R-CHOP Resistance in Diffuse Large B-Cell Lymphoma: Biological and Molecular Mechanisms
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for approximately 25% of NHL cases in the United States and 45.8% in China. The introduction of the R-CHOP regimen, which combines rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone, has significantly improved outcomes for DLBCL patients, with a 10-year overall survival (OS) rate of 43.5%. However, despite these advances, approximately 40% of patients experience relapsed or refractory disease, leading to poor survival outcomes, with a median OS of about 6.3 months. The mechanisms underlying R-CHOP resistance remain poorly understood, necessitating a deeper exploration of the biological and molecular pathways involved in DLBCL heterogeneity and treatment resistance.
Tumor Biology of R-CHOP Resistance
Cell of Origin
Gene expression profiling (GEP) has identified two major subtypes of DLBCL based on the cell of origin (COO): the activated B-cell-like (ABC) subtype and the germinal center B-cell-like (GCB) subtype, which account for approximately 50% and 30% of cases, respectively. The ABC subtype is characterized by the expression of genes such as interferon regulatory factor 4 (IRF4), FLICE-like inhibitory protein (FLIP), and B-cell lymphoma 2 (BCL-2), which are associated with B-cell proliferation and plasma cell differentiation. In contrast, the GCB subtype expresses genes like cluster of differentiation 10 (CD10), LIM domain only 2 (LMO2), and BCL-6, which are typical of germinal center B-cells. Notably, recurrent mutations in enhancer of zeste 2 (EZH2), phosphatase and tensin homolog (PTEN) deletions, BCL-2 translocations, and cREL amplifications are more common in the GCB subtype. Patients with the GCB subtype have a significantly better 3-year OS (85%) compared to the non-GCB subtype (69%) when treated with R-CHOP, suggesting distinct mechanisms of resistance in the ABC subtype.
Recent studies have further refined the classification of DLBCL based on genetic aberrations. For example, the ABC subtype can be divided into the MCD subtype (MYD88 and CD79B mutations), the BN2 subtype (BCL-6 fusions and NOTCH2 mutations), and the N1 subtype (NOTCH1 mutations). Similarly, the GCB subtype includes the EZB subtype (EZH2 mutations and BCL-2 translocations) and the BN2 subtype. Patients with the MCD or N1 subtype have significantly worse outcomes compared to those with the EZB or BN2 subtype, with 5-year OS rates of 26%, 36%, 65%, and 68%, respectively. These findings highlight the importance of targeted therapies, such as Bruton tyrosine kinase (BTK) inhibitors for the BN2 and MCD subtypes and immune checkpoint inhibitors for the N1 subtype.
Clonal Evolution
Clonal evolution plays a critical role in R-CHOP resistance in DLBCL. High-throughput sequencing (HTS) studies have identified three major patterns of clonal evolution: large global change, subclonal selection, and minimal or no change. Subclones harboring mutations in genes such as BCL-2 and proto-oncogene serine/threonine-protein kinase 1 (PIM1) are often selected during chemotherapy, leading to tumor progression. Additionally, non-synonymous gene mutations are associated with shorter median OS, suggesting that R-CHOP selects for subclones with mutations that favor resistance. For example, TP53 mutations are frequently observed in resistant subclones. Furthermore, rituximab treatment can lead to the loss of CD20 expression or mutations in the membrane-spanning four-domains, subfamily A, member 1 gene, contributing to resistance. Single nucleotide polymorphisms in FcrR can also diminish rituximab-induced antibody-dependent cellular cytotoxicity, further promoting resistance.
Tumor Microenvironment
The tumor microenvironment (TME) is a critical factor in DLBCL biology and treatment resistance. The TME consists of immune cells, stromal cells, and extracellular components, all of which influence the response to R-CHOP. Pretreatment TME characteristics, such as fibrosis, angiogenesis, and immune cell composition, can affect treatment outcomes. For example, CD37 deficiency, programmed cell death-ligand 1 (PD-L1), and CD47 upregulation are associated with immune evasion and poor prognosis in resistant cases. Notably, CD47 upregulation is more strongly associated with poor outcomes in the non-GCB subtype compared to the GCB subtype, indicating distinct TME features in different COO subtypes. The Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling pathway also plays a key role in regulating interactions between tumor cells and the TME, influencing angiogenesis, inflammation, immunosuppression, and oncogenesis.
Cell adhesion-mediated drug resistance (CAM-DR) is another mechanism of R-CHOP resistance. CAM-DR involves the protective adhesion of tumor cells to stromal cells, which can be mediated by ADAM metalloproteinase domain 12 (ADAM-12) upregulation and phosphatidylinositol 3-kinase (PI3K)-Akt signaling modulation. However, genetically identified non-malignant signatures of stromal-1 (fibrosis and myeloid infiltration) and stromal-2 (vessel formation) in pretreatment biopsies have been associated with favorable and unfavorable prognoses, respectively. Increased microvessel density has also been linked to inferior OS in DLBCL patients treated with R-CHOP.
Multi-Drug Resistance
Multi-drug resistance (MDR) is a major contributor to R-CHOP resistance in DLBCL. MDR is characterized by the acquired cross-resistance to a wide variety of structurally and functionally unrelated agents. The P-glycoprotein (Pgp) ATP binding cassette-1, encoded by the MDR-1 gene, acts as an ATP-dependent efflux pump, reducing intracellular cytotoxic drug concentrations and contributing to resistance. Polymorphisms in the MDR-1 gene further complicate this mechanism. Other transporters, such as multidrug-resistance-related protein-1 (MRP-1) and ATP-binding cassette subfamily G member 2 (ABCG2), have also been implicated in drug resistance. High expression of Pgp, MRP-1, or ABCG2 is associated with significantly poorer outcomes in DLBCL patients. Doxorubicin, vincristine, and prednisone, key components of the R-CHOP regimen, are substrates for Pgp and can induce MDR-1 expression, exacerbating resistance.
Targeting Molecular Mechanisms in R-CHOP Resistance
Double- or Triple-Hit Lymphoma and Double-Expressor Lymphoma
Double-hit lymphoma (DHL) and triple-hit lymphoma (THL) are high-grade B-cell lymphomas characterized by MYC/8q24 translocations accompanied by rearrangements of BCL-2/18q21, BCL-6/3q27, or both. Approximately 80-90% of DHLs and 19-34% of DLBCLs are also double-expressor lymphomas (DELs), which overexpress c-MYC and BCL-2. Patients with DHL, THL, or DEL generally have poor outcomes with R-CHOP, although the prognostic value of BCL-2, BCL-6, or MYC remains controversial. The dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin plus rituximab regimen has shown promise in patients with MYC translocation, although randomized controlled trials are needed to confirm this. Ibrutinib monotherapy has also demonstrated efficacy in relapsed/refractory DEL, and BCL-2 inhibitors such as ABT-199 have shown an overall response rate (ORR) of 38% in relapsed/refractory DLBCL. Lenalidomide combined with R-CHOP has improved outcomes in patients with MYC translocation, with a 2-year OS and disease-free survival (DFS) of 73% and 75%, respectively. Additionally, the XPO1 inhibitor has been shown to decrease c-MYC expression, and the combined use of BCL-2 and XPO1 inhibitors has been highly effective in eradicating DHL cells in vitro and prolonging host survival in vivo.
BCR Signaling Pathway
The B-cell receptor (BCR) signaling pathway is essential for B-cell activation, proliferation, and differentiation. Constitutive activation of PI3K and phosphatidylinositol-dependent kinase 1 (PDK-1) is critical for the survival of ABC-DLBCL cells with chronic active BCR signaling. The CARMA1–BCL-10–MALT-1 (CBM) complex, which includes caspase-associated recruitment domain 11 (CARD11), BIMP3, CARMA1, BCL-10, and mucosa-associated lymphoid tissue lymphoma translocation-1 (MALT-1), plays a key role in the upregulation of constitutive NF-kB activation in ABC-DLBCL. Chronic active BCR signaling is driven by frequent activating mutations in CD79B, CD79A, or CARD11. In contrast, GCB-DLBCLs often survive with a BCR-negative immunophenotype. Targeted inhibition of the BCR signaling pathway, such as BTK inhibitors, has shown promise in BCR-dependent ABC-DLBCL, although genetic assessments are recommended to confirm the specific molecular lesions.
PI3K-Akt Signaling Pathway
The PI3K-Akt signaling pathway is activated by the sequential phosphorylation of PI3K, phosphatidylinositol-4,5-bisphosphate (PIP2), and Akt/protein kinase B. PTEN, a major negative regulator of PI3K-Akt signaling, is frequently deficient in GCB-DLBCL, leading to dysregulation of this pathway. High levels of phosphorylated Akt are associated with poor prognosis in DLBCL patients treated with R-CHOP. Everolimus and temsirolimus, which target the mammalian target of rapamycin (mTOR), have shown efficacy in relapsed/refractory DLBCL, with an ORR of 30% reported in a phase II trial. Combined therapy targeting both BTK and PI3K has also demonstrated potential in overcoming resistance.
NF-kB Signaling Pathway
The NF-kB signaling pathway is activated through canonical and non-canonical pathways, both of which play roles in apoptosis and anti-apoptosis. Sustained NF-kB activity is a prominent survival feature in ABC-DLBCL, driven by aberrations in MYD88, BCL-10, CARD11, CD79A, CD79B, and other genes. Lenalidomide and its analog thalidomide, which inhibit NF-kB signaling by binding to cereblon, have shown promise in improving outcomes for non-GCB DLBCL patients when combined with R-CHOP.
JAK-STAT3 Signaling Pathway
The JAK-STAT3 signaling pathway regulates cell viability, immunosuppression, angiogenesis, and oncogenesis. STAT3 expression is detected in 37% of DLBCLs and 54% of ABC-DLBCLs, correlating with poor survival. The STAT3-BCL-2-IL-10 loop is implicated in R-CHOP resistance, and targeting JAK, STAT3, and IL-10 receptors has shown therapeutic potential. STAT3 antisense oligonucleotide AZD9150 has demonstrated efficacy in relapsed/refractory NHL.
Epigenetics
Epigenetic modifications, such as DNA methylation and histone acetylation, play a crucial role in DLBCL biology and drug resistance. Mutations in epigenetic modifiers like EP300, KMT2D, and SETDB1 are observed at both diagnosis and relapse, suggesting their role as driver mutations. Distinct epigenetic profiles contribute to the ABC and GCB phenotypes, and microRNAs are also involved in drug resistance. Epigenetic abnormalities are reversible, and histone deacetylase inhibitors like panobinostat have shown potential in selectively killing STAT3-positive DLBCL cells. DNA methyltransferase inhibitors and EZH2 inhibitors are also being investigated in clinical trials.
Conclusions
R-CHOP resistance in DLBCL is a complex phenomenon driven by diverse biological and molecular mechanisms. The COO, clonal evolution, tumor microenvironment, MDR, and epigenetic modifications all contribute to treatment resistance. Targeting specific molecular pathways, such as BCR, PI3K-Akt, NF-kB, and JAK-STAT3, offers promising therapeutic strategies. With a deeper understanding of DLBCL biology, more precise classification systems and tailored treatments can be developed to improve patient outcomes.
doi.org/10.1097/CM9.0000000000001294
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