The patient, a 72-year-old woman from a rural farming community in northeastern China, initially sought medical attention for progressive fatigue but was reluctant to undergo further diagnostic workup, including bone marrow examination, due to financial constraints and limited understanding of her condition. However, given her unexplained anemia (Hb 91 g/L) and marked thrombocytosis (502 × 10⁹/L), our team strongly recommended a comprehensive diagnostic evaluation, including morphology, immunophenotyping, cytogenetics, and molecular profiling (MICM), to establish an accurate diagnosis. We emphasized that empirical treatment without definitive diagnosis could obscure future bone marrow findings, complicating subsequent management. After detailed counseling, the patient and her family consented to proceed with the recommended investigations.

Following diagnosis, the proposed treatment regimen (e.g., azacitidine, luspatercept) posed significant financial burden, leading the patient and her family to request deferred intervention.

After thorough assessment of: Patient-specific factors (ECOG 1, stable hematologic parameters, no high-risk cytogenetics). Financial toxicity concerns (limited household income, lack of insurance coverage for novel therapies). Molecular risk-benefit analysis (SF3B1-dominant clone mitigating ASXL1-associated high-risk features). we adopted an active surveillance strategy, prioritizing affordability while maintaining safety. Given the patient’s remote residence and inability to travel frequently, we optimized monitoring through: Monthly CBC monitoring via local clinics to detect disease progression. Emergency financial assistance pathways. Deferred therapy unless clinical progression, with clear escalation thresholds to trigger reevaluation.

While WHO’s WHA71.8 resolution (1) advocates equitable access to essential medicines, its direct applicability to this case is limited, as the patient’s primary barrier was affordability of targeted therapies rather than systemic drug shortages. Instead, our approach aligns with NCCN Guidelines^® for lower-risk MDS/MPN, which endorse observation in asymptomatic patients with stable disease, particularly when treatment costs outweigh proven survival benefits in resource-limited settings (2). This pragmatic strategy balances diagnostic rigor, ethical autonomy, and socioeconomic realities, ensuring patient-centered care without compromising long-term outcomes.

The 2016 WHO classification mandated ≥15% ring sideroblasts for MDS/MPN-RS-T diagnosis, regardless of SF3B1 status (3). This morphological threshold missed 23% of SF3B1-mutated cases with <15% RS but similar clinical course (4).

Two evidence-based changes redefine diagnostic standards: Molecular primacy: SF3B1 mutation alone establishes diagnosis (RS threshold eliminated) (5). Nomenclature shift: “SF3B1-T” replaces “RS-T” to reflect molecular pathogenesis. Our patient’s presentation (10% RS, SF3B1 VAF 40.5%) aligns perfectly with this framework.

While JAK2 V617F excludes MDS/MPN-SF3B1-T diagnosis (5, 6), our patient’s rare R683G variant, lacks canonical MPN-driving potency, maintains diagnostic eligibility per 2022 WHO technical notes (5).

The diagnosis of MDS/MPN-SF3B1-T in this case required rigorous exclusion of overlapping entities through integrated clinicopathologic and molecular assessment:

Chronic Myelomonocytic Leukemia (CMML): Peripheral blood monocyte count remained persistently <1×10⁹/L. Absence of dysgranulopoiesis in bone marrow morphology, contrasting with CMML’s characteristic granulocytic dysplasia. Molecular exclusion: No mutations in SRSF2 or RAS pathway genes, which occur in >80% of CMML cases.

Other MDS/MPN Subtypes: Atypical CML (aCML): Ruled out by lack of prominent granulocytic hyperplasia (myeloid:erythroid ratio 1.27:1) and absence of CSF3R or SETBP1 mutations. MDS/MPN-U: Excluded by meeting definitive SF3B1-T criteria through the molecular precedence.

Essential Thrombocythemia (ET): Iron-stainable marrow iron stores and ring sideroblasts (10%) contradicted ET’s typical iron depletion. JAK2 p.R683G’s attenuated signaling precluded classical ET phenotype.

This systematic exclusion approach reinforces diagnostic specificity, particularly given the patient’s thrombocytosis and borderline ring sideroblast percentage. The 2022 WHO classification’s prioritization of SF3B1 mutation—even with subthreshold sideroblasts—was pivotal in resolving diagnostic uncertainty, while its emphasis on mutation combinations allowed for nuanced risk stratification despite phenotypic divergence from classic SF3B1-mutant disease. This integrative molecular-morphological framework enables precise categorization of biologically heterogeneous cases, overcoming limitations of prior morphology-centric systems.

The SF3B1 p.K700E mutation (VAF 40.5%), a hotspot variant disrupting spliceosome fidelity, drives erythroid failure. SF3B1 p.K700E induces aberrant splicing of iron metabolism genes. ABCB7 intron retention reduces mitochondrial iron exporter levels, promoting mitochondrial iron overload (7, 8). ALAS2 exon skipping impairs heme synthesis, uncoupling iron accumulation from hemoglobin production (7). Mispackaged pre-mRNAs trigger nuclear retention, depleting erythroid transcription factors (GATA1, KLF1) critical for differentiation (9).

SF3B1 mutations induce molecular iron mislocalization before morphological RS manifestation, justifying WHO 2022’s elimination of the 15% RS threshold (8). Dominant clonal status (VAF 40.5%) predicts superior response to luspatercept via SMAD2/3 pathway modulation (10).

This molecular hierarchy confirms SF3B1 mutations as both diagnostic biomarkers and therapeutic targets, independent of traditional morphological criteria.

The ASXL1 truncation drives myeloid neoplasms through PRC2-mediated epigenetic dysregulation and confers distinct therapeutic vulnerabilities.

PRC2 Disruption and Transcriptional Derepression

ASXL1 truncations destabilize the PRC2 complex causing global H3K27me3 loss at polycomb targets (e.g., HOXA9), activating leukemogenic pathways. Collapse of H3K4me2/H3K27me3 bivalent domains at differentiation promoters, priming stemness genes (11). ASXL1 mutations occurred more frequently in MDS/MPN overlap syndromes than classical MDS (12–14), retaining independent adverse prognosis with shorter OS and higher leukemic transformation risk (6, 13). This aligns with its incorporation in the IPSS-M model for cross-subtype risk stratification, validated in multi-institutional cohorts (4, 15). The shared prognostic impact underscores ASXL1’s role as a pan-myeloid driver of aggressive disease biology.

Mutation-Specific Therapeutic Vulnerabilities

Azacitidine Sensitivity: ASXL1 truncation increases BIM expression and enhances azacitidine-induced apoptosis, potentially through epigenetic derepression. Direct evidence of BIM promoter demethylation requires further validation. Venetoclax-Hypomethylating Agent (HMA) Synergy: The ASXL1 mutation disrupts H3K27me3-mediated epigenetic repression, leading to MCL-1 upregulation and consequent cancer cell dependence on MCL-1 for survival, which confers resistance to BCL-2-specific Venetoclax; hypomethylating agents (e.g., azacitidine) transiently suppress MCL-1 expression, forcing malignant cells to shift their survival dependency to BCL-2, thereby enabling Venetoclax to effectively trigger apoptosis through BCL-2 inhibition—this dual mechanism of “HMA-mediated MCL-1 suppression plus Venetoclax-induced BCL-2 blockade” synergistically overcomes the epigenetic resistance of ASXL1-mutated cells (16).

The JAK2 p.R683G mutation in the pseudokinase domain (JH2) exhibits distinct molecular characteristics compared to the canonical V617F variant, as evidenced by structural and functional studies:

JH2 Domain Destabilization

The R683G substitution destabilizes the JH2 pseudokinase domain by disrupting key hydrogen bonds within its inter-lobe linker region, as evidenced by molecular dynamics simulations showing a partially unfolded conformation (RMSD=2.3 Å versus wild-type). This structural perturbation significantly compromises JH2’s autoinhibitory capacity to constrain the JH1 kinase domain, while maintaining an intermediate activation state that permits constitutive signaling without full catalytic activation - a mechanism distinct from canonical V617F-mediated hyperactivation (17).

Attenuated Signaling Output

Given its impaired kinase activation, R683G likely induces weaker STAT5 phosphorylation than V617F, though quantitative comparisons require further experimental validation (18). Mechanistically, the unstable JH2 conformation allows transient kinase activation but fails to stabilize active JAK2 dimers required for sustained STAT signaling (17).

Thrombocytosis-Specific Phenotype

Clinical data from germline carriers demonstrate this mutation drives isolated thrombocytosis (median platelets 650×10⁹/L) without leukocytosis or splenomegaly (18). In somatic contexts, R683G associates with lower thrombotic risk than V617F (19), likely due to reduced inflammatory cytokine production (18).

Therapeutic Implications

The structural perturbation confers intermediate sensitivity to JAK inhibitors. In vitro testing shows R683G requires higher ruxolitinib concentrations for 50% inhibition compared to V617F (20). This resistance profile stems from impaired drug binding to the destabilized JH2 domain (17).

Impaired Substrate Recognition Underlies Divergent RTK Signaling Outcomes

The p.R149Q mutation in the CBL TKB domain (Arg149→Gln) disrupts a conserved salt bridge critical for maintaining the structural integrity of the phosphotyrosine-binding pocket (21). Molecular dynamics simulations demonstrate that this destabilization reduces binding affinity for activated receptor tyrosine kinases (RTKs) compared to wild-type CBL, impairing its ability to recognize and ubiquitinate specific RTK substrates (21). This defect primarily drives sustained RAS/MAPK activation through failed ubiquitination of RTKs such as EGFR and KIT, leading to their plasma membrane accumulation and prolonged RAS signaling (21).

Lineage-Specific Rewiring of JAK-STAT Signaling

The p.R149Q variant demonstrates lineage-specific effects on JAK2/STAT5 regulation, distinct from the constitutive activation typically observed in RING domain mutants (21): In Myeloid Precursors: Impaired CBL-JAK2 interaction redirects ubiquitination pathways. Mass spectrometry studies have shown enhanced K48-linked polyubiquitination mediated by the E3 ligase CHIP, leading to reduced JAK2 protein stability. This mechanism may contribute to attenuated JAK-STAT signaling in certain cellular contexts. In Megakaryocytic Lineage: While the molecular details remain to be fully characterized, preliminary evidence suggests that CBL mutations might maintain STAT5 activation through alternative mechanisms. The potential interaction between mutant CBL and hyperactive JAK2 variants (e.g., R683G) could represent a compensatory pathway, though quantitative assessment of phospho-STAT5 levels and functional validation in megakaryocytes are needed (22). This mutation highlights the growing recognition that CBL domain-specific mutations can orchestrate diverse signaling outcomes (21, 22). Although CBL’s role in MPN progression has been well-documented, emerging observations in overlap syndromes indicate that certain variants might unexpectedly limit proliferative signals through JAK2 destabilization. In our patient, the declining platelet counts could reflect a complex equilibrium between JAK2-driven thrombopoiesis and CBL-mediated regulatory mechanisms, though further studies are required to establish this causal relationship.

Splicing-epigenetic feedback: ASXL1 truncations destabilize PRC2-mediated H3K27me3 deposition at SF3B1-dependent splice sites (e.g., ABCB7 intron retention sites), amplifying mitochondrial iron overload compared to SF3B1 mutation alone (23).

Erythroid-specific toxicity: Co-expression in CD34+ cells reduces BFU-E colony formation vs SF3B1 alone, attributable to combined ALAS2 mis-splicing (SF3B1) and GATA1 transcriptional repression (ASXL1) (24).

Bidirectional Effects on Erythropoiesis: While ASXL1 truncations repress erythroid differentiation via GATA1/KLF1 silencing (contributing to anemia), concomitant H3K27me3 loss at proliferative loci (e.g., HOXA9) may paradoxically expand early erythroid progenitors. However, this pro-proliferative effect is functionally outweighed by differentiation blockade and SF3B1-mediated mitochondrial dysfunction, culminating in ineffective erythropoiesis.

Therapeutic implications: Despite erythroid suppression, ASXL1 truncation confers increased azacitidine sensitivity through BIM promoter demethylation (16), suggesting epigenetic therapy could bypass SF3B1-mediated resistance.

Structural-functional modulation: JAK2 R683G’s partial kinase activation (17) combines with CBL p.R149Q’s impaired RTK degradation to sustain RAS/MAPK signaling (22). Clonal competition dynamics: CBL p.R149Q destabilized JAK2 via enhanced ubiquitination, creating a negative feedback loop that restrained platelet production (22).

Bidirectional regulation: JAK2 R683G enhances EPOR signaling to increase erythroid precursor proliferation, while ASXL1 truncation represses KLF1-mediated differentiation (24). Phenotypic equilibrium: In murine models, co-expression results in net erythroid output comparable to WT (24), consistent with our patient’s stable hemoglobin.

The mutation network suggests two precision approaches supported by clinical trials:

JAK/MEK dual inhibition: Preclinical data show JAK2 R683G requires MEK co-targeting for complete pathway suppression (25).

Epigenetic priming with azacitidine-venetoclax combination: ASXL1 truncations enhance BIM-dependent apoptosis (azacitidine effect) while increasing MCL1 dependency (venetoclax target) (16), However, SF3B1-mediated mitochondrial iron overload may attenuate efficacy by reducing heme availability for apoptosis execution (caspase-9 activation requires heme) (10).

This molecular interplay (Figure 4) underscores WHO 2022’s diagnostic advance - mutational hierarchies (SF3B1 dominance) rather than individual mutations determine clinical trajectories. Our observations align with IPSS-M validation studies showing SF3B1 reduces ASXL1-associated AML risk (26).

This 72-year-old patient exhibited low-risk features, including minimal bone marrow blasts (0.12%), absence of TP53 mutations, and “Very Good” cytogenetics. Both IPSS-M (-1.26, LOW risk) (27) and IPSS-R (1.00, VERY LOW risk) (28) indicated favorable prognosis, with a median overall survival of 6 years and a 1-year AML transformation risk of 1.7%. The absence of high-risk molecular and cytogenetic markers supports a low-risk disease course.

The hierarchical clonal structure (SF3B1 VAF 40.5% vs. subclonal ASXL1/JAK2/CBL VAF 10–20%) reflects age-related clonal ecology (29). Jaiswal et al. demonstrated that in elderly individuals >70 years, clonal competition naturally selects for mutations with moderate fitness advantages (e.g., SF3B1 p.K700E) over more aggressive drivers like ASXL1 truncations (29). This evolutionary pressure may explain: ASXL1’s subclonal persistence: While ASXL1 mutations typically confer poor prognosis in younger cohorts (6), their impact in elderly patients may be modulated by clonal dynamics. In age-related clonal hematopoiesis (CHIP), lower VAF clones often exhibit slower expansion than dominant clones (29). which might transiently mitigate leukemic progression. However, specific thresholds linking ASXL1 VAF to AML risk require validation in dedicated cohorts. SF3B1-mediated lineage restriction: As highlighted in European LeukemiaNet guidelines (30), SF3B1 p.K700E creates an erythroid-dominant differentiation block, functionally constraining ASXL1’s pan-myeloid dysregulation—a phenomenon absent in ASXL1+/SF3B1- cases.

Kennedy & Ebert’s framework (31) provides critical context for interpreting multi-mutant phenotypes.

JAK2 p.R683G as “Passenger” in CHIP Context: At VAF 17.5%, this variant likely represents age-related clonal hematopoiesis rather than driver mutation, consistent with the “CHIP-to-MDS transition” model (29). Partial STAT5 activation aligns with CHIP-associated mutations that alter signaling without reaching leukemic thresholds (31).

CBL p.R149Q Functional Attenuation: Impaired FLT3 degradation mirrors the “incomplete penetrance” of CHIP variants (29), where subclonal mutations modify disease phenotype without dictating clinical trajectory.

Malcovati et al. (30) emphasize that elderly-specific risk factors (e.g., clonal hierarchy, comorbidity burden) supersede traditional molecular markers. IPSS-M’s superior performance in >70-year-olds (26) stems from its incorporation of clonal fraction data (VAF-Weighted Prognostication) —a principle strongly endorsed in ELN recommendations (30).

This study has key constraints: 1) Short 6-month follow-up precludes definitive assessment of ASXL1-associated leukemic risk (typical latency >18 months) (26, 29); 2) Single-case findings may not generalize across elderly populations with clonal heterogeneity (29); 3) Mechanistic insights derive from published models rather than patient-specific cellular validation (17, 22); 4) Socioeconomic-driven deferred treatment limits evaluation of mutation-specific therapies (16); 5) Lack of serial genomic profiling obscures clonal hierarchy dynamics (29). While highlighting molecular complexities, multi-center cohorts with extended surveillance and functional assays are needed to validate mutation interplay models in MDS/MPN.