treatment, the patient developed local recurrence in the small bowel mesentery at 24 months. A subsequent wedge resection was performed at 28 months to rule out primary lung cancer, which confirmed to be a metastatic GIST. Molecular analysis of the mesenteric and lung masses identified a duplication of A502-Y503 in KIT exon 9. As patients with this mutation respond less well to imatinib, the regimen was then changed to sunitinib 37.5 mg/day. A CT scan at 40 months showed residual tumors at the sites of previous operations with a suspicious metastatic lesion in the liver (Fig. 1b). A PDGFRA exon 18 D842V mutation was also detected in the plasma sample of this patient at the last follow-up. This mutation had not been identified in the tumor tissues. Case 3 A 82-year-old woman presented with palpable abdominal masses. She had a history of small bowel resection for GIST. The clinical and radiologic features of the masses were suggestive of hepatic metastases of GIST. She was started on imatinib 400 mg daily and achieved a partial response after 5 months of treatment. Even though the dose was reduced to 200 mg/day because of unacceptable side effects, the patient had a stable disease status over 30 months of follow-up. At 60 months, ctDNA only contained the primary tumor mutation in KIT exon 11 (P551A) without any detectable mutation in exons 9, 13, 14, 17, or 18 of KIT; exon 18 of PDGFRA; or exon 15 of BRAF (Fig. 1c).
Discussion There is an outstanding need for methods that can be used to detect mutations efficiently and effectively and thereby inform the selection of drugs that have validated roles against targeted
mutations. However, the mutations in primary tumor tissues may not reflect current molecular status in a patient with recurrent or metastatic disease [10]. Using BEAMing technology and allele-specific ligation PCR, two recent studies have recovered KIT/PDGFRA mutations from ctDNA in the plasma samples of patients with GIST [11, 5]. However, because they are designed to detect specific, commonly observed mutations in hot spot regions, negative test results cannot be interpreted as demonstrating the absence of mutations in the genes [10]. As compared with these two methods, NGS holds major advantages in terms of cost, turnaround time, and breadth of mutation coverage (i.e., the ability to simultaneously detect all mutation types for a large number of genes in a single assay) and might offer an opportunity for increasing sensitivity. In this report, we further validated the utility of ctDNA analysis for detecting mutations associated with acquired TKI resistance in patients with malignant or advanced GIST. It was possible to identify a primary tumor mutation present in ctDNA at an allele frequency of 1.58 %, and there was 100 % concordance between mutations found in the primary tumors and those found in the ctDNA. Of note, an additional KIT or PDGFRA mutation was detected in two of the plasma samples; the presence of which was associated with poor response to TKI. The identification of resistant mutations represents one of the most immediate clinical applications and would allow alternative therapies to be initiated early [12]. A highly significant example is the detection of KIT exon 17 substitution S821F in the plasma of case 1 during imatinib therapy, before relapse became detectable radiologically. To our knowledge, this is a novel mutation that has not been reported in GISTs. The present case suggests that it might be associated with decreased sensitivity to imatinib. In case 2, our patient’s tumors contained KIT exon 9 mutations, and he was found to have a secondary PDGFRA exon 18 D842V mutation. The D842V is the single most common PDGFRA mutation that confers resistance to both imatinib and sunitinib. Uncommonly, imatinib resistance also occurs through the mutation of a different kinase that is less sensitive to this drug. Previous reports have described a GIST with a primary KIT G565R mutation that acquired a secondary PDGFRA D842V mutation, as well as a tumor with a primary KIT mutation in exon 9 that developed an additional mutation in PDGFRA exon 14 [13, 14]. The ctDNA as a diagnostic, prognostic, and theranostic tool in cancer patients is an appealing approach, at least in theory, since it is noninvasive and easily repeated [15]. However, there are several hurdles to adopt ctDNA into clinical routine practice: (1) lack of standardization of techniques, (2) difficulty in controlling the pre-analytical phase to obtain robust and reproducible results, and (3) high cost restricting patients’ access [12, 15, 16].
Targ Oncol
Targ Oncol
Fig. 1
Clinical course of each patient and the mutations that were detected in tissue and plasma samples: a case 1, b case 2, and c case 3 (IM imatinib, SU sunitinib)
Overall, our results demonstrate that the analysis of ctDNA for drug-resistant mutations can complement current invasive biopsy procedures and provide promising biomarkers for TKI-resistant GISTs, thus allowing dose escalation of imatinib or switching to another TKI on a timely basis. However, our preliminary results need to be validated in independent cohorts of patients and further large prospective multicenter studies are needed to introduce ctDNA into the routine clinical practice.
7.
8.
9.
Conflict of interest The authors declare that they have no conflict of interest.
References 10. 1. Demetri GD, von Mehren M, Antonescu CR, DeMatteo RP, Ganjoo KN, Maki RG, Pisters PW, Raut CP, Riedel RF, Schuetze S, Sundar HM, Trent JC, Wayne JD (2010) NCCN task force report: update on the management of patients with gastrointestinal stromal tumors. J Natl Compr Cancer Netw 8(Suppl 2):S1–S41, quiz S42-44 2. Corless CL, Barnett CM, Heinrich MC (2011) Gastrointestinal stromal tumours: origin and molecular oncology. Nat Rev Cancer 11(12): 865–878. doi:10.1038/nrc3143 3. Dematteo RP, Ballman KV, Antonescu CR, Maki RG, Pisters PW, Demetri GD, Blackstein ME, Blanke CD, von Mehren M, Brennan MF, Patel S, McCarter MD, Polikoff JA, Tan BR, Owzar K (2009) Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumour: a randomised, double-blind, placebocontrolled trial. Lancet 373(9669):1097–1104. doi:10.1016/S01406736(09)60500-6 4. Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H, Roberts PJ, Eisenberg BL, von Mehren M, Fletcher CD, Sandau K, McDougall K, Ou WB, Chen CJ, Fletcher JA (2006) Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol 24(29):4764–4774 5. Maier J, Lange T, Kerle I, Specht K, Bruegel M, Wickenhauser C, Jost P, Niederwieser D, Peschel C, Duyster J, von Bubnoff N (2013) Detection of mutant free circulating tumor DNA in the plasma of patients with gastrointestinal stromal tumor harboring activating mutations of CKIT or PDGFRA. Clin Cancer Res 19(17):4854–4867. doi:10.1158/1078-0432.CCR-13-0765 6. Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF, Kingsbury Z, Wong AS, Marass F, Humphray S, Hadfield J, Bentley D, Chin TM, Brenton JD, Caldas C, Rosenfeld
11.
12.
13.
14.
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