The Chiba Cancer Center Tissue Bank includes 124 frozen tumor samples obtained from patients with OS that were treated at Chiba Cancer Center (Chiba, Japan) since 1993. Eligible cases were selected for the present study according to the following criteria: i) Diagnosed with conventional, high-grade OS occurring in the extremity, ii) pre-therapeutic biopsy samples were available, iii) age at diagnosis <25 years, and iv) follow-up was monitored for >5 years for survival cases. Finally, 50 tumor samples that satisfied these criteria were selected for study, Chiba OS (ChOS; Table SI). The present study was approved by the institutional review board of the Chiba Cancer Center (approval no. CCC19-14) and conformed to the provisions of the Declaration of Helsinki. Written informed consent was obtained from patients or legally authorized representatives.

Preoperative chemotherapy consisting of a MAP regimen was administered to all patients as described previously (14). If radiological evaluation by CT or MRI conducted during preoperative chemotherapy revealed enlargement of the primary tumor, ifosfamide was added to the MAP regimen. Definitive tumor resection was performed following preoperative chemotherapy, and chemotherapy response was evaluated histologically using resected tumor according to the relative area of necrosis. A ‘good response’ was defined as a necrotic area >90% of the tumor; ‘poor response’ was defined as necrotic tumor area <90%, as previously described (4,14).

All tumor specimens were obtained from pre-therapeutic biopsy samples. The tumor content of frozen samples was histologically confirmed as >80% tumor cellularity using paraffin sections (4-µm thick) obtained from the same biopsy. Genomic DNA was extracted using the standard proteinase K digestion method (15) or AllPrep DNA/RNA Mini kit (Qiagen KK). DNA concentration and purity were determined using a NanoDrop 100ND-1000 Spectrometer (Thermo Fisher Scientific, Inc.).

For aCGH analysis, 500 ng tumor or control DNA was fragmented and chemically labeled with Cy3- or Cy5-dye, respectively. Genomic DNAs extracted from normal placenta tissue was used as control DNA for aCGH. High-resolution aCGH was performed using the Agilent Human Genome CGH Microarray kit 4×44K (Agilent Technologies, Inc.), according to the manufacturers protocols, and analyzed using Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis (version 3.1; Agilent Technologies, Inc.). Data extraction from scanned microarray images was performed using Feature Extraction Software (versions 9.5.3.1, 10.7.3.1 and 11.0.1.1; Agilent Technologies, Inc.). Raw data were subsequently transferred to Agilent Genomic Workbench Software Version 6.5 (Agilent Technologies, Inc.) and further analyzed using ADM-2 algorithm with a threshold of 5.5. In order to find copy number variations (CNVs) that discriminated between patients who demonstrated good and poor responses, differential CNV analyses were performed with P-value <0.01 using Genomic Workbench Software. All final retained CNVs were validated using quality control data of the software. All genomic positions were defined according to the University of California Santa Cruz Human (version hg19; Genome Reference Consortium h37; genome.ucsc.edu/cgi-bin/hgGateway?db=hg19). Microarray data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (ncbi.nlm.nih.gov/geo/; accession no. GSE154540).

Targeted exome sequencing of 409 cancer-associated genes in tumor and non-tumor samples was conducted using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) (Thermo Fisher Scientific, Inc.). For each sample, 40 ng genomic DNA was amplified and used for library preparation using the Ion AmpliSeq™ Library 2.0–96LV and the Ion Barcode Adaptors 1–96 kits (both Thermo Fisher Scientific, Inc.). Then, the library was quantified using the Ion Library Quantification kit (Thermo Fisher Scientific, Inc.) and diluted to a final concentration of 8 pM. Targeted sequencing was performed using an Ion Proton sequencer (Thermo Fisher Scientific, Inc.) with an Ion PI Chip v2 according to the manufacturers instructions. The sequencing results were analyzed using Torrent Suite v5.0.4 software (Thermo Fisher Scientific, Inc.) to align the barcoded reads with the human reference genome (hg19) and to generate run metrics, including chip loading efficiency, total reads, and quality. Variant Caller v5.0 and Ion Reporter™ 5.2 software (Thermo Fisher Scientific, Inc.) were used for variant detection (allele frequency >5%) and annotation (The Single Nucleotide Polymorphism Database v146; ncbi.nlm.nih.gov/snp/), respectively. Each variant was confirmed visually using Integrated Genomics Viewer or validated using Sanger sequencing performed on an Applied Biosystems 3730 DNA analyzer with Sequencing Analysis Software v6.0 (both Applied Biosystems; Thermo Fisher Scientific, Inc.). The CNVs of the 409 genes were calculated by normalizing the amplicon coverage data from a tumor sample with that of paired non-tumor tissue. Each copy number ratio (tumor/normal) was demonstrated as a log2 ratio (log fold change). Sequence data have been deposited in the Japanese Genotype-phenotype Archive, hosted by the DNA Data Bank of Japan (accession no. JGAS000282).

Total RNA was prepared from human tissue and OS cell lines using AllPrep DNA/RNA Mini or RNeasy Mini kit (both from Qiagen KK) and reverse-transcribed using random primers and SuperScript II (Thermo Fisher Scientific, Inc.), according to the manufacturers protocols. The synthesized cDNA was subjected to PCR-based amplification using rTaq DNA polymerase (Takara Bio, Inc.). Primer sequences are listed in Table SII.

A Veriti PCR system (Thermo Fisher Scientific, Inc.) was used for PCR amplification. The resultant PCR products were separated on 2% agarose gel and visualized using a UV transilluminator (ATTO Corporation) after ethidium bromide staining. RT-qPCR analysis was conducted using an ABI Prism 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). TaqMan primers and probes for CDK4 (cat. no. HS00364847_m1) and β-actin (cat. no. 4310881E) were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.). RT-qPCR amplification was performed in a 20 µl reaction volume containing 10 µl 2X TaqMan Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 1 µl 20X TaqMan primers and probes and 2 µl cDNA. The thermocycling conditions were as follows: Decontamination for 2 min at 50°C, initial denaturation for 10 min at 95°C and 50 cycles of PCR for 15 sec at 95°C and 1 min at 60°C. The relative standard curve method was used for data quantification (16). All reactions were performed in triplicate.

All tumor samples were routinely decalcified and fixed with 10% buffered formalin for 12 h at room temperature and embedded in paraffin. Sections (4-µm thick) were deparaffinized and antigens were retrieved by autoclaving in 0.01 M citrate buffer (pH, 6.0) at 97°C for 60 min. Endogenous peroxidase activity was blocked by exposing the slides to 3% hydrogen peroxide for 15 min at room temperature. The primary antibody utilized was CDK4 (1:500; cat. no. AHZ0202; Invitrogen; Themo Fisher Scientific, Inc.). The slides were incubated for 20 min at room temperature with the primary antibody and subsequently labeled using the EnVision+/HRP system (Dako; Agilent Technologies, Inc.). Immunoreactions were visualized using diaminobenzidine (0.03%, 1 min at room temperature) by light microscope, and the sections were counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation) for 5 min at room temperature.

Human OS cell lines U2OS and SJSA1 were obtained from the American Type Culture Collection and maintained in DMEM or RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin (all Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO₂ at 37°C. For transient expression, CDK4 cDNA was amplified using PCR from human cDNA libraries incorporating a C-terminal 3X Flag-tag sequence, as described by Tatsumi et al (17) and ligated into the EcoRI and XhoI sites of the pCDNA3 vector (Thermo Fisher Scientific, Inc.). For CDK4 overexpression, OS cells were seeded in 6-well plates (80% confluency) and transfected with 2.5 µg pCDNA3-CDK4 or empty plasmid (control) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature. Following 24 h transfection, DNA damage and apoptosis were introduced by further incubation with 0–60 µM CDDP (Sigma-Aldrich; Merck KGaA) for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. In order to inhibit CDK4 activity, transfected cells were cultured with 0–15 nM PD 00332991 (CDK4/6 inhibitor palbociclib; Santa Cruz Biotechnology, Inc.) for 24 h in a humidified atmosphere of 5% CO₂ at 37°C.

Whole-cell lysates were separated using SDS-PAGE and transferred to PVDF membranes (Immobilon-P; EMD Millipore), as previously described (17). Membranes were incubated with primary antibodies (all 1:1,000) at room temperature for 2 h and then treated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,500) at room temperature for 1 h. Finally, signals were detected using a LAS-4000 Image Analyzer following incubation with ECL reagent (both GE Healthcare). The following antibodies were purchased from Cell Signaling Technologies, Inc.: Anti-CDK4 (cat. no. #12790), anti-Cyclin D1 (cat. no. #2978), anti-Bcl-2 (cat. no. #2870), anti-caspase-9 (cat. no. #9502), anti-E2F transcription factor 1 (E2F1; cat. no. #3742), anti-retinoblastoma (Rb; cat. no. #9309), anti-phosphorylated (phospho-)Rb (Ser807/811, cat. no. #9308), anti-phospho-Rb (Ser780, cat. no. #9307), anti-phospho-Rb (Ser807/811; cat. no. #9308), HRP-conjugated anti-rabbit (cat. no. #7074) and HRP-conjugated anti-mouse (cat. no. #7076). Anti-Flag (M2) antibody (cat. no. F3040) was purchased from Sigma-Aldrich (Merck KGaA), anti-poly(ADP-ribose) polymerase (PARP)-1 (cat. no. sc-8007) was purchased from Santa Cruz Biotechnology, Inc. and anti-actin (cat. no. 013-24553) was purchased from FUJIFILM Wako Pure Chemical Corporation.

Measurement of cell viability was conducted by water-soluble tetrazolium salt (WST)-8 assay (Dojindo Molecular Technologies, Inc.). Apoptosis progression caused by CDDP or palbociclib treatment was monitored by accumulation of cleaved caspase-9 and PARP1 via immunoblotting, as previously described (17).

Overall survival (OAS) was calculated using the Kaplan-Meier method, and differences in survival were assessed using the log-rank test and Cox proportional-hazards regression method. OAS was defined as the period from the date of diagnosis to that of death or the last follow-up and described with 95% confidence interval (CI). Comparisons were assessed using χ² and Fishers exact tests for categorical variables and Mann-Whitney tests for continuous variables. RT-qPCR data are presented as the mean (n=3)for triplicate experiments. Cell viability data are presented as the mean ± SD (n=4). All statistical tests were two-sided. Statistical analysis was performed using JMP^® version 9.0.2 software (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The 5-year OAS for 50 patients with OS was 73% (95% CI, 59–84%). Univariate analysis of clinical factors revealed that histological response to preoperative chemotherapy (‘chemotherapy response’) and metastasis at diagnosis were statistically significant prognostic factors associated with OAS (P=0.012 and P=0.0058, respectively), whereas chemotherapy regimen (MAP in the presence or absence of ifosfamide) was not associated with OAS (P=0.36) (Table I; Fig. 1). These results were confirmed by multivariate analysis (Table I).

Genome-wide aCGH analysis of the pretreatment of OS tissue samples was performed to identify CNVs associated with tumor chemosensitivity. Differential analyses of CNV between good (n=29) and poor responder (n=21) cohorts identified three loci that were significantly associated with chemosensitivity (P<0.01; Table II). Recurrent gain of chromosomes 12q14.1 and 19q13.32 were identified in 5 of 21 (24%) poor responders, whereas these gains were not identified in good responders (0/29, 0%; P=0.0096). A recurrent gain of chromosome 7q36.2-3 was also significantly observed in 8 of 29 (28%) good responders compared with poor responders (0/21; P=0.008). Meanwhile, there was no statistically significant recurrent copy number loss between good and poor responders.

It was hypothesized that one or multiple genes in loci with copy number gain, such as 12q14.1 and 19q13.32, contribute to chemo-resistance in poor responders. In order to address this, mRNA levels of 11 genes in gained regions 12q14.1 and 19q13.32 were compared between 24 tumors (13 good and 11 poor responders) with available RNA via semiquantitative RT-PCR. CDK4 in 12q14.1 was the only gene with higher transcript levels that were positively associated with copy number gain (Fig. S1).

In addition, aCGH confirmed that CDK4 was located in the minimal region of overlap in the gained region. Copy number analysis showed amplification or gain of the CDK4 locus in poor responders, which was not observed in good responders (Fig. 2A). In order to identify additional markers based on gene mutations, next generation sequencing (NGS)-based targeted exome sequencing of 409 cancer-associated genes was performed with seven and nine tumors from good and poor responder cohorts, respectively. Although 16 and 13 non-synonymous somatic gene mutations were found in the respective cohorts, no recurrent mutation was observed, except for CDK4 amplification or gain in poor responders (Table SIII). Increased copy number of CDK4 was also confirmed by calculation of relative read depth of the target exome sequences in three tumors from poor responders (Fig. 2B).

CDK4 transcript levels in primary OS tissue were assessed by RT-qPCR. As expected, higher CDK4 transcript levels were observed in four patients with 12q14.1 gain compared with those without 12q14.1 gain (Fig. 3A). CDK4 levels were significantly higher in poor (n=11) than in good responders (n=13; Fig. 3B; P<0.05). Immunohistochemistry analysis was employed to confirm positive nuclear staining of CDK4 in the OS tissue of patients with 12q14.1 gain (Fig. 3C). Thus, CDK4 was considered a strong candidate genomic prognostic marker and potential therapeutic target in poor responders.

Next, it was investigated whether higher expression of CDK4 in OS cells effectively decreases sensitivity to chemotherapeutic drugs, such as CDDP. Ectopic expression of CDK4 in OS (U2OS and SJSA1) cells was induced via pCDNA3-CDK4 transfection. Following 48 h transfection, increased protein levels of cyclin D1, a binding partner of CDK4, and Bcl-2, a pro-survival factor, were detected in U2OS and SJSA1 cells, accompanied by increased levels of exogenously introduced Flag-tagged CDK4 (Fig. 4A). Transfected cells were cultured in medium containing CDDP for 24 h to induce apoptosis. CDK4 overexpression caused a significant abrogation of the effect of CDDP on OS cell viability compared with control cells (Fig. 4B). Consistent with this observation, apoptotic cell death caused by CDDP was markedly decreased in OS cells overexpressing CDK4, as indicated by immunoblot detection of cleaved caspase-9 accumulation (Fig. 4C).

Finally, it was investigated whether overexpression of CDK4 sensitizes OS cells to CDK4/6 inhibitor palbociclib. When Flag-tagged CDK4 was ectopically overexpressed in U2OS cells, there was an increased phosphorylation of Rb at S807/811 and S780 under normal culture conditions (0 nM Palbociclib; Fig. 5A). Subsequent treatment with palbociclib attenuated Rb phosphorylation in a dose-dependent manner. Treatment of CDK4-overexpressing U2OS cells with palbociclib facilitated apoptosis in a dose-dependent manner, as evidenced by cleavage of PARP1 and caspase-9, compared with control cells. Palbociclib also caused a significant decrease in viability of CDK4-overexpressing U2OS cells (Fig. 5B).