A 39-year-old man with no previous medical conditions presented with persistent dull low back pain and numbness in his left leg following physical exertion in April 2022. He denied hypertension, coronary heart disease, or chronic illnesses, had no transfusion or family history of hematologic disorders, and reported a 20-year smoking history (approximately 20 cigarettes/day) with occasional alcohol use.

Lumbar spine magnetic resonance imaging (MRI) performed at the Third Hospital of Hebei Medical University demonstrated lumbar disc protrusion, osteophyte formation, and mild wedge deformity of the L1 vertebra. Oral medications (details unknown) provided minimal relief, and subsequent intravenous therapy at a local clinic yielded only transient improvement.

By mid-June 2022, the patient’s back pain and leg symptoms worsened, prompting admission to the orthopedics department of our hospital. Chest computed tomography (CT) and magnetic resonance imaging (MRI) unexpectedly revealed a mediastinal mass (Figures 1, 2). On June 24, video-assisted thoracoscopic resection of the mass was performed. Postoperative histopathology showed tumor cells positive for myeloperoxidase (MPO) and a Ki-67 proliferation index of approximately 60%. Immunohistochemistry favored a diagnosis of myeloid sarcoma (Figure 3).

Approximately one month after surgery, his back and leg pain recurred and progressed, accompanied by a 15-kg weight loss over six months, poor appetite, and sleep disturbance. On July 27, 2022, he was admitted to the hematology department. Physical examination showed a chronically ill appearance, localized spinal tenderness, and mild bilateral lower-extremity edema. Vital signs were: temperature 36.0 °C, pulse 108/min, and blood pressure 151/100 mmHg.

Laboratory findings included WBC 3.04 × 10^9/L, hemoglobin 79 g/L, and platelets 12 × 10^9/L. Peripheral smear exhibited immature cells and marked thrombocytopenia. Coagulation testing indicated a hypercoagulable state with D-dimer 5333 ng/mL and elevated fibrin/fibrinogen degradation products (FDP). Lactate dehydrogenase (LDH) was 1806.6 U/L, and serum ferritin was substantially increased (Table 2).

Bone marrow aspiration performed on July 27 revealed hypercellularity (nucleated cell ratio ~80%), a granulocyte–erythroid ratio of 65:1, and abnormal promyelocytes accounting for 98% of nucleated cells. The cytoplasm was packed with coarse reddish-purple granules, occasionally containing both primary and secondary granules (Figure 4).

Flow cytometry analysis on July 29 showed that abnormal cells comprised 96.2% of total nucleated cells, expressing CD13, CD33, MPO, CD38, CD64, with partial expression of CD117 and CD123, and negative for HLA-DR and CD34—an immunophenotype highly suggestive of acute promyelocytic leukemia (APL) (Figure 5). Bone marrow biopsy findings were consistent with this interpretation.

Differential Diagnosis, Investigations and Treatment:

Considering the morphologic and immunophenotypic features together with significant coagulopathy, APL was strongly suspected. All-trans retinoic acid (ATRA) induction therapy was initiated on July 30. On the third day of ATRA therapy, the WBC count rose to 13.29 × 10^9/L, accompanied by fever and hypoxemia. Differentiation syndrome was suspected; ATRA was temporarily discontinued, and hydroxyurea plus dexamethasone were administered.

After clinical stabilization, ATRA was resumed on August 3, combined with arsenic trioxide (ATO) dual induction therapy.

However, subsequent molecular analysis revealed unexpected results. PCR testing for the PML::RARA fusion gene and FISH analysis for RARA rearrangement were both negative. Comprehensive screening for leukemia-associated fusion genes and karyotype analysis showed no abnormalities, except for detection of a heterozygous DNMT3A frameshift mutation by next-generation sequencing. As these molecular findings did not support classical APL, the diagnosis was revised to acute myeloid leukemia (AML), and idarubicin plus cytarabine (IA regimen) chemotherapy was started on August 13.

Follow-up bone marrow examination after chemotherapy demonstrated a reduction of abnormal immature granulocytes to approximately 34%, but the therapeutic response was unsatisfactory. Flow cytometry indicated granulocytic maturation arrest at the myelocyte stage.

Given persistent diagnostic ambiguity, multiple multidisciplinary consultations were convened. Bone marrow samples were sent to Lu Daopei Hospital for whole-transcriptome sequencing (WTS). At the RNA level, viral integration sequences were detected. Bioinformatic analysis confirmed Torque teno mini virus (TTMV) insertion into the RARA gene, forming a TTMV::RARA fusion. RT-PCR amplification validated this finding, leading to the definitive diagnosis of TTMV::RARA-positive APL (Figure 6).

The confirmed diagnosis was TTMV::RARA fusion-positive acute promyelocytic leukemia, a rare molecular subtype. This case displayed distinct features including extramedullary involvement (vertebral and mediastinal myeloid sarcomas) and typical APL morphology and immunophenotype, despite repeated negative conventional molecular assays. The final diagnosis was achieved only through RNA-based comprehensive sequencing, emphasizing the value of advanced genomic technologies for detecting cryptic RARA fusions.

Tragically, by the time of definitive diagnosis, the patient had lost confidence in further treatment and elected to discontinue therapy. Follow-up revealed that he died several months later.

This case highlights the diagnostic challenges posed by variant APLs and underscores the importance of high-throughput sequencing in identifying rare RARA fusion events, such as TTMV::RARA, especially in patients with APL-like morphology but negative conventional molecular results.

The TTMV::RARA fusion gene is a newly identified molecular subtype of APL, with fewer than 10 cases reported to date (3, 4). TTMV (Torque teno mini virus) is a widely distributed human commensal virus. The mechanism of TTMV::RARA fusion involves insertion of TTMV into intron 2 of the RARA gene, resulting in transcription and translation of a viral (TTMV)–human (RARA) fusion protein (3). This fusion protein retains the ligand- binding domain (LBD) of RARA, but its 5′ end is replaced by viral sequences, disrupting the normal function of RARA. Consequently, myeloid differentiation is blocked and proliferation promoted, ultimately leading to leukemia development (3, 4).

The clinical presentation of this patient shared several similarities with previously reported TTMV::RARA APL cases, including a marked tendency toward extramedullary infiltration. This patient initially presented with myeloid sarcomas involving the vertebrae and mediastinum, which is uncommon in APL. Myeloid sarcoma is a solid tumor formed by infiltrating immature myeloid cells in extramedullary tissues; it is often associated with AML and may occur before, during, or after AML diagnosis (9). In this case, myeloid sarcoma appeared when the complete blood count showed only mild anemia and the peripheral smear was unremarkable, indicating that extramedullary disease was already present at an early stage and thus increased the difficulty of initial diagnosis.

The diagnostic course of this case exemplifies the difficulties encountered in “APL- like” leukemia when routine diagnostic tests are limited. The patient’s bone marrow morphology (numerous abnormal promyelocytes) and flow cytometric immunophenotype (dim CD45, CD13+, CD33+, MPO+, HLA- DR−, CD34−) were highly consistent with APL, accompanied by a hypercoagulable state (markedly elevated D- dimer), all strongly suggesting APL. However, standard PML:: RARA fusion gene detection (PCR) and RARA rearrangement FISH testing were negative, preventing a definitive diagnosis. Although sequencing detected a DNMT3A mutation, indicating the presence of a myeloid malignancy commonly associated with adverse risk in AML (10), this still could not account for the classic morphological features of APL, leading clinicians to revise the diagnosis to AML and modify the treatment plan and to discontinue ATRA/ATO therapy.

This process highlights the limitations of conventional molecular testing. Current clinical testing for PML:: RARA primarily targets known breakpoint regions or common fusion types. For extremely rare fusions such as TTMV::RARA, produced by viral integration, routine targeted PCR or FISH methods often fail to detect the rearrangement. In this case, only high- throughput whole- transcriptome sequencing successfully identified and confirmed the fusion gene (11, 12). This suggests that for patients with morphology and flow cytometry highly suggestive of APL but with negative routine molecular tests, particularly those with extramedullary involvement, clinicians should be alert to the possibility of variant RARA fusion genes and consider comprehensive molecular testing such as whole- transcriptome sequencing to avoid missed diagnoses and inappropriate treatment interruption.

During early ATRA treatment, the patient developed leukocytosis and differentiation syndrome–like manifestations, indicating some degree of differentiation response. Notably, recent evidence indicates that TTMV::RARA–positive APL may be responsive to ATRA/ATO–based therapy. Xu reported that patients harboring TTMV::RARA achieved clinical and molecular responses with ATRA/ATO, suggesting preserved sensitivity to differentiation therapy when appropriately treated (8). In the present case, ATRA/ATO therapy was initiated but discontinued once the diagnosis was revised to AML, which likely contributed to the unfavorable outcome. Therefore, the poor clinical course observed here should not be interpreted as definitive evidence of intrinsically adverse prognosis associated with TTMV::RARA APL. Taken together, these findings suggest that TTMV::RARA APL may not inevitably confer a poor prognosis and that timely recognition and continuation of ATRA/ATO–based therapy may result in more favorable outcomes. Future research is needed to explore optimized treatment strategies for such patients, including intensified induction chemotherapy, targeted therapy, or hematopoietic stem cell transplantation.

This case also underscores the critical role of the laboratory in diagnosing difficult hematologic malignancies. When morphology reveals “critical value”- level abnormal promyelocytes, laboratory physicians must promptly communicate with clinicians and initiate rapid diagnostic pathways. At the same time, laboratory professionals should recognize that morphology is not the gold standard and should collaborate actively in performing stepwise molecular testing. When routine tests yield negative results, more comprehensive high- throughput sequencing should be recommended to assist in definitive diagnosis and therapeutic decision- making. Given the rarity of TTMV::RARA, its incidence may be underestimated, prompting consideration of how to develop optimized bioinformatic strategies and detection methods to improve early detection rates of this fusion and to ensure timely initiation or continuation of potentially effective differentiation therapy.