Double-hit and double-protein-expression lymphomas: aggressive and refractory lymphomas

Review

Double-hit and double-protein-expression lymphomas: aggressive and refractory lymphomas Clémentine Sarkozy, Alexandra Traverse-Glehen, Bertrand Coiffier

Double-hit lymphoma (DHL) is a subgroup of aggressive lymphomas with both MYC and BCL2 gene rearrangements, characterised by a rapidly progressing clinical course that is refractory to aggressive treatment and short survival. Over time, the definition was modified and now includes diffuse large B-cell lymphoma (DLBCL) with MYC translocation combined with an additional translocation involving BCL2 or BCL6. Some cases that have a similar clinical course with concomitant overexpression of MYC or BCL2 proteins were recently characterised as immunohistochemical doublehit lymphomas (ie, double-protein-expression lymphomas [DPLs]). The clinical course of these DPLs is worse than so-called standard DLBCL but suggested by some studies to be slightly better than DHL, although there is overlap between the two categories. Present treatment does not allow cure or long-term survival in patients with genetic or immunohistochemical double-hit lymphomas, but several new drugs are being developed.

Introduction Double-hit lymphoma (DHL) was initially described as a subgroup of diffuse large B-cell lymphoma (DLBCL) with both MYC and BCL2 gene rearrangements.1–4 Anecdotal cases were reported as early as 1998,1 but the first patients were described by Kanungo and colleagues2 in 2006 and by Le Gouill and co-workers3 in 2007. Initially, cases arising in de-novo DLBCL and other aggressive lymphomas or secondary cases arising during the evolution of follicular lymphoma were combined in analyses. Later, the definition of DHL was narrowed to include only de-novo DLBCL.4 Clinically, DHL has dismal survival outcomes, since it progresses rapidly and is refractory to even aggressive therapies. Outcome of patients with DHL was not improved by the addition of rituximab to chemotherapy, unlike socalled standard DLBCL, which can be cured in 60% of patients with chemotherapy and rituximab.5 As for refractory lymphomas, much effort has been made to better characterise DHL to develop new therapeutic possibilities.6 The definition of DHL has further evolved to include aggressive B-cell lymphomas with concurrent abnormalities of MYC and BCL2 in addition to translocations of MYC and BCL2 or MYC and BCL6.7 The problem became more complex as some cases with a similar clinical course were reported to have overexpression of both MYC and BCL2.6 The clinical course of these immunohistochemical DHLs (ie, double-protein-expression lymphomas [DPLs]) is worse than standard DLBCL but different from DHL.8 These new categories of lymphoma, defined by histological and biological characteristics, represent a diagnostic and therapeutic challenge for pathologists and clinicians. In this Review, we describe the clinical and molecular features of these lymphoma subtypes, address when and how these patients should be identified, and discuss therapeutic approaches.

The MYC oncogene and lymphoma MYC was identified 32 years ago and was originally described as the cellular homologue of the viral oncogene www.thelancet.com/oncology Vol 16 November 2015

(v-MYC) of the avian myelocytomatosis retrovirus.9 It is located on chromosome 8q24.10,11 Belonging to a family of transcription factors, including N-MYC, S-MYC, and L-MYC, it encodes the nuclear phosphoprotein MYC, which has both gene-activating and gene-repressing functions depending on its binding partners and the cellular context. The MYC transcription factor regulates 10–15% of the human genome, affecting diverse cellular processes such as apoptosis (eg, by stabilisation of P53 and inhibition of BCL2), proliferation, growth, cell-cycle control, cell migration, and metabolism. Deregulated expression of MYC has oncogenic potential.11 Initially described in Burkitt’s lymphoma,12 MYC deregulation is implicated in 20% of all human malignancies and is often associated with poor prognosis. Deregulation of MYC is an important driver of oncogenic transformation, enhancing cell proliferation, growth, angiogenesis, genomic instability, and cellular metabolism, and inhibiting cell differentiation. MYC is upregulated by translocation or other mechanisms, such as gene gain or amplification, mRNA overexpression, aberrant somatic mutation, or microRNA deregulation (table 1), resulting in gene deregulation and protein accumulation. When overexpressed in mouse B cells, Myc can induce lymphomas, but the tumour type depends on the stage of B-cell differentiation at which Myc is expressed and on the genomic context.11 The oncogenic activity of MYC and its target genes depends on the tumour type. The translocations t(8;14)(q24;q32) and variants t(2;8) or t(8;22) involving MYC and an immunoglobulin gene are characteristic of, and were first identified in, Burkitt’s lymphoma.13 In this lymphoma subtype, MYC rearrangement occurs only with an immunoglobulin gene, more often at the heavy-chain locus (14q32) than the light-chain kappa (2p12) or lambda (22q11) loci, and is usually the sole chromosomal aberration or associated with few additional alterations in genes such as TP53, TCF3, or IDH3. However, MYC rearrangement also occurs in other lymphomas, such as DLBCL, follicular lymphoma, mantle-cell lymphoma, and occasionally in de-novo acute

Lancet Oncol 2015; 16: e555–67 Haematology Department (C Sarkozy MD, Prof B Coiffier PhD) and Service d’Anatomie Pathologique (Prof A Traverse-Glehen PhD), Hospices Civils de Lyon, Lyon, France; and University Lyon 1, Lyon, France (C Sarkozy, Prof A Traverse-Glehen, Prof B Coiffier) Correspondence to: Prof Bertrand Coiffier, Haematology Department, Centre Hospitalier Lyon-Sud, 69310 Pierre-Benite, France [email protected]

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Diffuse large B-cell lymphoma

B-cell lymphoma unclassifiable

BCL2 rearrangement

30–40%

78%

BCL6 rearrangement

30%

ND

5–17%

MYC translocation

35–90%

Burkitt’s lymphoma 0% 0% 90–100%

BCL2–MYC rearrangement

58–85%

47–60%

0%

BCL6–MYC rearrangement

5–8%

ND

0%

7–16%

ND

BCL3–MYC rearrangement

0%

BCL2–BCL6–MYC rearrangement

16%

ND

ND

Translocation partners

Most frequently non-IG (35–53%)

Most frequently non-IG (38%)

IG (100%)

MYC copy-number gains (3–4 copies)

21–38% 2%

MYC amplification (>4 copies) MYC aberrant somatic mutations

32%

ND

ND

ND

Rare

20%

Frequent

IG=immunoglobulin. ND=no data.

Table 1: Frequency of common genetic abnormalities in aggressive lymphomas

lymphoblastic lymphoma or leukaemia and plasmablastic lymphoma. In DLBCL, MYC is the third most commonly translocated oncogene (in 5–17% of cases).14 By contrast with Burkitt’s lymphoma, in other aggressive lymphomas, MYC rearrangement is a secondary event with complex chromosomal abnormalities, involving immunoglobulin genes (50–65%; usually light chain) or nonimmunoglobulin gene (eg, BCL6, BCL11A, PAX5, IKAROS) partners, and is frequently associated with translocation of BCL2 or BCL6 (DHL), or both (triple-hit lymphoma [THL]). Most cases of DHL have MYC and BCL2 rearrangements (58–85%), rather than rearrangements involving MYC and the BCL6 (5–8%) or BCL3 (7–16%) loci.15–18 With immunoglobulin genes, the translocation breakpoints occur at the 5ʹ end of MYC for a heavy-chain gene (IGH) and the 3ʹ end for a light-chain gene (IGL).19 Other MYC abnormalities in DLBCL are copy-number gains (3–4 copies; 21–38%), amplifications (>4 copies; 2%), and aberrant somatic mutations (32%).15,20 The mutation hotspots are concentrated in activationinduced cytidine deaminase recognition motifs within 2 kb from the transcription start site, suggesting similarities with the mechanisms at the initiation of MYC rearrangement.21 MYC aberrant somatic mutations are more frequently seen in cases with MYC rearrangement. The chromosomal translocation t(14;18)(q32;q21) juxtaposes the antiapoptotic oncogene BCL2 (18q21) with the IGH enhancer (14q32), resulting in deregulated expression of BCL2. It is the most frequent translocation in germinal centre-like DLBCL (30–40%) but not as frequent in the activated B-cell subtype (<30%).8 Translocation at 3q27, where the BCL6 gene is located, is also a common genetic abnormality in DLBCL (table 1).

Histological presentation Few studies have analysed the pathological features of DHL, which include a range of morphological features (figure 1) that are shared with DLBCL and B-cell lymphoma e556

unclassifiable (BCL-U), which in turn have features intermediate between Burkitt’s lymphoma and DLBCL.16,22 Patients with concurrent MYC and BCL2 abnormalities often present with features or a history of follicular lymphoma, suggesting that additional deregulation of MYC via formation of a secondary MYC translocation is a means of high-grade transformation in this lymphoma subtype.23 Additionally, two series14,24 have shown that patients with DLBCL with immunoblastic morphology have MYC translocations more frequently than patients with DLBCL without immunoblastic morphology (33–39% vs 7%). By contrast with other DLBCLs, the translocation partner of MYC in immunoblastic DLBCL is always IGH. CD10 is frequently expressed in patients with immunoblastic DLBCL who have MYC rearrangement (62% vs 15% of those without such rearrangements). BCL-U is a provisional 2008 WHO category of aggressive lymphoma defined by morphological characteristics and was initially introduced to define a category for intermediate Burkitt’s lymphoma discovered molecularly by gene expression profiling.25 It remains a heterogeneous category, including suspected Burkitt’s lymphoma cases for which at least one essential reason not to make the diagnosis exists.25 Compared with Burkitt’s lymphoma, BCL-U typically has more cellular pleomorphism with irregular nucleoli and fewer intermediate-sized cells, few nucleoli and either no or partial so-called starry sky appearance, absence of amphophilic cytoplasm, and coagulative necrosis, whereas DHL cells have no specific characteristics. Like Burkitt’s lymphoma, most cases are CD10 positive and half are BCL2 positive. Ki67 positivity is usually less than 95%. MYC rearrangement can be detected in 35% of these cases, and 60% have a concurrent BCL2 or BCL6 rearrangement26 with a complex karyotype in addition to MYC rearrangement (table 1). As in DLBCL and by contrast with Burkitt’s lymphoma, MYC is frequently translocated to IGL or to a non-immunoglobulin gene partner in BCL-U. The frequently reported occurrence of MYC and BCL2 rearrangement in BCL-U is biased because, in some studies, this double-hit criterion has been used to define BCL-U. Major pathological studies of DHL have focused on the cell of origin (germinal centre or activated B cell), BCL2 expression, and proliferation index. Most cases of BCL2–MYC DHL are of germinal centre origin (CD10+, BCL6+, MUM1–) and are highly proliferative. By contrast, BCL6–MYC DHL is more frequently reported to be of activated B-cell origin, more likely to be immunoblastic, contains extranodal sites, infrequently expresses BCL2, and is cytogenetically less complex than BCL2–MYC DHL.27 THLs are defined as lymphomas with translocation or rearrangement of both MYC and BCL2, in addition to another gene, usually BCL6. These cases are rare and very few studies are available. A review of published cases28 shows that the morphology is heterogeneous, with a small subset of patients www.thelancet.com/oncology Vol 16 November 2015

Review

presenting with concurrent or a history of follicular lymphoma. Of 11 cases reported, all patients were men, and seven (64%) had de-novo disease. Ten (91%) had aggressive lymphoma (five had BCL-U, four had DLBCLs, and one had DLBCL with concurrent follicular lymphoma), and one (9%) had low-grade follicular lymphoma.

MYC protein overexpression and MYC–BCL2 double expression MYC protein overexpression occurs in 29–47% of DLBCL cases, independent of the presence of MYC rearrangement, Haematoxylin and eosin staining

and prognosis is particularly poor if this MYC overexpression is accompanied by BCL2 protein overexpression.8,15,29,30 Although MYC protein expression occurs in nearly half of DLBCL cases, the number of positive cells varies widely, and the prognostic value depends on how MYC overexpression is defined (ie, the cutoff for the percentage MYC-positive cells). Generally, a cutoff of more than 40% positive cells is used to define MYC overexpression.10 Additionally, several studies have attempted to correlate MYC overexpression with the presence of MYC rearrangement by fluorescence in-situ hybridisation (FISH) analysis using break-apart and Ki67

MYC expression

DLBCL with immunoblastic morphology

DLBCL with immunoblastic morphology

BCL-U with morphological features intermediate between Burkitt’s lymphoma and DLBCL

DLBCL with centroblastic morphology

Figure 1: Heterogeneous morphology and phenotype in four aggressive lymphomas with MYC protein expression and MYC translocation BCL-U=B-cell lymphoma unclassifiable. DLBCL=diffuse large B-cell lymphoma. These lymphomas have distinct morphology in haematoxylin and eosin staining (magnification ×200). Corresponding proliferation index assessed by Ki67 and MYC expression by immunohistochemistry (magnification ×400) are shown.

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fusion probes.30 The detection of a high number of MYCpositive cells correlates well with gene rearrangement;29 however, a small number of MYC-positive cases (19%) do not have MYC gene alterations.15,29 Furthermore, not all patients with MYC rearrangement overexpress the protein,29 although this is controversial and the data are contradictory because of different cutoff values to define overexpression of MYC and methods used. Most studies using a cutoff of 40–70% MYC-positive cells have shown MYC overexpression to be associated with MYC gene rearrangement; nevertheless, the more cells with positively stained nuclei, the higher the probability of detecting MYC rearrangement.31 However, the use of immunohistochemistry to define protein overexpression and correlate with gene alterations has several issues, including heterogeneous staining and low specificity or positive predictive value compared with FISH analysis, despite good sensitivity and negative predictive value.31 The association between MYC overexpression and gene rearrangement remains to be clarified. 20 years ago, the adverse prognosis of MYC overexpression was noted to worsened when it coexists with BCL2 overexpression (ie, in DPLs).32 Importantly, DPL is more common than, and different from, DHL and THL, even though 80–90% of patients with DHL or THL also express both MYC and BCL2.29,33 Roughly 19–34% of DLBCL and 50% of BCL-U cases are initially diagnosed as DPLs.8,15,29,33,34

Molecular studies Most gene expression profile analyses concur that the majority of DHLs originate from germinal centre cells.4,14,35–37 Such analyses showed that all MYC-translocated lymphomas have similar gene expression profiles to singlehit lymphomas (which only contain MYC rearrangements) and DHLs, and share several gene expression patterns with lymphomas with immunoglobulin–MYC translocations but not non-immunoglobulin–MYC translocations.14 Comparison of single-hit lymphomas and DHLs showed no difference in MYC translocation partner (immunoglobulin vs non-immunoglobulin), genomic complexity, and MYC expression.17 Next-generation sequencing studies have identified several overlapping mutations between Burkitt’s lymphoma and DLBCL. TP53 mutations are frequent in MYC–BCL2 DHL but not in MYC–BCL6 DHL, suggesting a difference at the molecular level that has pathological implications for the role of p53 and potential synergistic effects with BCL2 overexpression.38 TCF3 and ID3 mutations initially described in Burkitt’s lymphoma have also been reported in BCL-U and DLBCL with MYC rearrangement.39 Moreover, ID3 mutations are recurrent events in DHL and are observed at a frequency intermediate between that of DLBCL and Burkitt’s lymphoma, suggesting that there might be a common lymphomagenesis pathway for a subset of patients with these diseases. e558

Detection of MYC rearrangement or overexpression In view of its prognostic effects, detection of MYC aberrations is becoming increasingly important in routine clinical practice, but identification of cases that need to be further investigated is difficult. Although most cases are of a germinal centre origin and have a high Ki67 index, the presence of MYC deregulation by translocation or other mechanisms cannot be identified by the morphology or immunophenotype but require classical cytogenetics by karyotyping or FISH. Both methods might miss the translocation or rearrangement, but FISH seems to be more sensitive than karyotyping.40 FISH studies can be done in paraffin-embedded tissues; break-apart probes are the most sensitive but can result in false negatives. Although there is no consensus, MYC and BCL2 gene analysis by FISH (ideally with a MYC break-apart probe followed by BCL2 and then BCL6 probes) and protein expression detection by immunohistochemistry are recommended in all patients with DLBCL.40–43 The MYC–IGH dual-fusion probe can also be useful in cases that are negative for MYC rearrangements by FISH (10%) if more than 50% of cells stain positive for MYC using immunohistochemistry.

Clinical presentation Patients with DHL do not have a specific clinical presentation (table 2) that is distinct from other DLBCLs or aggressive non-Hodgkin lymphoma.41,46 However, various studies report higher frequency of stage III/IV disease, bone marrow involvement, elevated lactate dehydrogenase (LDH), extranodal disease including CNS involvement, and higher International Prognostic Index (IPI) score in DHL than in other lymphoma types.3,16,22,35,36,44,47,48 The relation between DHL and DPL was investigated in two retrospective studies8,29 comparing clinical characteristics and outcome. Green and colleagues8 included 193 patients with DLBCL, of whom 54 (28%) had DPL (using cutoffs of >40% and >70% positive cells, respectively, to define MYC and BCL2 overexpression by immunohistochemistry), and 11 (6%) had DHL by FISH. 9 (90%) of 10 of these patients with DHL, who could be assessed for protein expression, also had overexpression of MYC and BCL2 by immunohistochemistry. No significant differences were noted among initial clinical characteristics of DHL and DPL, except for the germinal centre subtype (91% of DHL vs 37% of DPL). Johnson and colleagues29 used a cutoff of more than 40% positive cells and 50% positive cells, respectively, to define MYC and BCL2 overexpression. 55 (18%) of 307 patients had DPL and 10 (77%) of 13 DHL (defined by FISH) tested also had concurrent overexpression of both MYC and BCL2. Patients with DHL more frequently had higher LDH, higher IPI, and lower performance status than those with DPL, but both groups had similar disease stage and number of www.thelancet.com/oncology Vol 16 November 2015

Review

The poor prognosis related to MYC rearrangement varies with its translocation partners, and various studies report a poor outcome for immunoglobulin translocations but not non-immunoglobulin translocations.16,17,44 However, in other reports, outcome was not affected by the translocation partner (immunoglobulin vs nonimmunoglobulin) of MYC rearrangement.14 In older reports, including those of patients treated without rituximab, the median overall survival was less than 6 months, and patients with THL had poorer overall survival than those with DHL.3,44 In the rituximab era, the median overall survival remains short, ranging from 5 months to less than 2 years (table 3). Some studies have suggested that few initial characteristics could be used as prognostic markers, and two reports16,37 showed that elevated LDH, two or more extranodal sites, bone marrow or CNS involvement, and IPI greater than 2, but not histological subtype, were associated with poor outcome. In a large study of 129 patients,22 performance status at diagnosis and bone marrow involvement were the strongest prognostic markers. Median event-free survival and overall survival were 8 months and 18 months, respectively, and were similar across disease stage (except for stage I in complete response) and age. In the largest published study in the rituximab era of patients with DHL (n=311), median progression-free survival and overall survival were 10·9 months and 21·9 months, respectively;36 advanced stage, LDH more than three times the upper normal limit, CNS involvement, and leukocytosis were prognostic factors for overall survival in multivariate analysis. Studies focusing on the outcome of THL are rare and consist mainly of case reports. Treatment response and

extranodal sites. An activated B-cell phenotype was reported in 76% of patients with DPL and 36% of patients with DHL, whereas 24% of patients with DPL and 64% of patients with DHL had a germinal centre phenotype. In another large-scale analysis, Hu and colleagues33 described DPL treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and used the same cutoff values for protein overexpression as Green and colleagues8 (ie, 40% positive staining for MYC and 70% for BCL2). Of 466 patients, 157 (34%) had DPL and ten (3%) had DHL. Furthermore, 124 (32%) of 384 cases that did not have MYC or BCL2 gene rearrangements had overexpression of MYC and BCL2. In accord with the results of Johnson and colleagues, this study also found that patients with DPL more frequently had an activated B-cell subtype (66% vs 39% for other patients). Compared with patients with DLBCL that did not have overexpression of MYC and BCL2, patients with DPL had worse performance status, more stage III/IV diseases, multiple extranodal sites, higher IPI, and higher proliferation index, but no differences in sex, TP53 mutations, LDH, or tumour size.

Clinical outcome of patients with DHL or DPL Although MYC rearrangement alone is usually associated with a poor outcome for patients with DLBCL,8,15,18,30,33,43,49–53 some exceptions exist.8,29,30,54,55 Horn and colleagues30 showed that, BCL2 or BCL6 rearrangements did not affect progression-free survival or overall survival. One study found that patients with germinal centre subtype DLBCL with BCL2 translocations have a worse prognosis following treatment with R-CHOP, irrespective of MYC status.54 Number of cases

Stage III/IV LDH elevated

CNS

Bone marrow

Extranodal disease

Ki67

Germinal Centre (IHC)

IPI >1

Le Gouill et al3

16 DHL

100%

100%

50%

93%

87% >1

70–90%

100%

Lin et al4

23 DHL

>75%

NR

NR

75%

74%

60–100%

NR

Johnson et al16

54 DHL

76%

50%

NR

59%

35%

NR

NR

70%

Niitsu et al35

19 DHL

100%

100%

NR

84%

89%

50–90%

84%

89%

Tomita et al44

27 DHL*

96%

93%

56%

70%

93%

<90%

89%

87%

Landsburg et al40

17 DHL

88%

94%

NR

60%

53%

80%

90%

79%

Oki et al22

129 DHL

84%

69%

10% (tested)

42%

49% >1

85% (40–100)†

93%

87%

NR

58%

NR

83%

NR

Petrich et al36

311 DHL

Aukema et al14

47 DHL

Cohen et al45 Johnson et al29 Green et al8 Hu et al33

81%

76%

NR

NR

NR

7%

29 DHL

93%

NR

55 DPL 13 DHL

63% 77%

55%‡ 86%

54 DPL 11 DHL

56% 82%

157 DPL

67%

81% (aaIPI3) NR

41%

60%

NR

NR

NR

21%

NR

90% (50–100)†

NR

NR NR

NR NR

21% 29%

65% (>90%)†‡ 93%

24%‡ 64%

76%‡ 92%

56% 73%

NR NR

NR NA

20% >1 27%

NR NR

37% 91%

41% 64%

64%

NR

NR

28% >1

74% (>70%)†

34%

49%

Median 90%

100%

Johnson et al and Green et al compared initial characteristics of DPL and DHL cases. Other reports focused on either DHL or DPL cases. LDH=lactate dehydrogenase. CNS=CNS involvement. IPI=International Prognostic Index. DHL=double-hit lymphoma. DPL=double-protein-expression lymphoma. aaIPI3=age-adjusted IPI, score 3. NR=not reported. *22 cases at diagnosis. †The range of Ki67 expression is shown in parentheses. ‡Significant difference between DPL and DHL. 29

8

Table 2: Clinical characteristics of DPL and DHL at presentation

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DPL or DHL

Regimen

Median OS or PFS

Prognostic factors and comments

Le Gouill et al3

DHL

R-CHOP, COPADM, ASCT, ALLO

N (%)* 16 (NA)

OS 5 months for all patients

NA

Tomita et al44

DHL

CHOP, CODOX-M/IVAC, or hyperCVAD, with or without rituximab

27† (NA)

OS 6 months

Triple-hit lymphoma is a marker of poor prognosis

Johnson et al16

DHL

High-dose chemotherapy, CHOP, R-CHOP

54 (4%)

OS 1·4 years with rituximab, 0·4 years without rituximab

IPI, BM, BCL2, IG–MYC; benefit of rituximab but not of high-dose chemotherapy

Li et al37

DHL

R-CHOP

57 (NA)

OS 18·6 months

LDH, BM, CNS, IPI

Horn et al30

DHL and DPL

R-CHOP

21 (4·7%)

3-year EFS 15·6%, 3-year OS 41·6% (combined IHC and FISH score high)

Combined IHC and FISH score: 15% of patients had highrisk score defined as MYC high, BCL2 high, and BCL6 low

Oki et al22

DHL

R-CHOP, R-hyperCVAD, DAEPOCH-R

129 (NA)

3-year EFS 33%, OS 4%

ECOG performance status, BM

Petrich et al36

DHL

R-CHOP, R-hyperCVAD, DA-EPOCH-R, R-CODOX-M/IVAC

311 (NA)

PFS 10·9 months, OS 21·9 months for all regimens

LDH, stage, CNS, leucocytosis; intensive chemotherapy improves PFS but not OS; ASCT and ALLO: no effect on OS

Sun et al48

DHL

R-CODOX-M/IVAC

32 (NA)

2-year PFS 41% 2-year OS 53%

ASCT and ALLO: 2-year OS 82%

Cohen et al45

DHL

R-CHOP, DA-EPOCH-R, R-HyperCVAD, R-CODOXM/R-IVAC

29 (NA)

PFS 8 months OS 12·5 months

CR and age

Pederson et al17

DHL

R-CHOP (81%)

23 (10%)

1-year OS 60%

No effect of DHL on OS. IG–MYC rearrangement as a marker of poor prognosis

Johnson et al29

DPL DHL

R-CHOP

55 (19%) 14 (5%)

5-year OS 36% 5-year PFS 27%

DHL have poorer OS than DPL

Green et al8

DPL DHL

R-CHOP

54 (21%) 11 (6%)

OS 24 months OS 13 months

Prognostic value of MYC and BCL2 coexpression is independent of COO or IPI score

Valera et al15

DPL

R-CHOP

32 (15%)

PFS 18 months, OS roughly 30 months MYC overexpression is an independent prognostic factor, not BCL2

Friedberg et al56

DPL

R-CHOP + iodine-131 tositumomab

13 (20%)

2-year PFS 58%

Prognostic value of MYC and BCL2 coexpression is independent of COO or IPI score

Perry et al57

DPL

NA

47 (44%)

2-year OS 58%, EFS roughly 52%

The poor prognostic value of MYC and BCL2 coexpression is independent of IPI score and COO in a COX regression model

Hu et al33

DPL

R-CHOP

55 (18%)

5-year OS 30% PFS 27%

DPL is a better prognostic factor than COO

DPL=double-protein-expression lymphoma. DHL=double-hit lymphoma. OS=overall survival. PFS=progression free survival. R-CHOP=rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. COPADM=cyclophosphamide, vincristine, prednisolone, doxorubicin, and methotrexate. ASCT=autologous stem cell transplant. ALLO=allogeneic transplant. NA=not available. CODOX-M/ IVAC=cyclophosphamide, doxorubicin, vincristine, methotrexate/ifosfamide, etoposide, and high-dose cytarabine. HyperCVAD=hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone. IPI=International Prognostic Index. BM=bone marrow involvement. IG=immunoglobulin. LDH=lactate dehydrogenase. CNS=CNS involvement. EFS=event-free survival. IHC=immunohistochemistry. FISH=fluorescence in-situ hybridisation. DA-EPOCH-R=rituximab and dose-adjusted etoposide, prednisone, vincristine, and doxorubicin. ECOG=Eastern Cooperative Oncology Group. CR=complete response. COO=cell of origin. *The percentage of patients with the disease in the study is shown in parentheses. †Some patients had DHL or DPL diagnosed at relapse.

Table 3: Outcome of chemotherapeutic regimen for DHL and DPL in retrospective series

prognosis are poor worldwide, and more than 90% of patients relapse or are refractory to first-line regimen, with overall survival shorter than 1 year.28 The prognostic value of double-protein expression has been assessed in various retrospective analyses. Green and colleagues8 showed that DPL was significantly associated with lower complete response, shorter overall survival, and shorter progression-free survival than was de-novo DLBCL, independently of IPI and cell of origin. Johnson and colleagues29 showed that MYC and BCL2 coexpression, but not MYC overexpression alone, was associated with poor overall survival in patients with DLBCL. In multivariate analysis, the outcome remained significantly worse for overall survival but not progression-free survival, and was e560

independent of IPI and cell of origin. This poor outcome remained when DHL (5%) were excluded, but DPL had a better outcome than DHL (figure 2). Hu and colleagues33 showed that patients with DPL who were given R-CHOP also had a lower complete response rate, shorter 5-year overall survival, and shorter 5-year progression-free survival than did patients with non-DPL. This poor outcome of DPL was independent of cell-of-origin subtypes, IPI score, presence of B-cell symptoms, and presence of TP53 mutation. Furthermore, DPL had a noticeable predilection for the activated B-cell subtype, and this subtype was no longer associated with inferior overall survival or progressionfree survival after excluding all DPL cases. Therefore, in www.thelancet.com/oncology Vol 16 November 2015

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Treatment strategies in DHL and DPL The poor outcome of DHL and DPL is related to chemotherapy refractoriness. Overall and progression-free survival curves show the poor outcome after relapse. Nevertheless, some patients who achieve complete response after first-line treatment achieve substantial overall survival times after reaching complete response with first-line treatment, which should be the ultimate goal.45 DHL and DPL might have different pathological mechanisms. For DHL, the genetic alterations induce proliferation and loss of control from homoeostatic genes. Therefore, targeting the MYC and BCL2 pathways might successfully control proliferation and induce responsiveness to therapeutic agents. By contrast, patients with DPL might not have genetic alterations in MYC and BCL2 but do have protein overexpression, suggesting that alternative pathways are involved and therapeutic inhibition of these alternative pathways is required.

100

Classical DLBCL (n=236) MYC+/BCL2+ (n=55) DHL (n=14)

80 Overall survival (%)

most studies, the adverse outcome for patients with the activated B-cell subtype seems to be related to the presence of MYC plus BCL2 overexpression. This adverse outcome for DPL was not confirmed in a randomised study by Molina and colleagues.58 In patients included in a prospective randomised trial (RICOVER30), a combined immunohistochemistry and FISH score based on MYC rearrangements, MYC and BCL2 overexpression, and reduced BCL6 expression identified a subset (15% of the trial patient population) of patients with particularly poor prognosis. Perry and colleagues57 showed that of 106 patients treated with R-CHOP-like regimens, 17 patients had DPL using the cutoff value of 30% for BCL2 and 50% for MYC. By contrast with other reports, these 17 patients more frequently had a germinal centre subtype (46·0%) than non-germinal centre subtype (41·5%) but also had poor overall survival (median around 4·5 years vs not reached for other categories), whereas patients with overexpression of either MYC or BCL2 had an intermediate outcome. This predictive value was discriminant in the germinal centre subgroup but not in patients with the non-germinal centre subtype. These results were validated in an independent cohort of 205 patients with DLBCL who were given R-CHOP, including 47 with DPL.

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Figure 2: Double-protein-expression lymphoma has an intermediate outcome between DHL and classical DLBCL DHL=double-hit lymphoma. DLBCL=diffuse large B-cell lymphoma. Reproduced with permission from Johnson and colleagues.28

classical DLBCL,5 and 2-year progression-free survival and overall survival were only 25% and 41%, respectively. For patients with DPL, 5-year overall survival was 36% and 5-year progression-free survival was 32%, compared with 71% and 65%, respectively, for patients with classic DLBCL.29,33 Perry and colleagues57 reported a risk of death that was nine times greater for patients with DPL than for patients with non-DPL DLBCL who were given R-CHOP. With respect to risk of CNS disease, one study59 showed that intrathecal prophylaxis lowers the risk of developing CNS disease at 3 years, and another group further showed that intravenous high-dose methotrexate is more efficient than intrathecal prophylaxis; thus, the introduction of two cycles of high-dose methotrexate after a rituximab plus doxorubicin-based strategy is recommended.60 As R-CHOP fails to achieve good responses or to reduce the numbers of early relapses or refractory disease, alternative therapies are being investigated. The incorporation of iodine-131 tositumomab consolidation was assessed in the SWOG SO433 trial, in which 13 patients with DPL had a 2-year progression-free survival (58% [95% CI 27–80]) that was inferior to that of patients with non-DPL (estimate 73% [59%–83]); these results are nevertheless encouraging in view of other retrospective series.56

Intensive strategies in de-novo DHL and DPL R-CHOP regimen in patients with de-novo DHL and DPL Although better than CHOP alone, the R-CHOP regimen does not seem to give satisfying results in all DHL series. Johnson and colleagues16 showed that, for 11 patients with DHL who received R-CHOP, the median overall survival was 1·4 years, compared with 0·4 years for patients who received CHOP. Oki and colleagues22 showed that, for 57 (45%) of 127 patients with DHL who were given R-CHOP, only 23 (41%) patients achieved complete response, a much lower proportion than that in www.thelancet.com/oncology Vol 16 November 2015

One study61 has shown that rituximab and dose-adjusted etoposide, prednisone, vincristine, and doxorubicin (DA-EPOCH-R), an intensive regimen, gives comparable outcomes for patients with DLBCL or BCL-U that had MYC rearrangements relative to patients with DLBCL without MYC rearrangements (4-year event-free survival 83% vs 76%). Moreover, this regimen also improves outcome in patients with DHL compared with treatment with R-CHOP,22 with higher complete response rate (68% vs 40%), longer progression-free survival (67% vs 25% at e561

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2 years; 67% vs 20% at 3 years), and longer overall survival (76% vs 41% at 2 years; 76% vs 35% at 3 years). Petrich and colleagues36 showed that 64 patients given DA-EPOCH-R had better overall and progression-free survival than 100 patients who received R-CHOP. Additionally, other intensive regimens such as R-hyperCVAD (rituximab plus hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) or R-CODOX-M/IVAC (rituximab plus cyclophosphamide, doxorubicin, vincristine, methotrexate/ifosfamide, etoposide, and high-dose cytarabine) have higher complete response rates and longer progression-free survival than R-CHOP.36 However, thus far these analyses have been retrospective and therefore have inherent limitations that compromise definitive conclusions. Compared with DA-EPOCH-R, the R-hyperCVAD regimen resulted in higher complete response rates (68%) but shorter progression-free survival (32% at 2 and 3 years) and overall survival (44% at 2 years and 40% at 3 years). In the same study, R-ICE (rituximab plus ifosfamide, carboplatin, and etoposide), R-DHA (rituximab plus aracytine and high-dose dexamethasone), R-bendamustine, and R-CEOP (rituximab plus epirubicin, vincristine, prednisone, and etoposide) had very poor results (event-free survival <10%; complete response 60%), but some patients in this study had a history of lowgrade lymphoma and were more challenging to treat than those with true DLBCL.22 Importantly, in this study of 20 patients with localised disease, six (30%) had early progression, suggesting a more aggressive disease than classical localised DLBCL. In the largest retrospective analysis (n=311),36 this intensified regimen of R-hyperCVAD/MA (rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone/methotrexate, cytarabine) was given to 21% of patients and was associated with longer progression-free survival (median 21·6 vs 7·8 months [p=0·001]) but not with longer overall survival relative to R-CHOP. However, no differences were seen between the intensive regimens (figure 3). In a retrospective analysis of 32 patients with DHL,48 R-CODOX-M/IVAC was given to 25 (78%) patients (the others received R-CHOP-like regimens), of whom 20 (80%) reached partial response or better and nine (36%) had a complete response. Consolidation haemopoietic stem cell transplant treatment was given to 19 (86%) of 22 patients who achieved complete remission after induction therapy (11 autologous stem cell transplant, seven matched unrelated donor transplant, and one matched sibling allogeneic transplant). Median overall survival and progression-free survival after diagnosis for all patients were 28 months and 11 months, respectively. The proportion of patients without disease progression at 2 years was 41%, and 53% of all patients were alive at 2 years. Of the patients who received a transplant, 82% were alive at 2 years, and 60% did not have disease progression at 2 years. This suggests that if the lymphoma can be controlled until transplantation, survival can be e562

prolonged. However, progression before transplantation remains an issue, and Li and colleagues37 showed no difference in complete response between R-CHOP (n=19) and a more aggressive regimen (mainly R-hyperCVAD; n=30) before autologous stem cell transplantation. In summary, several retrospective analyses and one trial suggest that regimens that are more intensive than R-CHOP might improve outcome for patients with DHL. Fewer studies have investigated intensive therapy in patients with DPL. In the LYSA LNH 03-2B trial,58 patients with DLBCL were randomised between standard R-CHOP and the more aggressive R-ACVBP (rituximab, doxorubicin, cyclophosphamide, vindesine, bleomycin, and prednisone) regimen. R-ACVBP improved the outcome of patients with the non-germinal centre subtype compared with R-CHOP, but no difference in outcome of patients with DPL was reported. However, the authors noted that most DPLs are of the non-germinal centre cell subtype, and patients with non-germinal centre cell subtype DLBCL fared worse than those with germinal cell subtype DLBCL, but statistical power was insufficient to test differences in patients with DPL because of small patient numbers.

Autologous stem cell transplant and allogeneic transplant For DHL, most reports have not shown any benefit of autologous stem cell transplantation or allogeneic transplantation in patients who achieved complete response with initial therapy,16,22,36,37 but this finding remains inconclusive because of the selection bias associated with retrospective analyses. However, in a study48 of 16 patients given CODOX-M/IVAC-R followed by autologous transplant, 13 (82%) achieved 2-year overall survival. The SWOG S9704 study62 prospectively randomised patients with aggressive non-Hodgkin lymphoma to receive consolidation with further R-CHOP or an autologous stem cell transplant. A subset analysis of three patients with DHL and 16 patients with DPL showed that autologous transplantation seems to improve outcome (estimated 2-year progression-free survival 63% vs 19%; overall survival 63% vs 27%). However, this analysis was limited by small numbers compounded by the fact that nearly a third of patients with MYC overexpression did not receive consolidation at all because of early progression or death.63

Salvage strategies Alongside the high frequency of chemotherapyrefractory disease and the poor benefit of stem cell transplant in first complete response, chemotherapybased salvage strategies are similarly disappointing for this disease. Cuccuini and colleagues64 first reported that classical salvage treatment with R-ICE or R-DHAP (rituximab plus ribdexamethasone, cytarabine, and cisplatin) followed by an intensification with BEAM (BCNU, etoposide, cytarabine, and melphalan) and autologous stem cell transplant was not successful in 28 patients with DLBCL who had MYC rearrangements. www.thelancet.com/oncology Vol 16 November 2015

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21 of these patients had DHL, and 4-year progressionfree survival (18% vs 42% [p=0·0322]) and overall survival (29% vs 62% [p=0·0113]) were significantly lower than those of 133 patients without MYC rearrangements. Oki and colleagues22 showed that 3-year overall survival after progression was 7% for patients with DHL, and 15% for patients who also received a stem cell transplantation.

Development of new agents Agents modulating the transcription of MYC, BCL2, or BCL6 Epigenetic alterations—caused by somatic mutations in genes encoding histone modifiers such as MLL2, EZH2, EP300, and CREBBP—have a key role in DLBCL pathology. Bromodomain proteins can recognise specific histone modifications and function as epigenetic readers to contribute to the pathogenesis of DLBCL, and preclinical reports of bromodomain inhibitors are encouraging.65 For example, one study66 investigating resistance to rituximab identified the nuclear factor CYCLON and showed that it cooperates with MYC to

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promote lymphoma growth in mice and that knockdown of CYCLON increased sensitivity to rituximab. Furthermore, a small-molecule inhibitor of BET bromodomain protein (JQ1) had the same effect in vitro by inhibiting the binding of the BRD4 co-activator (BET family) to chromatin and suppressing MYC-driven transcription. Therefore, epigenetic-based treatments with BET bromodomain inhibitors (eg, BAY1238097 [NCT02369029] and CPI-0610 [NCT01949883]) might be promising, especially to treat rituximab resistance. Other reports have confirmed that JQ1 can induce cell death, cell-cycle arrest, or senescence of DLBCL cells (both germinal cell centre and activated B-cell subtypes), and has activity in preclinical mouse models.67 The activity of JQ1 alone, or in combination with other epigenetic modifiers or targeting agents, is being studied in clinical trials, but no data have yet shown activity in DHL. OTX015, another BET bromodomain inhibitor, has shown promising activity and good safety in preliminary results of a phase 1 trial (NCT01713582). In-vitro studies have further shown that OTX015 targeted NF-κB/TLR/JAK/STAT signalling pathways, genes

R-CHOP (n=63) R-hyperCVAD (n=38) DA-EPOCH-R (n=57) R-CODOX-M/IVAC (n=41) Other treatments (n=24)

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Figure 3: Effect of different regimens on progression-free survival and overall survival for patients with double-hit lymphoma DA-EPOCH-R=rituximab and dose-adjusted etoposide, prednisone, vincristine, and doxorubicin. R-CHOP=rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. R-CODOX-M/IVAC=rituximab plus cyclophosphamide, doxorubicin, vincristine, methotrexate/ifosfamide, etoposide, and high-dose cytarabine. R-hyperCVAD=rituximab plus hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone. Reproduced with permission from Petrich and colleagues.36

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regulated by MYC and E2F1, cell-cycle regulation, and chromatin structure. Furthermore, OTX015 showed synergistic activity with mTOR and BTK inhibitors in vitro.68 In-vitro and in-vivo data from lymphoma models have shown a synergistic effect of the histone deacetylase inhibitor ricolinostat with carfilzomib (an irreversible proteasome inhibitor) through several stress-related mechanisms.69 MYC transcriptional activity can be abolished in mice with small-molecule inhibitors, resulting in eradication of KRAS-driven lung adenocarcinoma. However, none of the small-molecule inhibitors of MYC–MAX dimerisation has shown clinical effect because of their short halflives.70 Mycro3, a MYC–MAX dimerisation inhibitor, was developed for pancreatic cancer and showed a curative effect in mice models of pancreatic cancer.71 This agent seems to have good bioavailability and might be promising for treatment of DHL or DPL.

Agents targeting the expression of MYC, BCL2, and BCL6 proteins No specific data for the efficacy of drugs such as lenalidomide and BTK inhibitors that target subtypes of DLBCL (ie, activated B-cells or germinal centre cells) in patients with DPL are available at present. Heat shock protein H1/105 (HSPH1) is a chaperone that is overexpressed in DLBCL, and its expression is associated with MYC expression and lymphoma aggressiveness. HSPH1 knockdown induced MYC, BCL2, and BCL6 downregulation and inhibited proliferation of lymphoma cells and decreased neoangiogenesis in a lymphoma mouse model.72 The BCL2 family also represents a target for DHL. Navitoclax (ABT-263), a BCL2 inhibitor, showed anticancer activity in a phase 1 trial of patients with lymphoid malignancies, but toxicities with severe thrombocytopenia were reported.73 Venetoclax (ABT-199), a BCL2 inhibitor designed to reduce platelet toxicity, showed promising antitumour activity in a xenograft model of DLBCL (a phase 1 trial, NCT01594229, is underway) and impressive response rates and progression-free survival in patients with chronic lymphocytic leukaemia,74 but resistance was reported that could be related to expression of MCL1, an MYC target gene encoding an apoptotic regulator that is important in survival of MYC-positive B-cell lymphoma

cells.75 Targeting of PI3K/AKT/mTOR pathways reduces MCL1 expression and acts synergistically with ABT-737, thus decreasing the possible resistance.76 Cyclindependent kinase (CDK) inhibitors can also be used to target MCL1.77 Dinaciclib, a CDK inhibitor in phase 1 and 2 development for multiple myeloma and chronic lymphocytic leukaemia (NCT01515176), targets CDK9 and has been shown to have preclinical activity in cell lines and mouse models.77,78 Activity of venetoclax has been validated in DHL and THL cell lines, and coadministration of venetoclax and JQ1 greatly suppressed THL cell growth.79 Inhibitors of BCL6 have shown promising preclinical activity in DLBCL models80 and might prove useful for MYC–BCL6 DHL. Other therapies targeting MYC-dependent cancer metabolism could also be used in DHL and DPL. Agents targeting glucose metabolism, which is upregulated in cells overexpressing MYC, are being developed. An example of such an agent is AZD3965, a specific inhibitor of the monocarboxylate transporter (MCT1), which causes the accumulation of lactate, lowers cellular pH, and inhibits glycolysis and lymphoma growth in mice. AZD3965 is being tested in a phase 1 trial (NCT01791595) for DLBCL and other solid cancers. Aberrant MYC protein expression induces aurora kinases A and B expression, and analysis of data from the Lymphoma/Leukaemia Molecular Profiling Project showed that expression of these kinases is associated with MYC and BCL2 expression in DLBCL. In a mouse model with aggressive DLBCL coexpressing MYC and BCL2, the combination of the aurora kinase inhibitor alisertib with rituximab and vincristine induced synthetic lethality and potential cure.81 MYC is also expressed in other solid malignancies, and new agents targeting the MYC pathway or expression have been studied in these tumour types. AZD1208, an inhibitor of PIM (a kinase that has been linked to MYC-driven tumourigenesis), has been investigated in a mouse model for prostate cancer where it induced inhibition of proliferation and apoptosis.82 To develop and assess new therapeutics, a new mouse model of humanised MYC–BCL2 DHL that recapitulates the pathological and clinical aspects of chemotherapyresistant and rituximab-resistant human DHL has been developed.83

Conclusion Search strategy and selection criteria We searched MEDLINE through PubMed and Scopus from Jan 1, 2000, to April 1, 2015, using the terms “double-hit lymphoma”, “double-protein expression lymphoma”, “MYC”, “MYC overexpression”, “BCL2 overexpression”, and “Burkitt-like lymphoma”. We also searched the personal database (on EndNote X7) of the corresponding authors of these manuscripts, which accumulated more than 40 000 references on lymphoma over the past 30 years. Only articles in English were reviewed. The final reference list was generated on the basis of importance, originality, and relevance to the subject.

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DHL are rare variants of DLBCL; its outcome is very poor with both R-CHOP and intensified regimens, and stem cell transplantation does not seem to improve this outcome. Identification of patients with DHL at clinical diagnosis remains a challenge; although stage III/IV disease, bone marrow or CNS involvement, high LDH, high IPI, and extranodal disease are frequently reported, these markers are not specific and FISH analysis is still required for diagnosis. Concordant with MYC and BCL2 biology, DHLs originate from the germinal centre. DPL www.thelancet.com/oncology Vol 16 November 2015

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cases are five to ten times more frequent than DHL and are more likely to present an intermediate outcome than DHL. Additionally, their initial clinical presentation is less aggressive than that of DHL. In view of this poor outcome, DHL and DPL cases should be identified at diagnosis for all patients with DLBCL. Besides diagnostic difficulties, therapeutic options are not ideal, and new strategies based on targeted therapy with kinase inhibitors, monoclonal antibodies, BET inhibitors, or other agents (eg, BCL2 and CDK inhibitors) are needed. Data from phase 2 and 3 studies targeting DHL and DPL should be available soon to improve therapeutic options for these patients. Contributors All authors contributed to the preparation, writing, and review of this report. Declaration of interests CS reports honoraria from Gilead Sciences outside the submitted work. BC reports personal fees from Astra-Zeneca outside the submitted work. AT-G declares no competing interests. Acknowledgments We thank Gilles Salles, who has reviewed this manuscript and given advice. This manuscript has been edited for language by Andrew Lane, Lane Medical Writing (paid by the authors). References 1 Kramer MHH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood 1998; 92: 3152–62. 2 Kanungo A, Medeiros LJ, Abruzzo LV, Lin P. Lymphoid neoplasms associated with concurrent t(14;18) and 8q24/c-MYC translocation generally have a poor prognosis. Mod Pathol 2006; 19: 25–33. 3 Le Gouill S, Talmant P, Touzeau C, et al. The clinical presentation and prognosis of diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC rearrangement. Haematologica 2007; 92: 1335–42. 4 Lin P, Medeiros LJ. High-grade B-cell lymphoma/leukemia associated with t(14;18) and 8q24/MYC rearrangement: a neoplasm of germinal center immunophenotype with poor prognosis. Haematologica 2007; 92: 1297–301. 5 Coiffier B, Thieblemont C, van den Neste E, et al. Long-term outcome of patients in the LNH-98.5 trial, the first randomized study comparing rituximab-CHOP to standard CHOP chemotherapy in DLBCL patients: a study by the Groupe d’Etudes des Lymphomes de l’Adulte. Blood 2010; 116: 2040–45. 6 Li S, Seegmiller AC, Lin P, et al. B-cell lymphomas with concurrent MYC and BCL2 abnormalities other than translocations behave similarly to MYC/BCL2 double-hit lymphomas. Mod Pathol 2015; 28: 208–17. 7 Aukema SM, Siebert R, Schuuring E, et al. Double-hit B-cell lymphomas. Blood 2011; 117: 2319–31. 8 Green TM, Young KH, Visco C, et al. Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol 2012; 30: 3460–67. 9 Vennstrom B, Sheiness D, Zabielski J, Bishop JM. Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29. J Virol 1982; 42: 773–79. 10 Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer 2008; 8: 976–90. 11 Dang CV. MYC on the path to cancer. Cell 2012; 149: 22–35. 12 Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM. Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 1982; 79: 7824–27. 13 Greenough A, Dave SS. New clues to the molecular pathogenesis of Burkitt lymphoma revealed through next-generation sequencing. Curr Opin Hematol 2014; 21: 326–32.

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