Clinical and pathological findings
The patient was a 37-year-old, previously healthy male who presented with a dry cough. He first admitted to his family doctor and was pointed out to have a 5 cm diameter left cervical tumor, then 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 (Figure 1A). Peripheral blood examination showed the followings: Hb level 16.2 g/dL; leucocyte count 9.9 x 109/L, and platelet count 293 x 109/L. Serum alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (bhCG), and lactate dehydrogenase levels were 1921 ng/mL, 511 mIU/mL, and 390 IU/L, respectively. A computed tomography (CT) scan revealed a 19.5 cm x 10.8 cm heterogeneously enhancing anterior mediastinal mass and a 4.3 cm left cervical mass (Figure 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 (Figure 2A, B). Immunohistochemical staining of these tumor cells revealed immunoreactivity with AFP and Glypican-3 (Figure 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 bhCG 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 (Figure 2E). These cells were negative for myeloperoxidase by immunostaining (Figure 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 (Figure 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, 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.
Cytogenetic analysis and whole-exome sequencing analysis
To clarify the possible clonal relationship between the GCT and AML, we performed cytogenetic and WES analysis 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; while, no analyzable metaphases were obtained in the GCT sample. Therefore, cytogenetic analysis did not provide us enough information to clarify the clonal relationship between both tumors. In the WES analysis, we detected 16 somatic mutations in the GCT sample, including 15 single nucleotide variants (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, PTEN, RLF, DLG2, YY2, PCLO, GOLGA8J, EDRF1, and ASF1A were observed in both tumors; and TP53, PTEN, RLF, DLG2, and YY2 showed relatively higher variant allele frequencies than PCLO, GOLGA8J, EDRF1 and ASF1A (Figure 3). 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.