Assessment of Predictive Biomarkers of the Response to Pazopanib Based on an Integrative Analysis of High-grade Soft-tissue Sarcomas: Analysis of a Tumor Sample from a Responder and Patients with Other Soft-tissue Sarcomas

Abstract

Background

Soft-tissue sarcomas are a rare group of malignant tumors that usually are treated with surgical excision and radiation therapy, but recently, pazopanib, an oral tyrosine kinase inhibitor, has been used in patients with metastases who do not respond to standard chemotherapy regimens. Based on patients with advanced soft-tissue sarcomas who had received prior chemotherapy, several clinical studies have reported the survival and sensitivity (approximately 5% to 10% sensitive) of patients with soft-tissue sarcomas treated with pazopanib. Recently, next-generation sequencing (NGS) technologies have been used to provide a wide genetic information and to develop personalized medicine in cancer treatment. However, there are few reports and no genetic analyses of patients with soft-tissue sarcomas who had a complete response (CR) to pazopanib.

Questions/purposes

We described the clinicopathologic features of a patient with a rare, advanced soft-tissue sarcoma who achieved a CR to pazopanib treatment. Furthermore, integrative analyses using NGS and arrays were performed to elucidate characteristic alterations, including gene mutations, copy number changes, and protein expression that were associated with response to pazopanib. Additionally, functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified alterations were associated with oncogenic abilities and drug responses.

Methods

In a sample from a 70-year-old woman with an advanced soft-tissue sarcoma treated for 1 month with 800 mg of oral pazopanib daily, CT scans demonstrated a CR to treatment. To our knowledge, there have been no patients with soft-tissue sarcomas among several clinical trials of pazopanib that have achieved a CR and therefore, our patient is considered to be extremely rare. We performed an integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases (RTK) using tumor samples from a patient with a CR matched to normal samples. From here on we will refer to this patient as having a CR, although a short term high-grade partial response may be more accurate. These analyses were performed using NGS and the phosphoreceptor tyrosine kinase (phospho-RTK) array. As a validation study, we also performed target sequencing using three samples from patients with long-term stable disease and two samples from patients with progressive disease who responded to pazopanib treatment. In addition, characteristic gene alterations that were identified according to the response to pazopanib in one patient with a CR, in three patients with long-term stable disease, and in 27 patients with high-grade soft-tissue sarcomas with different histologic subtypes and different responses to pazopanib were verified by quantitative real-time polymerase chain reaction. We conducted a focus formation assay to evaluate the transforming activities of these genomic alterations.

Results

In the patient with a CR to pazopanib, we identified several somatic mutations including Fms related receptor tyrosine kinase 1 (FLT1) p.G38S, platelet-derived growth factor receptor alpha (PDGFRA) p.T83S, and platelet-derived growth factor receptor beta (PDGFRB) exon 13 skipping. Amplification at chromosome 12q13-14 encompassing GLI family zinc finger 1 (GLI1) and cyclin-dependent kinase-4 (CDK4) was also detected. Furthermore, an elevated PDGFRB phosphorylation level was observed in the tumor. In target sequencing analyses in five patients, one of three patients with long-term stable disease had 12q13-14 amplification. The mRNA expression of GLI1, CDK4, and pazopanib targets including PDGFRA, PDGFRB, vascular endothelial growth factor receptor (VEGFR)1-3, and stem cell factor receptor (KIT) in samples from the patient with a CR, and 27 patients with high-grade soft-tissue sarcomas was verified. The expression of GLI1 was characteristically increased in the patient with a CR and in those with long-term stable disease relative to other patients with soft-tissue sarcomas. Overexpression of GLI1 showed strong transforming potential in 3T3 cells. Moreover, the overexpression of GLI1 upregulated the expression of the PDGFRB protein and promoted phosphorylation, which was dose-dependently inhibited by pazopanib. However, inhibition of GLI1-induced transformation by pazopanib was limited in the focus formation assay; therefore, mechanisms other than PDGFRB activation may contribute to transformation.

Conclusions

We identified several gene alterations that might be associated with a CR and long-term stable disease in patients who received pazopanib for advanced soft-tissue sarcomas. We therefore believe that this distinct molecular profile warrants further investigation to identify predictive biomarkers of the response to pazopanib.

Clinical Relevance

Our findings identify molecular mechanisms that possibly explain the high sensitivity of soft-tissue sarcomas to pazopanib and may lead to the development of predictive biomarkers and novel therapies in patients with this and other types of soft-tissue sarcomas.

Introduction

Soft-tissue sarcomas are rare malignant tumors consisting of more than 50 histologic subtypes [2, 4]. Recently, the FDA approved pazopanib, an oral multi-target angiogenesis inhibitor, to treat patients with advanced soft-tissue sarcoma who have received prior chemotherapy, based on a multicenter, international, double-blind, placebo-controlled Phase III clinical trial that evaluated the safety and efficacy of pazopanib in 369 patients [23]. Pazopanib is a novel tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and stem cell factor receptor (KIT) [20].

Several clinical studies have reported the survival benefit of pazopanib in patients with soft-tissue sarcoma [10, 15, 23]. In terms of overall responses to pazopanib treatment in patients with soft-tissue sarcomas, the PALETTE study revealed that 0% of their patients (0 of 246) showed a complete response (CR), 6% (14) demonstrated a partial response, 67% (164) had stable disease, and 23% (57) had progressive disease [23]. In the placebo group in the PALETTE study, 0% of the patients (0 of 123) showed a CR, 0% (0) had a partial response, 38% (47) had stable disease, and 57% (70) had progressive disease [23]. The results of a study from the Japanese Musculoskeletal Oncology Group [15] with 125 enrolled patients revealed that no patients achieved a CR. Furthermore, to our knowledge, no studies have investigated the genetic factors in patients with soft-tissue sarcoma who have CRs to pazopanib Therefore, patients with soft-tissue sarcoma who have a CR to pazopanib seemed to be extremely rare.

In this study, we described the clinicopathologic features of a patient who had a high-grade soft-tissue sarcoma. The pathological diagnosis was a pleomorphic sarcoma; this patient had an advanced stage with multiple metastases (two in lung and chest wall) and had a CR (or a short-term high-grade partial response) to pazopanib. The patient, a 70-year-old woman, showed a CR to pazopanib after 1 month of treatment. The responses of these tumor lesions were evaluated by CT scans. We then used surgical samples of the patient’s primary tumor to conduct an integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of tyrosine kinase proteins to investigate factors associated with the high anti-tumor activity of pazopanib. As a validation study of the integrative analysis, we also performed target sequencing and quantitative real-time PCR (qPCR) using 27 soft-tissue sarcoma samples classified according to the patient’s response to pazopanib. Finally, to verify the functions of our identified factors, functional assays were performed.

Materials and Methods

Clinicopathological Information and Response to Pazopanib from Patient Samples

In our institution, 13 patients with soft-tissue sarcoma were treated with pazopanib from September 2015 to August 2019. We tracked these 13 patients’ clinicopathological features, including their pazopanib responses (Table 1). These included the patient with a CR (or short term high-grade partial response), three patients with long-term stable disease (if stable disease was present for more than 6 months, the patients were considered to have benefitted from the treatment [15]), and nine patients with progressive disease despite pazopanib treatment (Table 1). Of these patients, we collected frozen tumor samples from eight, but all 13 patients had formalin-fixed paraffin-embedded tumor samples available for analysis. These samples, consisting of both frozen and formalin-fixed paraffin-embedded, were used for an integrative analysis and target sequencing (see Table 1; Supplemental Digital Content 1, http://links.lww.com/CORR/A372).

Table 1.

Clinical and analytical data of advanced soft tissue sarcoma cases with pazopanib treatment

Number.	Case number	Gender	Age	Primary lesion	Histological diagnosis	Pazopanib response	Status of frozen samples	Best response	Analysis/validation
									NGS	Quantitative-PCR
1	1 (CR)	F	76	Axilla	Pleomorphic sarcoma	CR	Available	CR	WES/TS	Analysis
2	2	F	21	Heart	Myxoid sarcoma	Long SD (8 months)	Available	SD	Target Seq	Analysis
3	3	M	50	Chest wall	Leiomyosarcoma	Long SD (7 months)	Available	SD	Target Seq	Analysis
4	4	M	52	Thigh	MPNST	Long SD (7 months)	Not available	PR	Target Seq	Not analysis
5	5	M	50	Lower leg	High-grade myxofibrosarcoma	PD (3 months)	Available	SD	Target Seq	Analysis
6	6	F	72	Retroperitoneum	Undifferentiated pleomorphic sarcoma	PD (1 month)	Available	PD	Target Seq	Analysis
7	7	F	79	Forearm	Undifferentiated pleomorphic sarcoma	PD (3 months)	Available	PR	Not analysis	Analysis
8	8	F	80	Buttock	Undifferentiated pleomorphic sarcoma	PD (1 month)	Available	PD	Not analysis	Analysis
9	9	M	74	Thigh	Leiomyosarcoma	PD (3 months)	Available	SD	Not analysis	Analysis
10	-	F	55	Retroperitoneum	Leiomyosarcoma	PD (4 months)	Not available	SD	Not analysis	Not analysis
11	-	F	38	Buttock	Undifferentiated pleomorphic sarcoma	PD (4 months)	Not available	SD	Not analysis	Not analysis
12	-	M	62	Inguinal	Undifferentiated pleomorphic sarcoma	PD (2 months)	Not available	PD	Not analysis	Not analysis
13	-	M	70	Lung	Myxoid sarcoma	PD (2 months)	Not available	PD	Not analysis	Not analysis

Step1 analysis performed WES/TS using frozen samples (Number 1_a case).

Step 2 analysis performed Target Seq using formalin-fixed paraffin-embedded samples (Number 2 - 6_5 cases).

NGS = next generation sequencing; CR = complete response; WES = whole exome sequencing; TS = transcriptome sequencing; SD = stable disease; Target Seq = TruSight tumor 170; MPNST = malignant peripheral nerve sheath tumor; PR = partial response; PD = progressive disease; Long SD (long-term SD) = more than 6 months SD.

As a validation study, we evaluated the mRNA expression of the selected genes and pazopanib target genes using samples from 27 patients with a high-grade soft-tissue sarcoma (see Table 1; Supplemental Digital Content 1, http://links.lww.com/CORR/A372). The samples were comprised of the above-mentioned eight samples from patients who had an soft-tissue sarcoma and were treated with pazopanib, as well as 19 other samples from patients with one of the six histological subtypes of soft-tissue sarcoma (Fig. 1).

Fig. 1.

An analysis chart of the patients in this study is shown.

Histological diagnosis was made with immunohistochemical analysis based on the WHO classification of tumors [4]. These soft-tissue sarcomas were collected and analyzed using a protocol approved by the institutional review boards at the National Cancer Research Institute (No. 2015-202) and Juntendo University (No. 2018135). After consent was given, samples of the resected tumor specimen and surrounding normal tissue as the source of matched normal (germline) DNA were obtained from the Department of Orthopedic Surgery, Graduate School of Medicine, Juntendo University.

Study Overview

Integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases (RTK) was performed using CR-paired samples (tumor and normal) to investigate characteristic genetic alterations associated with a CR to pazopanib (Fig. 2, step 1). In the validation study based on the pazopanib responses, target sequencing analyses (TruSight Tumor 170, Illumina Inc, San Diego, CA, USA) were performed using five paraffin embedded samples that were treated with pazopanib, including three patients with long-term stable disease and two patients with progressive disease (Fig. 2, step 2). In the further validation study based on pazopanib responses and high-grade soft-tissue sarcomas, we performed quantitative PCR to evaluate mRNA expression of characteristic genetic alterations (GLI1, CDK4) and the pazopanib target genes (PDGFRA, PDGFRB, VEGFR1-3, and KIT) across 27 patients with high-grade soft-tissue sarcomas and a patient with CR (Fig. 2, step 3).

Fig. 2.

A patient with a CR was analyzed using an integrative analysis with whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of RTKs (Step 1). Additionally, five high-grade sarcomas that were treated with pazopanib (three patients with long-term stable disease and two with progressive disease) were analyzed using target sequencing (TruSight tumor 170, Illumina) (Step 2). In the validation study, eight patients with soft-tissue sarcoma who were treated with pazopanib (response: CR, stable disease, and progressive disease) and 19 patients with soft-tissue sarcoma including several histologic subtypes were evaluated to verify characteristic genetic alterations (GLI1 and CDK4) and pazopanib targeting gene (PDGFRA [platelet-derived growth factor receptor alpha], PDGFRB [platelet-derived growth factor receptor beta], VEGFR1-3 [vascular endothelial growth factor receptor 1-3], and KIT [stem cell factor receptor]) (Step 3). Characteristic gene alterations that were associated with response to pazopanib were verified by a functional analysis (Step 4). A color image accompanies the online version of this article.

To elucidate the transforming activities of characteristic genetic alterations (GLI1, CDK4), associations between pazopanib and proliferation of GLI1-overexpressing cells were assessed using functional analyses consisting of in vitro and in vivo assays (Fig. 2, step 4).

Patient with Complete Response to Pazopanib

A soft-tissue mass was found in the right axilla of a 70-year-old female patient (Fig. 3A-C). A histologic image showed a high-grade pleomorphic sarcoma (Fig. 4). Multiple metastases of the left lung and left chest wall were found on a chest CT scan (Fig. 5A-D). She was treated with resection of the right axillary tumor to alleviate pain and prevent tumor self-destruction (meaning, that it would grow and erode through the skin). Because of the patient’s age and performance status, it was thought that she would not tolerate standard chemotherapy, therefore as an alternative, pazopanib, which had been approved for advanced soft-tissue sarcoma in patients who had received prior chemotherapy was considered. She was treated with oral pazopanib 800 mg daily as first-line systemic therapy, in October 2015, without prior chemotherapy. Her first chest CT scan after 1 month showed CR (or a short-term high-grade partial response) of metastases of the right lung and right chest wall (Fig. 5A-D). However, because of liver dysfunction, pazopanib was discontinued for 4 weeks. During this period, her lung metastases reappeared, and pazopanib treatment was restarted with a reduced dose (200 mg to 400 mg daily). She was re-treated with oral pazopanib 400 mg daily for 5 days; however, liver dysfunction recurred, and the treatment was stopped. Finally, after withdrawal for approximately 3 weeks, she received oral pazopanib 200 mg daily for 6 weeks; however, the size tumor lesions in the lung remained unaffected. The treatment was terminated in February 2016 because of general malaise as per the wishes of the patient. She died of disease progression in March 2016.

Fig. 3.

A soft-tissue mass of the right axilla is seen in a 70-year-old female patient. MRI demonstrates a 7-cm subcutaneous mass that was enhanced by gadolinium. (A) A T1-weighted image is shown. (B) A T2-weighted image is shown from a different angle. (C) A gadolinium-enhanced T1-weighted image is shown.

Fig. 4.

The histologic diagnosis was a high-grade pleomorphic sarcoma, which is shown here. A color image accompanies the online version of this article.

Fig. 5.

CT images of soft-tissue cross sections show images of (A) the right intercostal muscle and (B) the right lung. After 1 month of treatment with pazopanib (800 mg daily), CT images of (C) the right intercostal muscle and (D) the right lung were taken. Both metastatic lesions disappeared, indicating a complete response.

Integrative Analysis

We performed an integrative analysis [14, 18] including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases using matched tumor and normal samples (Table 1). This integrative analysis investigated genetic and proteomic alterations including genomic mutations, copy number alterations, and the phospho-RTK protein expression changes in patient samples to elucidate the factors associated with the high anti-tumor activity of pazopanib (see Supplemental Digital Content 2, http://links.lww.com/CORR/A373). The samples were obtained from the resected right axillary lesion before chemotherapy.

Furthermore, we performed target sequencing (TruSight Tumor 170, Illumina) using five samples from patients with soft-tissue sarcomas who were classified according to their response to pazopanib (Table 1). The TruSight Tumor 170 was performed using whole genome sequencing, mutational analysis, transcriptome sequencing, expression analysis, and detection of fusion genes and exon skipping (see Supplemental Digital Content 2, http://links.lww.com/CORR/A373).

Western Blotting

We performed western blotting of PDGFRB, PDGFB, and phosphorylated PDGFRB using paired protein samples of CR to pazopanib to confirm expressions of phospho-RTK and related proteins. We also performed western blotting using antibodies to GLI1, phospho-PDGFRB, PDGFRB, and PDGFB in the GLI1-transfected 3T3 cells to confirm expression of the transfected GLI1 gene and investigate the expression of the pazopanib target proteins. Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently blotted on a nitrocellulose membrane. The membrane was incubated with antibodies against platelet derived growth factor receptor beta (PDGFRB; [clone 42G12] LS-B3667, LSBio, Seattle, WA, USA; 1:50 dilution), platelet-derived growth factor beta (PDGFB; ab178409, Abcam, Cambridge, UK; 1:1000 dilution), phospho-PDGFRB ([Tyr751] C63G6, Cell Signaling, Danvers, MA, USA; 1:1000 dilution), glioma-associated oncogene (GLI1; ab134906, Abcam, Cambridge, UK; 1:1000 dilution), or glyceraldehyde 3-phosphate dehydrogenase ([6C5] sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000 dilution), followed by incubation with a horseradish peroxidase-conjugated secondary mouse antibody (GE Healthcare Life Sciences, Marlborough, MA, USA; 1:1000 dilution) or rabbit antibody (GE Healthcare Life Sciences; 1:1000 dilution). The expression of protein was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Construction of a Retroviral Vector with Random Barcodes

We constructed a retroviral vector with random barcodes to evaluate associations between transforming potentials and the transfected genes. The random barcodes provided both confirmation and quantification of transfections. Plasmids encoding wild-type human cDNA for FLT1, PDGFRA, PDGFRB, and GLI1 were isolated with PCR and ligated into a pcx5bleo vector [13]. cDNAs encoding the FLT1, PDGFRA, and PDGFRB mutants were generated using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) and ligated into pcx5bleo.

Focus Formation Assay

We performed focus formation assays using the 3T3 cells infected with recombinant retroviruses that expressed the wild-type cDNAs (wild-type GLI1, FLT1, PDGFRA, and PDGFRB), mutant cDNAs (FLT1 p.G38S, PDGFRA p.T83S, and PDGFRB exon 13 skipping), and GFP (a negative control) to verify the transforming potential of genes identified by integrative analyses. The recombinant plasmids and packaging plasmids (Takara Bio, Shiga, Japan) were introduced into HEK293T cells to obtain recombinant retroviral particles. For the focus formation assay, 3T3 cells were infected with ecotropic recombinant retroviruses using 4 μg⁄mL of Polybrene (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours and cultured in Dulbecco's Modified Eagle Medium (DMEM)-F12 supplemented with 5% calf serum for up to 2 weeks. For the focus formation assay of pazopanib, GLI1-expressing 3T3 cells and Green Fluorescent Protein (GFP)-negative control cells were used. Six-well plates were seeded with 2.0 × 10² cells per well, followed by treatment with 10 nM to 100 μM of pazopanib in the presence of 5% calf serum for 14 days. Cell transformation was assessed through either phase-contrast microscopy or staining with Giemsa solution. The focus number was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to that of the negative control.

Quantitative Real-time PCR

We performed quantitative real-time PCR (qPCR) to elucidate associations between gene expression and clinicopathological features including the pazopanib responses. The mRNA expression levels of both genes identified by an integrative analysis (GLI1 and CDK4) and the pazopanib target genes (PDGFRA, PDGFRB, VEGFR1-3, and KIT) were measured using qPCR in the patient with a CR and 27 high-grade sarcoma samples (see Table 1; Supplemental Digital Content 1, http://links.lww.com/CORR/A372). Quantitative real-time PCR was performed using TaqMan assays (20 × Primer Probe mix; Applied Biosystems, Foster City, CA, USA) corresponding to PDGFRA (Assay ID Hs00998018 _m1), PDGFRB (Assay ID Hs01019589_m1), GLI1 (Assay ID Hs00171790_m1), CDK4 (Assay ID Hs00364847_m1), MDM2 (Assay ID Hs00540450 _s1), VEGFR1 (Assay ID Hs01052961 _m1), VEGFR2 (Assay ID Hs00911700 _m1), VEGFR3 (Assay ID Hs01047677 _m1), KIT (Assay ID Hs00174029 _m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID Hs02758991_g1). All qPCR reactions were performed with TaqMan Fast Advanced Master Mix (Applied Biosystems) on an Applied Biosystems StepOnePlus Real-Time PCR system in accordance with standard protocols. The amount of each target gene relative to the housekeeping gene was determined using the comparative threshold cycle method. All assays were performed in triplicate.

In Vivo Animal Models

We performed in vivo animal model studies to clarify the antitumor activity of pazopanib in the GLI1-transfected cells. All animal studies were conducted in accordance with the protocols approved by the Animal Ethics Committee of the National Cancer Research Center, Tokyo, Japan. Before injection, 3T3 cells (1.0 × 10⁶) were mixed in phosphate-buffered saline with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at a 1:1 ratio. The cell suspension was injected subcutaneously (200 µL/mouse) into the back of 6-week-old female BALB/c nude mice (CREA Japan, Tokyo, Japan). In accordance with our previous study which similarly evaluated the drug efficacy using a 3T3 xenograft model, five age- and sex-matched mice of this strain were randomly assigned to each group [12]. When tumors reached approximately 100 mm³ to 150 mm³ in size, mice were randomized into two groups. Either 100 mg/kg of pazopanib or a vehicle control was administered by oral gavage using a stomach feeding tube. Both treatments were administered once daily for 5 days per week. Tumor volume was used as the endpoint to directly evaluate the drug efficacy with regard to tumor growth inhibition. The tumor volume (mean ± SD) in each group is expressed in cubic millimeters and was calculated using the formula π/6 × (largest diameter) × (smallest diameter)². Tumor size assessment was completed by observers (SK, TU, SM) blinded to the study groups of the animals and their treatments. The mice were euthanized when the tumor reached 1.5 cm in diameter and the collected data was analyzed.

Results

Elevated Levels of PDGFRB Phosphorylation in a Tumor Sample from a Patient with Complete Response to Pazopanib

The RTK array analysis showed that PDGFRB had higher phosphorylation levels than the other RTK proteins in the tumor sample that had a CR to pazopanib (Fig. 6A). The tumor sample showing CR to pazopanib had higher phosphorylation levels of PDGFRB in 42 RTKs (PDGFRB; mean value: 1.67 (SD 0.04); p < 0.01). In western blotting regarding the PDGFRB, PDGFB, and phosphorylated PDGFRB blots as confirmation assays, the tumor sample showed higher protein expression and protein phosphorylation than the control samples did (Fig. 6B).

Fig. 6.

This figure shows a comparison of protein levels in normal and tumor tissue. (A) This image shows protein phosphorylation of receptor tyrosine kinases, as screened by a Human Phospho-RTK array kit (R&D Systems, Minneapolis, MN, USA), using matched samples of tumor and control tissues in a patient with a CR. EGFR and PDGFRB demonstrated elevated protein phosphorylation in tumor samples. PDGFRB, PDGFB, and PDGFRB phosphorylation increased in tumor tissues in comparison with those in the control tissue. (B) This western blot compares tumor and normal tissue. PDGFRB, PDGFB, and PDGFRB phosphorylation increased in tumor tissue in comparison with the control tissue.

Molecular Profiling Identifies 12p13-14 Amplifications and RTK Mutations Including FLT1, PDGFRA, and PDGFRB from Samples with Responses to Pazopanib

In the whole-exome sequencing, somatic mutations in RTKs including FLT1 p.G38S and PDGFRA p.T83S were identified in the patient’s sample (Table 2). Based on the whole-exome sequencing data, copy number alterations were measured in the paired samples. We detected amplification on chromosome 12p13-14 as qualitative analysis. That region did not include the pazopanib-targeted genes such as VEGFRs, PDGFRs, and KIT, which were located other chromosomes, but did include GLI1 and CDK4 (Fig. 7). RNA sequence reads were analyzed and we found PDGFRB exon 13 skipping, which is a potential target of pazopanib (see Fig. 1; Supplemental Digital Content 3, http://links.lww.com/CORR/A374). Target sequencing of samples from five patients with an advanced soft-tissue sarcoma showed one of the three patients with long-term stable disease had 12p13-14 amplification that was also detected in the patient with a CR with PDGFRA amplification (Table 3). In these assays, we found RTK mutations including mutations within FLT1, PDGFRA, and PDGFRB in samples with CR to pazopanib. The 12p13-14 amplifications including GLI1 and CDK4 were identified in both CR and long-term stable disease samples.

Table 2.

DNA sequencing

Gene name	HGVS.p	HGVS.c	Total read #	Mutation read #	VAF%	Polyphen-2	SIFT	Transcript_ID
PFKP	p.Arg575Gly	c.1723C>G	328	135	41.1	0.99		NM_002627
ALDH18A1	p.Ile32Phe	c.94A>T	155	32	20.6	0	0.12	NM_002860
ATM	p.Arg23*	c.67C>T	197	46	23.3	0.73	1	NM_000051
STYK1	p.Gly42Val	c.125G>T	510	145	28.4	0		NM_018423
FLT1	p.Gly38Ser	c.112G>A	169	29	17.1	0.79	0.11	NM_002019
MYO3B	p.Gln874His	c.2622G>T	521	215	41.2	0.017	0.33	NM_138995
NRP2	p.Gly584Val	c.1751G>T	329	103	31.3	1		NM_003872
PASK	p.Glu323Asp	c.969G>T	222	59	26.5	0.005	0.34	NM_015148
PDGFRA	p.Thr83Ser	c.247A>T	427	88	20.6	0.002	1	NM_006206
PDK4	p.Ile361Val	c.1081A>G	368	151	41	0	1	NM_002612
PHKA2	p.Val512Leu	c.1534G>C	400	63	15.7	0.013	0.4	NM_000292
RPS6KA3	p.Leu506Val	c.1516C>G	709	155	21.8	0.009	0.11	NM_004586
GK	p.Ile542Met	c.1626A>G	288	58	20.1		0.13	NM_001205019
NEK10	p.Gly1164fs	c.3492delG	894	154	17.2			NM_152534

The list of somatic mutations (kinase) in the case. To elucidate the factor of high anti-tumor activates to pazopanib, DNA sequencing was performed using the paired tumor and normal samples. The samples were obtained from resected lesion at right axillar and were pre-chemotherapy samples. Somatic mutations of kinase family were identified based on obtained reads of DNA-seq. When referring to main target tyrosine kinase genes including VEGFR, FGFR, PDGFR and KIT to pazopanib, FLT1 p.G38S and PDGFRA p.T83S were identified as somatic mutations in sample that had high sensitivities to pazopanib; HGVS = Human Genome Variation Society; SIFT = Sorting Intolerant from Tolerant; PFKP = phosphofructokinase, platelet; ALDH18A1 = aldehyde dehydrogenase 18 family member A1; ATM = ATM serine/threonine kinase; STYK1 = serine/threonine/tyrosine kinase 1; FLT1 = Fms related receptor tyrosine kinase 1; MYO3B = myosin IIIB; NRP2 = neuropilin 2; PASK = PAS domain containing serine/threonine kinase; PDGFRA = platelet-derived growth factor receptor alpha; PDK4 = pyruvate dehydrogenase kinase 4; PHKA2 = phosphorylase kinase regulatory subunit alpha 2; RPS6KA3 = ribosomal protein S6 kinase A3; GK = glycerol kinase; NEK10 = NIMA related kinase 10.

Fig. 7.

12q13-14 gene amplification showed the co-occurrence of GLI1 and CDK4.

Table 3.

Copy number alterations and mRNA expression of advanced soft tissue sarcoma cases with pazopanib treatment

Case number	Pazopanib response	Copy number alteration	Quantitative-PCR and/or CNA of GLI1
1 (CR)	CR	CDK4 AMP, GLI1 AMP	Very high
2	Long SD	PDGFRA AMP (2.69), 12q13-14 (CDK4 AMP (3.66), MDM2 AMP (5.74))	Very high
3	Long SD	No copy number alteration	Low
4	Long SD	No copy number alteration	Low
5	PD	EGFR AMP (2.01)	Low
6	PD	PDGFRA AMP (6.40), KIT AMP (5.28)	Low

CR = complete response; PD = progressive disease; long SD (long-term SD) = SD over 6 months;

AMP = amplification; CNA = copy number alteration.

High GLI1 and CDK4 mRNA Expression Levels were Associated with Responses to Pazopanib

From qPCR analysis of GLI1, CDK4, and the pazopanib target genes in the patient with a CR and 27 high-grade sarcoma samples, as quantitative differences, the expression of GLI1 was greatly elevated in the patient with an CR (Number 1: the expression value was 355.9) and a patient with long-term stable disease (Number 2: the expression value was 742.6) compared with the other specimens (the mean expression value of Number 3-28 was 1.3) (Fig. 8A). For CDK4, the patient with a CR (Number 1) and the patient with long-term stable disease (Number 2) demonstrated the second- and third-highest expression levels. The sample from the patient with the highest expression of CDK4 also showed elevated MDM2 expression, and this tumor was histologically diagnosed as a dedifferentiated liposarcoma (Fig. 8A). With respect to the expression of PDGFRB, the sample from our patient with an CR (Number 1) showed moderate mRNA expression compared with the other 26 patients with high-grade soft-tissue sarcoma (Fig. 8A). However, a sample from a patient with long-term stable disease (Number 2) with higher expression of GLI1 had higher PDGFRB mRNA expression, despite the absence of PDGFRB amplification in the copy number analysis. These assays revealed that high mRNA expression levels of GLI1 and CDK4 might be associated with good responses to pazopanib regardless of expression of the pazopanib target genes (PDGFRA, PDGFRB, VEGFR1-3, and KIT) (Fig. 8B).

Fig. 8.

This chart shows the mRNA expression of GLI1, CDK4, and pazopanib target genes (PDGFRA and PDGFRB) in samples from a patient with a CR (Number 1), a patient who had long-term stable disease with 12q13-14 amplification (Number 2), and 25 controls with high-grade soft-tissue sarcoma. (A) The expression of GLI1 was greatly increased for both the patient with a CR (Number 1) and the patient with long-term stable disease (Number 2), relative to controls. The patient with a CR (Number 1), the patient with long-term stable disease (Number 2), and a patient with dedifferentiated liposarcoma (Number 27) demonstrated elevated expression of CDK4; the patient with dedifferentiated liposarcoma (Number 27) also had elevated expression of MDM2. The expression of PDGFRB in the patient with a CR was reduced relative to that of controls. However, a patient with long-term stable disease who had greater GLI1 expression demonstrated greater expression of PDGFRA mRNA despite the absence of PDGFRB amplification in the copy number analysis. (B) The second chart shows the mRNA expression of pazopanib target genes (VEGFR1-3, and KIT) in samples from a patient with a CR (Number 1), a patient who had long-term stable disease with 12q13-14 amplification (Number 2), and 25 controls with high-grade soft-tissue sarcoma. There were no characteristic expressions regarding both pazopanib responses and histological subtypes in VEGFR1-3, and KIT.

Based on both an elevated phosphorylation level of PDGFRB in our patient with an CR and higher mRNA expression of PDGFRB in our patient with long-term SD, as well as the target profile of pazopanib (VEGFR, PDGFR, and KIT), we focused on the phosphorylation of PDGFRB as a factor influencing the tumor’s sensitivity to pazopanib.

GLI1 Overexpression Has Transforming Potential and Promotes Phosphorylation of PDGFRB

In focus formation assays using the 3T3 cells infected with recombinant retroviruses of identified gene alterations, we observed accumulated foci in the GLI1-transfected cells, indicating strong transforming potential associated with the expression of GLI1 (Fig. 9A). With the sample analysis we had, we could not demonstrate a difference in transforming activities among FLT1 wild type, PDGFRA wild type, PDGFRB wild type, FLT1 p.G38S, PDGFRA p.T83S, PDGFRB exon 13 skipping, and GFP (see Fig. 2; Supplemental Digital Content 4, http://links.lww.com/CORR/A375). Interestingly, western blotting revealed that GLI1 and PDGFRB expression, as well as PDGFRB phosphorylation, increased in the GLI1-transfected 3T3 cells compared to those in the negative control (GFP) (Fig. 9B). These results suggest that overexpression of GLI1 leads to upregulation of the expression and phosphorylation of PDGFRB.

Fig. 9.

These images show focus formation and levels of PDGFRB expression and phosphorylation in 3T3 cells. (A) Plasmids containing GLI1 and GFP (empty control) were transfected into 3T3 cells. GLI1–transfected cells had increased focus formation relative to the negative control. (B) This image shows a western blot of 3T3 cells transfected with GLI1 or GFP (empty control). GLI1–transfected cells had higher levels of PDGFRB expression and PDGFRB phosphorylation than the control did (GFP). A color image accompanies the online version of this article.

Pazopanib Inhibited Proliferation and PDGFRB Phosphorylation in GLI1-Overexpressing Cells

In the GLI1-transfected 3T3 cells, pazopanib treatment inhibited PDGFRB phosphorylation in a dose-dependent manner ranging from 1 nM to 1 µM, whereas the expression of PDGFRB in these cells was not inhibited by pazopanib (Fig. 10A). Cell proliferation was partially suppressed by 1 µM of pazopanib treatment (Fig. 10B). In further evaluation in vivo regarding responses to pazopanib, pazopanib treatment demonstrated no difference in tumor volume compared with control mice (1060 mm³ versus 780 mm³; p = 0.53) (Fig. 10C).

Fig. 10.

This image shows the effects of pazopanib on the expression and phosphorylation of PDGFRB in a GLI1-transfected 3T3 cell line. (A) Western blotting showed that PDGFRB phosphorylation stimulated by GLI1 transfection was inhibited by pazopanib in a dose-dependent manner. (B) Using a 3T3 focus formation assay, we evaluated the efficacy of pazopanib in inhibiting proliferation. (C) We evaluated the effect of pazopanib on GLI1-overexpressing cells in vivo. We calculated tumor volumes of the GLI1-transfected 3T3 cell xenografts in BALB/c nude mice treated with a vehicle or pazopanib. There were no differences in the weight of the mice between the drug treatment and vehicle groups. Representative resected tumors from vehicle-treated mice and 100 mg/kg pazopanib- treated mice after 6 weeks of administration showed a difference in tumor size. These results revealed that the tumors in the pazopanib treatment group were not different in tumor size compared to mice treated with vehicle alone. A color image accompanies the online version of this article.

Discussion

The FDA has approved pazopanib, a multikinase inhibitor, to treat patients with an advanced soft-tissue sarcoma who have previously been treated with chemotherapy [10, 15, 23]. Several clinical studies have reported the benefits and sensitivity (approximately 5% to 10% sensitive) of using pazopanib in patients with an soft-tissue sarcoma [10, 15, 23]. However, to our knowledge, there are few reports and no genetic analyses of patients with an soft-tissue sarcoma who achieved a CR to pazopanib [10, 15, 23]. To elucidate the factors associated with the high anti-tumor activity of pazopanib, this study has generated integrative and functional analyses using samples that were obtained from an advanced soft-tissue sarcoma patient who achieved a CR to pazopanib (or a short-term, high-grade partial response). This study ultimately revealed that a GLI1 amplification and an elevated PDGFRB phosphorylation level were characteristic alterations associated with high antitumor activity of pazopanib.

Limitations

There are three limitations of this study. Several clinical studies using huge cohorts have reported sensitivities to pazopanib and revealed low sensitivities to pazopanib in patients with soft-tissue sarcoma [10, 15, 23]. Additionally, patients with soft-tissue sarcoma who have demonstrated a CR to pazopanib remain rare, and there are no genetic analyses of patients with a CR [10, 15, 23]. However, although the identified characteristic gene alternations were validated with patients who had responses to pazopanib, this study analyzed just one patient with a CR by integrative and functional analyses. Additionally, this patient did not receive any prior chemotherapy. Although the patient achieved a CR in the short-term (or a short-term high-grade partial response), the patient appeared to have difficulty tolerating the drug due to adverse events, including liver toxicity, and subsequently died after withdrawal of the treatment.

With respect to the functional assay, the GLI1-transfected 3T3 cells led to an upregulation of the PDGFRB expression and PDGFRB phosphorylation. Furthermore, in the GLI1-transfected 3T3 cells, pazopanib treatment inhibited the PDGFRB phosphorylation in a dose-dependent manner. However, in vivo, we could not demonstrate that pazopanib treatment decreased the tumor volume compared with that in the control mice; there were no differences in tumor volumes compared with those in the control group. Considering the gene expression findings shown in our results, a GLI1 overexpression may overcome the effect of an RTK overexpression (Table 3). These results suggest that a GLI1 expression is one oncogenic factor and a response mechanism to pazopanib, although other oncogenic factors remain to be elucidated. Therefore, further studies are required to elucidate these CR functions.

Platelet derived growth factor receptor is a member of a family of transmembrane receptors with tyrosine kinase activity [19]. PDGF isoforms stimulate the growth, survival, and motility of mesenchymal cells and certain other cell types [1, 6], and they have important functions during embryonic development and in the control of tissue homeostasis in adults [6]. Overactive PDGF-PDGFR signaling is associated with the development of several malignant diseases, and tumor growth may be promoted through PDGFR phosphorylation because of autocrine PDGF stimulation, overexpression, or hyperactivation of PDGFR, PDGFR autophosphorylation by PDGFR mutations, or PDGF stimulation of angiogenesis in the tumor [7]. In immunohistochemical analyses of PDGFA, -B, -C, and –D and PDGFRA and -B in tumors in patients with soft-tissue sarcomas, high expression of PDGFB and the co-expression of PDGFB and PDGFRA were independent, negative prognostic markers of disease-specific survival [11]. Furthermore, a high expression of PDGFB and PDGFRB was correlated with higher grading [9, 21]. Based on the findings of these previous studies, alterations of the expression of PDGFRB appear to be associated with malignant soft-tissue sarcoma.

Our assays measured the mRNA expression levels of GLI1, CDK4, and pazopanib targets including PDGFRA, PDGFRB, VEGFR1-3, and KIT using both the CR sample and high-grade spindle cell sarcoma samples, demonstrating a greatly elevated expression of GLI1 and CDK4 in our patient with a CR compared with the other samples from patients with other soft-tissue sarcomas. In addition, samples from one of the patients with long-term SD revealed elevated expression of GLI1, CDK4, and MDM2. Based on previous studies, well-differentiated liposarcoma, dedifferentiated liposarcoma, and some alveolar rhabdomyosarcomas are cytogenetically characterized by amplification of chromosome 12q13-15, including GLI1, CDK4, and MDM2 [3-5, 8]. These findings suggest that this might be an effective modality in the management of dedifferentiated liposarcoma. Therefore, further studies are warranted to elucidate detailed functional mechanisms. Of the soft-tissue sarcomas in the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) database (https://www.cbioportal.org/), 4% of samples (16 of 439) had GLI1 amplification; nine had dedifferentiated liposarcomas, two had well-differentiated liposarcomas, two had sarcomas not otherwise specified, one had an intimal sarcoma, one had a spindle cell rhabdomyosarcoma, and one had a fibrosarcoma [22] (see Fig. 3; Supplemental Digital Content 5, http://links.lww.com/CORR/A376). In terms of 12q13-15 amplification, pazopanib exerted antitumor activities in dedifferentiated liposarcoma models in vivo [8]. A recent clinical trial proved pazopanib had a potential benefit in a subset of patients with liposarcomas and soft-tissue sarcomas [17]. In particular, a sample from our patient with an CR had 12q13-14 amplification including GLI1 and CDK4 but not MDM2. Three samples of patients in the above-mentioned 16 MSK-IMPACT trial had only GLI1 and CDK4 amplification, with the remaining showing GLI1, CDK4, and MDM2 amplification [22] (see Fig. 3; Supplemental Digital Content 5, http://links.lww.com/CORR/A376). The histologic subtypes of the three tumors were spindle cell rhabdomyosarcomas, dedifferentiated liposarcoma, and sarcoma not otherwise specified. We indicated that these three patients (0.7%, 3 of 439) had similar genetic profiling with the CR patient, and we surmise that the three patients might potentially respond to pazopanib treatment.

Both our in vitro and in vivo inhibitor assays using GLI1-transfected 3T3 cells revealed that pazopanib inhibited PDGFRB phosphorylation in a dose-dependent manner. However, the inhibition of GLI1-induced transformation by pazopanib was limited in the 3T3 focus formation assay, suggesting that mechanisms other than PDGFRB activation contribute to GLI1-mediated transformation.

GLI1 is associated with the Hedgehog pathway [16]. With respect to the association between GLI1 and PDGFRB, our clinical data (a patient with long-term stable disease and one with a CR) and our assays both demonstrated that GLI1 overexpression led to increased PDGFRB expression and phosphorylation. However, this increased expression of PDGFRB was not accompanied by increased expression of mRNA (data not shown), suggesting that GLI1 transfection did not affect PDGFRB transcription. This finding is consistent with our mRNA expression data, where the PDGFRB expression level in our patient with a CR was intermediate among the high-grade sarcoma samples. Increased phosphorylation without transcriptional upregulation in our patient with a CR may have been the result of post-transcriptional regulation, such as translational regulation by miRNA or protein degradation by the ubiquitin-proteasome system. However, functional details remain unknown. Additionally, it is possible that PDGFRB activation may not contribute to GLI-1 mediated transformation. Further studies are needed to elucidate the molecular functions and pathways involved in the antitumor activity of pazopanib in sarcomas with 12q13-14 amplification associated with the overexpression of GLI1 and CDK4.

In summary, we identified an extremely rare instance of a CR to pazopanib; the patient had a high-grade soft-tissue sarcoma (an advanced tumor with multiple metastases) and demonstrated a complete response after 1 month of treatment with pazopanib. In our validation and functional analysis that included whole-exome sequencing, transcriptome sequencing, and protein phosphorylation profiling, several factors were associated with tumorigenicity. Additionally, characteristic gene alteration (GLI1 and CDK4 amplification) in our patient with a CR was also identified in soft-tissue sarcomas in a large clinical sequence database (MSK-IMPACT) [8], indicating that the frequency of this characteristic gene alteration in soft-tissue sarcoma is extremely low. Although we did not identify the molecular mechanism explaining the high sensitivity of this sarcoma to pazopanib, this study was a first report of integrative analyses of a CR to pazopanib. We believe our findings, including characteristic gene alternations, especially a GLI1 amplification associated with responses to pazopanib, may provide critical information for future validation studies, including the development of novel biomarkers and the elucidation of mechanisms regarding responses to pazopanib. Additional studies might verify the roles of our identified genetic alterations in soft-tissue sarcoma, and these findings may be compared with genetic profiles incorporating several clinicopathological factors including histological subtypes in soft-tissue sarcomas to deliver personalized medicine. Finally, with further information and validation of these concepts, we hope that physicians will be able to choose suitable drugs for patients based on the defined genetic alterations in soft-tissue sarcoma, which will begin a new era in this field.