TP53 and PTEN mutations were shared in concurrent germ cell tumor and acute megakaryoblastic leukemia

This study was approved by the Research Ethics Committee of the Faculty of Medicine, University of Miyazaki. GCT samples (the left cervical mass) and AML samples (bone marrow) were obtained from the patient with written informed consent.

Cytogenetic analyses were performed by G-banding on GCT and AML samples, and interphase fluorescence in situ hybridization (FISH) on the frozen stocked GCT sample. In FISH analysis, human 6p22/6q22 probe, 8 centromere/21q22 probe, and 12p12/12q14 probe (Chromosome Science Labo Inc., Sapporo, Japan) were used. Frozen cells were thawed and washed by PBS. After treatment of 0.075 mol/L KCL for 20 min at room temperature. Cells were fixed 3 times with methanol:acetic acid = 3:1 and fixed cells were spread on slides. Probes were applied to the cell spreads, covered with cover slips and simultaneously denatured at 70 °C for 5 min and hybridized overnight at 37 °C. The hybridized slide was washed and counterstained with 4′,6-diamidino-2-phenylindole and mounted in anti-fade solution. Separate fluorochrome images were captured using a Leica DC 350FX cooled CCD camera (Leica, Wetzlar, Germany) mounted on a Leica DMRA2 microscope using Leica CW4000 FISH software. The images were analyzed using Leica CW4000 karyo software (Leica).

Genomic DNA from GCT and AML samples was extracted using the QIAamp DNA Mini kit. WES analysis of GCT and AML samples were performed using the patient’s buccal mucosa as a germline control, as previously described [11]. SureSelect Human All Exon v6 kits (Agilent Technologies) were used for exome capture according to the manufacturer’s instructions. Sequencing data were generated using the Illumina NextSeq 500 platform with a standard 150-bp paired-end read protocol, as previously described [11]. Sequence alignment and mutation calling were performed using the Genomon pipeline (https://​github.​com/​Genomon-Project), as previously described. Putative somatic mutations with (i) Fisher’s exact P value < 0.01; (ii) > 2 variant reads in tumor; (iii) allele frequency in tumor ≥0.035; and (iv) allele frequency in germline < 0.035 were adopted and filtered by excluding (a) synonymous single nucleotide variants (SNVs); (b) variants only present in unidirectional reads; and (c) variants occurring in repetitive genomic regions. These candidate mutations were further filtered by removing known variants listed in NCBI dbSNP build 131, the 1000 Genomes Project (October 2014 release), National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) 6500, and the Human Genome Variation Database, unless they were listed in the COSMIC database (v70). Finally, all detected mutations were manually checked by Integrative Genomics Viewer (IGV) and their allele frequencies were calculated using pysam’s pileup function (version 0.14.1).

The patient was a 37-year-old Japanese, previously healthy male who presented with a dry cough. He first visited his family doctor and was pointed out to have a 5-cm diameter left cervical tumor, following which, he was referred to our hospital. Examination revealed tachycardia (107/min) and elastic hard left cervical mass with a 5 cm diameter. A chest X-ray revealed a well-circumscribed bilateral hilar mass with a maximum dimension of 20.5 cm, and dullness of the right costal pleural angle (Fig. 1a). Peripheral blood examination showed the following: Hb level 16.2 g/dL; leucocyte count 9.9 × 10⁹/L, and platelet count 293 × 10⁹/L. Serum alpha-fetoprotein (AFP) (normal range: 0–8.5 ng/mL), beta-human chorionic gonadotropin (βhCG) (normal range: 0–4 mIU/mL), and lactate dehydrogenase levels (normal range: 119–213 IU/L) were 1921 ng/mL, 511 mIU/mL, and 390 IU/L, respectively. A computed tomography (CT) scan revealed a 19.5 cm × 10.8 cm heterogeneously enhancing anterior mediastinal mass and a 4.3 cm left cervical mass (Fig. 1b). A surgical biopsy of the left cervical mass showed heterogenous features including immature cartilages, immature mesenchymal cells, columnar epithelium cells, and yolk sac tumor-like components (Fig. 2a, b). Immunohistochemical staining of these tumor cells revealed immunoreactivity with AFP and Glypican-3 (Fig. 2c, d). He was diagnosed with non-seminomatous GCT, and was treated with BEP therapy (bleomycin, etoposide, and cisplatin). After starting the therapy, the serum βhCG level promptly decreased, but there was no reduction in the size of the mediastinal mass. Thrombocytopenia started 15 days after BEP therapy and persisted for 1 week. To evaluate its cause, bone marrow (BM) examination was performed. The BM aspirate showed that 74% of all nucleated cells were blasts, which were medium to large in size with round nuclei, and one to three nucleoli (Fig. 2e). These cells were negative for myeloperoxidase by immunostaining (Fig. 2f), and were positive for CD7 (79.6%), CD13 (82.6%), CD33 (81.1%), CD34 (99.1%), CD41a (99.1%), and CD117 (44.5%) by flow cytometry. BM biopsy showed hypercellular marrow, and blasts were positive for von Willebrand factor (Fig. 2g, h). The cause of cytopenia was revealed to be AMKL. Induction chemotherapy with idarubicin and cytosine arabinoside was administered for AMKL. He achieved first complete remission with enough platelet recovery. The chemotherapy for AML had no effect on the GCT, and the mediastinal mass enlarged. We therefore continued therapy for GCT with 2 courses of TIP (paclitaxel, ifosfamide, and cisplatin), 1 course of TGO (paclitaxel, gemcitabine, oxaliplatin), and finally another course of BEP therapy. These treatments did not reduce the size of the mediastinal or cervical masses. AMKL relapsed during the TIP therapy for GCT, and thrombocytopenia, which required platelet transfusion every other day, continued during the therapy. Despite these treatments, he died 6 months after his initial diagnosis.

To clarify the possible clonal relationship between the GCT and AML, we performed cytogenetic and WES analyses of GCT and AML samples. In the cytogenetic analysis, the AML sample revealed a hyperdiploid karyotype: 63XXY,+Y,+ 1,-2,-4,-5, add(6)(p21),+ 8,-9,-11,-13,-17,-18,-19 in 4/20 metaphases and 46XY in 16/20 metaphases (Fig. 3). As no analyzable metaphases were obtained in the GCT sample, we performed two-color FISH analysis on the GCT sample using each pair probes for chromosome 6p22/6q22, 8 centromere/21q22, and 12p12/12q14. In the FISH analysis, trisomy 8, tetrasomy 8, trisomy 21, and tetrasomy 21 were detected in 11/82, 8/82, 16/84, and 15/84 mGCT cells, respectively (Fig. 4). In addition, 16/84 mGCT cells possessed three signals of both 6p22 and 6q22, and 21/85 cells showed three signals of both 12p12 and 12q14.

In the WES analysis, we detected 16 somatic mutations in the GCT sample, including 15 SNVs and one deletion, and 18 somatic mutations in the AML samples, including 17 SNVs and one deletion. Among them, mutations in 9 genes, specifically TP53(c.G836A), PTEN(c.492 + 1G > A), RLF(c.4563_4567del), DLG2(c.C140T), YY2(c.G813A), PCLO(c.T13947G), GOLGA8J(c.G992A), EDRF1(c.C3172T), and ASF1A(c.T231A) were observed in both tumors and at the same nucleotide. Their detailed nucleotide changes and variant allele frequency (VAF) in each tumor are shown in Table 1. TP53, PTEN, RLF, DLG2, and YY2 showed relatively higher VAFs than PCLO, GOLGA8J, EDRF1, and ASF1A (Table 1, Fig. 5). In our case, the TP53 mutation (p.G279E) occurred in the DNA binding domain and the PTEN mutation (exon5:c.492 + 1G > A) occurred in the splicing donor site of intron 5, which codes for the phosphatase domain (Fig. 6).