Abstract

   The ETV6::PDGFRB fusion gene is commonly reported in chronic
   myelomonocytic leukemia with eosinophilia, yet patients with
   ETV6::PDGFRB presenting myeloid and lymphoid neoplasms successively
   have not been reported. Here, we report the first case of a 35-year-old
   man with myeloid and lymphoid neoplasms harboring an ETV6::PDGFRB
   fusion gene who demonstrated poor response to imatinib. The patient was
   diagnosed with an ETV6::PDGFRB fusion gene myeloid neoplasm on initial
   diagnosis at our hospital. After 5 months of treatment with imatinib,
   he was diagnosed with T-cell lymphoblastic lymphoma. ETV6::PDGFRB
   turned negative after increasing the dose of imatinib, but enlarged
   superficial lymph nodes reappeared the following year. Notably, the
   patient exhibited a worse response to imatinib treatment. This study
   describes this rare case and speculates on a possible mechanism.

   Keywords: ETV6, PDGFRB, fusion, myeloid/lymphoid neoplasms, imatinib

Introduction

   The ETV6::PDGFRB fusion gene is commonly associated with myeloid
   neoplasms with monocytosis (such as CMML), and with eosinophilia.[44]^1
   The PDGFRB gene, located on 5q33, encodes PDGFRβ, a type III tyrosine
   kinase comprising an extracellular domain consisting of five
   immunoglobulin (Ig)-like domains, a single-transmembrane helix domain,
   a juxtamembrance domain, and an intracellular tyrosine kinase
   domain.[45]^2 PDGFRB can fuse with >30 partner genes (ETV6 being the
   most commonly seen) and result in a constitutive activation of the
   tyrosine kinase domain, despite extracellular signals, and give rise to
   an uncontrolled proliferation of blood cells, mostly later-stage
   myeloid cells, and less frequently immature B- or T-lymphoid
   cells.[46]^2 The ETV6::PDGFRB fusion gene with lymphoid blasts is
   rarely seen and less frequently reported in the
   literature.[47]^1^,[48]^2

   In the past, the aim of treatment was to limit organ damage by
   controlling the eosinophil count using drugs like prednisone,
   hydroxyurea, and IFNα. Since imatinib, a TKI first approved for the
   treatment of chronic myeloid leukemia by inhibiting ABL kinase
   activity, was discovered to block PDGF receptors at even lower
   concentrations in 2002, the prognosis of PDGFRB-rearrangement neoplasms
   was revolutionized.[49]^3 Patients with ETV6::PDGFRB exhibit an
   excellent response to imatinib, with much less frequent transform to
   blastic crisis phase.[50]^3^,[51]^4 It is challenging for clinicians to
   treat patients with poor response to target therapy, and even disease
   transformation can occurr.

   Here, we report a rare case of ETV6::PDGFRB that manifested initially
   as a myeloid neoplasm and was treated with imatinib, but then an
   invasion of lymphoblastic T cells was found on lymph node (LN) biopsy.
   The patient got better after a course of chemotherapy and an increasing
   dose of imatinib, but enlarged superficial LNs reappeared the following
   year. Notably, the patient exhibited a worse response to imatinib
   treatment. This study describes this rare case and speculates on a
   possible mechanism, displays the complexity of targeting fusion genes,
   and highlights the need for a comprehensive understanding of the
   molecular basis of neoplasms and personalized therapeutic strategies.

Case Presentation

   A 35-year-old man with a history of IgA nephropathy was followed up in
   our hospital. A bone marrow (BM) aspiration biopsy was done when a
   significant leukocytosis (WBCs 26×10^9/L) and eosinophilia (nucleated
   cells [eosinophils] 12.5%) was observed on complete blood count. A
   marked myeloid hyperplasia with 5% myeloblasts and 3% eosinophils was
   found in the BM aspirate smear, while lymphoid was hypoplasia with only
   1.5% of mature lymphocytes counted ([52]Figure 1A). A positive
   ETV6::PDGFRB fusion gene and negative BCR/ABL was found on RTq-PCR,
   PDGFRB gene breakage of 90% of cells was detected by FISH ([53]Figure
   1B), and karyotype analysis showed 46, XY, del(12) (p13p11.2),
   indicative of a diagnosis of myeloid neoplasms with ETV6::PDGFRB fusion
   gene. After hydroxyurea (Qilu Pharma, China; 1.5 g/day) for 4 days,
   followed by imatinib (Hansoh Pharma, China) 200 mg/day, his WBCs and
   eosinophils had normalized within a month, then imatinib was decreased
   to 100 mg/day. Five months after initial diagnosis, an enlargement of
   LNs was found after a cold in his bilateral neck, axilla and groin.
   Ultrasound examination showed the max LN was 3.7×1.5 cm in zone II of
   the right neck with a regular shape and clear border. Biopsy of the max
   LN revealed intermediate blastoid infiltration by neoplastic
   lymphocytes. Immunohistochemical analysis of lymphoma cells showed
   CD34^+, CD10^+, CD3^+, CD7+, and Tdt^+ scattered, leading to a
   diagnosis of T-LBL. At the same time, the BM aspirate showed a
   hypocellularity with 11.5% blasts ([54]Figure 1C), and 24% PDGFRB
   breakage cells on FISH ([55]Figure 1D). The karyotype was normal at
   this time.

Figure 1.

   [56]Figure 1
   [57]Open in a new tab

   (A and B) Bone marrow at first diagnosis; (C and D) bone marrow during
   lymph-node invasion of lymphoblastic T cells; (E and F) bone marrow at
   complete response. (A, C and E) Wright’s stain (100×, 10×). (B, D and
   F) Interphase FISH results: red (CSF1R), green (PDGFRB), 2 yellow
   (PDGFRB no breaking), 1 red, 1 green, 1 yellow (PDGFRB breaking
   detected).

   Imatinib was increased to 400 mg/d, and induction chemotherapy with
   PCIOD (asparaginase, cyclophosphamide, idarubicin, vindesine,
   dexamethasone) was begun. Shrunken LNs after chemotherapy, but a severe
   pulmonary fungal infection occurred in the process, and an impaired
   renal function arose, then imatinib was decreased to 200 mg/day (after
   1 month of 400 mg/day). WBC and eosinophil counts reached normality
   gradually. The BM aspirate was normal, and hypocellularity and PDGFRB
   breakage detection in BM was negative on FISH 1 year after initial
   diagnosis and remained negative on follow-up ([58]Figure 1E and [59]F).
   However, 14 months after initial diagnosis, an enlargement of his LNs
   was detected in his bilateral neck, axilla, and groin again. Maximum
   sizes were 1.4–1.8×0.6–0.9 cm on ultrasound. Impacted by the COVID-19
   situation in our city, 200 mg/day imatinib was continued. Enlarged LNs
   kept stable relative to 1 month before manuscript submission (20 months
   after initial diagnosis). For further treatment decisions and
   understanding the pathogenesis of this disease, more experiments were
   done.

Methods

   Serial BM and LN samples were collected for FCM, FISH, and DNA and RNA
   sequencing at three time points: (A) first diagnosis of
   myeloid/lymphoid neoplasm, abbreviated as diagnosis; (B) lymphoblastic
   T-cell invasion in LNs, abbreviated as invasion; and (C) 1 year after
   diagnosis, when PDGFRB breakage cells in BM had turned negative on
   FISH, abbreviated as CR. FCM immunophenotyping analyses were performed
   using 10-color flow cytometry (Navios, Beckman Coulter, USA). FISH
   analyses were performed on unstimulated interphase cells using
   dual-color break-apart probes (D-FISH, Jinpujia, China; CSF1R signal
   red, PDGFRB signal green, two yellow signals represent PDGFRB without
   breakage, one red, one green, and yellow signals represent one PDGFRB
   breakage detected) according to the manufacturer’s instructions.
   Targeted DNA sequencing and RNA sequencing were performed with the
   NovaSeq 6000 sequencing platform (Illumina, USA) and high-throughput
   Sanger sequencing using an ABI 3730 automated DNA sequencer (Applied
   Biosystems, USA). Total genomic DNA and RNA was extracted from BM and
   LN samples for target sequencing, and 38 genes commonly mutated in
   myeloid neoplasms were investigated with a custom-designed
   next-generation sequencing approach for library construction (Yuanqi,
   China). RNA-sequencing libraries were prepared using the TruSeq
   Stranded Total RNA Library Prep Kit (Illumina, USA) following the
   manufacturer’s instructions. P< 0.05 and log[2]-fold change >2 were
   considered to be significantly differentially expressed. GO and KEGG
   pathway enrichment analysis was performed using KOBAS
   ([60]http://kobas.cbi.pku.edu.cn) and DAVID
   ([61]https://david.ncifcrf.gov).

Results

   At diagnosis, FCM analysis showed one progenitor-cell population with
   CD34par+CD38+CD117+CD7-CD56par+cMPO+CD123dimHLA-DR+cCD3- in the BM
   sample ([62]Figure 2A). Upon invasion, progenitor-cell populations were
   CD34+CD38+CD117par+CD7+CD56+cMPOpar+CD123+HLA-DR+cCD3- in the BM sample
   ([63]Figure 2B) and CD34+CD38+CD117±CD7+CD56+cMPOfew+ CD123+
   HLA-DR-cCD3+ in the LN sample ([64]Figure 2C). Once it was that found
   blast cells coexpressed cMPO and cCD3 simultaneously in the LN sample
   upon invasion, a CD117 gate was used, and the results showed
   CD117-positive cells (red cells, accounting for 13.93% of nuclear
   cells) were positive for cMPO and cCD3 (a mixed T and myeloid
   phenotype), CD117 negative cells were cMPO-negative and cCD3-positive
   (blue cells, accounting for 69% of nuclear cells) (T-LBL phenotype)
   ([65]Figure 2C). In LNs during invasion, the imprint cytology image
   showed intermediate blastoids with intermediate-to-condensed chromatin
   and scant cytoplasm ([66]Figure 2D). FISH detected a break in 64% of
   the cells from the invaded LNs ([67]Figure 2E).

Figure 2.

   [68]Figure 2
   [69]Open in a new tab

   (A and C) Flow-cytometry histograms at different stages. (A) Bone
   marrow at first diagnosis; (B) bone marrow at times of lymph-node
   invasion of lymphoblastic T cells; (C) lymph node during lymph-node
   invasion of lymphoblastic T cells; (D) imprint of lymph-node invasion;
   (E) interphase FISH results of lymph node during lymph-node invasion of
   lymphoblastic T cells.

   NPM1 and TET2 mutations were positive at diagnosis and negative at
   invasion in the BM sample, while TET2 mutation was positive at invasion
   in LN samples. NOTCH3 p. R2031H mutation kept positive but changed the
   VAF at diagnosis, while VAFs of CHD8, MUC16 and PLCG2 remained close to
   50% in samples at diagnosis and invasion. Additionally, MED12 mutation
   was detected at invasion in the LN sample ([70]Table 1). RNA sequencing
   demonstrated the same break points in ETV6::PDGFRB fusion
   (12:12006495:+-5:149510225:-) between the BM sample and LN sample.

Table 1.

   Major gene mutations detected in the disease course
   2021/9/14 2022/3/28 2022/3/28 2022/9/14
   Stage Diagnosis Invasion Invasion CR
   Sample BM BM LN BM
   Method Target DNA-seq and RNA-seq Sanger-seq Target DNA-seq and RNA-seq
   Target DNA-seq and RNA-seq
   Gene NPM1 NPM1 NPM1 NPM1
   AA change W288fs W288fs W288fs W288fs
   VAF% 1 0 0 0
   Gene TET2 TET2 TET2 TET2
   AA change L1151V L1151V L1151V L1151V
   VAF% 30 0 32 0
   Gene NOTCH3 NOTCH3 NOTCH3 NOTCH3
   AA change R2031H R2031H R2031H R2031H
   VAF% 35 + 70 73
   Gene MED12 / MED12 MED12
   AA change V319FS/S849FS / V319FS/S849FS V319FS/S849FS
   VAF% 0 / 61/12 0
   [71]Open in a new tab

   Abbreviations: BM, bone marrow; LN, lymph node; seq, sequencing.

   RNA sequencing revealed some important expression changes in BM samples
   between diagnosis and CR ([72]Figure 3). GO functional enrichment
   analysis revealed that stem cell–related genes had higher expression at
   diagnosis, such as embryonic organ development and morphogenesis,
   cell-fate commitment, and signaling pathways regulating pluripotency of
   stem cells. Neutrophil-related genes in terms of neutrophil activation,
   neutrophil-mediated immunity, and neutrophil degranulation were more
   highly expressed during CR. Additionally, KEGG functional analysis
   revealed that some signaling pathway conduction had also changed, with
   more gene expression related to the NOTCH pathway, MAPK signaling, PI3K
   signaling, and less gene expressions related to NF-B signaling at
   diagnosis.

Figure 3.

   [73]Figure 3
   [74]Open in a new tab

   Gene-expression changes between diagnosis and CR detected by RNA
   sequencing. This shows the five most significantly changed expression
   pathways, as well as GO terms. Some pathways we focused, increase or
   decrease were indicated as diagnosis of CR.

Discussion

   ETV6::PDGFRB was first reported in a CMML patient in 1994, and in the
   2022 WHO and ICC classification, neoplasms associated with ETV6::PDGFRB
   were classified as myeloid/lymphoid neoplasm with eosinophilia and
   tyrosine kinase gene fusions (M/LN-eo-TK), a specific entity and an
   updated name.[75]^5–7 Although the category name includes myeloid and
   lymphoid neoplasms, ETV6::PDGFRB-associated neoplasms with both myeloid
   and lymphoid cells in one patient had never been reported in the
   literature. Fusions of PDGFRB with partners like DIAPH1, SART3, RABEP1,
   and CBORF204 were reported to be associated with chronic myeloid
   neoplasms in BM and T-cell lymphoid neoplasms in LNs.[76]^8^,[77]^9 The
   ETV6 gene, located on 12p13, encodes a transcriptional repressor
   playing a crucial role in hematopoietic regulation and can also fuse
   with >30 partners in myeloid and lymphoid leukemia, among which
   ETV6::PDGFRB in CMML and ETV6::RUNX1 in acute lymphoblastic B-cell
   leukemia (ALL) are well known.[78]^10 ETV6::LYN and ETV6::NCOA2 have
   been reported to be related to T-cell lymphoid and myeloid-mixture
   leukemia.[79]^11^,[80]^12

   PDGFRB-rearranged patients demonstrated good response to imatinib at
   100–400 mg/day: 96% response rate, 90% 10-year overall survival,[81]^13
   and median duration of 6.6 years.[82]^14 In non-AML cases, a low
   imatinib dosage (100–200 mg) is sufficient to induce and maintain
   long-term hematological, cytogenetic, and molecular remissions.[83]^4
   In blastic crisis patients, whether to proceed with HSCT can only be
   made on an individual basis.[84]^15 In one study, all three patients
   with allogeneic HSCT relapsed. Patients may only be offered TKI
   monotherapy, rather than intensive chemotherapy.[85]^8 Three patients
   with chronic myeloid neoplasms and lymphoblastic T-cell lymphoma
   neoplasms received chemotherapy: two had a poor outcome, and the other
   transplant patient survived >3 years with complete molecular
   remission.[86]^8^,[87]^9 For this young male patient in this study, the
   neoplasm cells were controlled by 200 mg/day imatinib, but were partly
   controlled by 200 mg/day imatinib again. The dose of imatinib is very
   important, and may show better outcomes with 200–300 mg/day imatinib
   continuously. Overall, there was a poor response to imatinib, whether
   at diagnosis or used alone after chemotherapy.

   The existence of ETV6::PDGFRB fusion within the cells with T-cell and
   myeloid-cell biomarkers in the LNs revealed the same origin as the
   tumor cells in BM. CD34+CD117+CD7+CD56+ cell populations appeared
   simultaneously in invasive BM, and lymphatic samples confirmed it. Once
   the N-terminal part of the transcription repressor ETV6 is joined to
   the kinase domain of PDGFRB, the kinase domain is constitutively
   activated despite PDGF, resulting in the increased activation of
   several signaling pathways, including PI3K, STAT, MAPK and NF-B.[88]^16
   NF-KB is critical for the cell growth and differentiation of
   ETV6::PDGFRB.[89]^17 NF-B signaling was not activated in this case,
   revealing that further mechanisms may go beyond the known role of
   ETV6::PDGFRB. Deregulated NOTCH3 and pre-TCR signaling contribute to
   the development of leukemia and lymphoma.[90]^18 Mutated MED12 can
   increase NOTCH signaling in CLL patients and was mutated in 10%
   pediatric T-ALL.[91]^19^,[92]^20 The mutated NOTCH3 and activated NOTCH
   signaling in this case may be associated with the T-cell phenotype,
   especially in LN cells that have passed the pre-TCR signaling process.

   ETV6::PDGFRB is capable of transforming lymphocytes in mice expressing
   it, although it is usually displayed as a myeloproliferative disease in
   mouse models.[93]^21^,[94]^22 The break point in intron 8 of PDGFRB was
   special in the ETV6::PDGFRB fusion gene in this case snd in intron 10
   in most cases, resulting in the retention of the fifth Ig domain
   expressed by exon 9 and exon 10, which may also be associated with the
   special phenotype of this case, likely abnormal increased activating
   ability of PDGFRB2.[95]^2 Notably, the karyotype at diagnosis favored a
   cryptic variant translocation, which was consistent with the atypical
   break point of PDGFRB. Several mechanisms have been proposed for the
   transformation of leukemia lineage, including stem-cell plasticity in
   which common myeloid/lymphoid progenitor cells were present expression
   of myeloid and B/T-lymphoid antigen, and clonal selection in which
   subclones with different phenotypes exist such that major clones could
   change under selection of drugs.[96]^23^,[97]^24 ETV6::NCOA2 fusion can
   cause lymphoid specification while sustaining myeloid potential, and
   then cooperate with mutated NOTCH1, resulting in a T/My MAL.[98]^12
   This case manifested as a phenotype of T/My MAL in the disease process.
   Both stem-cell plasticity and clonal selection were assumed to
   function, and in the background of multiple gene mutations, drugs like
   imatinib play an important role in accelerating the manifestation of
   T-LBL.

   Fortunately, the LN size was still under control, but for this young
   patient, choosing whether to increase doses of imatinib, second/third
   generation of TKIs, or combine with chemotherapy while LNs enlarge
   again is challenging. TKI targets the kinase domain of ETV6::PDGFRB
   protein and blocks the constitutive activation of signaling
   pathways.[99]^25 Due to the atypical activated signaling pathway in
   this patient, single administration of TKIs may have had limited
   efficacy. Combined with target therapy on NOTCH signaling, chemotherapy
   and HSCT may be tried in the future.[100]^26 It is regretful that
   imatinib plasma levels were not measured in this patient, and the LN
   was not biopsied during the second enlargement. Meanwhile, there are
   some limitations of this study, such as the potential influence of
   individual genetic variability on treatment response and the
   limitations of using a single case to draw broad conclusions, which
   could be partly controlled by expanding case numbers in future studies.

Funding Statement

   Shuiqing Liu: Young Talent Project of Xinqiao Hospital (2022YQB076).
   Xiaojuan Deng: General Project of Chongqing Medical Scientific Research
   (2021MSXM140).

Abbreviations

   LN, lymph node; CMML, chronic myelomonocytic leukemia; TKI, tyrosine
   kinase inhibitor; BM, bone marrow; WBC, white blood cell; RTq-PCR,
   real-time quantitative polymerase chain reaction; FISH, fluorescence in
   situ hybridization; T-LBL, T-cell lymphoblastic lymphoma; FCM, flow
   cytometry; CR, complete response; GO, Gene Ontology; KEGG, Kyoto
   Encyclopedia of Genes and Genomes; VAF, variant allele frequency; WHO,
   World Health Organization; ICC, International Consensus Classification;
   HSCT, hematopoietic stem-cell transplantation; T/My MAL, T/myeloid
   mixed phenotype acute leukemia.

Ethics Approval

   This study was approved by the Medical Ethics Committee of the Second
   Affiliated Hospital of Army Medical University.

Consent to Participate

   The patient provided their written informed consent to participate in
   this study.

Consent for Publication

   All authors have seen and approved the final version of the manuscript
   being submitted.

Author Contributions

   All authors made a significant contribution to the work reported,
   whether in the conception, study design, execution, acquisition of
   data, analysis, and interpretation, or all these areas, took part in
   drafting, revising, or critically reviewing the article, gave final
   approval to the version to be published, have agreed on the journal to
   which the article has been submitted, and agree to be accountable for
   all aspects of the work.

Disclosure

   The authors declare no competing interests in this work.

References