Page 350 - Haematologica Vol. 109 - July 2024

CASE REPORT
Neoantigen-specific T-cell response after donor lymphocyte infusion associates with favorable outcome in a patient with i(12p) germ cell tumor, acute leukemia and sarcoma of the same clonal origin
A 22-year-old man was admitted to our hospital for bleeding diathesis with anemia and severe thrombocytopenia. He reported dry cough, fever, weight loss, and asthenia. Rare blast cells were observed on the blood smear. A 8.7 cm anterior mediastinal mass was seen on computed tomog- raphy (CT) scan. The levels of α-fetoprotein and lactate dehydrogenase were 45.7 μg/L and 7,380 U/L, respectively and the b-human chorionic gonadotropin level was normal. Biopsy through mediastinotomy revealed a placental alka- line phosphatase (PLAP)-positive mixed germ cell tumor (GCT), in which atypical large cells, later determined to be CD61-positive, were focally identified (Figure 1A-C). In ad- dition to the seminoma, there were mature and immature teratomatous components represented by disorganized respiratory epithelium and a poorly differentiated mes- enchymatous tissue, respectively. The bone marrow was massively infiltrated by similar, CD34-negative, CD61- and CD43- positive cells, with multilobulated nucleus and eo- sinophilic cytoplasm (Figure 1D, E). Staining for CD45, PLAP as well as lymphocytic and epithelial markers was negative. Cytogenetic analysis of the bone marrow showed a complex karyotype including polyploidy, +der(3) and an isochro- mosome i(12)p. Given these results, synchronous acute megakaryocytic leukemia (AML) and primary mediastinal mixed GCT was diagnosed, an association carrying a dismal prognosis.1 Inspired by a report of combined chemotherapy targeting both tumoral components in a pediatric patient,2 we designed a regimen based on cytarabine, mitoxantrone, etoposide phosphate, and cisplatin. After the first cycle of chemotherapy, bone marrow was seen to be leukemia-free, with a normal karyotype. The patient received three cycles of this chemotherapy regimen, with an intrathecal chemo- therapy (methotrexate, cytarabine, and methylprednisolone) during the second cycle. Two additional cycles of etoposide phosphate and cisplatin were given at reduced doses due to hematotoxicity and infectious complications.
After completion of chemotherapy, the leukemia was in complete remission, the mediastinal mass had decreased in metabolism (SUV max 5.1 vs. 7.2) and size (4.7x.4.7cm vs. 8.6x5.8cm), and the α-fetoprotein level was normal. The patient underwent hematopoietic stem cells transplantation (HSCT) from a mismatched unrelated donor (human leuko- cyte antigen [HLA] compatibility 8/10, with incompatibility to 1 HLA-A and 1 DQB1 allele), with a dose of 0.37x106/kg CD34+ donor cells harvested from bone marrow (1.3x108/kg
nucleated cells). Conditioning consisted of anti-thymocyte globulins 10 mg/kg/d for 5 days, cyclophosphamide 60 mg/ kg/d for 2 days, etoposide phosphate 34 mg/kg, methyl- prednisolone 1 g for 2 days, and total body irradiation (total dose 12 Gy). The patient required numerous blood transfu- sions and experienced hemorrhagic cystitis but returned home 2 months after transplantation. A medullary biopsy performed at 3 months showed no residual leukemia.
Six months after HSCT, in the context of recurrent chest pain, a 18F-fluoro-2-deoxy-D-glucose positron emssion tomography (PET)/CT showed a 60% increase in size of the residual mediastinal mass with abnormal metabolic activity. α-fetoprotein and b-human chorionic gonadotropin levels were normal. Histopathological examination of the surgically resected mass showed a fusiform sarcoma with angiomatous differentiation (Figure 1F). The cells were negative for both CD61 and PLAP. The post-operative course was complicated by mediastinitis, pleural effusion, and lung edema.
Due to the appearance of mixed chimerism 10 months after HSCT, we administered two donor lymphocyte infusions (DLI) (5x105 and 6x106 CD3+ cells/kg). Complete donor chimerism was obtained and persistent complete hematological remis- sion was confirmed. Almost 10 years after HSCT, the patient has no evidence of neoplastic disease, and an excellent performance status without any limitation in daily activities. In order to explore the possible relation between the three tumors, we performed tumor exome sequencing. Forty-eight somatic mutations were detected in the GCT, 17 in the leu- kemia and 331 in the sarcoma. Phylogenetic reconstruction revealed 13 shared genetic events, suggesting a common clonal origin (Figure 2A). One of these was a driver splice site mutation in TP53 (c.920-1 G>A), reported as COSM6917 in the COSMIC v76 database. The variant allele frequency (VAF) of this mutation was >2 times higher than median VAF of all other somatic mutations in the tumors (Figure 2B), suggesting a probable loss of the other allele and the lack of wild-type TP53 in tumor cells. Another splice site muta- tion, also on chromosome 17, involved CDK12. Other shared genetic alterations included five loss of heterozygosity (LOH) events, including PTEN LOH.
Given the relatively high number of mutations detected in the sarcoma, we sought to identify whether mutation-specific anti-tumor T-cell responses played a role in the extraor- dinary course of this patient. We used somatic mutations from the three tumors to predict HLA class I-restricted
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