Clinical utility of recently identified diagnostic, prognostic, and predictive molecular biomarkers in mature B-cell neoplasms

Abstract

Genomic profiling studies have provided new insights into the pathogenesis of mature B-cell neoplasms and have identified markers with prognostic impact. Recurrent mutations in tumor-suppressor genes (TP53, BIRC3, ATM), and common signaling pathways, such as the B-cell receptor (CD79A, CD79B, CARD11, TCF3, ID3), Toll-like receptor (MYD88), NOTCH (NOTCH1/2), nuclear factor-κB, and mitogen activated kinase signaling, have been identified in B-cell neoplasms. Chronic lymphocytic leukemia/small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, Waldenström macroglobulinemia, hairy cell leukemia, and marginal zone lymphomas of splenic, nodal, and extranodal types represent examples of B-cell neoplasms in which novel molecular biomarkers have been discovered in recent years. In addition, ongoing retrospective correlative and prospective outcome studies have resulted in an enhanced understanding of the clinical utility of novel biomarkers. This progress is reflected in the 2016 update of the World Health Organization classification of lymphoid neoplasms, which lists as many as 41 mature B-cell neoplasms (including provisional categories). Consequently, molecular genetic studies are increasingly being applied for the clinical workup of many of these neoplasms. In this review, we focus on the diagnostic, prognostic, and/or therapeutic utility of molecular biomarkers in mature B-cell neoplasms.

Main

Mature B-cell neoplasms represent a heterogeneous group of disorders that often have overlapping clinical presentations or morphologic findings or both and therefore can present diagnostic challenges. Furthermore, patients with B-cell neoplasms that show relatively homogeneous pathologic findings can have highly variable clinical outcomes and presenting challenges in clinical management. Recent comprehensive genomic profiling studies have improved our understanding of B-cell lymphomagenesis and have identified novel molecular biomarkers with a diagnostic and prognostic value.

Whereas some B-cell neoplasms such as hairy cell leukemia have a single molecular abnormality (ie BRAFV600E) in >95% of cases, other B-cell neoplasms, such as diffuse large B-cell lymphoma (DLBCL), are shown to be genetically highly heterogeneous containing a number of different molecular aberrations. In addition, the same gene may be involved in more than one type of B-cell neoplasm with variable distribution of mutations or clonal predominance among different neoplasms. For example, BRAF mutations in hairy cell leukemia are restricted to codon V600 and tend to represent the dominant clone as judged by a high variant allele frequency. In comparison, BRAF mutations in chronic lymphocytic leukemia involve exon 11 in addition to codon V600 (exon 15) and can represent a subclonal population. Similarly, in an appropriate clinicopathologic context, BRAF mutations are diagnostic of hairy cell leukemia, but not of chronic lymphocytic leukemia or plasma cell neoplasms. EZH2 mutations in DLBCL primarily involve codon 646 as compared with mutations involving multiple exons in myeloid neoplasms. Within a given tumor type, different biomarkers are shown to be associated with different clinicopathologic features and help explain heterogeneity in outcomes. In patients with chronic lymphocytic leukemia, mutations in MYD88 are seen more commonly in early/untreated cases, whereas mutations in SF3B1, NOTCH1 and TP53 are seen more commonly in progressive/treated cases.¹ Gene mutations can be associated with a specific subtype within a given lymphoma, allowing their use as a surrogate marker for that subtype. Mutations in CARD11, CD79A/B, and MYD88 are more common in activated B-cell-like DLBCL as compared with germinal center B-cell-like DLBCL, whereas EZH2 mutations are almost restricted to germinal center B-cell-like DLBCL. Interestingly, in DLBCL MYD88 mutations show a site preference being more common in neoplasms arising in immune privileged sites, such as central nervous system (~75%) and testis (~70%), compared with DLBCL involving lymph nodes (~15%) and GI tract (~10%).² In addition to diagnostic and prognostic significance, the mutation profiles also predict response to therapy. Lack of response to standard chemotherapy in patients with chronic lymphocytic leukemia with TP53 mutations and the modifying effect of CXCR4 and MYD88 mutation status on response to ibrutinib in Waldenström macroglobulinemia patients justify pre-treatment screening for these mutations. The emergence of gene mutations during the course of disease, for example, BTK and/or PLCG2 mutations in chronic lymphocytic leukemia post-ibrutinib treatment, provides an important basis for treatment failure.

These important genetic insights are reflected in the evolving classification of B-cell neoplasms as well as consensus patient management guidelines.^{3, 4, 5, 6} With increasing integration of molecular biomarkers into the care of patients with B-cell neoplasms, there is a need to develop better tools for diagnosis, identification of novel targets, and monitoring response to therapy. Here, we provide a comprehensive review of recently identified molecular biomarkers in mature B-cell neoplasms and the existing evidence determining their potential clinical utility. Description of well-established cytogenetic biomarkers is kept to a minimum in order to focus on the newly identified molecular biomarkers. Similarly, background information about the lymphoma subtypes has been limited to pertinent details only.

Genomic features and mutated genes of non-Hodgkin lymphomas

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma)

Chronic lymphocytic leukemia/small lymphocytic lymphoma represents 18.6% of NHL cases in the United States.⁷ Chronic lymphocytic leukemia is characterized by a highly heterogeneous clinical course. Several clinical and biological prognostic factors have been identified, such as the Rai and Binet clinical staging systems, specific cytogenetic alterations (−11q, −13q, −17p, +12), mutational status of the immunoglobulin heavy-chain variable region genes (IGHV), and protein expression (CD38, ZAP-70).⁸ A number of mutations have been reported in 2–10% of newly diagnosed chronic lymphocytic leukemia cases, including mutations in the tumor-suppressor genes TP53, ATM, or BIRC3; NOTCH1; and SF3B1 involved in the splicing machinery^{9, 10, 11, 12, 13, 14, 15} (see Table 3). TP53, NOTCH1, and SF3B1 mutations are enriched in patients with specific clinical presentations or high-risk disease,¹⁶ and are associated with decreased overall survival, and differences in the risk of progression and response to treatment^{17, 18, 19, 20, 21} (Tables 1, 2, 3 and Figure 1).

Table 1 Clinical significance of the main driver mutations in chronic lymphocytic leukemia

Table 2 Morphological, immunophenotypic, cytogenetic, and molecular characteristics of the main subtypes of B-cell lymphoma

Table 3 Function and location of the main genes mutated in B-cell lymphomas

Figure 1

Clinical impact of recurrent driver alterations in mature B-cell non-Hodgkin lymphomas. BL: Burkitt lymphoma; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; LPL: lymphoplasmacytic lymphoma; MCL: mantle cell lymphoma; NMZL: nodal marginal zone lymphoma; SMZL: splenic marginal zone lymphoma.

Somatic hypermutations and IGHV region gene utilization

Using the widely accepted (although arbitrary) cutoff of 2% mutations in the IGHV genes, mutated (>2%) and unmutated chronic lymphocytic leukemia cases show a distinctive behavior. Patients with unmutated chronic lymphocytic leukemia have a worse clinical outcome as these neoplasms are associated with adverse prognostic factors. Furthermore, usage of VH3-21 is an adverse prognostic marker that is independent of IGHV mutation status.^{4, 22} Unmutated chronic lymphocytic leukemia cases are associated with high levels of expression of CD38 (≥30%) and ZAP-70 (≥20%), which can be used as surrogates for unmutated status.

Ataxia-telangiectasia mutated

Missense Ataxia-telangiectasia mutated (ATM) mutations leading to kinase inactivation are present in 12% of chronic lymphocytic leukemia patients and may be important in predicting treatment failure²¹ (Figure 1 and Table 1). Deletion of chromosome 11q was recognized initially as a recurrent karyotypic abnormality acquired during the course of disease in patients with progressive chronic lymphocytic leukemia. ATM mutations are identified in 20% of patients with advanced chronic lymphocytic leukemia, and are associated with bulky lymphadenopathy and a poorer outcome in relatively young (<55 years) patients.¹⁶ Mutations are associated with a shorter time to first treatment, disease progression (23.5 months for 100% of ATM-deleted patients and 30 months for 62.5% of patients with ATM point mutations), as well as a shorter treatment-free interval (64.2 months in patients with ATM alterations)²³ (Table 1 and Figure 1). ATM inactivation (via mutation or deletion) is associated with refractoriness to chemotherapy through failure to activate p53 and p21^{24, 25, 26} (Tables 1, 2, 3 and Figure 1). Small molecules that inhibit the MDM2–p53 interaction increase p53 levels and induce apoptosis in chronic lymphocytic leukemia cases with deletion of ATM.²⁷ Similar approaches may be useful therapeutically.

Baculovial IAP repeat containing 3

The majority of Baculovial IAP repeat containing 3 (BIRC3) (previously known as API2 or cIAP2) mutations described in chronic lymphocytic leukemia are of the inactivating type: whole-gene deletions, frameshifts, or nonsense mutations resulting in truncation of cIAP2 prior to the C-terminal RING domain.^{28, 29, 30} BIRC3 inactivation leads to constitutive activation of the non-canonical nuclear factor-kB (NF-κB) pathway thought to be responsible for resistance to treatment in this subset of chronic lymphocytic leukemia patients^{30, 31, 32, 33, 34} (Figure 1 and Tables 1, 2, 3).

BIRC3 mutations are present in 4% of newly diagnosed chronic lymphocytic leukemia patients and are in up to 25% of patients with relapsed and fludarabine-refractory chronic lymphocytic leukemia.²⁹ These mutations are also associated with other adverse prognostic factors that predict chemo-refractoriness and poorer prognosis^{9, 29, 31} (Figure 1 and Tables 1, 2, 3). As a result, a recent review classified patients with chronic lymphocytic leukemia containing BIRC3 aberrations as being very high risk, with recommended therapeutic strategies including p53-independent drugs, BTK inhibitors, and allogeneic stem cell transplantation.³⁵

MYD88

MYD8 8 mutations have been detected in 1.5–4% of chronic lymphocytic leukemia cases, identifying a population of patients with favorable outcome:^{36, 37, 38} diagnosis at a younger age (≤50 years), association with low expression of CD38 and ZAP-70, and a higher frequency of mutated IGHV.^{36, 39} MYD88 mutations in chronic lymphocytic leukemia patients are associated with a good prognosis and confer an OS similar to an age- and gender-matched population of patients without chronic lymphocytic leukemia³⁶ (Table 1).

NOTCH1 mutations

NOTCH1 mutations in chronic lymphocytic leukemia are mainly represented by frameshift or nonsense events that cluster within exon 34, including the highly recurrent c.7544_7545delCT deletion (80–95% of all mutations), that disrupt the C-terminal PEST domain of the protein. NOTCH1 mutations cause impaired degradation of protein and result in sustained NOTCH signaling^{40, 41} with consequent deregulation of cell metabolism and cell-cycle progression³⁹ (Figures 2 and 3). Recently, mutations in the 3′ untranslated region (UTR), predicted to result in alternative splicing, have been associated with an adverse prognosis in chronic lymphocytic leukemia patients.³⁸

Figure 2

Schematic representation of the key pathways activated in B-cell mature neoplasms. (a) BCR signaling pathway. The BCR is composed of a membrane immunoglobulin (IgM) bound to CD79A and CD79B. CD79A is a transmembrane protein with a cytoplasmic domain that contains multiple phosphorylation sites including a conserved dual tyrosine binding motif, termed immunotyrosine-based activation motif (ITAM). CD79A has several cysteine residues, one of which forms covalent bonds with CD79b. Association of the CD79A/B heterodimer with the immunoglobulin heavy chain is required for surface expression of the BCR. BCR antigen binding induces the recruitment of Src family kinases (SYK and LYN) and signaling initiation through the phosphorylation of the ITAM domains. Subsequently, the Bruton’s tyrosine kinase (BTK), phospholipase Cϒ2 (PLCϒ2), and phosphoinositide 3 kinase (PI3K) are activated. BTK leads to phosphorylation of PLCϒ2, promoting the production of diacylglycerol (DAG) and IP3, which IP3 activate protein kinase Cβ (PKCβ), leading to CARD11 phosphorylation, and the recruitment of the molecules MALT1 and BCL10 necessary for CBM multiprotein complex composition. This finally leads to activation of the IKKβ kinase and NF-kB signaling initiation. Mutations affecting CD79A and CD79B ITAM domains have been described in enhancing BCR signaling through a decrease of BCR internalization after its ligand activation. CARD11 mutations also sustain NF-kB activation promoting spontaneous multimerization of the complex, and NF-κB signaling pathway activation in a BCR-independent manner. TCF3 promotes BCR signaling by transactivating the immunoglobulin heavy- and light-chain genes, thereby increasing surface BCR expression, and by repressing the PTPN6 gene, which encodes the BCR signaling inhibitor SHP-1. (b) NOTCH1 signaling pathway. NOTCH1 signaling is initiated when a ligand from the Jagged or Delta families binds the receptor and induces a cascade of proteolytic processes that results in the release and nuclear translocation of the NOTCH1 intracellular domain (NCID). This, in conjunction with the transcription factor CBF1/RBP-Jk, leads to the activation or repression of its target genes. NOTCH1 truncating mutations remove sites of recognition for the E3 ligase FBW7 that targets NOTCH1 for ubiquitin-mediated proteasomal degradation. Thus, NOTCH1 mutations lead to an impairment of the degradation of NOTCH1 protein and stabilization of its active form. NOTCH1 signaling pathway activation leads to deregulation of several pathways, including those controlling cell metabolism and cell-cycle progression. (c) TOLL-like receptor pathway. Upon ligand binding, Toll-like receptors (TLR) aggregate and initiate intracellular signaling by engaging various cytoplasmic adaptors, including MYD88. The MYD88 gene encodes for an adapter protein that acts as a signal transducer in the IL-1, IL-18, and Toll-like receptor signaling pathways, as part of the innate immune response. The MYD88 protein consists of an N-terminal death domain, a linker region, and a C-terminal TIR domain, which may mediate contact with TLR TIR domains upon signaling activation. MYD88 mutations results in uncontrolled formation of the MYD88/IRAK complex, recruitment of TRAF6, constitutive phosphorylation of TAK1, and NF-κB signaling activation. (d) RAS/MAPK/ERK/pathway: the BRAF protein is a member of the serine–threonine kinase RAF family (comprising RAF-1/CRAF, ARAF, and BRAF). Under physiological conditions, the ligand-receptor binding activates that pathway, triggering the recruitment of the adaptor molecules Grb2 to the inner part of the cell membrane and its binding to the guanine nucleotide-exchange factor SOS. Activated SOS displaces guanosine diphosphate from RAS, promoting the formation of guanosine triphosphate–RAS, which recruits BRAF to the cell membrane where it is phosphorylated. In its active form, phosphorylated BRAF can form BRAF-CRAF heterodimers that in turn lead to activation of MEK1 and MEK2, that phosphorylate extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). Phosphorylated ERK1 and ERK2 are translocated to the nucleus, where they activate different transcription factors of target genes that promote cell-cycle progression and survival. BRAF mutants can phosphorylate ERK as monomers and in a RAS and ligand-independent manner. Thus, BRAFV600E in Hairy cell leukemia constitutively activates the RAF–MEK–ERK pathway leading to enhanced survival of the neoplastic cells.

Figure 3

Schematic representation of key hotspot mutations in driver genes in chronic lymphocytic leukemia. (a) Schematic representation of the TP53 and main hotspot mutations in chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). The p53 protein is divided into five domains: an RNA polymerase transactivating domain, a proline-rich domain, a DNA-binding domain, a tetramerization domain, and a carboxy-terminal regulatory domain. TP53 DNA-binding domain (DBD) is divided into three loops: L1 (codons 112–141), L2 (codons 163–195), and L3 (codons 236–251), and two loop–sheet–helix regions (codons 119– 135; 272–287). TP53 inactivating mutations usually cluster in the DNA-binding domain DBD (encoded by exons 4–8). The main hotspot mutations in CLL are R175H, H193, S241, R248W, R249Q, R273H. (b) Schematic representation of the NOTCH1 protein and main hotspot mutation in CLL. The two base-pair deletion in codon 2514 (c.7541_7542delCT) in exon 34 accounts for 80–95% of all NOTCH1 mutations in CLL. This frameshift mutation disrupts the C-terminal PEST domain of the protein leading to the generation of a more active protein, impairing NOTCH1 proteasomal degradation by removing sites of recognition for the E3 ligase FBW7, necessary for NOTCH1 for ubiquitin-mediated degradation. (c) Schematic representation of SF3B1. Somatically acquired mutations in SF3B1 generally represent missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted three hotspots (codons 662, 666, and 700).

NOTCH1 mutations are present in 5–10% of newly diagnosed chronic lymphocytic leukemia cases and are enriched in chronic lymphocytic leukemia patients with evidence of progressive disease (15–20%).^{13, 14, 15} NOTCH1 mutations predict a shorter overall survival (median overall survival 54.8 vs 74.6 months), progression-free survival (2.0 vs 26.4 months),¹⁴ and time to treatment¹⁷ (Figure 1 and Table 1). NOTCH1 mutations also have been identified as a predictive marker for patients who are less likely to respond to the addition of rituximab to fludarabine-based therapeutic regimens.¹⁷ Patients with chronic lymphocytic leukemia that carry NOTCH1 mutations have an increased risk of transformation to DLBCL, also known as Richter syndrome.^{13, 39, 42} In up to 90% of cases of Richter syndrome NOTCH1 mutations have been identified¹⁹ (Figure 1 and Tables 1, 2, 3). Targeted therapy using NOTCH signaling inhibitors (ϒ-secretase inhibitors)⁴³ is available for chronic lymphocytic leukemia patients, but further studies are needed to confirm the utility of these drugs in this clinical setting.

Splicing factor 3b, subunit 1

Mutations in splicing factor 3b, subunit 1 (SF3B1) have been identified in 4–12% of chronic lymphocytic leukemia patients, with a higher frequency (17–24%) at the time of disease progression^{20, 44} (Tables 1, 2, 3). SF3B1 is located on chromosome 2q33.1 and encodes a critical component of the RNA splicing machinery that ensures successful transcription and functional diversity of protein species using alternative spicing. Mutations in SF3B1 are somatically acquired, are generally represented by missense nucleotide changes, and are clustered in selected tandem repeat protein motifs (known as HEAT repeats) of the 155 kDa SF3B1 protein. There are three mutation hotspots at codons 662, 666, and 700 (Figure 3). SF3B1 mutations are associated with adverse biological parameters,²⁰ and correlate with shorter time to treatment (3.8 vs 8.0 years), 5-year overall survival (64.7% vs 86.7%), fludarabine-refractory disease, and poorer OS.^{13, 18, 45} SF3B1 mutations are also associated with del(11q) (20.3% vs 7.5%) and are less frequently present in chronic lymphocytic leukemia associated with del(13q) (7.5% vs 11.2%), contributing to the prognostic stratification of these cytogenetic subgroups of patients²⁰ (Figure 1 and Tables 1, 2, 3).

Tumor-suppressor p53

A deleterious tumor-suppressor p53 (TP53) alteration is the most informative predictor of poor outcome in chronic lymphocytic leukemia patients. TP53 mutations predict for shorter overall survival, progression-free survival, time to first treatment, and are associated with a poorer response or refractoriness to fludarabine-containing regimens.⁹ TP53 codons 248 and 273 represent the most common hotspots in chronic lymphocytic leukemia and are affected by missense (73.9%) and nonsense (4.1%) mutations⁴⁶ (Table 1 and Figure 3). TP53 mutations have been found in 4–10% of patients with untreated chronic lymphocytic leukemia,^{47, 48} and are frequently present in association with NOTCH1 and SF3B1 mutations (16.1% and 14%, respectively).⁴⁹ TP53 mutations are enriched in IGHV unmutated cases, and are detected with a significantly lower frequency in chronic lymphocytic leukemia cases carrying del(11q) or trisomy 12.¹⁷ TP53 mutations and deletions of 17p13 are closely associated. Deletion of 17p is found in 4–9% of chronic lymphocytic leukemia patients at diagnosis or at initiation of first treatment,^{47, 50, 51, 52} and always includes band 17p13, where the tumor-suppressor gene TP53 is located. In the study originally performed by Rossi et al,⁵³ the median overall survival for patients with TP53 mutations was 79.6 months and 5-year overall survival was 66.2% compared with 85.2% in patients without TP53 mutations. Five-year overall survival for patients with del17p13 was 65.8% (median, overall survival 104.2 months) compared with 86.9% for patients without del17p13. Furthermore, TP53 inactivation is associated with progression and transformation to aggressive DLBCL, being present in 40% of patients who develop Richter syndrome. It also represents an independent predictor of overall survival after Richter syndrome transformation^{18, 19} (Figure 1 and Tables 1, 2, 3).

TP53 mutations predict refractoriness to chemotherapy independently of concomitant del17p13. Median time to chemo-refractoriness in the cohort studied by Rossi et al⁵³ for patients treated with fludarabine-based regimens was 6.3 months in TP53 mutated patients versus 72.7 months for patients without TP53 mutations. The results of the CLL8 trial of a large series of untreated chronic lymphocytic leukemia patients, which evaluated first-line therapy with fludarabine and cyclophosphamide versus fludarabine and cyclophosphamide with rituximab among patients with untreated disease⁵³ confirmed that 17p13 deletions and TP53 mutations are associated with treatment refractoriness, and represent independent adverse markers for fludarabine-based treatments, for all efficacy end points⁴⁹ (Figure 1).

Currently, the National Cancer Center Network (NCCN) guidelines and the European Research Initiative on Research in chronic lymphocytic leukemia (ERIC) guidelines recommend assessment for TP53 abnormalities in all de novo chronic lymphocytic leukemia patients, in outside-clinical trial patients who would be eligible to an allogeneic stem cell transplantation or other intensive therapies, as well as in previously treated patients with wild-type TP53 at the initial diagnosis if further therapy is required.^{4, 54} The last update of the NCCN guidelines suggested ibrutinib and idelalisib as therapeutic choices in patients with de novo or relapsed chronic lymphocytic leukemia with TP53 mutations or del(17p).⁴ The ERIC recommendations⁵⁴ indicate that patients with TP53 mutations may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens obtain a high proportion of complete responses, but with short duration. It is encouraged that these patients be included in new clinical trials with novel therapeutic agents.

BTK and PLCG2 mutations are mechanisms of resistance to Ibrutinib treatment

Bruton tyrosine kinase (BTK) is transcriptionally upregulated and constitutively active in chronic lymphocytic leukemia. Ibrutinib, a BTK inhibitor, irreversibly binds BTK at the C481 residue, inactivates the kinase, and induces chronic lymphocytic leukemia cells to undergo apoptosis through inhibition of the BCR signaling pathway. The NCCN guidelines⁴ suggest treatment with ibrutinib in patients with relapsed chronic lymphocytic leukemia, as ibrutinib therapy is associated with partial or complete responses in 71% of patients.⁵⁵ Resistance to ibrutinib often involves a mutation of a cysteine residue where ibrutinib binding occurs. Functional analysis has shown that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The S707Y mutation in PLCG2, located immediately downstream to BTK in the BCR signaling pathway, has a gain-of-function effect owing to disruption of an autoinhibitory SH2 domain. The R665W and L845F mutations in PLCG2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity⁵⁶ (Table 3).

Subclonal mutations in chronic lymphocytic leukemia

In addition to specific clonal aberrations, many subclonal aberrations have been identified in chronic lymphocytic leukemia cases. Several studies show that subclonal mutations are associated with a poorer prognosis because clonal evolution is a key feature of cancer progression and relapse. Tumor subclones can exhibit different somatic mutations in the same gene and can expand over time. Jethwa et al⁵⁷ demonstrated the presence of convergence in chronic lymphocytic leukemia, and also brought evidence that most mutations in chronic lymphocytic leukemia are subclonal and found in 68% of the cases. Whole-exome studies had previously shown the presence of subclonal driver mutations, such as TP53 and SF3B1, expanding over time and representing an independent risk factor for rapid disease progression.⁵⁸ Previously unrecognized chronic lymphocytic leukemia driver genes, such as RPS15 and IKZF3, and new potentially targetable pathways also have been identified in subclonal populations. Thus, early detection, monitoring and quantification of subclones in chronic lymphocytic leukemia are clinically relevant.

Splenic Marginal Zone Lymphoma

Splenic marginal zone lymphoma is an indolent small B-cell lymphoma involving the spleen, splenic hiliar and abdominal lymph nodes, and bone marrow that exhibits a striking clinical variability. Ten-year survival varies from 42 to 95% in different studies,^{59, 60, 61} and adverse clinical prognostic factors are related to high tumor burden and poor performance status. Whole-exome sequencing of splenic marginal zone lymphoma cases has shown mutations in genes involved in marginal zone differentiation (NOTCH2 in 25%, KLF2 in 21% and others).^{36, 40, 41, 62, 63} Specific genetic and molecular features, such as deletion of 7q, unmutated IGHV genes, NOTCH2 and KLF2 mutations, and TP53 inactivation (16%),⁶⁴ have been associated with histological signs of progression in splenic marginal zone lymphoma^{65, 66, 67} (Figure 1 and Table 2). Additional recurrently mutated genes have been described: MAP3K14 (15%), MYD88 (13%), BIRC3 (10%), IKBKB (8%), TNFAIP1 (7%), ARID1A (6%), TRAF3 (5%), and KTM2D/MLL2 (4.7%).

BIRC3 and TRAF3

BIRC3 and TRAF3 inactivating mutations (frameshift or nonsense substitutions) have been described in up to 10% and 5% of splenic marginal zone lymphoma and extranodal MZL, respectively,²⁹ leading to non-canonical (BIRC3, TRAF3, MAP3K14) NF-κB pathway constitutive activation.^{29, 34, 41} Inactivating mutations in TRAF3 lead to elimination of the C-terminal MATH domain of the protein that provides a docking site for MAP3K14 and is required for MAP3K14 recruitment to BIRC3 degradation. Thus, TRAF3 and BIRC3 mutations cause MAP3K14 stabilization in the cytoplasm and constitutive activation of the non-canonical NF-κB signaling pathway²⁹ (Figure 2).

KLF2

Truncating KLF2 mutations, including frameshift, nonsense substitutions, and splice-site mutations, have been identified in up to 20% of splenic marginal zone lymphoma cases. The aberrant transcripts retain intronic sequences and lose their coding potential,⁶² thereby disrupting the entire protein or removing the zinc-finger domains of the nuclear localization signal.⁶⁸ KLF2 mutations that cluster in the C2H2 domain (C terminus) are an independent poor prognostic factor in splenic marginal zone lymphoma patients, and are found in association with del(7q) (53% with KLF2 mutation vs 11% without mutation), unmutated IGHV genes (50% vs 7%), and other gene mutations including NOTCH2, TNFAIP3, and ARID1A. KLF2 mutations confer a higher risk of patients requiring treatment, including splenectomy, and predict for shorter time to treatment, higher risk of histological transformation, and shorter overall survival⁶⁴ (Figure 1 and Table 2).

NOTCH1/2 and TP53

Splenic marginal zone lymphoma cases carry alterations of genes belonging to the NOTCH pathway in 30–40% of cases.⁴¹ The NOTCH2 gene, located on chromosome 1p13–p11, is one of the most frequently mutated genes in splenic marginal zone lymphoma (20–25%), and predicts for a worse outcome.^{40, 41} NOTCH2 mutations in splenic marginal zone lymphoma have been associated with increased risk of histological transformation, shorter relapse-free survival, and shorter time to histological transformation and/or death (32.6 vs 107.2 months in NOTCH2 wild-type patients)⁴⁰ (Figure 1 and Table 2). NOTCH1 also is mutated in 5% of splenic marginal zone lymphoma.⁴¹

TP53 mutations have been identified in 16% of splenic marginal zone lymphoma cases, representing an independent prognostic factor for shorter time to first treatment, progression-free survival, and overall survival⁶⁴ (Figure 1 and Tables 3 and 4).

Table 4 Clinical significance of the main driver mutations in mature B-cell lymphoid neoplasms

Somatic hypermutation of IGH and VH family utilization

About 50% of splenic marginal zone lymphoma cases exhibit somatic hypermutations of the IGHV genes. The absence of somatic mutations in the IGHV genes has been associated with histological signs of progression.^{65, 66, 67} Bias in VH1-2 usage has been found in mutated and unmutated cases, suggesting that splenic marginal zone lymphoma is derived from a highly selected B-cell population. In one study, utilization of three IGHV genes accounted for about 50% of cases: IGHV1-2, 24.9%; IGHV4-34, 12.8%; and IGHV3-23, 8.1%.⁶⁹

Hairy Cell Leukemia and Hairy Cell Leukemia Variant

BRAF

A recurrent mutation involving the proto-oncogene BRAF, located at chromosome 7p24, was recognized in 2011 as a causal genetic event in hairy cell leukemia. To date, BRAF mutations have been detected in >97% of hairy cell leukemia patients,^{70, 71, 72} and BRAF mutation testing is described as essential in the 2016 NCCN guidelines 2016.^{4, 5} BRAFV600E is a point mutation which results in substitution of thymine for adenine at position 1799 on exon 15 and a change of amino acid 600 from valine (V) to glutamate (E). The overall effect is constitutive activation of the RAS–RAF–MEK–ERK signaling pathway (Figure 2). The availability of BRAF and MEK inhibitors, originally developed for the treatment of BRAF-mutated metastatic melanoma,⁷³ offers a new therapeutic opportunity for hairy cell leukemia patients who become refractory to purine analog treatment and/or anti-CD20 monoclonal antibody (rituximab),⁷⁴ as mentioned in the NCCN guidelines update.⁴

Most (>85%) of hairy cell leukemia cases have VH genes with somatic hypermutation, indicative of a post-germinal center stage of maturation. Different studies have shown that hairy cell leukemia cases with unmutated IGH genes predict for shorter patient survival.⁷⁵ Thus, molecular analysis to detect IGHV mutational status is considered useful under certain circumstances in the NCCN guidelines.⁴

MAP2K1

Hairy cell leukemia variant bears morphologic resemblance to hairy cell leukemia, but is no longer considered to be closely related. Distinguishing hairy cell leukemia from hairy cell leukemia variant is important because patients with hairy cell leukemia variant are often resistant to conventional hairy cell leukemia therapy. The neoplastic cells of hairy cell leukemia variant have nucleoli, unlike classical hairy cell leukemia, and are usually negative for CD25, CD123, and CD200. There is no distinct chromosomal abnormality in hairy cell leukemia variant, but del(17p13) and TP53 mutations are frequent. Mutations of the IGHV, particularly involving the VH4-34, are observed in two-thirds of cases and are associated with higher disease burden at diagnosis, poor response to single agent cladribine, and shorter overall survival.⁷⁶ Whole-exome sequencing has documented mutations of MAP2K1 in 48% of hairy cell leukemia-variant cases and classical hairy cell leukemia with IGHV4-34 family usage in one study,⁷⁷ whereas BRAF mutations have not been shown in hairy cell leukemia variant to date.⁷⁰ MAP2K1 encodes the dual specificity kinase MEK1, which is a direct effector of BRAF and directly upstream of ERK1/2 in the MAPK pathway, inducing an increase in basal enzymatic activity and cell proliferation⁷⁷ (Figure 2).

Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia

Lymphoplasmacytic lymphoma is defined as a B-cell neoplasm composed of lymphocytes, plasmacytoid lymphocytes, and plasma cells that does not meet criteria for other types of small B-cell lymphoma. Waldenstrom macroglobulinemia is defined as lymphoplasmacytic lymphoma involving the bone marrow and associated with a serum monoclonal IgM paraprotein of any level. About 95% of all cases of lymphoplasmacytic lymphoma are also Waldenström macroglobulinemia and therefore the molecular data regarding lymphoplasmacytic lymphoma are derived primarily from the study of Waldenström macroglobulinemia cases.

MYD88

Somatic mutations affecting the myeloid differentiation primary response 88 gene (MYD88), located on chromosome 3p22, were described initially by Ngo et al⁷⁸ in 2011 and the reported frequency of mutation in Waldenström macroglobulinemia ranges from 67% to nearly 100%.⁷⁹ MYD88 L265P mutations are also common^{79, 80, 81, 82} in IgM, monoclonal gammopathy of undetermined significance (45–87%), suggesting that this mutation may be an early oncogenic event. Relatively few cases of the non-Waldenström macroglobulinemia type of lymphoplasmacytic lymphoma have been assessed and MYD88 mutations have been shown in a subset of cases, but less frequently than in Waldenström macroglobulinemia. Mutations result in an L265P missense substitution, affect the C-terminal TIR domain of the proteinm, and result in uncontrolled formation of the MYD88/IRAK complex, recruitment of TRAF6, constitutive phosphorylation of TAK1, and activation of NF-κB signaling^{78, 79, 83} (Figure 2). The presence of MYD88 mutation has prognostic significance as patients with mutation respond better to ibrutinib than patients who are wild type.⁵ MYD88 L265P mutations have been proposed as a diagnostic biomarker, helpful in the differential diagnosis of Waldenström macroglobulinemia from other overlapping B-cell neoplasms such as MZLs,^{84, 85} as stated by the NCCN guidelines⁴ (Figure 1 and Tables 3 and 4). However, MYD88 L265P is not specific as this mutation has been identified in 4–13% of splenic marginal zone lymphoma, 7–9% of MALT lymphoma,^{78, 86} 1.5–4% of chronic lymphocytic leukemia,^{20, 39} and in DLBCL, particularly activated B-cell-like DLBCL (30–40%) and DLBCL of the central nervous system and testis (70–75%). The last update of the NCCN guidelines included MYD88 L265P testing of the bone marrow as being essential (instead of useful) in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia diagnosis. Furthermore, NCCN guidelines recommend testing for non-L265P MYD88 mutations in patients with Waldenström macroglobulinemia in whom MYD88 L265P mutation is not detected in an adequate sample.⁵

CXCR4

The chemokine (C-X-C motif) receptor type 4 (CXCR4) is the second most common gene mutated in Waldenström macroglobulinemia, in about 30% of cases, and the gene is located at chromosome 2q22. Nonsense and frameshift mutations clustered in the C-terminus of the protein interfere with normal receptor internalization required to downregulate CXCR4-mediated signaling. As a result, there is an increase in the number of membranous CXCR4 receptors, leading to increased activation of the signaling pathway. CXCR4 mutations correlate with a subgroup of Waldenström macroglobulinemia patients with higher disease activity including higher disease burden, higher serum IgM levels, a greater extent of bone marrow infiltration, and a higher frequency of lymphadenopathy.⁸⁷ CXCR4 mutations do not negatively impact overall survival,⁸⁷ but are associated with resistance with BTK inhibitors⁸⁸ (Figure 1 and Table 2). Strategies targeting both MYD88 signaling and CXCR4 may be needed in Waldenström macroglobulinemia patients with CXCR4 mutations.^{88, 89}

Multiple Myeloma

Multiple myeloma, also known as plasma cell myeloma, is a neoplasm of plasma cells that is driven by RAS pathway mutations and MYC translocations.^{90, 91} Immunoglobulin genes are clonally rearranged and there is a high load of somatic hypermutation, suggesting that plasma cells are derived from a post-germinal center, antigen-driven B cell.

Cytogenetics subgroups and gene expression signature

The main clinically relevant molecular subgroups of multiple myeloma are defined by balanced translocations involving the IGH locus (Table 5). IGH translocations are present in 55–70% of multiple myeloma and there are five major recurrent oncogenes are involved in these translocations that account for about 75% of all translocations (40% of multiple myeloma overall): CCND1/11q13), 15–25%; MAF/16q23, 2–9%; FGFR3/MMSET/4p16.3, 15%; CCND3/6p21, 3%; and MAFB/ 20q11, 2% (Table 5). Non-translocated cases of multiple myeloma are mostly hyperdiploid and usually show gains involving odd numbered chromosomes, 3, 5, 7, 9, 11, 15, 19, and 21.³

Table 5 Main translocations present in mature B-cell neoplasms

Important independent negative prognostic indicators in patients with multiple myeloma include high risk t(4;14) and the MAF translocations t(14;16) and t(14;20), deletion of 17p/TP53 sequences and increased serum levels of β2-microglobulin. The t(4;14)(p16.3;q32) juxtaposes IGH with FGFR3 and identifies a subgroup of high-risk multiple myeloma patients with shorter overall survival and progression-free survival.⁹² The NCCN guidelines recommend assessment for the following as a part of the initial work up: deletion of 17p13, deletion of chromosome 13, amplification of chromosome 1, t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23).⁶ In addition to cytogenetic markers of prognosis, gene expression profiling studies allow identification of a high-risk molecular signature based on the expression of either 70 or 17 genes. The ratio of mean expression levels of upregulated genes mapped to chromosome 1q divided by mean expression levels of downregulated genes mapped to chromosome 1p, define a high-risk score present in 13% of patients, with shorter durations of complete remission and shorter progression-free survival and overall survival.⁹³ The NCCN guidelines state that gene expression profiling, although not used currently in the clinical practice, may be helpful in selected patients to estimate the aggressiveness of disease and individualize treatment.⁶

Specific mutations

Different genes have been found to be mutated in multiple myeloma cases with an appreciable frequency: KRAS, 21.2%; NRAS, 19.4%; DIS3, 10.36%; TP53, 8%; FAM46C, 5.4%; BRAF, 6.7%; TRAF3, 3.7%); IRF4, 3.2%; LTB, 3.0%; CYLD,2.4%; and HIST1H1E, 2.6%.⁹⁴

The RAS/MAPK pathway is the most frequently mutated pathway, being altered in up to 43.2% of patients, with no correlation with overall survival.⁹⁴ Activating mutations of KRAS and NRAS are thought to represent an early event in progression, mediating the transition from monoclonal gammopathy of undetermined significance to multiple myeloma. Other events involved in disease progression are secondary IGH or IGK/IGL translocations, deletion or mutation of TP53 (17p13), translocations involving MYC, gains of chromosome 1q, loss of chromosome 1p, FGFR3 mutations in tumors with t(4;14), and inactivation of p18INK4c or RB1. CXCR4 mutations are found in 20% of IgM monoclonal gammopathy of undetermined significance, but are absent in IgG or IgA monoclonal gammopathy of undetermined significance cases, identifying IgM monoclonal gammopathy of undetermined significance as being more closely related to lymphoplasmacytic lymphoma/Waldenström macroglobulinemia or other B-cell lymphomas and segregated from the uniformly wild-type MYD88 IgG and IgA monoclonal gammopathy of undetermined significance cases that are more closely related to multiple myeloma.³ Mutated genes correlate with cytogenetic subgroups. Activating mutations of FGFR3 occur in 17% of the t(4;14) group. The t(4;14) translocation results in the ectopic expression of the receptor tyrosine kinase (RTK), fibroblast growth factor receptor 3 (FGFR3). Activated FGFR3 can be inhibited by tyrosine kinase inhibitors such as CHIR-258, representing a therapeutic target, as demonstrated by Trudel et al.⁹⁵

CCND1, TP53, ATM, and ATR mutations have been associated with an adverse outcome. CCND1 mutations, present in 12% of the t(11;14) subgroup, predict shorter overall survival (2-year overall survival 38.1% vs 80%).⁹⁴ Del(17p) and TP53 mutations also have a significant negative impact on outcome, showing an increased frequency at disease progression (26%), as well as treatment refractoriness and other high-risk phenotypes (eg plasma cell leukemia). ATM mutations have been associated with an impaired progression-free survival (median, 15.4 vs 26.6 months) and shorter OS (2-year OS, 50% vs 80.3%). Similarly, ATR mutations predict poorer prognosis (1.5% of cases; median progression-free survival 23.9 vs 26.6 months; 2-year OS, 67% vs 80%).⁹⁴

IRF4 and EGR1 mutations appear to have a positive impact on survival. IRF4 K123R mutations correlate with improved progression-free survival (2-year progression-free survival, 71% vs 54%; 2-year overall survival, 100% vs 79%), as well as mutations in EGR1 (median progression-free survival 35.1 vs 26.2 months; 2-year overall survival, 100% vs 78%); EGR1 mutations have been described in hyperdiploid cases.⁹⁴ Different treatments with small molecules targeting these mutations are a potential therapeutic option, and are being tested in different clinical trials. Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors trigger p53-dependent and -independent anti-multiple myeloma activity.⁹⁶ Deregulated RAS/MAPK and NF-κB pathways in multiple myeloma represent major potential therapeutic targets.

TP53 mutation status also might drive therapeutic decisions regarding consolidation and maintenance strategies in multiple myeloma patients.⁹⁷ Nooka et al⁹⁷ suggested improvement in progression-free survival and overall survival among 17p deleted patients by using consolidation and maintenance therapy with lenalidomide, bortezomib, and dexamethasone, after high-dose chemotherapy and autologous stem cell transplantation in high-risk multiple myeloma patients compared with other strategies.

Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a low-grade B-cell lymphoma arising in various extranodal organs, such as the stomach, lungs, salivary glands, ocular adnexae, and skin. MALT lymphoma is often associated with chronic inflammation or autoimmune diseases and accounts for about 8% of all cases of NHL.³ Immunoglobulin heavy- and light-chain genes are rearranged and show somatic hypermutation of the immunoglobulin gene variable regions, consistent with derivation from a post-germinal center memory B-cell.

Specific translocations

The t(11;18)(q21;q21) occurs at different frequencies in MALT lymphoma arising at different sites, and leads to the generation of the BIRC3 (API2)–MALT1 (mucosa-associated lymphoid tissue lymphoma translocation) fusion gene⁹⁸ (Table 5). BIRC3 contains three amino-terminal baculovirus IAP repeats (BIRs), a central caspase recruitment domain (CARD), and a carboxy-terminal zinc-binding RING finger domain. Breakpoints in BIRC3 occur downstream of the third BIR domain, upstream of the C-terminal RING domain, with 91% occurring just before the CARD domain. MALT1 is composed of an N-terminal death domain, followed by two Ig-like domains and a caspase-like domain. Four breakpoints have been identified in the introns upstream of the caspase-like domain. The translocation leads to the generation of a BIRC3-MALT fusion transcript comprising the N-terminal region of BIRC3 with three intact BIR domains and the C-terminal MALT1 region containing an intact caspase-like domain, which activates nuclear factor-kB (NF-κB).⁹⁹ This translocation is most common in lung and stomach MALT lymphomas. In the stomach, translocations correlate with advanced stage disease and are less likely to respond to Helicobacter pylori eradication. The t(14;18)(q32;q21) translocation brings the MALT1 gene under the control of the enhancer region of IGH, causing its overexpression.^{99, 100} This translocation seems to occur more frequently in non-gastrointestinal MALT lymphomas, such as the lungs and ocular adnexae. The t(3;14)(p14.1;q32) occurs in MALT lymphomas of the thyroid gland, ocular adnexae, and skin. The t(1;14)(p22;q32) translocation occurs in approximately 5% of intestinal MALT lymphomas, The translocation juxtaposes BCL10 with the IGH locus and deregulates BCL10 expression.⁹⁹

NF-κB activation

MALT lymphoma is characterized constitutive activation of the NF-κB pathway^{29, 34, 41} resulting from translocations or specific mutations. As previously mentioned, BIRC3 and TRAF3 mutations in about 5% of cases are responsible for non-canonical NF-κB pathway activation. Similarly, MALT lymphoma-associated chromosome translocations are capable of activating both canonical and non-canonical NF-κB pathways.¹⁰¹ Furthermore, TNFAIP3 (A20) inactivation by deletion and/or mutation abolishes the auto-negative feedback fostering NF-κB activation. Recurrent mutations in other NF-κB regulators such as KMT2D/MLL2 (22%), NFKBIE (17%), BCL10 (14%), MYD88 (7–9%), NOTCH1 (8%), and NOTCH2 (8%) have been recently identified in MALT lymphomas.^{102, 103}

Nodal Marginal Zone Lymphoma

Specific gene mutations have been identified in nodal marginal zone lymphoma, an uncommon B-cell lymphoma that accounts for about 2% of all cases of NHL. The diagnosis of nodal marginal zone lymphoma relies mainly on morphological demonstration of marginal zone differentiation in the absence of extranodal or splenic disease. The immunophenotype of nodal marginal zone lymphoma is generally not specific; only a couple of molecules (MNDA and IRTA1) have been proposed as diagnostic markers, whereas B-cell receptor, JAK/STAT, NF-κB, NOTCH, and Toll-like receptor signaling pathways have been proposed to be the main deregulated pathways and potential targets for therapy. Given the significant overlap with other CD5-, CD10- low-grade B-cell lymphomas, the NCCN guidelines underline the potential utility of MYD88 and BRAF mutations status assessment in the differential diagnosis of nodal marginal zone lymphoma with Waldenström macroglobulinemia and hairy cell leukemia, respectively.⁴

The immunoglobulin genes are clonally rearranged with a predominance of mutated VH3 and VH4 families.¹⁰⁴ A nodal marginal zone lymphoma molecular signature was described recently¹⁰⁵ identifying features in common with other types of MZL (NOTCH2 and KLF2 mutations in 20 and 17% of nodal marginal zone lymphoma cases, respectively) and specific mutated genes such as PTPRD. PTPRD encodes the receptor-type-protein-tyrosine-phosphatase-d, a tumor-suppressor gene involved in cell growth regulation. PTPRD is somatically mutated in solid tumors, promoting tumorigenesis through Y705-phospho STAT3 activation.¹⁰⁶ Truncating mutations (splice-site variant, missense substitutions) affecting the tyrosine phosphatase domain of the protein have been reported in 14.3% of nodal marginal zone lymphoma cases and appear to be specific for this entity.¹⁰⁵

Inactivating mutations of KMT2D/MLL2, a tumor-suppressor histone-modifier gene located on chromosome 12q13, have emerged as the most frequent mutations in nodal marginal zone lymphoma, in 34% of cases in one study.¹⁰⁷ Nonsense and frameshift- mutations affecting a residue within either the catalytic SET domain, the FYRC domain (FY-rich carboxy-terminal domain) or PHD zinc-finger domains have been described¹⁰⁷ (Figure 1 and Tables 3 and 4).

Follicular Lymphoma

Follicular lymphoma is a neoplasm composed of follicle center B-cells (centrocytes and centroblasts), usually with at least a partial follicular pattern, and accounts for 20% of all lymphomas worldwide.^{3, 4} Follicular lymphoma is highly associated with the t(14;18)(q32;q21) present in about 90% of all cases (Table 5). TNFRSF14 mutations have been described in 46% of the cases, and recurrent mutations of histone modifiers such as KMT2D (63%), CREBBP (55%), EP300 (14%), EZH2 (89%), ARIDIA (9%), have been identified as early events in pathogenesis.¹⁰⁸

Alterations deregulating cell-cycle progression and DNA-damage responses (CDKN2A/B, MYC, TP53), as well as aberrant somatic hypermutation, are the most frequent genetics events found in transformed follicular lymphoma (tFL).^{109, 110} Biallelic loss of CKN2A/B is the most frequent finding (46%), followed by MYC genetic deregulation (25–33% of tFL) and TP53 mutations (17.9%).¹⁰⁹ Mutations in other genes such as B2M (13%), CD58 (5.1%), CCND3 (1%), GNA13, S1PR2, and P2RY8 have also been reported at lower frequencies.^{109, 111}

Specific translocations

Most cases of follicular lymphoma carry the t(14;18)(q32;q21), which places the BCL2 gene at 18q21 adjacent to enhancer sequences of the IGH at 14q32, resulting in overexpression of bcl-2.^{112, 113} FISH techniques can detect BCL2 translocations in up to 90% of follicular lymphoma, but more sensitive techniques, such as PCR can also be used to assess minimal residual disease (MRD). Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 or downstream and are mainly clustered in either the major breakpoint region (mbr). There are also smaller clusters of breakpoints in the intermediate breakpoint cluster region (icr) and the minor breakpoint cluster region (mcr). Using polymerase chain reaction (PCR) analysis, BCL2/JH involving the mbr can be detected in 60 to 70% of follicular lymphoma cases, respectively.^{114, 115} BCL6 gene rearrangements also occur in 15–20% of cases of follicular lymphoma.

Chromatin modifier genes are the hallmark of germinal center-derived tumors

Results from NGS studies have shown that genes encoding chromatin modifiers such as histone acetyltransferases (CREBBP, EP300), and histone methyltransferases (KDM2C/D, EZH2) are mutated at a high frequency in germinal-center-derived tumors, as has been reviewed by Lunning et al.¹⁰⁸ Gain-of function mutations in EZH2, a H3K27 methyltansferase, lead to accumulation of the trimethylated form of the protein and repression of cell-cycle inhibitors (CDKN1A and CDKN2A/B genes).^{116, 117} EZH2 mutations have been identified in 89% of follicular lymphoma cases and are thought to be a common early event in pathogenesis.^{77, 118} EZH2 mutations are potentially targetable with selective EZH2 inhibitors.^{75, 76} These data suggest that chromatin modifier mutations could be the hallmark of germinal center-derived tumors (Table 3).

CDKN2A/B

The tumor-suppressor genes CDKN2A and CDKN2B encode proteins that participate in DNA damage responses (via the p14-ARF/p53 pathway) as stabilizers of the tumor-suppressor p53, and cell-cycle regulation as negative regulators of cell-cycle G1 progression (via the RB/p16 tumor suppressive pathway). Biallelic loss of CDKN2A/B has been demonstrated to be specifically acquired during follicular lymphoma transformation. Genomic alterations affecting these loci (copy number losses, nonsense mutations and copy number loss of heterozygosity have been reported in up to 46% of tFL.¹⁰⁹

MYC and TP53

Genetic lesions deregulating MYC are important in disease progression. Chromosomal translocations and copy number gains/amplifications have been reported in 25% and 33% of tFL cases.¹⁰⁹ TP53 mutations also identify a subgroup of patients with high-risk follicular lymphoma. About 6% of follicular lymphoma cases carry TP53 mutations which are associated with older age at diagnosis, higher International Prognostic Index score, and shorter overall survival and progression-free survival (Figure 1 and Table 2).¹¹⁹ Transformed follicular lymphoma cases are enriched for TP53 mutations (17.9%)¹⁰⁹ and therefore TP53 mutation analysis is useful for prognostication and therapeutic decision making.

Large B-Cell Lymphoma with Irf4 Rearrangement

Chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin loci were recently described as a novel recurrent aberration in mature B-cell lymphoma. The t(6;14)(p25;q32)/IRF4-IGH has been identified in 8% of germinal-center lymphomas and is associated with a young age at presentation and a favorable outcome. These cases were predominantly germinal center B-cell-like DLBCL or follicular lymphoma grade 3, with strong expression of IRF4/MUM1 and BCL6, and lack of PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks.¹²⁰ Thus, the t(6;14)(p25;q32) identifies a new provisional entity in the 2016 update of the WHO classification under the name 'large B-cell lymphoma with IRF4 rearrangement'³ (Table 5).

Pediatric-Type Follicular Lymphoma

Pediatric-type follicular lymphoma has been included as an entity in the 2016 WHO update. These tumors usually occur in children, but rarely adults are affected. Patients with pediatric-type follicular lymphoma usually present with localized disease and have an indolent clinical course. Therefore, surgical excision alone, without chemotherapy, is often adequate for patient management.

Pediatric-type follicular lymphoma is characterized by large, expansile, highly proliferative follicles composed of large or blastoid follicular center cells. The proliferation rate as shown by Ki-67 is often high and these tumors often do not express BCL2 protein and do not carry BCL2, BCL6 or MYC rearrangements.^{121, 122, 123}

The gene mutation findings in pediatric-type follicular lymphoma are distinctive. This entity is characterized by frequent mutations in the MAPK pathway, and absence of mutations that are common in the usual type of follicular lymphoma, such as mutations in epigenetic modifiers like CREBBP and KMT2D.¹²⁴ Activating mutations in MAP2K1 are most common, in 43% of cases in one study. These mutations occur in exons 2 and 3 of MAP2K1, which encodes MEK1 protein, affecting the negative regulatory region and catalytic domain of the protein, respectively. Other mutations in this pathway also occur uncommonly, including MAPK1 or RRAS.¹²⁵ TNFRSF14 is the second gene most frequently mutated in pediatric-type- follicular lymphoma, being found in 29% of the cases.^{124, 125}

Predominantly Diffuse Follicular Lymphoma with 1p36 Deletion

Another distinctive group of follicular lymphoma is also recognized in the 2016 WHO update, designated as 'predominantly diffuse follicular lymphoma with 1p36 deletion'.³ These tumors usually present as large inguinal masses. Histologically, these tumors have a diffuse pattern, are often low grade, usually express CD23 in addition to germinal center B-cell antigens, usually lack BCL2 rearrangements, and commonly exhibit 1p36 deletions.¹²⁶ At the genetic level, these tumors carry STAT6 mutations at a high frequency, over 82% in one study, a substantially higher frequency than observed in usual cases of follicular lymphoma.¹²⁷

Mantle Cell Lymphoma

Mantle cell lymphoma is an aggressive small B-cell neoplasm that accounts for approximately 4% of NHL cases.³ The t(11;14)(q13;q32) juxtaposes CCND1 at 11q13 with IGH on the derivative chromosome 14 and plays a key role in mantle cell lymphoma pathogenesis. Mantle cell lymphoma shows a poor prognosis with a median overall survival of about 3 years, and is characterized by continuous relapses and increasing resistance to chemotherapy.¹²⁸ Nevertheless, a subset of mantle cell lymphoma cases carry mutated IGHV^{129, 130} and do not express SOX11.^{131, 132} Patients with these tumors follow a more clinically indolent course with stable disease, even in the absence of chemotherapy.^{133, 134}

A number of recurrently mutated genes have been described in mantle cell lymphoma, including ATM (45%), MEF2B (37%), CCND1 (18–35%), CDKN2A/B/C (25%), WHSC1 (25%), MAP3K14 (24%), RB1 (26%), TP53 (7–20%), CDK4 (20%), and PLCG (20%), POT1 (20%), KNT2C/MLL3 (16%), KMT2D/MLL2 (14%), BIRC3 (15%), NOTCH1 (12%), NOTCH2 (5.2%), CARD11 (5.5%), and RELA (2%). Only, CCND1, NOTCH1, NOTCH2 and TP53 mutations have been shown to correlate with poorer overall survival in mantle cell lymphoma patients (Figure 1 and Table 2).^{39, 133, 135, 136, 137, 138, 139}

CCND1 rearrangement

The specific chromosomal translocation t(11;14)(q13;q32), that leads to cyclin D1 overexpression in mantle cell lymphoma includes many different breakpoints. Approximately 30–50% of breakpoints are located in a 2-kilobase region referred to as the major translocation cluster (MTC).^{140, 141, 142} The translocation partner is IGH at chromosome 14q32¹⁴⁰ (Table 5). The breakpoints within IGH occur in a chromosomal region 5' of the joining genes (JH), joining the CCND1 to the enhancer of IGH complex at the 3' site of the JH region. More breakpoints also have been localized within the close vicinity of the 5'-end of the CCND1 gene. Thus, multiple breakpoints are present over a large region, and, therefore, many probes are necessary to cover all breakpoints in a PCR-based assay.

BIRC3

BIRC3 mutations were identified in approximately 15% of cases of mantle cell lymphoma, in association with MAP3K14-dependent NF-κB pathway activation and ibrutinib insensitivity.¹⁴³ Transcriptome sequencing of wild-type BCR signaling inhibitor-sensitive mantle cell lymphoma cell lines revealed chronic activation of the BCR-driven classical nuclear factor κB pathway, whereas insensitive cell lines carrying BIRC3 mutations displayed activation of the alternative NF-κB pathway.¹⁴³ These results suggest that BIRC3 aberrations in mantle cell lymphoma may result in decreased sensitivity to ibrutinib, and that protein kinase MAP3K14 could be a potential therapeutic target in BIRC3-mutated lymphomas¹⁴³ (Figure 1 and Tables 2 and 3).

CCND1

CCND1 mutations, resulting in cyclin D1 overexpression, are associated with a higher proliferation index, shorter overall survival, and ibrutinib resistance^{118, 144} (Figure 1, Tables 2 and 3).

Two different genomic events can lead to an increased expression of cyclin D1. First, genomic deletions and point mutations of the 3′UTR region result in the generation of shorter and more stable mRNA transcripts lacking the destabilizing AU-rich elements. Second, deletions or point mutations in the translated exons of CCND1 can be seen in 22% of mantle cell lymphoma cases and are associated with an adverse prognosis^{118, 145} (Figure 1 and Table 2). Recent large-scale genomic studies identified a hotspot for recurring somatic mutations in exon 1 of CCND1 in 18–35% of cases^{136, 138, 139} (Table 2). The three most frequent CCND1 mutations (E36K, Y44D, and C47S) in mantle cell lymphoma cell lines induce a decrease in protein proteolysis through the ubiquitin–proteasome pathway, leading to the stabilization of CCND1 in the nucleus. These mutations are associated with increased resistance to ibrutinib¹⁴⁴ (Figure 1 and Tables 3 and 4).

TP53

TP53 mutations have been identified in 7–20% of mantle cell lymphoma cases,^{132, 136, 137} in the same hotspots described previously in the literature for chronic lymphocytic leukemia, and are associated with a poorer prognosis.^{146, 147} Halldorsdottir et al¹³⁷ reported the largest series of mantle cell lymphoma cases in which the frequency of TP53 mutation and the prognostic impact of TP53 and 17p deletions were studied. Their series was composed of 119 cases of mantle cell lymphoma, including 84 classical and 34 blastoid variant, in which assessment of exons 4–8 showed non-silent TP53 mutations affecting the classical hotspots in 14% of cases.^{147, 148} Missense mutations were most frequent followed by nonsense mutations; most mutations were transitions, predominantly GC>AT transitions at CpG methylated sites, most commonly in exons 5 and 8. The codons most commonly mutated were at positions 176, 248, and 273. The presence of TP53 mutations was associated with significantly decreased overall survival (13 vs 43 months) (Figure 1 and Table 2). The presence of 17p deletions did not appear to have any prognostic impact on mantle cell lymphoma. Some groups had previously described the presence of TP53 mutations or 17p deletions in association with the clinically aggressive blastoid variant of mantle cell lymphoma,^{138, 149} but this observation was not confirmed in the study performed by Halldorsdottir et al,¹³⁷ in which classic and blastoid mantle cell lymphoma cases had similar mutation frequencies (13% vs 18%). Targeted therapy directed at TP53 mutations could be an option in patients with aggressive clinical behavior and poorer outcome.

Somatic hypermutations and IGHV utilization

Most cases of mantle cell lymphoma carry unmutated IGHV genes, but up to 40% of cases show somatic hypermutation. Mutated IGHV genes correlate with a more indolent clinical course. Biased use of the VH genes also has been reported in mantle cell lymphoma. Thorselius et al¹⁵⁰ described restricted usage of three individual VH genes: VH4-34 (22%), VH3-21 (16%), and VH5-51 (12%). The finding of preferential VH gene usage supports the hypothesis that a subset of mantle cell lymphoma cases is an antigen-driven process occurring in B cells expressing specific VH genes, thus implicating Ig specificity in mantle cell lymphoma development. VH3-21 usage occurs in unmutated cases and correlates with a better prognosis and a reduced number of chromosomal imbalances, compared with mantle cell lymphoma utilizing other immunoglobulin VH genes.¹⁵¹

Diffuse Large B-Cell Lymphoma

DLBCL is the most common type of lymphoid malignancy worldwide, accounting for about one-third of all NHL cases.⁴ DLBCL is characterized by an aggressive clinical course, and exhibits marked heterogeneity in clinical, morphologic, and molecular findings.

Gene expression profiling studies identified three distinct molecular subgroups of DLBCL with different outcomes and pathogenic mechanisms:^{85, 151, 152, 153} germinal center B-cell like (94% 5-year overall survival), activated B-cell like (68% 5-year overall survival),^{154, 155} and an unclassifiable group. Subsequently, the latter group was shown to include cases of primary mediastinal large B-cell lymphoma (64% 5-year overall survival).^{151, 152, 153} In activated B-cell-like DLBCL the NF-κB pathway is activated and NGS studies identified, non-synonymous missense substitutions affecting CD79A/B (20%), CARD11 (10%), and other pathway genes as the explanation^{156, 157, 158} (Figure 2). Many other mutations have been described in DLBCL, with diverse clinical implications, and gene mutations correlate in part with cell-of-origin classification: STAT6 (36%), TNFAIP3 (36%), PIK3CA/D (40%), PIM1 (25%), KMT2C/MLL3 (23%), SOCS1 (17%), PRDM1 (12%), KMT2D/MLL2 (24%), PTEN (22%), TNFRSF14 (22%), CCND3 (18%), and MEF2B (18%).¹⁵⁹ TP53 mutations identified in 20% of DLBCL cases of either germinal center B-cell like or activated B-cell like type are important because their presence is an independent predictor of worse outcome.^{160, 161} MYD88 mutations (30–40%) are increased in activated B-cell-like DLBCL in association with an adverse clinical presentation and poor outcome.^{78, 162, 163, 164} In contrast, germinal center B-cell-like DLBCL is associated with mutations involving histone modification and acetylation (EZH2) and MYC translocations. Heterozygous somatic gain-of-function point mutations affecting the EZH2 Y641 residue are also specific for the germinal center B-cell-like DLBCL subtype, and are present in 30% of the cases^{77, 165} (Table 3).

Specific translocations

In DLBCL, translocations involving BCL2 or MYC are present in 20% and 5–10% of cases, respectively, whereas BCL6 translocations involving many different partners are present in about 30% of cases. IG-associated BCL6 translocations occur in 5–10% of DLBCL.^{166, 167} A number of rare IG locus-associated translocations involving other partner genes have been identified in DLBCL, including BCL3, BCL8, BCL11A, CCNE1, CCND3, EBF1, FOXP1, IRF8, NFKB2, RCK, SPIB, and TNFSF13. Most translocations that involve MYC in DLBCL involve one of the immunoglobulin loci, but other partner loci, in particular the BCL6 locus and a chromosomal region in 9p13 in the vicinity of the PAX5 gene¹⁶⁸ (Table 5) can also be involved. Importantly, the co-occurrence of MYC translocation with translocations involving BCL2 or BCL6 or both, so-called double or triple hit lymphoma, identified a subset of highly aggressive B-cell lymphomas that can have DLBCL-like or Burkitt-like morphology.¹⁶⁹ The 2016 update of the WHO includes a provisional new category designated as high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements for these neoplasms.³

CD79A/B and CARD11

CD79A and CD79B mutations are present in up to 20% of activated B-cell-like DLBCL cases.¹⁵⁷ Non-synonymous missense substitutions affecting the CD79A and CD79B ITAM domains enhance BCR signaling, suppress the negative regulation of LYN kinase, and decrease BRC internalization after its ligand activation.¹⁵⁷ CARD11 mutations also sustain NF-κB activation in activated B-cell-like DLBCL in 10% of the cases.^{156, 158} CARD11 encodes a multi-domain protein that takes part in a signaling complex required for NF-κB signaling downstream of the BCR receptors. CARD11 mutants promote spontaneous multimerization of the complex, association with BCL10 and IKKB activation, leading to NF-κB signaling pathway activation in a BCR-independent manner.¹⁵⁶

Thus, chronic active BCR signaling in activated B-cell-like DLBCL may be targeted by inhibitors of upstream components of the signaling pathway, such as BTK, SYK, SRC family kinases, PKCβ, and PI3Kϒ.¹⁷⁰ There are currently several ongoing clinical trials using kinase inhibitors that show promising results for DLBCL targeted therapy in relapsed or refractory cases. Ibrutinib, a selective BTK inhibitor, showed selective toxicity in activated B-cell-like DLBCL cells and good results in phase I/II trials.¹⁷¹ PI3K can also be targeted with a specific inhibitor, idelalisib (GS-1101), that showed AKT and NF-κB downstream signaling inhibition and activated B-cell-like DLBCL lymphoma cell death in pre-clinical experiments.¹⁷¹ Inhibition of IRAK4, downstream of MYD88, also has been shown to be effective in NF-κB inactivation in DLBCL and Waldenström macroglobulinemia cells harboring MYD88 mutations (Table 3).

MYD88

Some studies have suggested that MYD88 L265P mutations identify a subgroup of patients with DLBCL, mainly of activated B-cell-like origin, with a poor outcome. In a study performed by Rovira et al¹⁶² MYD88 mutations were seen in 22% of the cases, most of which were activated B-cell-like type. Mutated tumors occur more often in older patients with more advanced stage disease, frequent extranodal involvement, and higher International Prognostic Index scores. MYD88 L265P mutations correlated with a worse clinical outcome and shorter OS (52% 5-year OS vs 75%)¹⁶² (Table 2 and Figure 1). Therefore, MYD88 mutational status represents a useful prognostic indicator in DLBCL patients, allowing the identification of a subgroup with extranodal involvement and poor outcome. Moreover, somatically acquired MYD88 mutations have been shown to promote NF-κB and JAK–STAT3 signaling in activated B-cell-like DLBCL.^{163, 172} Therefore, patients with activated B-cell-like DLBCL with MYD88 L265P mutations may benefit from therapies targeting IRAK4 alone or in combination with agents targeting the B-cell receptor,¹⁵⁷ NF-κB,^{173, 174} or JAK–STAT3 pathways.¹⁶³

TP53

TP53 mutational status is an independent prognostic indicator of poor survival in patients with DLBCL.^{160, 161, 175, 176, 177} Clinically, TP53 mutations are associated with a younger age at diagnosis, high serum LDH level, bulky tumors, and high international prognostic index risk group¹⁶¹ (Figure 1 and Table 2). The domain location of TP53 mutations plays a critical role in determining clinical outcome.¹⁶¹ TP53 mutations occur in about 20% of DLBCL patients and mutations in exons 5–9 affecting the DNA-binding domain resulting in a loss of function have been associated with a significant worse overall survival in DLBCL patients.^{160, 161, 178} Mutations in loop 1, loop 3, and LSH (codons 272–287), which are essential for DNA binding,^{179, 180} correlate with a poorer median and 5-year OS. In contrast, TP53 mutations in loop 2 have minimal impact on overall survival¹⁶¹ (Figure 3). TP53 mutational status also allows stratification of patients with germinal center B-cell-like DLBCL into two different prognostic subgroups, predicting for poorer overall survival in the TP53 mutated subset.

Others have proposed that TP53 mutations may confer a selective survival advantage for lymphoma cells during chemotherapy, enhancing resistance to drug-induced apoptosis,¹⁸¹ and predict poorer overall survival and progression-free survival in DLBCL patients treated with R-CHOP.¹⁸² Thus, TP53 mutations contribute to the risk stratification of patients with DLBCL, representing a potential therapeutic target in patients with DLBCL.^{183, 184}

Burkitt Lymphoma

In Burkitt lymphoma, t(8;14)(q24;q32) involves MYC at chromosome 8q24 and the IGH locus at chromosome 14q32 and accounts for approximately 90% of all cases (Table 5). There are also two variant translocations, t(2;8)(q12;q24) and t(8;22)(q24;q11) in Burkitt lymphoma. In t(8;14), MYC resides on the derivative chromosome 14 but in the variant translocations MYC resides on the derivative chromosome 2 (IGK) or 22 (IGL), respectively. MYC consists of three exons, among which exons 2 and 3 include the coding sequence for the protein. The negative regulatory exon 1 and intron 1 sequences are removed in some lymphomas because the t(8:14) breakpoints tend to be in the first intron.¹⁴⁷ Translocations occur in a head to head (5′–5′) fashion between the two loci of MYC and IGH. The IGH enhancer elements affect the transcription of MYC resulting in MYC overexpression.¹⁸⁵ Primers flanking the breakpoints in exon 2 of MYC and for the joining and constant regions of IGH (CH1 exons of IGHG or in the sixth joining segment of IGHJ) can be used to detect the t(8:14).¹⁸⁶ MYC translocations also can be found in 2–5% of DLBCL, a subset of cases of B prolymphocytic leukemia and chronic lymphocytic leukemia, and rare cases of follicular lymphoma and mantle cell lymphoma.^{187, 188, 189, 190}

EBV status likely impacts the occurrence of genetic alterations in Burkitt lymphoma. In the endemic cases, EBV-positive MYC/Ig breakpoints originate from aberrant somatic hypermutation, while in the EBV-negative sporadic cases, MYC translocations mostly involve the Ig switch regions of the IGH locus.^{191, 192} In EBV-positive tumor cells, the growth-transforming program of viral gene expression (latency III) is extinguished, and only the EBV nuclear antigen (EBNA)1 is expressed via the alternative latency I program. Gene expression profiling studies in EBV-positive and -negative BL found significant differences in the expression of viral micro-RNAs and in selected target genes: LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, STK10/STK33L. These findings demonstrated significant differences in the transcriptional profiles of EBV-positive and EBV-negative Burkitt lymphoma cases, suggesting that EBV infection may play a critical role, by itself, in Burkitt lymphoma L pathogenesis, precluding the need for additional mutations.¹⁹³

NGS studies have disclosed the importance of the BCR pathway in the pathogenesis of Burkitt lymphoma.^{194, 195} Activation of the MYC proto-oncogene at 8q24 via chromosomal translocation with one immunoglobulin locus is almost universal, but recent studies have shown the existence of regulatory pathways cooperating with MYC in Burkitt lymphoma pathogenesis.¹⁹⁶ The most frequently mutated genes in Burkitt lymphoma are TP53 (69%), ID3 (58%), MYC (40%), CCND3 (38%), CDK4 (17%), GNA13 (10.5%), CREBBP (6%), PIK3R1 (2%), and tumor-suppressor genes such as ARID1A (7%), and SMARCA4 (21%).¹⁹⁴

Schmitz et al¹⁶⁵ reported mutations affecting the TCF3 gene or disrupting its negative regulator, ID3, in 70% of Burkitt lymphoma cases. These alterations result in a constitutive BCR tonic activation, and thus provide a potential mechanism for PI3K activation in human Burkitt lymphoma.^{165, 197} TCF3 is a member of the helix–loop–helix transcription factor family. TCF3 activates transcription by binding to regulatory E-box sequences on target genes, as heterodimers or homodimers, and is inhibited by heterodimerization with inhibitor of DNA-binding (class IV) helix–loop–helix proteins. Thus, inhibitor of DNA-binding (ID) proteins, such as ID3, can bind TCF3 via the helix–loop–helix motif common to both and inhibit TCF3 function. ID3 mutations affecting the helix–loop–helix domain are mainly represented by nonsense and frameshift mutations, as well as somatic missense mutations (encoding a R606Q change),¹⁸⁹ with a silencing effect on the gene.¹⁹⁸ The overall consequence of ID3 and TCF3 mutants in Burkitt lymphoma is an impairment of TCF3/ID3 inhibitory heterodimerization, leading to a tonic BCR signaling in an antigen-independent manner upon release from the ID3-TCF3 heterodimer (Figure 2 and Table 3).

Burkitt-Like Lymphoma with 11q Aberration

Burkitt-like lymphoma with 11q aberration was introduced in the 2016 update of the WHO classification of lymphoid neoplasms³ as a provisional entity, identifying a subgroup of lymphomas with morphologic and clinical features of Burkitt lymphoma, lacking MYC translocations. Burkitt-like lymphoma cases are frequently found in immunodeficient hosts, in association with a specific pattern of aberrations in chromosome 11 (interstitial gains including 11q23.2–q23.3 and telomeric losses of 11q24.1–qter).^{116, 117}

Genomic Features of Hodgkin Lymphoma

The paucity of Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (HL) and lymphocyte predominant cells in nodular lymphocyte predominant HL cells has made the study of recurrent genetic alterations involved in pathogenesis highly challenging. Mutations in activators or inhibitors of apoptosis such as TP53, FAS, caspase 8, caspase 10, FAS-associated via death domain (FADD), BCL-2 agonist of cell death (BAD) or ataxia-telangiectasia mutated (ATM) as well as translocations involving BCL2, have been found in HRS cells and HL cell lines supporting the hypothesis that rescue of HRS cells from apoptosis is probably a key event in HL pathogenesis.¹⁹⁹

A consistent feature of HRS cells in classical HL is constitutive activation of the NF-κB pathway, which can be the result of different types of genetic alterations. Genomic gains and amplifications of REL and translocation involving BCL3 are observed in 50% and 10% of HL cases, respectively, leading to upregulation of the pathway. Mutations in the negative regulators NFKBIA, NFKBIE, and TNFAIP3 (A20) have been described in 20–60% of HL cases, also contributing to NF-κB pathway upregulation.¹⁹⁹ Both the canonical and non-canonical pathways of NF-κB are also activated by members of the TNFR family, such as CD30, CD40, TNFRSF13B or TACI, BCMA or TNFRSF17 and RANK (TNFRSF11A).

The JAK–STAT pathway is frequently activated by both genetic lesions and by autocrine and/or paracrine signaling. Many cytokines activate members of the JAK family, inducing the phosphorylation of STAT factors that dimerize and translocate to the nucleus, acting as transcription factors. Furthermore, genomic gains of JAK2 are frequently seen in HRS cells, as well as mutations in the negative regulator suppressor of cytokine signaling 1 (SOCS1).¹⁹⁹ Mutations of protein tyrosine phosphatase non-receptor 1 (PTPN1), an inhibitor of the JAK–STAT pathway, occur in about 20% of cases.²⁰⁰ The PI3K–Akt pathway, and the erk pathway, as well as AP1 and multiple receptor tyrosine kinases have also been shown to be deregulated in HRS cells.¹⁹⁹

The tumor microenvironment and interaction with HRS cells is also important in pathogenesis. HRS cells overexpress PD-L1 and PD-L2, usually as a result of amplification or translocation involving chromosome 9p24.²⁰¹ Translocations involving CIITA, a coactivator of the promoter of MHC class II at chromosome 16p13.13, also occur in about 20% of cases of classical HL.²⁰²

Emerging Applications of Molecular Testing in Lymphoma

MRD monitoring

The assessment of MRD by real-time quantitative polymerase chain reaction-based techniques has emerged as a widely feasible and standardized tool for direct assessment of therapy-induced reduction of tumor burden and regrowth after treatment in lymphoma, with much improved sensitivity compared with conventional staging procedures. With the exception of follicular lymphoma, where MRD is typically assessed by amplifying the t(14;18) translocation, IGH-based MRD is the most established and widely employed method for MRD detection and monitoring in lymphoid neoplasms

During early B- and T-cell differentiation, the germline variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin and T-cell receptor genes rearrange and each lymphocyte thereby obtains a particular combination of V–(D–) J segments. Clonal antigen receptor gene rearrangements can be amplified by PCR: IGH, IGK, IGL, TRG, TR delta, and TRB gene rearrangements can be analyzed in a multiplex approach as stated in the BIOMED-2 consensus.²⁰³ Discrimination between monoclonal and polyclonal PCR products is required and can be achieved by heteroduplex analysis or fluorescent gene scanning.²⁰⁴ However, PCR amplification of IGH or T-cell receptor gene rearrangements does not allow sequencing of the tumor clonotype, which is sometimes necessary for MRD determination.^{205, 206} Patient-specific clonal rearrangements of the IGH or T-cell receptor genes can be tracked by sequencing the specific PCR products and designing clone-specific primers, which represents a laborious and time-consuming practice.

Conventional PCR-based MRD detection has a number of limitations, including failure of marker identification in somatically hypermutated tumors and cases with low tumor percentage. Recently, NGS-based methods have been developed to overcome these limitations and are emerging as a reliable tool to quantify the T-cell receptor and IGH repertoires.^{207, 208, 209} Different studies comparing NGS-based methods and allele-specific oligonucleotides PCR for MRD assessment have shown excellent concordance between the results obtained using both methods.^{209, 210, 211} Thus, NGS approaches may have some advantages in terms of achieving a higher level of sensitivity (up to 1 × E−06), avoiding the use of patient-specific reagents.^{209, 210} NGS techniques for MRD assessment have also extended our current understanding of genetic diversity and disease biology. With increasing adoption of NGS-based methodologies in routine clinical testing, NGS-based MRD monitoring of lymphoma is likely to increase.

Liquid biopsy

With the emergence of targeted therapy, genotyping tumor tissue in search of somatic genetic alterations for actionable information is currently routine practice in clinical oncology. Although this information has been obtained traditionally by assessment of a tissue biopsy specimen, biopsy specimens are difficult to obtain serially, and are prone to sampling bias. Cells undergoing apoptosis or necrosis are shed into the bloodstream, and the load of circulating cell-free DNA correlates with tumor staging and prognosis.²¹² Nowadays, genotyping circulating cell-free DNA for somatic genomic alterations found in tumors is possible, as well as detection and quantification of somatic mutations that can be tracked in liquid biopsies in solid tumors. Therefore, circulating tumor DNA or circulating cell-free DNA can provide information about the tumor clonal heterogeneity in a non-invasive way, also allowing the study of changes over time.

Regarding lymphomas, plasma circulating cell-free DNA genotyping has been shown to be a useful tool for detecting clonal somatic mutations in DLBCL, and for tracking clonal evolution and emergence of treatment-resistant clones in DLBCL. Rossi et al²¹³ performed a longitudinal analysis to identifying DLBCL-associated mutations in pre-treatment circulating cell-free DNA from DLBCL patients using ultra-deep targeted next-generation sequencing. These mutations were cleared in subsequent plasma samples from R-CHOP responding patients, and persisted among patients with resistant disease. Furthermore, new mutations among resistant clones were identified. Circulating cell-free DNA genotyping is rapidly emerging as a minimally invasive diagnostic tool to detect and track somatic mutations and emergence of treatment-resistant clones.

Conclusion

An unprecedented number of novel diagnostic, prognostic and predictive molecular biomarkers have recently been identified in B-cell lymphomas, several of which have already been included in the last update of the NCCN guidelines and 2016 WHO classification of lymphoid neoplasms⁴ (Table 2). Novel targeted therapies based on the new molecular alterations are promising to change the therapeutic landscape for patients with B-cell mature neoplasms.² At the same time, advances in clinical laboratory technologies are enabling ultrasensitive screening of multiple biomarkers in a variety of sample types in routine clinical care of a broad spectrum of mature B-cell neoplasms. In conclusion, accelerated biomarker discovery, prompt integration in consensus evidence-based clinical practice guidelines, and development of clinical laboratory tools continue to drive the integration of molecular biomarkers in modern lymphoma care.