Leukemia & Lymphoma, November 2014; 55(11): 2428–2437 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.883075

REVIEW

Moving beyond rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone for diffuse large B-cell lymphoma Mark Roschewski, Kieron Dunleavy & Wyndham H. Wilson

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Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma (NHL). While the de facto treatment standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) is curative in most cases, it is ineffective for a significant proportion of patients, particularly those with intermediate and high-risk disease. Efforts to improve upon the results of R-CHOP have principally explored dose intensification of chemotherapy and resulted in considerable additive toxicity without clear benefit. DLBCL is not a uniform disease, however, and can be dissected into distinct molecular subtypes by gene expression profiling. These subtypes are characterized by distinct oncogenic mechanisms of activation and addictions to aberrant intracellular signaling pathways. Novel therapeutic agents that target these pathway addictions are emerging, and may have specific activity within molecular subtypes of DLBCL. To move beyond R-CHOP for all patients with DLBCL, targeted therapies added to the most effective chemotherapy platforms must be studied within the context of molecularly defined subsets. Keywords: Lymphoma and Hodgkin disease, molecular genetics, chemotherapeutic approaches

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma (NHL) constituting 30–35% of cases, and affects patients of all ages [1]. DLBCL is curable, but a significant number of patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) are not cured with initial therapy [2]. Pretreatment prognostic factors that predict outcome have historically been based on clinical features such as chronologic age and surrogates of tumor burden, as captured in the International Prognostic Index (IPI) [3]. Although the IPI has validated prognostic value, it does not take into account how biological mechanisms may contribute to treatment

failure. DLBCL is a molecularly diverse group of disease with disparate clinical and pathological features, many of which reflect underlying oncogenic mechanisms (Table I) [4]. Gene-expression profiling (GEP) has identified at least three molecular subtypes of DLBCL with transcriptional profiles reflective of their postulated cell-of-origin [5–7]. Nextgeneration sequencing (NGS) techniques of whole genome, exome and transcriptome sequencing have further revealed the molecular complexity of DLBCL tumors [8–11]. Systematic mutational discovery with NGS provides a platform to elucidate the functional relationship of somatic mutations and cellular signaling pathways. Emerging biologic discoveries create opportunities to translate mechanistic hypotheses to the clinic. Efforts to move beyond R-CHOP in DLBCL have principally relied on therapeutic intensification, which is generally limited to younger (ⱕ 60 years) and fit patients [12–14]. Given the aging population and the greater incidence of DLBCL in older patients, it is of critical importance to develop alternative and more effective therapies. In the genomic era, innovative clinical trial designs that detect responses in subsets of patients with unique disease mechanisms are required [15]. In this review, we reflect on the development of R-CHOP and highlight biologically rational targeted agents within the context of molecular subtypes of DLBCL that promise to move the field beyond R-CHOP for all age groups.

Review criteria PubMed and MEDLINE databases were searched to identify articles published from 1975 to October 2013. Only articles and abstracts published in English were considered for this review. Search terms included: “diffuse large B-cell lymphoma,” “R-CHOP,” “DLBCL,” “ABC DLBCL,” “GCB DLBCL,” “BCR signaling,” “MYC” and “gene expression profile.” Articles were also identified through searches of the authors’ own files. This is not a systematic review but is a selection of references. The final reference list was generated on the basis of originality and relevance to the scope of this review.

Correspondence: Wyndham H. Wilson, Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10 Room 4N115, Bethesda, MD 20892, USA. E-mail: [email protected] Received 16 December 2013; accepted 9 January 2014

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Beyond R-CHOP

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Table I. Underlying biology of DLBCL subsets. Molecular subtype

Oncogenic mechanisms

Activated B-cell DLBCL

NF-κB activation CD79B mutations CARD11 mutations MYD88 mutations A20 deletions BCL-2 translocation EZH2 mutations PTEN deletions Loss of PTEN expression NF-κB activation REL amplification JAK2 mutations 9p24 amplification CIITA translocations MYC translocations BCL-2 translocations BCL-6 translocations

Germinal center B-cell DLBCL

PMBL

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B-cell lymphoma, unclassifiable, with features between DLBCL and Burkitt lymphoma

Therapy targets BCR pathway CBM complex IRAK-4 JAK/STAT pathway BCL-2 EZH2 PI3K/Akt pathway BCL-6 JAK/STAT pathway PD-1 pathway

MYC BCL-2

DLBCL, diffuse large B-cell lymphoma; PMBL, primary mediastinal B-cell lymphoma; NF-κB, nuclear factor-κB; BCR, B-cell receptor; CBM, CARD11–MALT1–BCL-10; JAK, janus activated kinase; STAT, signal transduction and activator of transcription; PTEN, phosphatase and tensin homolog; PI3K, phosphatidylinositol 3-kinase; PD-1, programmed death-1.

Pathobiology: the first essential element The modern era emphasizes biologically based pathology classifications in lymphoid tumors, which are mandatory for optimal clinical research. The National Cancer Institute working formulation, used in the United States until the early 1990s, was biologically agnostic and categorized lymphomas by morphology alone and clinical behavior [16]. In contrast, the Kiel classification, used in Europe, incorporated immunologic techniques to subdivide lymphomas on the basis of B-cell or T-cell lineage [17]. It was not until the Revised European-American Lymphoid (REAL) classification, published in 1994, that a clinical–biological foundation based on the combination of morphologic, immunologic and genetic features of the cell and its postulated normal counterpart within the immune system was formally incorporated into the classification of lymphomas [18]. Recent focus in the diagnostic criteria of the World Health Organization (WHO) classification of lymphomas has been toward the incorporation of clinical features, morphology, immunohistochemistry and ever-evolving genetic data [19]. DLBCL is clinically and biologically diverse, but until recently it was not possible to subdivide it into distinct disease entities because of overlapping morphology and pathologic features. The application of large-scale GEP allows morphologically indistinguishable DLBCL tumors to be subdivided into at least three distinct cell-of-origin subtypes: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal B-cell lymphoma (PMBL) [5–7]. The molecular subtypes of DLBCL originate from B-cells at different stages of development, have distinctive mechanisms of oncogenic activation and have different prognoses with therapy [20,21]. The genes associated with GCB DLBCL include known markers of germinal center differentiation, while the genes that define ABC DLBCL are genes that normally regulate plasma cell differentiation. Thus, it is postulated that GCB DLBCL arises from germinal center B cells, whereas ABC DLBCL arises from post-germinal center B cells that are blocked during plasmacytic differentiation. Two studies using GEP have confirmed that PMBL is a

third molecular subtype of DLBCL with a unique biological identity that shares many features with nodular sclerosing Hodgkin lymphoma (NSHL) [6,22]. Frequently, PMBL and NSHL tumors contain thymic remnants, suggesting that they derive from a post-thymic B cell.

Chemotherapy: the foundation of treatment Combination chemotherapy regimens first demonstrated curative potential in patients with advanced diffuse histiocytic lymphoma (now known as DLBCL) in the 1970s [23]. Since that time, anthracyclines have been established as the essential drug class for DLBCL, and are included in all upfront regimens (Table II). Despite promising single-arm studies, the empiric addition of agents did not improve the cure rate, and the first-generation combination regimen CHOP has been the standard platform since a randomized phase III study of patients with advanced intermediategrade and high-grade lymphomas compared three doseintensive regimens to CHOP and demonstrated no difference in overall survival (OS) [24]. No significant improvements beyond CHOP were realized in DLBCL until the anti-CD20 monoclonal antibody R was successfully added to CHOP by the Groupe d’Etude des Lymphomes de l’Adulte (GELA) in a randomized study of patients with DLBCL over the age of 60 [25]. In this study, R-CHOP demonstrated an improvement in 2-year event-free survival (EFS) rate compared to CHOP alone (57% vs. 39%, p ⬍ 0.001), and this early benefit translated to a sustained improvement in the OS rate at 10 years (43.5% vs. 27.6%, p ⫽ 0.005) [2]. The MabThera International Trial (MInT) Group study was a randomized study of R-CHOP compared to CHOP in younger patients (aged 18–60 years) with favorable prognostic features. In this study R-CHOP demonstrated excellent results, with 79% of patients free of relapse at 3 years (79% [95% confidence interval (CI) 75–83] vs. 59% [54–64]) and an advantage in OS at 3 years (93% [90–95] vs. 84% [80–88]) [26]. Longer follow-up at 6 years demonstrated durable benefit in EFS with R of almost 20% (74.3% vs. 55.8%, p ⬍ 0.0001) [27].

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Table II. Treatment outcome in untreated DLBCL.

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Therapy

Phase

Study group

Event-free survival

Overall survival

36.5% vs. 20% at 10 years* 74.3% vs. 55.8% at 6 years

43.5% vs. 27.6% at 10 years 90.1% vs. 80% at 6 years

74.8% vs. 75.4% at 2 years†

80.8% vs. 82.7% at 2 years

GELA

Aged 60–80, stage II–IV Aged 18–60, bulky stage I or stage II–IV, aaIPI ⫽ 0–1 Aged ⱖ 18, bulky stage I or stage II–IV Aged 60–80, aaIPI ⱖ 1

56% vs. 60% at 3 years

72% vs. 69% at 3 years

III

DSHNHL

Aged 18–60, aaIPI ⫽ 2–3

61.4% vs. 69.5% at 3 years

77% vs. 84.6% at 3 years

III III

GELA US Intergroup

81% vs. 67% at 3 years 69% vs. 55% at 2 years

92% vs. 84% at 3 years 74% vs. 71% at 2 years

II

CALGB

Aged 18–59, aaIPI ⫽ 1 Aged 15–65, stage II–IV, aaIPI ⫽ 2 or 3 (aggressive NHLs) Aged ⱖ 18, stage II–IV

81% at 5 years

II

NCI

94% at 5 years (GCB), 58% at 5 years (non-GCB) 93% at 5 years

R-CHOP vs. CHOP R-CHOP vs. CHOP

III III

GELA DSHNHL

R-CHOP21 vs. R-CHOP14 R-CHOP21 vs. R-CHOP14 R-MegaCHOEP* vs. R-CHOP R-ACVBP vs. R-CHOP R-CHOP with ASCT vs. R-CHOP DA-EPOCH-R

III

UK NCRI

III

DA-EPOCH-R

Patient group

Aged ⱖ 18, bulky stage I, or stage II–IV (PMBL)

97% at 5 years

DLBCL, diffuse large B-cell lymphoma; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone; R-CHOP21, R-CHOP delivered every 21 days; R-CHOP14, R-CHOP delivered every 14 days; R-MegaCHOEP, rituximab and dose-escalated CHOP plus etoposide; R-ACVBP, rituximab, doxorubicin, cyclophosphamide, vindesine, bleomycin and prednisone; ASCT, autologous stem cell transplant; DA-EPOCH-R, dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine and prednisone; GELA, Groupe d’Etude des Lymphomes de l’Adulte; DSHNHL, Deutsche Studiengruppe für Hochmaligne Non-Hodgkin’Lymphome; UK NCRI, United Kingdom National Cancer Research Institute; CALGB, Cancer and Leukemia Group B; NCI, National Cancer Institute; aaIPI, age-adjusted IPI; NHL, non-Hodgkin lymphoma; PMBL, primary mediastinal B-cell lymphoma; GCB, germinal center B-cell-like. *This regimen requires the use of stem cell reinfusion. †Progression-free survival.

Taken together, the GELA study and the MInT study established R-CHOP as the de facto standard in DLBCL for all age groups. Moving beyond the R-CHOP platform for DLBCL has been difficult, in part because it has relied on therapeutic intensification without modification for underlying tumor biology. Prior to rituximab, the approach of delivering “dose-dense” CHOP every 14 days (CHOP14) demonstrated improvements in OS compared to CHOP every 21 days (CHOP21) in a phase III randomized study of patients with DLBCL over the age of 60 [28]. On the basis of these results, bi-weekly CHOP was considered by some to be the standard delivery method for DLBCL, even without verification in the R era. Two recent studies have suggested that the advantage of “dose-dense” CHOP is obviated by the use of R. First, a randomized, multicenter, phase III study was performed in patients with newly diagnosed DLBCL aged ⱖ 18 years, comparing the conventional R-CHOP21 with R-CHOP14 [29]. After a median follow-up of 46 months, no difference in 2-year OS was observed in the R-CHOP14 arm compared to R-CHOP21 (82.7% [95% CI 79.5–85.9] vs. 80.8% [95% CI 77.5–84.2]). The GELA group also performed a randomized study of R-CHOP14 compared to R-CHOP21 in patients with DLBCL aged 60–80 with at least one adverse prognostic indicator in the IPI [30]. With a median follow-up of 56 months, no difference in 3-year EFS was observed with R-CHOP14 compared to R-CHOP21 (56% [95% CI 50–62] vs. 60% [95% CI 55–66]), but it did result in more hematologic toxicity. Thus, no discernible benefit has been demonstrated with R-CHOP14 in any subgroup of DLBCL. In the pre-R era, high dose therapy requiring peripheral blood stem cell (PSBC) support demonstrated benefit in patients with high-risk disease, prompting its evaluation in the modern era in patients deemed at risk to fail standard therapy. The Deutsche Studiengruppe für Hochmaligne Non-Hodgkin’Lymphome (DSHNHL) group tested doseescalated CHOP plus etoposide with R (R-MegaCHOEP) in a series of early phase studies in patients under the age of

60 years with features of high risk, defined as an elevated lactate dehydrogenase (LDH) level [31]. Compared to historical controls, these results suggested an improvement in both EFS (72.7% vs. 47.2%, p ⫽ 0.013) and OS (78.7% vs. 55.0%, p ⫽ 0.045) for the group treated with dose intensification. On the basis of these results, the DSHNHL group performed an open-label phase III multicenter randomized study of R-MegaCHOEP with PSBC support versus R-CHOP with etoposide delivered in 14-day cycles (R-CHOEP14) in 275 patients aged 18–60 with high-risk features, defined by the IPI score [13]. In this study, the 3-year difference in EFS between the two groups was not significant (69.5% vs. 61.4%, p ⫽ 0.14), at a median of 42 months. All patients treated with R-MegaCHOEP experienced grade 4 leukopenia, and 75% of them had a grade 3 or 4 infection as a result of the dose intensification. Autologous stem cell transplant (ASCT) as consolidation therapy in first remission in the rituximab era was also evaluated in a multicenter randomized study of patients with a variety of aggressive lymphomas with high-risk features determined by the age-adjusted IPI (aaIPI) [14]. In this study, patients who responded to five cycles of induction therapy (CHOP or R-CHOP) were randomized to three more cycles of induction or one more cycle of induction therapy followed by a consolidative ASCT. Despite a 2-year estimated improvement in progression-free survival (PFS) (69% vs. 55%, p ⫽ 0.005), no improvement in OS was noted 2 years after the ASCT (74% vs. 71%, p ⫽ 0.30) [14]. Due to the lack of survival benefit, considerable long-term toxicities such as myelodysplastic syndromes [32] and lack of generalizability to all patients with DLBCL, ASCT consolidation in first remission is an unacceptable standard of care in the R era. Similar encouraging results were seen in the pre-R era by the GELA with the polydrug combination of doxorubicin, cyclophosphamide, vindesine, bleomycin and prednisone (ACVBP) with intensive central nervous system prophylaxis, compared to standard CHOP, in patients over the age

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Beyond R-CHOP of 60 years and with a variety of aggressive lymphomas [33]. In this study, the 5-year OS was improved with ACVBP over CHOP (46% vs. 38%, p ⫽ 0.036). To assess the results in the modern era, a randomized study was performed of R added to ACVBP (R-ACVBP) versus R-CHOP in patients under 60 years old and with low-intermediate risk IPI [12]. R-ACVBP was superior to R-CHOP in both 3-year EFS rates (81% vs. 67%, p ⫽ 0.0035) and 3-year OS (92% vs. 84%, p ⫽ 0.007). The overall survival benefit demonstrated by R-ACVBP in young patients with low-intermediate IPI scores in this study is important, as it confirms that therapeutic improvements beyond R-CHOP are conceptually possible with intensive therapy. However, severe toxicities were more common in the R-ACVBP regimen (42% vs. 15%), and it remains agnostic to molecular subtypes of DLBCL. The hematologic toxicity of R-ACVBP limits its use for older patients and restricts its use as a universal platform from which to add novel targeted therapies. The pharmacodynamically derived dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine and prednisone (DA-EPOCH) regimen was developed from in vitro modeling of drug resistance, and employs infusional drug scheduling with topoisomerase II targeting [34]. The infusional nature and requirements for dose adjustments with the DA-EPOCH-R regimen present more logistical issues than R-CHOP, but the serious toxicity profile is similar, allowing its use in all age groups. In a phase II study at the National Cancer Institute (NCI), 80% and 79% of patients were progression-free and alive, respectively, at the median follow-up of 5 years [35]. The Cancer and Leukemia Group B (CALGB) cooperative group performed a multicenter phase II study of patients aged 23–83 years with newly diagnosed DLBCL treated with DA-EPOCH-R, and reported a 5-year time to progression (TTP) and OS of 81% and 88%, respectively [36]. In this study, the outcomes were also assessed within the GCB and non-GCB molecular subtypes: TTP was 100% in GCB and 67% in non-GCB DLBCL at 62 months [36]. The CALGB has recently completed accrual on a randomized phase III comparison of DA-EPOCH-R and R-CHOP with analysis of outcome within the molecular subtypes of DLBCL [37]. The results of this randomized study should shed light on the most appropriate treatment platform within molecular subtypes of DLBCL.

Entering the modern era: activated B-cell-like and germinal center B-cell-like diffuse large B-cell lymphoma The IPI score was developed in the pre-R era, and does not discriminate on the basis of biologic differences between patients with DLBCL. Molecular subtypes of DLBCL, though, demonstrate groups with different prognosis in addition to distinct underlying biology. Retrospective analysis of patients treated with R-CHOP showed that GCB DLBCL had a better prognosis than ABC DLBCL, with a 3-year OS of 84% compared to 56%, respectively (p ⬍ 0.001) [20]. The basis for this difference is likely due to an overexpression of nuclear factor-κB (NF-κB) target genes that are associated with tumor proliferation and treatment refractoriness.

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ABC DLBCL is characterized by constitutive activation of the NF-κB pathway via multiple mechanisms [38,39]. Staudt et al. first demonstrated the concept of differential sensitivity within molecular subtypes, when they showed that inhibitors of the IκB kinase (IKK) complex were selectively toxic to ABC DLBCL cell lines [40]. Bortezomib is a proteasome inhibitor that blocks degradation of the NF-κB inhibitory protein, IκBα [41]. Dunleavy et al. applied this concept to a clinical study in which bortezomib was combined with DA-EPOCH in patients with relapsed/refractory DLBCL and molecular DLBCL subtypes defined by GEP [42]. Patients with ABC DLBCL had a significantly higher response rate (83% vs. 13%; p ⫽ 0.0004) and median OS (10.8 vs. 3.4 months; p ⫽ 0.0026) compared to GCB DLBCL. These findings provided the initial proof-of-concept that genetically distinct DLBCL subtypes are selectively sensitive to targeted therapies. The concept was further supported in a study of bortezomib combined with R-CHOP in patients with untreated DLBCL, in which the 2-year PFS of GCB DLBCL and non-GCB DLBCL was similar [43]. Ongoing clinical studies testing R-CHOP with and without bortezomib in patients with non-GCB DLBCL are further exploring these findings. A randomized phase III study in the UK is testing R-CHOP compared to R-CHOP with bortezomib (RB-CHOP) in unselected cases of DLBCL [44], and a randomized phase II study of R-CHOP with and without bortezomib in patients with non-GCB DLBCL is ongoing in the USA [45]. Lenalidomide also may have selective activity in ABC DLBCL. Lenalidomide kills ABC DLBCL cell lines by directly inhibiting interferon regulatory factor 4 (IRF4), which is a direct target of NF-κB transcription factors (Figure 1) [46]. In a study of patients with relapsed DLBCL, lenalidomide demonstrated differential efficacy for non-GCB DLBCL, with a response rate of 52.9% in patients with non-GCB DLBCL compared to 8.7% in patients with GCB DLBCL (p ⫽ 0.004) [47]. Two phase I studies have been completed in patients with DLBCL treated with lenalidomide added to R-CHOP, and demonstrated an acceptable toxicity profile, including in patients over the age of 70 [48,49]. A randomized phase II study comparing R2CHOP to R-CHOP is currently enrolling patients of all molecular DLBCL subtypes [50]. Notably, the distinction between GCB DLBCL and ABC DLBCL does not yet guide therapy decisions, and the best practical method(s) for identifying these subtypes remain unclear. While GEP remains the “gold standard,” it has practical limitations for clinical practice, and is not routinely available. Technologic advances that allow GEP to be performed on formalin-fixed, paraffin-embedded (FFPE) tissue are promising but require further validation [51].

Targeting activated B-cell-like diffuse large B-cell lymphoma The mechanisms of constitutive NF-κB activation in ABC DLBCL are varied, and the most effective method of targeting the NF-κB pathway may differ across individual patients. Responses are determined by functional dependence on signaling pathways, and the positions of molecular lesions

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Figure 1. The NF-κB pathway is constitutively activated in ABC DLBCL leading to survival of the cancer cell, but the molecular mechanisms underlying NF-κB activation differ across tumors. Lenalidomide is an immunomodulatory agent with multiple targets that results in downstream inhibition of the NF-κB pathway. The position of the molecular lesions resulting in NF-κB activation in individual patients may be important in more selectively targeted therapies. Ibrutinib is an oral BTK inhibitor that is effective in ABC DLBCL tumors that rely on chronic active BCR signaling, but does not appear to work in tumors that have CARD11 mutations downstream of BTK. Ibrutinib seems to work in tumors with MYD88 mutations if CD79B mutations are coexistent, but is ineffective in patients with MYD88 mutations without CD79B mutations. Sotrastaurin is an oral PKCβ inhibitor that also appears to selectively work in tumors that harbor underlying CD79B mutations. NF-κB, nuclear factor-κB; ABC, activated B-cell; DLBCL, diffuse large B-cell lymphoma; BTK, Bruton’s tyrosine kinase; BCR, B-cell receptor. Figure courtesy of Louis M. Staudt.

within these pathways are critical in predicting the clinical response to targeted agents for ABC DLBCL. Signaling through the B-cell receptor (BCR) is a normal process central to B-cell development. Antigen-independent (tonic) BCR signaling is mediated by phosphatidylinositol 3-kinase (PI3K) [52], and antigen-dependent (chronic active) BCR signaling involves a separate cascade of kinases [53]. After antigen stimulation, CARD11, BCL10 and MALT1 (CBM) form a signaling complex of adaptor proteins that leads to downstream activation of NF-κB, and CARD11 mutations are found in ˜10% of cases of ABC DLBCL (Figure 1) [54]. A promising therapeutic target in lymphomas that rely on chronic active BCR signaling is Bruton’s tyrosine kinase (BTK). Staudt et al. demonstrated that targeting BTK has in vitro activity specific for ABC DLBCL, and was ineffective in cell lines with CARD11 mutations [53]. Point mutations in the B-cell co-receptor CD79B, seen in 21% of cases of ABC DLBCL, also result in “chronic active” BCR signaling that is sensitive to upstream inhibition of the NF-κB pathway [53]. An alternative mechanism of NF-κB activation specific for ABC DLBCL occurs via stimulation of Toll-like receptors (TLRs). Point mutations in MYD88 have been identified in almost 30% of cases of ABC DLBCL, but are rarely observed in GCB DLBCL [55]. MYD88 mutations activate a complex of interleukin-1-associated kinases (IRAK1 and IRAK4) that lead to the production of interleukin-6 (IL-6) and IL-10 with resultant constitutive activation of NF-κB and janus activated kinase (JAK) pathways (Figure 1) [55].

Ibrutinib is a first-in-class inhibitor of Bruton’s tyrosine kinase (BTK) that has demonstrated activity in ABC DLBCL. In a phase I study of patients with relapsed/refractory B-cell malignancies, patients with DLBCL responded to treatment with ibrutinib at various dose levels [56]. A phase II multicenter study tested ibrutinib at 560 mg in heavily pretreated patients with relapsed/refractory DLBCL [57]. Study objectives were to assess whether ibrutinib had differential activity in ABC versus GCB DLBCL, in addition to the role of MYD88, CARD11 and CD79 mutations in predicting clinical responses. Seventy patients were enrolled and there were 29 patients with ABC, 20 with GCB and 21 unclassified/unknown patients, based on GEP. Overall, 23% of the patients responded; 41% of those with ABC and 5% with GCB DLBCL (p ⫽ 0.007), supporting the hypothesis that ABC DLBCL is selectively addicted to chronic BCR signaling [53]. The relationship between individual mutations and overall response rate was also assessed in a pilot mutational analysis of pretreatment and post-treatment biopsies. Clinical responses were observed in patients with ABC DLBCL with both wild-type CD79B (10/29, 34%) and mutated CD79B (5/7, 71%), indicating that ibrutinib does not require a BCR mutation. Interestingly, 80% (4/5) of patients with both mutant CD79B and MYD88 responded, whereas patients with wild-type CD79B and mutant MYD88 did not respond, suggesting a MYD88 independent pathway for NF-κB activation. As hypothesized, patients with CARD11 mutations (n ⫽ 4) did not respond, suggesting that these tumors rely on downstream NF-κB signaling. These results are based on

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Beyond R-CHOP very small numbers, but suggest that BTK is an important target in ABC DLBCL and that clinical responses are dependent on the position of specific mutations. These interesting findings require confirmation in larger series, however. A multi-institutional phase IB trial testing the safety of ibrutinib in combination with R-CHOP for patients with untreated B-cell lymphomas has shown encouraging results, with no unanticipated toxicities observed [58]. An international randomized phase III study of R-CHOP with or without ibrutinib in patients with newly diagnosed non-GCB DLBCL has begun enrollment [59]. Protein kinase Cβ (PKCβ) is a serine/threonine kinase amplified through the BCR signaling pathway and another potential therapeutic target in DLBCL [60]. Enzastaurin is an oral PKCβ inhibitor that demonstrated promising early results in DLBCL, but did not meet its primary endpoint of delaying disease-free survival when added as monotherapy to R-CHOP in patients with high-risk DLBCL [61]. Sotrastaurin, another selective PKCβ inhibitor, has also been evaluated in preclinical models, and mutations in CD79B correlated with sensitivity while CARD11 mutations conferred drug resistance [62]. Sotrastaurin is currently being tested in an international multi-institutional phase I study in patients with relapsed and refractory DLBCL who harbor either a CD79A or a CD79B mutation [63]. The JAK/signal transducer and activator of transcription (STAT) signaling pathway represents another potential therapeutic target for ABC DLBCL, due to the constitutive activation of STAT3 that has recently been correlated with poor overall survival with R-CHOP in this molecular subtype [64]. ABC DLBCLs that demonstrate elevated activity of STAT3 activity demonstrate higher NF-κB activity than those with low STAT3 activity, and selective inhibition of STAT3 signaling is effective in ABC but not GCB cell lines [65,66]. Knockdown of STAT3 expression in mouse models suppresses the growth of ABC DLBCL tumors, validating STAT3 as a target specific to this molecular subtype [67]. Pacritinib (formerly SB1518) is an oral selective JAK2 inhibitor that showed in vitro activity in JAK2-dependent DLBCL cell lines and has demonstrated a favorable safety profile in a phase I study of 34 patients with relapsed/refractory lymphomas, including DLBCL [68]. Numerous other small molecule inhibitors with selectivity for ABC DLBCL are in various stages of clinical development [69–72], and clinical trials investigating their effectiveness in this DLBCL molecular subtype with the worst prognosis are essential.

Targeting germinal center B-cell-like diffuse large B-cell lymphoma GCB DLBCL has a better prognosis than ABC DLBCL, but targeted therapies are needed with selective activity in this molecular subtype. The anti-apoptotic regulator, BCL-2, and the master transcriptional regular of the germinal center reaction, BCL-6, may be amenable to therapeutic exploitation with novel agents. Next-generation sequencing studies have also identified mutations of genes encoding histone modifiers such as CREBBP, EZH2 and MLL2 that serve as rational therapeutic targets [4,73–75].

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BCL-6 is the master regulator of the germinal center reaction and a transcriptional repressor of many target genes involved in cell growth and metabolism, proliferation, apoptosis and the DNA damage response [76,77]. In GCB DLBCL, BCL-6 is overexpressed through chromosomal translocations of the BCL-6 locus with a promoter, as well as multiple somatic mutations. The net result of BCL-6 overexpression is inhibition of apoptosis and DNA damage response, coupled with unchecked proliferation and unregulated cell growth and metabolism. Inhibition of topoisomerase II by etoposide leads to down-regulation of BCL-6 expression through ubiquitin-mediated protein degradation and possibly transcriptional inhibition [78]. This raises the possibility that regimens that more effectively inhibit topoisomerase II would be more effective in GCB DLBCL. Since ABC DLBCL is associated with increasing age [79], one would expect the benefit of etoposide to be concentrated in younger patients. In a population-based study of the Danish Lymphoma Database, R-CHOEP14 was more effective than R-CHOP14 in patients aged 18–60 with high-risk IPI scores [80]. The advantage seen with the DA-EPOCH-R regimen in GCB DLBCL may also be through its maximal inhibition of BCL-6; it incorporates two topoisomerase II inhibitors and optimizes topoisomerase II inhibition through a prolonged 96 h infusion and pharmacodynamic dose adjustment with subsequent cycles [34]. The targeted inhibitor 79-6 specifically disrupts the activity of BCL-6, selectively inhibits co-repressor proteins and induces apoptosis in DLBCL cell lines that were dependent on BCL-6 [81]. Tumor shrinkage was observed in a DLBCL xenograft mouse model following treatment with 79-6, supporting rational development of BCL-6 inhibitors with selectivity for GCB DLBCL [82]. BCL-2 represents a novel therapeutic target that is overexpressed in both GCB and ABC DLBCL, albeit through different mechanisms. BCL-2 expression as a result of t(14:18) is exclusively found in GCB DLBCL [83], and NGS studies have shown BCL-2 to be the most highly mutated gene in GCB DLBCL [84]. Navitoclax (ABT-263) is a BCL-2 inhibitor that demonstrated significant clinical activity in chronic lymphocytic leukemia, but also co-inhibited BCL-xL, leading to dose-limiting thrombocytopenia [85]. ABT-199 is a second-generation BCL-2 inhibitor that has demonstrated activity in early patient studies in a variety of B-cell lymphomas, including DLBCL, without significant thrombocytopenia [86,87]. ABT-199 in combination with bendamustine and R is currently being tested in patients with relapsed DLBCL [88]. The PI3K signaling pathway also represents a rational therapeutic target in GCB DLBCL. Deletions of phosphatase and tensin homolog (PTEN) and amplification of the mir17–92 microRNA (MIHG1 locus cluster on chromosome 13) are molecular mechanisms specific for GCB DLBCL that promote cell proliferation [89,90]. PTEN inactivation was investigated in primary DLBCL samples and found in 55% of cases of GCB DLBCL, but in only 14% of cases of nonGCB DLBCL [90]. PTEN status was inversely correlated with activation of the PI3K/Akt pathway, suggesting constitutive activation of this pathway [90]. A pan-PI3K inhibitor demonstrated selective toxicity in PTEN-deficient GCB DLBCL

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cell lines that was not observed in PTEN-positive GCB DLBCL cell lines [90]. These results give a strong indication that PTEN status could represent a predictive biomarker for cases of GCB DLBCL amenable to inhibition of the PI3K/Akt pathway. Missense mutations within exon 15 of the EZH2 gene have been identified through NGS and found to be specific for lymphomas of germinal center origin [75]. Primary GCB DLBCL samples were analyzed, and 21.7% were found to harbor this EZH2 mutation, while no mutations were found in the ABC DLBCL samples analyzed [75]. The EZH2 mutation appears to work as a gain-of-function mutation, resulting in enhanced trimethylation of a specific histone in B-cell malignancies [91,92]. The functional consequence of this point mutation in NHL is increased histone methyltransferase activity, with resultant increase in proliferation [93]. DLBCL cell lines treated with selective inhibitors of EZH2 exhibit cell-cycle arrest and apoptosis [94,95], and an oral EZH2 histone methyltransferase inhibitor is in early clinical testing [96].

Primary mediastinal B-cell lymphoma: where are we therapeutically? PMBL, a disease that predominantly affects young females, is the third molecular subtype of DLBCL. It arises in the mediastinum from a thymic B cell and shares many clinical and biological characteristics with classical HL. Due to a lack of prospective studies, there has been little consensus on what the optimal therapeutic approach to PMBL should be. Early studies suggested that mediastinal radiotherapy was a critical component of curative therapy, and most strategies today continue to include it [97]. Studies also suggested that more aggressive chemotherapy platforms were associated with an improved outcome [98]. Nonetheless, most patients with PMBL today receive the de facto DLBCL standard, R-CHOP, typically followed by consolidative radiation. The most robust assessment of R-CHOP in PMBL to date was a subgroup analysis of the MInT study of R-CHOP based treatment [99]. Among 44 patients with PMBL the EFS rate was 78% at 34 months, and importantly, 73% of patients received radiotherapy. Although high cure rates in PMBL can be achieved with combined modality therapy, it is apparent from the collective experience in HL that mediastinal radiation has devastating late-term sequelae, particularly an excess risk of breast cancer [100,101]. To obviate the need for routine radiation, Dunleavy and colleagues at the NCI investigated DA-EPOCH-R in PMBL [102]. They treated 51 patients, and the EFS and OS rates were 93% (95% CI 81–98) and 97% (95% CI 81–99) at 5 years’ follow-up. Importantly, only two patients (4%) required radiation, suggesting that this is a highly curable strategy that eliminates the need for routine radiation and removes the risk of its long-term toxicities. To provide confirmatory evidence of this strategy, an international study of DA-EPOCH-R is ongoing in children with PMBL; a group in whom standard approaches have been associated with disappointing survival rates compared to other subtypes of DLBCL.

Novel strategies in PMBL should test targeted agents in combination with the most effective immunochemotherapy platforms, and aim to reduce toxicity while maintaining high cure rates. In 30–50% of cases of PMBL, a region on chromosome 9p24 is amplified – this contains several critical targets such as JAK2 as well as PD-L1 and PD-L2 [103,104]. PD-L1 and PD-L2 are ligands of the programmed cell death-1 (PD-1) pathway that may reflect an ineffective T-cell immune response [105,106]. Rearrangements of the major histocompatibility complex (MHC) class II transactivator (CIITA) are found in 38% of cases of PMBL, and these CIITA gene fusions also result in overexpression of PD-1 ligands [107]. Inhibitors of the JAK/STAT pathway and neutralizing antibodies of PD-1 are rational targeted agents for investigation in PMBL.

Diffuse large B-cell lymphoma with a MYC rearrangement Cases of DLBCL that harbor an underlying MYC rearrangement (MYC⫹ DLBCL) represent a subset of DLBCL with aggressive behavior and poor overall outcomes when treated with R-CHOP [108,109]. Using fluorescence in situ hybridization (FISH), between 6 and 14% of newly diagnosed DLBCLs will have evidence of a MYC rearrangement [108,109]. The British Columbia Cancer agency retrospectively analyzed 135 patients with DLBCL treated with R-CHOP, and found a markedly worse 5-year overall survival of MYC⫹ DLBCL compared to those without the rearrangement (33% vs. 72%) [108]. In a similar retrospective review of 303 patients treated with R-CHOP, a markedly worse 2-year OS (35% vs. 61%) was observed with MYC⫹ DLBCL. Preliminary data suggest that DA-EPOCH-R might be an effective chemotherapy platform for the treatment of MYC⫹ DLBCL [110], and these findings are currently being prospectively evaluated in an ongoing multicenter phase II study of patients with newly diagnosed MYC⫹ DLBCL [111]. Since FISH testing is expensive, time-consuming and not available in all centers, the overexpression of MYC using immunohistochemistry on FFPE tissue has been evaluated, and is associated with a poor prognosis with R-CHOP [112]. In a series of 77 patients, the cut-off of ⬎ 50% nuclear staining of tumor cells identified all cases with an underlying MYC translocation (n ⫽ 15) in addition to other cases without a MYC translocation, suggesting alternative mechanisms of dysregulation. Given the broad range of MYC protein expression, prospective validation of the optimal cut-offs and method for MYC status determination in larger series will be critical. It is currently unknown whether the presence of a MYC rearrangement alone is responsible for the poor prognosis, or whether additional oncogenic alterations such as BCL-2, BCL-6 or CCND1 (“double-hit” lymphomas) that cooperate with MYC dysregulation are the reason for poor outcomes seen with R-CHOP [113,114]. Concurrent expression of BCL-2 and MYC on immunohistochemistry represents a subset of DLBCL with an inferior prognosis, but it is unclear whether this identifies a molecularly distinct group [115–118].

Beyond R-CHOP Direct therapeutic targeting of the MYC protein has been challenging, because it is not amenable to inhibition by small molecules. MYC transcription relies on the regulatory function of bromodomain proteins such as BRD4 [119], however, and small molecules that inhibit the BET family of bromodomain proteins are in development as an indirect method of targeting MYC⫹ tumors [119].

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Conclusions and future directions R-CHOP therapy remains the de facto standard for most patients with newly diagnosed DLBCL. It is highly effective for young patients with a low-risk IPI score, but specific molecular subsets of DLBCL at high risk for treatment failure with R-CHOP are becoming evident. Efforts to improve upon R-CHOP have principally explored dose intensification of chemotherapy in young and fit patients. Hematopoietic stem cell transplant (HSCT) in first remission does not confer an OS benefit, and dose-dense administration of R-CHOP does not provide clear benefit for any subgroup. Highly intensive regimens such as R-ACVBP have demonstrated a survival benefit in young patients with favorable features, but widespread application is limited by its toxicity. DLBCL is a heterogeneous group of diseases with welldefined molecular subtypes that exhibit distinct oncogenic mechanisms, cellular pathway addictions and differential clinical responses to therapy. Achieving the goal of personalized medicine in the genomic era is likely to spawn from exploitation of the biological differences between subsets. Numerous small molecules that target driver mutations and pathway addictions of distinct DLBCL subtypes are at various stages of clinical development. Clinical trials of novel agents should be enriched with the patients most likely to benefit, and evaluate the mechanisms of resistance. Ultimately, the addition of novel targeted agents to the most effective chemotherapy platform within molecular subtypes is the most promising and rational method of moving beyond R-CHOP to effect a cure in patients of all ages with DLBCL.

Acknowledgements All research support comes from the intramural research program of the NIH. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.

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