Case Report: Exome sequencing reveals recurrent RETSAT mutations and a loss-of-function POLDIP2 mutation in a rare undifferentiated tongue sarcoma

Pathological findings

The tumor mass measured grossly 7 × 7 × 5.5 cm on examination following resection. On microscopic examination, the tumor was composed of mostly spindle cells arranged in short fascicles or a vague storiform pattern. Frequent mitotic figures and prominent tumoral necrosis were seen. No osseous, chondroid or rhadomyomatous differentiation was seen on haematoxylin and eosin staining (Figure 1–Figure 3). The closest margins were 2 mm and located at the postero-inferior, right and left margins.

Figure 1. Sarcoma forming cellular fascicles is seen in the stroma of the tongue, focally invading the intrinsic muscle (left upper corner).

The surface squamous epithelium shows no dysplastic field change or connection with sarcoma. (H&E stain; magnification, ×20).

Figure 2. Tumor necrosis (pink foci) was prominent in the undifferentiated sarcoma.

(H&E stain; magnification, ×40).

Figure 3. The sarcoma tumor cells possessed enlarged, elongated, pleomorphic and hyperchromatic nuclei, distinct nucleoli and small amount of cytoplasm.

Mitotic figures were frequently noted. No osseous, chondroid or rhadomyomatous differentiation was seen. (H&E stain; magnification, ×400).

Further immunohistochemical studies showed that the tumor cells were positive for vimentin and p16; had focal weak positivity for SMA; had moderate positivity for TLE-1; and were negative for p63, EMA, AE1/AE3 antibodies, c-Kit, S100, CD31, CD34, desmin, myogenin, melan A, HMB45 antibody, ALK-1, human herpesvirus 8 (HHV8), CD99 (MIC-2) and SRY-box 10 (SOX10). Calponin was equivocal. No loss of integrase interactor 1 (INI1) staining was noted. PCR for high-risk human papillomavirus (HPV) DNA was negative. The Molecular Break-Apart FISH test for SS18 translocation was negative. Overall the pathological features demonstrated a high-grade sarcoma with no definite line of differentiation, consistent with an undifferentiated sarcoma.

Mutational profile of the patient’s tumor

WES at >200x depth (Illumina HiSeq 4000) was performed to determine the genetic aberrations underlying this rare form of head and neck cancer. Normal DNA from the patient’s blood was also subjected to WES at the same depth. Surprisingly, WES revealed only 19 non-synonymous mutations in the tumor. The very low rate of non-synonymous mutations is uncommon in HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC)⁵, and is slightly lower than that of reported sarcomas.

The somatically mutated genes were, in the order of allele frequencies of these 19 mutational events, POLDIP2 (DNA polymerase delta interacting protein 2), tubulin gamma complex associated protein 3TUBGCP3,, mutated in colorectal cancers (MCC), TUBA3D (tubulin alpha 3d), DDX11 (DEAD/H-box helicase 11), CWF19L2 (CWF19 like 2, cell cycle control), ZNF91 (zinc finger protein 91), RETSAT (retinol saturase) (2 mutations), PRR21 (proline rich 21), TAS2R46 (taste 2 receptor member 46), FAM186A (family with sequence similarity 186 member A), HEATR5A (HEAT repeat containing 5A), VPS4B (vacuolar protein sorting 4 homolog B), PRAMEF12 (PRAME family member 12), FAM170B (family with sequence similarity 170 member B), BBS4 (Bardet-Biedl syndrome 4), ARHGAP5 (Rho GTPase activating protein 5) and ATAD3B (ATPase family, AAA domain containing 3B) (Table 1). Strikingly, based on the functional annotation of these mutated genes, five of the top seven mutated genes with high allele frequencies (>10%) were known to be involved in DNA replication, and mitosis (Table 1). This suggests that major somatic mutations of this rare tumor appear to affect DNA replication and mitosis, consistent with an aggressive phenotype. Of note, the patient’s tumor carried a 93% allele frequency of the POLDIP2 S28fs mutation, which is a hotspot mutation in multiple cancers. In addition, the MCC gene was also mutated in this rare tumor (MCC p.G20S) at a high allele frequency of 28%, indicating its likely role as a driver event for tumorigenesis. Interestingly, the PRR21 mutation is also found to be mutated at high frequencies in TCGA HNSCC cohort with a hotspot mutation S86Gfs*291 and M48Tfs*329 mutation, while in this tumor, PRR21 mutation occurred at G114C, a position near to these two hotspot mutations in HNSCC.

Most strikingly, among all the somatic mutations identified in this patient, we found that recurrent somatic events of RETSAT (RETSAT p.A533V and p.G536R mutations), a gene known to be important for the promotion of adipogenesis and normal adipocyte differentiation. It is plausible that RETSAT mutations may affect adipocyte adipogenesis and differentiation, related to the sarcoma features of this rare tumor.

Gene copy number analysis was also performed and a total of 221 somatic copy number alterations (CNA) events were identified (Figure 4 and Supplementary Table 1). The somatic CNA detected in this patient did not harbor any of these common HNSCC CNA events including losses of chr. 3p and 8p, as well as focal amplification or gains of chr. 3q26/28, 5p15, and 8q24⁵. The common CNA events in sarcomas including aberrations of the MDM2-p53 and p16-cyclin dependent kinase 4 (CDK4)-retinoblastoma-associated protein pathways, deletion of TP53 (tumor protein p53), CDKN2A (cyclin dependent kinase inhibitor 2A), as well as chr. 12q13-q15 and CDK4 amplification were also absent in this patient⁶. These results suggest that this unique tumor is likely distinct from HNSCC and adult soft tissue sarcomas.

Figure 4. A Circos plot showing somatic genetic variations.

An ideogram of a normal karyotype is shown in the outermost ring. Chromosomes are segmented into contiguous bins of 1 Mb in size. The second ring from outside shows CNVs at corresponding chromosomal positions. Distinct heights of red dots indicate different inferred copy numbers. Regions that are not covered by any dots have a copy number of two. The third ring shows SNV density. Purple dots with different heights indicate SNV densities of the corresponding regions. The innermost ring shows positions of genes with non-synonymous mutations found in the undifferentiated sarcoma. CNV, copy number variation; SNV, single nucleotide variant.

Multiple germline mutations in key tumor suppressor genes

Given the young age of this patient, we also carefully examined potential aberrations in his germline mutational profile by WES of his blood DNA. Importantly, we found that this patient carried multiple germline mutational events of TP53, including a missense mutation of TP53 p.P72R (100% allele frequency), 2 mutations at the immediate 5’-UTR, likely in the promoter region of TP53 (c.-112G>A; and c.-123C>G; both with 100% allele frequencies), as well as two other intron variants with unknown effects (Table 2). The TP53 germline events likely explain the early age of onset for cancer for this patient. In addition, a 5’-UTR mutation, likely at the promoter region of CDKN2A, was identified (c.*193G>C), with an allele frequency of 100%. A CASP8 (caspase 8) missense mutation, CASP8 p.L14R, was also found in this patient’s blood with a 46.7% allele frequency. However, the potential function of this mutation is unclear. Lastly, this patient carries as many as 23 germline variants of NOTCH1; however, none of these variants are non-synonymous mutations, and the effects of these variants are largely unknown. No telomerase reverse transcriptase promoter or exon mutation was found. Lastly, no germline gene copy number alterations were observed for any critical HNSCC-associated oncogenes or suppressor genes (Supplementary Table 2)

Tissue collection

Tumor tissue and blood sample were collected from the patient under written informed consent according to The Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee, Hong Kong SAR (protocol number CRE-2015.396). The tumor mass was freshly frozen for DNA extraction using the QIAGEN DNeasy Blood and Tissue kit. Blood DNA was extracted from patient’s buffy coat in a similar manner. DNA samples were then quantified and quality-assessed using a bioanalyzer.

Exome sequencing and variant calling

Genomic DNA of the patient’s tumor and buffy coat were used for WES using the Agilent SureSelect Human All Exon V5 Kit, with sequencing performed using the Illumina HiSeq 4000 platform (Macrogen, Korea) with a goal coverage of ×200 for tumor and ×100 for the blood (buffy coat) sample of the same patient.

Upon sequencing, all reads (FASTQ files) were mapped to the hg19 human reference genome assembly with Burrows-Wheeler Alignment Tool (version 0.7.12). Variant calling of single nucleotide variants (SNVs) and indels was performed using the Genome Analysis Toolkit (version v3.4.0) HaplotypeCaller pipeline. Called variants were annotated with SnpEff (version 4.3), the exome sequencing data have been deposited into European Nucleotide Archive (ENA) with accession number PRJEB25783. Somatic mutations were defined as mutations found in the tumor tissue of the patient but not in the patient’s blood. All identified recurrent mutations were confirmed in Integrative Genomics Viewer (IGV) (version 2.4) as a final check of the calling (100% accurate).

Copy number alterations (CNAs)

CNAs were analyzed from segmented WES data using the Control-FREEC copy number and genotype caller (version 11.0) in both somatic and germline mode. In both modes, captured genomic regions were characterized using the SureSelect Human All Exon V5 bed file. Somatic CNA analyses were run by referencing tumor WES data to the paired blood WES data, whereas germline CNA analysis were run by referencing blood WES data to the hg19 human genome assembly. Identified regions were annotated with ANNOVAR (version 2017-07-17) and SnpEff. Segments with CNA were manually confirmed with an IGV check. Genetic variations were visualized using Circos (version 0.69) with “chromosomes_unites = 1000000”.

Immunohistochemical staining

The patient’s tumor tissue was FFPE-preserved and sectioned (5–8 µm) for immunohistochemical and H&E staining. Immunohistochemical staining was performed with the following antibodies: Melan A (M7196, Dako, Denmark), HMB34 (M0634, Dako, Denmark), ALK-1 (M7195, Dako, Denmark), HHV8 (NCL-HHV8-LNA, Novo, UK), MIC-2 (M3601, Dako, Denmark), SOX-10 (ACI3099, Biocare, USA), Calp (M3556, Dako, Denmark), INI1 (612110, DB transduction, USA), Vimentin (M0725, Dako, Denmark), p16 (805-4713, Ventana, USA), SMA (M0851, Dako, Denmark), p53 (M7001, Dako, Denmark), TLE1 (401M-16, Cell Marque, USA), p63 (M7317, Dako, Denmark), EMA (MS348, Thermo, UK), AE1/3 (M3515, Dako, Denmark), c-kit (A4502, Dako, Denmark), S100 (NCL-S100p, Leica, UK), CD31 (M0823, Dako, Denmark), CD34 (NCL-L-END, Leica, UK), Desmin (M0760, Dako, Denmark) and Myogenin (M3559, Dako, Denmark). Results were examined by an experienced pathologist for the presence or absence of the target proteins. Pictures were taken under a light microscope at ×20, ×40 and ×400 magnification.

HPV analysis

The tumor cells from the formalin-fixed, paraffin-embedded sections were isolated for DNA extraction. The DNA was subjected to PCR analysis using the consensus primers GP5+/6+, as previously documented²³.