Chromosomal translocations involving the immunoglobulin. MYC/BCL2 Double-Hit High-Grade B-Cell Lymphoma REVIEW ARTICLE

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1 REVIEW ARTICLE MYC/BCL2 Double-Hit High-Grade B-Cell Lymphoma Shaoying Li, MD,* Pei Lin, MD,w Ken H. Young, MD,w Rashmi Kanagal-Shamanna, MD,w C. Cameron Yin, MD, PhD,w and L. Jeffrey Medeiros, MDw Abstract: Double-hit lymphoma (DHL) has been defined by others as a B-cell lymphoma with MYC/8q24 rearrangement in combination with a translocation involving another gene, such as BCL2, BCL3, or BCL6. The most common form of DHL has translocations involving MYC and BCL2, also known as MYC/BCL2 DHL. In recent years, a number of case series of MYC/BCL2 DHL have been published. Most cases of MYC/BCL2 DHL morphologically resemble diffuse large B-cell lymphoma (DLBCL) or B- cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma. These tumors are of B-cell lineage, have a germinal center B-cell immunophenotype with a high proliferation rate, and a complex karyotype. Patients with these tumors have an aggressive clinical course and poor prognosis despite high-intensity chemotherapy. More recently, studies have suggested expanding the spectrum of MYC/BCL2 DHL to include cases that have concurrent MYC and BCL2 cytogenetic abnormalities, but not necessarily translocations. In addition, overexpression of MYC and BCL2 has been shown in an appreciable subset of DLBCL tumors. These tumors show overlap with MYC/ BCL2 DHL, but are not equivalent. In this review, we discuss the clinicopathologic, immunophenotypic, cytogenetic, and prognostic features of MYC/BCL2 DHL. Key Words: double-hit, B-cell lymphoma, MYC/8q24, BCL2/ t(14;18)(q32;q21) (Adv Anat Pathol 2013;20: ) Chromosomal translocations involving the immunoglobulin (IG) genes are common in B-cell non-hodgkin lymphomas. 1,2 The t(14;18)(q32;q21) is present in 80% to 90% of cases of follicular lymphoma and also in 20% to 30% of cases of de novo diffuse large B-cell lymphoma (DLBCL). 3 The t(14;18) juxtaposes BCL2 at 18q21 with the promoter of the IGH gene on the derivative chromosome 14q32 driving BCL2 overexpression. A primary function of BCL2 is to inhibit cellular apoptosis. MYC is involved in a number of translocations, including t(8;14)(q24;q32), t(2;8)(p12;q24), and t(8;22)(q24;q11), which juxtapose MYC at 8q24 with either the IGH, k, orl genes, respectively, upregulating MYC expression. 4,5 MYC translocation is a hallmark of Burkitt lymphoma (BL), with t(8;14)(q24;q32) present in approximately 80% of cases and the variant translocations involving the IG light chain loci in approximately 20% of cases. MYC also can partner with non-ig genes. MYC rearrangement, partnered with either IG or non-ig genes, is observed in 5% to 22% of DLBCL and is rearranged with variable frequency in other highgrade B-cell lymphomas, such as B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (BCLU) MYC also can be rearranged in small B-cell lymphomas that undergo transformation, such as follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma and mantle cell lymphoma. 11,12 MYC has many physiological functions including proliferation, apoptosis, metabolism, and differentiation. A recent study has suggested that MYC is a nonlinear amplifier of gene expression. 13 In recent years, cases of double-hit lymphoma (DHL) have become well recognized. DHL is broadly defined as B-cell lymphoma with a MYC translocation combined with an additional translocation involving other genes, such as BCL2, BCL3, orbcl6. 14 The most common type of DHL carries MYC/8q24 rearrangement and IGH@BCL2/ t(14;18)(q32;q21), referred to by others in the literature as simply, double-hit B-cell lymphoma, or MYC/BCL2 or M/B DHL, or BCL2 + /MYC + lymphoma. We believe that specifying the specific molecular abnormalities in DHL conveys important biological information and therefore in this review we will refer to these neoplasms as MYC/BCL2 DHL. MYC/BCL2 DHL is the most common form of DHL. There are multiple studies of MYC/BCL2 DHL cases reported in the literature, although different authors have focused on various clinical or therapeutic aspects of affected patients. As a result, many cases of MYC/BCL2 DHL that have been reported lack sufficiently detailed clinical or pathologic information. In this study, we systemically review the literature on MYC/BCL2 DHL and discuss disease frequency, clinical and morphologic features, immunophenotype, cytogenetic findings, therapy, and prognosis of affected patients. We also discuss recent studies that have suggested expanding the spectrum of MYC/BCL2 DHL to include cases that have concurrent MYC and BCL2 cytogenetic abnormalities, but not necessarily translocations, as well as tumors that overexpress MYC or BCL2 as shown by immunohistochemistry. From the *Department of Pathology, Microbiology, and Immunology, Division of Hematopathology, Vanderbilt University School of Medicine, Nashville, TN; and wdepartment of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX. The authors have no funding or conflicts of interest to disclose. Reprints: L. Jeffrey Medeiros, MD, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Unit Holcombe Blvd, Houston, TX ( ljmedeiros@ mdanderson.org). All figures can be viewed online in color at pathology.com. Copyright r 2013 by Lippincott Williams & Wilkins FREQUENCY MYC/BCL2 DHL was once thought to be uncommon, representing <1% of all lymphomas and approximately 4% of high-grade B-cell lymphomas reported in the older literature More recent studies in which MYC and BCL2 abnormalities were sought systematically, however, have shown that MYC/BCL2 DHL is more common than had been appreciated. Pedersen et al 18 studied 157 patients with DLBCL or BCLU. All 157 cases were analyzed by Adv Anat Pathol Volume 20, Number 5, September

2 Li et al Adv Anat Pathol Volume 20, Number 5, September 2013 using fluorescence in situ hybridization (FISH) with MYC and BCL2 break-apart probes and 11% of the cohort was MYC/BCL2 DHL. This subset represented 7% of de novo lymphomas and 21% of transformed lymphomas. 18 Barrans et al 6 investigated 303 previously untreated DLBCL patients who had no evidence of underlying follicular lymphoma using interphase FISH for MYC, BCL6, and t(14;18)/bcl2 rearrangements. Specifically, MYC rearrangements were shown in 35 (14%) of 245 biopsy specimens, including 26 (11%) MYC/BCL2 DHL and 7 (3%) MYC, BCL2, and BCL6, so-called triple-hit lymphoma. Foot et al 10 performed a prospective study of 162 patients with non-burkitt high-grade B-cell non-hodgkin lymphoma. MYC, IGH@BCL2, and BCL6 status were determined by FISH probes and DHL was shown in 18 (11%) cases, including both MYC/BCL2 DHL and MYC/BCL6. Overall, it seems that the frequency of MYC/BCL2 DHL is approximately 10%. Therefore, the frequency of MYC/ BCL2 DHL was underestimated, likely for at least 2 reasons. The first, and likely most important, is that traditionally cases of aggressive B-cell lymphoma have not been routinely tested by conventional cytogenetics or FISH. Secondly, translocations can be difficult to recognize using conventional karyotyping methods in a subset of tumors. 19 CLINICAL FEATURES For this review, we have focused on studies of MYC/ BCL2 DHL in the literature in which Z10 cases were reported. The results of these studies are summarized in Table 1. 15,17,18,20 26 MYC/BCL2 DHL occurs mainly in older patients and slightly more often in men. The median age at diagnosis has ranged from 51 to 65 years, which is older than patients with BL (fourth decade), but slightly younger than patients with DLBCL (seventh decade). 21,25,27 A subset of patients has a history of or concurrent lowgrade B-cell lymphoma, most often follicular lymphoma, and the frequency of MYC/BCL2 DHL seems to be increased in patients with follicular lymphoma. MYC/ BCL2 DHL is truly rare in children, adolescents, and young adults. One of 52 MYC/BCL2 DHL patients in a study from MD Anderson Cancer Center was 18 years old. 26 In a study of nodal DLBCL in the pediatric population by Gualco et al, 28 MYC/BCL2 DHL was identified in one of 16 patients. In another study of 97 children with aggressive B-cell lymphomas, no cases of MYC/BCL2 DHL were identified. 29 Most patients with MYC/BCL2 DHL present with advanced stage disease (Ann Arbor III/IV). Extranodal sites of involvement are frequent, with the bone marrow and central nervous system being involved most commonly. Virtually, any extranodal site can be involved. The gastrointestinal tract has been reported to be a common site of disease in several studies, 15,21,22,24 26 although no cases of MYC/BCL2 DHL were identified in a study of 101 highgrade B-cell lymphomas of the gastrointestinal tract by Choi et al. 30 An elevated serum lactate dehydrogenase (LDH) level is very common in patients with MYC/BCL2 DHL. Most patients have a high-intermediate or high International Prognostic Index (IPI). Snuderl et al 25 compared MYC/BCL2 DHL patients with IPI-matched DLBCL patients and found that the former group had a higher median LDH level at presentation (727 vs. 366 U/L) and higher frequency of bone marrow involvement (59% vs. 23%). TABLE 1. Clinical Features of MYC/BCL2 Double-Hit Lymphoma IPI HI-H: n/n (%) >1 Extranodal Site: n/n (%) CNS + : n/n (%) BM + : n/n (%) Elevated LDH: n/n (%) Stage III/IV: n/n (%) History or Concurrent Low-Grade Lymphoma: n/n (%) Sex: M/F Age: Median (Range) Total DHL Cases References Macpherson et al /7 6/13 (46) 12/13 (92) 8/10 (80) 9/13 (69) NA 8/13 (62) 9/10 (90) Kanungo et al (29-72) 10/4 0 NA 13/14 (93) 11/14 (79) 5/14 (36) 9/14 (64) NA Le Gouill et al (36-73) 10/6 4/16 (25) 16/16 (100) 16/16 (100) 15/16 (94) 8/16 (50) 14/16 (88) 13/16 (81) Bertrand et al (45-81) 5/5 1/0 (10) 7/10 (70) 9/9 (100) NA NA NA 5/9 (56) Johnson et al NA 32/22 20/54 (37) 41/54 (76) 27/54 (50) 32/45 (71) NA 19/54 (35) 38/54 (70) Niitsu et al (29-79) 10/9 0 19/19 (100) 19/19 (100) 16/19 (84) 4/19 (21) 12/19 (63) 17/19 (89) Tomita et al (36-79) 12/15 6/27 (22) 22/23 (96) 25/27 (93) 16/23 (70) 56/27 (56) 15/23 (65) 20/23 (87) Snuderl et al (32-91) 11/9 6/20 (30) 18/19 (95) 18/18 (100) 10/17 (59) 5/11(45) 6/20 (30) 17/20 (85) Li et al 26 * 52* 55 (18-76) 33/19 10/52 (19) 37/52 (74) 37/45 (82) 27/47 (57) 7/30 (23) 27/52 (55) 30/52 (63) Pedersen et al /9 9/17 (53) 14/17 (82) 14/17 (82) 4/17 (24) NA 8/17 (47) 11/17 (64) *Data summarized for 52 traditionally defined DHL cases. BM + indicates bone marrow involvement; CNS +, central nervous system involvement; DHL, double-hit lymphoma; HI-H, IPI high-intermediate to high; IPI, International Prognostic Index; LDH, lactate dehydrogenase; n, positive cases; N, total cases tested r 2013 Lippincott Williams & Wilkins

3 Adv Anat Pathol Volume 20, Number 5, September 2013 MYC/BCL2 Double-Hit Lymphoma MORPHOLOGIC FEATURES Cases of MYC/BCL2 DHL show a spectrum of morphologic features (Table 2), with most cases categorized as either BCLU or DLBCL (Fig. 1). 17,21,24 27 Much less often, cases of MYC/BCL2 DHL can be classified as follicular lymphoma, usually grade 3 and often associated with areas of DLBCL, B-lymphoblastic lymphoma/leukemia (TdT + ), or composite lymphoma. The composite lymphomas reported have consisted of BCLU associated with follicular lymphoma, DLBCL associated with follicular lymphoma, and DLBCL associated with B-lymphoblastic lymphoma/ leukemia. 24,26 A small subset of MYC/BCL2 DHL are difficult to classify using the current classification system. These tumors are composed of small to intermediately sized cells with blastoid features, resembling in part B-lymphoblastic lymphoma/leukemia, but having a mature B-cell immunophenotype (TdT ). 31 In 3 studies of follicular lymphoma in which a systematic search for MYC/8q24 rearrangements and t(14;18)(q32;q21)/igh@bcl2 was performed the frequency of concurrent MYC and BCL2 rearrangement ranged from 2% to 8% Follicular lymphomas with concurrent MYC and BCL2 rearrangement are most often grade 3, and a subset has been described to have blastoid features. Vaidyanathan et al 35 reported 3 cases of follicular lymphoma with blastoid morphologic features and identified 27 additional cases in the literature: half of these tumors had concurrent MYC rearrangement and were MYC/BCL2 DHLs. Cases of low-grade follicular lymphoma with concurrent MYC and BCL2 rearrangements also have been reported. 32 Rare cases of plasma cell myeloma with coexistent MYC and BCL2 rearrangements also have been reported. 18 Follow-up studies of low-grade lymphomas and plasma cell myeloma with concurrent MYC and BCL2 rearrangements are currently lacking. IMMUNOPHENOTYPIC FEATURES Cases of MYC/BCL2 DHL are positive for pan-b-cell markers and most cases express monotypic Ig light chain. The intensity of surface CD20 and Ig light chain expression is decreased in some cases as compared with normal germinal center B cells. CD19 expression also can be decreased in MYC/BCL2 DHL. These changes are best appreciated by flow cytometry immunophenotypic analysis. 24,36 Harrington et al 37 have suggested that the combination of decreased CD19 and CD20 expression carries a specificity of 100% for the diagnosis of MYC/BCL2 DHL among CD10 + high-grade B-cell lymphomas. MYC/BCL2 DHLs are negative for CD2, CD3, and CD7; most cases are also CD5 negative, but CD5 + cases have been reported rarely. Virtually, all cases of MYC/BCL2 DHL have a germinal center B-cell immunophenotype. CD10 is positive in 76% to 100% of cases, and BCL6 is positive in 63% to 96% of cases in various studies (Table 2). BCL2 is often positive by immunohistochemical analysis, in 80% to 100% of cases (Table 2 and Fig. 2). In BCL2-negative cases, mutations in the BCL2 gene in a subset of MYC/BCL2 DHL preclude recognition of the epitope recognized by the commonly used anti-bcl2 antibody 124 (Dako). 17 In one study, Johnson et al sequenced 3 cases of MYC/BCL2 DHL negative for BCL2 using the 124 antibody and identified BCL2 mutations in 2 tumors. Both mutated cases were positive for BCL2 by immunohistochemistry using the E17 (Epitomics) antibody. MYC/BCL2 DHL cases usually have a high TABLE 2. Morphologic and Immunophenotypic Features of MYC/BCL2 Double-Hit Lymphoma References Total DHL Cases DLBCL BCLU B-ALL/LBL FL-3 Other CD10 + : n/n (%) BCL2 + : n/n (%) BCL6 + : n/n (%) Ki-67 Macpherson et al NA NA NA NA NA NA 13/13 (100) NA NA Kanungo et al /12 (100) 12/12 (100) NA 70%-99% Le Gouill et al /16 (100) 11/11 (100) 6/7 (86) 70%-90% Bertrand et al NA NA NA NA Johnson et al /43 (79) 23/28 (82) NA Z80% in 14/20 Niitsu et al /19 (84) 19/19 (100) 12/19 (63) 50%- > 90% Tomita et al 24 20/27* (BCLU + FL-LG) 18/19 (95) 19/20 (95) 11/15 (73) 47%-91% in 14 Snuderl et al /19 (90) 18/18 (100) 17/19 (89) 25%-100% 47/48 (98) 40/46 (87) 22/23 (96) 40%-99% Li et al (DLBCL + FL-3B), 1 (DLBCL + B-LBL) Pedersen et al /17 (76) 14/17 (82) NA 15%-100% *Only 20 cases of lymphoma available, 4 of 27 are leukemic presentations with no detailed information. B-ALL/LBL indicates B-lymphoblastic leukemia/lymphoma; BCLU, B-cell lymphoma, unclassifiable, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma; DHL, double-hit lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; LG, low grade; n, positive cases; N, total cases tested. r 2013 Lippincott Williams & Wilkins 317

4 Li et al Adv Anat Pathol Volume 20, Number 5, September 2013 FIGURE 1. Morphologic spectrum of 4 cases of MYC/BCL2 double-hit lymphoma (DHL). A, B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. B, DLBCL with high mitotic and apoptotic activity. C, Grade 3B follicular lymphoma. D F, A case of composite lymphoma with components of TdT + (not shown) B-lymphoblastic lymphoma (D) and TdT (not shown) DLBCL (E). The B-lymphoblastic lymphoma was CD20 dim + to and DLBCL was CD20 + (F). A C, 400; D and E, 1000; F, 200. proliferation index as assessed by immunostaining for Ki-67. The median percentage of Ki-67 positive cells is approximately 90% in 123 well-documented cases in the literature. Although the range is wide, very few cases of MYC/BCL2 DHL have a proliferation rate that is <50%. 18,24 26 MYC expression evaluated by immunohistochemical analysis has been reported in several recent studies of MYC/BCL2 DHL (Figs. 2, 4). Using a cutoff of 40% to 50%, MYC was expressedin10of14casesin1study, 38 9 of 11 cases in another study, 39 andall8casesassessedinastudybykluketal. 40 PATHOGENESIS By definition, all cases of MYC/BCL2 DHL have concurrent MYC/8q24 rearrangement and t(14;18)(q32;q21)/igh@ r 2013 Lippincott Williams & Wilkins

5 Adv Anat Pathol Volume 20, Number 5, September 2013 MYC/BCL2 Double-Hit Lymphoma FIGURE 2. Immunophenotypic and fluorescence in situ hybridization (FISH) findings in 2 cases of MYC/BCL2 double-hit lymphoma (DHL). A F, A case of MYC/BCL2 DHL associated with typical MYC translocation t(8;14). The neoplastic cells (A: H&E, 400; B: H&E, 1000) are clonal with a CD45 +, CD20 +, PAX5 +, CD10 +, and BCL2 + (C: 200), MUM1, BCL6 + immunophenotype, and a high proliferation index of 70% to 80% by Ki-67 expression (D: 400). The lymphoma cells show 100% positivity for MYC immunostain (E: 400), and typical MYC translocation t(8;14) by FISH (F). G L, Another case of MYC/BCL2 DHL associated with alternative MYC translocation t(8;22). The neoplastic cells (G: H&E, 400; H: H&E, 1000) are clonal with a CD45 +, CD20 +, PAX5 +, CD10 +, and BCL2 + (I: 200x), MUM1, BCL6 + immunophenotype, and a high proliferation index of 70% to 80% by Ki-67 expression (J: 400). The lymphoma cells show 95% positivity for MYC immunostain (K: 200), and alternative MYC translocation t(8;22) (L). BCL2 that can be detected by conventional cytogenetic analysis or FISH. MYC, an oncogene located at 8q24, functions as a transcription factor controlling the expression of many genes, directly or indirectly, and is essential for many cellular physiological functions. 5,13 MYC rearrangement upregulates MYC expression. 5 MYC is more commonly partnered with the IG loci, but up to 50% of MYC/BCL2 DHL cases have a non-igh partner (Table 3). 15,17,18,20 22,26 Johnson et al 17 have suggested that MYC/BCL2 DHL cases more often resemble DLBCL when MYC is partnered with a non-ig locus. Others 23 have shown that the most common non-ig partners include BCL6, BCL11A, PAX5, and IKAROS; these genes are known to be important in lymphomagenesis. BCL2, located at 18q21, encodes an antiapoptotic protein. The t(14;18)(q32;q21) juxtaposes BCL2 with the IGH gene enhancer at 14q32, resulting in BCL2 overexpression (Figs. 2, 4). BCL2 also has a role in physiological DNA repair. It is hypothesized that the coexistence of MYC and BCL2 translocations in MYC/BCL2 DHL results in markedly increased cell proliferation and decreased in apoptosis, resulting in rapid tumor growth. Cooperation between MYC and BCL2 in contributing to lymphomagenesis has been shown in transgenic mouse models 42 and humanized mouse models Conventional cytogenetic analysis of cases of MYC/BCL2 DHL often show a complex karyotype, with Z3 structural and/or numerical abnormalities, possibly related to genomic instability and impaired DNA repair mechanisms in these tumors. The functions of the additional cytogenetic aberrations and their roles in lymphomagenesis and prognosis are unclear. However, genetic complexity correlates with poorer prognosis in a number of tumor types. In 2 studies of high-grade B-cell lymphoma, genetic complexity appeared to contribute to the dismal prognosis of affected patients. 52,53 Approximately 80% to 85% of cases of MYC/BCL2 DHL arise in patients de novo, whereas 15% to 20% of cases are thought to have progressed from follicular lymphoma. In patients with previous follicular lymphoma, it seems reasonable to hypothesize that MYC rearrangement occurs as a secondary event resulting in high-grade transformation InaseriesreportedbyTomitaetal, 24 2patientshadacomposite lymphoma composed of low-grade follicular lymphoma and high-grade B-cell lymphoma. t(14;18)(q32;q21)/ IGH@BCL2 was identified in the low-grade follicular lymphoma component, but concurrent MYC and BCL2 translocations were detected in the high-grade B-cell lymphoma component. These observations support the concept that MYC rearrangement is a secondary event in MYC/BCL2 r 2013 Lippincott Williams & Wilkins 319

6 Li et al Adv Anat Pathol Volume 20, Number 5, September 2013 BCR Signaling BCL2 CARD11, BCL10, MALT1 Apoptosis JAK/STAT3 NF-KB PI3K/Akt P53, etc MYC Proliferation & Survival Oncogenic mirnas: mir-17-92, mir-29 Tumor Suppressor mirnas: let-7, mir-26a, mir-15a, mir- 16-1, mir-34a, mir150, etc FIGURE 3. Illustration of pathways in which MYC and BCL2 induced B-cell lymphomagenesis: MYC and BCL2 interact with multiple different pathways with a net effect of increased cell proliferation and decreased apoptosis. DHL. The frequent involvement of IG light chain loci and a non-ig genes as partners also suggests that MYC rearrangement in MYC/BCL2 DHL is likely a secondary event. However, concurrent MYC and BCL2 rearrangements have been detected in follicular lymphomas, raising the possibility that MYC rearrangement may arise concomitantly or precede t(14;18) in some patients. For example, Snuderl et al 25 reported a case of follicular lymphoma associated with t(14;18)(q32;q21) that also had blastoid morphologic features and MYC translocation. Similarly, in a study from MD Anderson Cancer Center, 26 MYC/BCL2 DHL arose shortly after or nearly coincided with the diagnosis of low-grade follicular lymphoma in 6 patients, suggesting that a subset of tumor cells was carrying a MYC translocation in otherwise morphologically low-grade follicular lymphoma. Perhaps an additional genetic event may be required for transformation to occur in a follicular lymphoma carrying both MYC and BCL2 abnormalities. As has been suggested by others, perhaps there are 2 (or even more) pathways to MYC/BCL2 DHL: 1 from a clinically overt or subclinical follicular lymphoma, and the other directly from B cells with IGH@BCL2 that have not attained malignant potential. 14 TREATMENT AND PROGNOSIS As shown in Table 4, patients with MYC/BCL2 DHL have been treated with different regimens, including traditional chemotherapy regimens and more aggressive chemotherapy regimens with or without stem cell transplant. The most common traditional regimens that have been used are R-CHOP (rituximab, cyclophosphamide, daunorubicin, vincristine, prednisone) or CHOP. More aggressive chemotherapy regimens used include Hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone), R-Hyper-CVAD, and others (Table 4). In a study of BCLU in which approximately 40% of cases were MYC/BCL2 DHL, the progression-free survival of tumors with MYC rearrangement was significantly worse in patients treated with R-CHOP than in those treated with R-Hyper-CVAD. 59 However, other studies of MYC/BCL2 DHL have not shown significant prognostic differences between different treatment regimens. As shown in Table 4, the median overall survival of MYC/BCL2 DHL patients is very poor, ranging from 2 months to 1.5 years, regardless of the therapeutic regimen employed. 15,17,20 26 Johnson et al 17 reported a number of features in patients with MYC/BCL2 DHL that correlated with a more favorable prognosis. These features included: absence of bone marrow involvement, low IPI score, conventional DLBCL morphologic features, absence of BCL2 protein expression, non-ig MYC translocation partner, and treatment with a rituximab-based chemotherapy regimen. In the study of Johnson et al, 17 low IPI score and DLBCL r 2013 Lippincott Williams & Wilkins

7 Adv Anat Pathol Volume 20, Number 5, September 2013 MYC/BCL2 Double-Hit Lymphoma TABLE 3. MYC Rearrangement Partners and Cases With Copy Number Changes Involving MYC and BCL2 MYC Partners: n/n (%) Cases With Extra Copies of MYC and/or BCL2 No RA, Extra Copies of Both BCL2 RA + Extra Copies of MYC MYC RA + Extra Copies of BCL2 DHL Only by FISH Cases With Karyotype References Total Cases IGH j k Non-IG Macpherson et al /12 (42) 0 6/12 (50) Kanungo et al /10 (30) 1/10 (10) 6/10 (60) Le Gouill et al /8 (38) 1/8 (13) 3/8 (38) 1/8 (13) 8 8 Bertrand et al /8 (100) Johnson et al /54 (30) 3/54 (6) 11/54 (20) 24/54 (44) 54 Niitsu et al /19 (37) 2/19 (12) 2/19 (12) (no FISH) Tomita et al /27 (52) 4/27 (15) 9/27 (33) 0 27 Snuderl et al /11 0 9/11 (82) 2/11 (18) Li et al /21 (33) 2/21 (10) 9/21 (43) 3/21 (14) Pedersen et al /14 (21) 2/14 (14) 1/14 (7) 8/14 (57) 0 17 Li et al DHL indicates double-hit lymphoma; FISH, fluorescence in situ hybridization; IG, immunoglobin; n, positive cases; N, total cases tested; RA, rearrangement. morphology were independent predictors of overall survival. Pedersen et al 18 also found that the MYC translocation partner gene was important, with an IG partner associated with an inferior prognosis in MYC/BCL2 DHL. In a study from MD Anderson Cancer Center, 26 IPI score, bone marrow involvement, elevated serum LDH level, Z2 extranodal sites of disease, and central nervous system involvement were associated with a worse overall survival. However, morphologic features (DLBCL vs. non- DLBCL) and BCL2 expression did not correlate with prognosis. Furthermore, there was no statistically significant difference in median overall survival and progression-free survival between patients who received R-CHOP versus those treated with R-Hyper-CVAD, or between patients with or without stem cell transplant. However, only 11 patients received autologous or allogeneic stem cell transplant in this study. It has been generally accepted, based on the results in earlier studies, that patients with MYC/BCL2 DHL have a worse prognosis compared with patients with DLBCL with or without MYC rearrangement (Fig. 4). An abstract at the 2012 American Society of Hematology Annual Meeting, however, reported an opposite result. 60 Copie-Bergman and colleagues identified 53 patients with DLBCL associated with MYC rearrangement in their cohort of 776 CD20 + de novo DLBCL patients treated with R-chemotherapy. These 53 patients included 21 MYC simple-hit and 32 MYC double-hit or triple-hit (MYC/BCL2, MYC/BCL6, or MYC/BCL2/BCL6) cases. In both univariate and multivariate analyses, MYC simple-hit but not double-hit had an adverse prognostic effect on survival. However, as the study has only been presented in abstract form, information is not described in detail. Although more studies are needed to further clarify the prognostic significance of MYC rearrangement in high-grade B-cell lymphomas, including DHL cases, in most studies published to date patients with MYC/ BCL2 DHL have had disease that is highly resistant to current standard chemotherapy regimens. Therefore, novel therapeutic strategies are needed for this patient population. ATYPICAL MYC/BCL2 DHL The presence of concurrent translocations that involve MYC/8q24 and BCL2/18q21 are thought to result in upregulation of MYC and BCL2 expression in MYC/BCL2 DHL. However, MYC and BCL2 can theoretically be activated through mechanisms other than translocation, including gene amplification, copy number changes, or transcriptional or translational upregulation via micro- RNA or other regulatory components in their signaling pathways (Fig. 3). We have encountered cases of high-grade B-cell lymphoma in which concurrent MYC and BCL2 abnormalities are present, but translocations per se are not identified in either MYC, BCL2, or both genes. These cases can be grouped into 3 types: (1) MYC translocation coexisting with extra copies of BCL2, but lacking t(14;18)/igh@bcl2; (2) t(14;18)/igh@bcl2 coexisting with extra copies of MYC, but without evidence of MYC translocation; and (3) extra copies of MYC and BCL2 without translocations involving either gene. For the sake of convenience, we have designated these tumors atypical MYC/BCL2 DHL. In a previous study by Li et al, 8 cases of atypical MYC/BCL2 DHL were reviewed simultaneously with traditional MYC/BCL2 r 2013 Lippincott Williams & Wilkins 321

8 Li et al Adv Anat Pathol Volume 20, Number 5, September 2013 A 100 B All others (n=309) 80 All others (n=309) OS (%) PFS (%) p<.0001 DP (n=157) 20 p<.0001 DP (n=157) C 100 Months D 100 Months 80 Non-DH (n=373) 80 Non-DH (n=373) OS (%) PFS (%) DH (n=10) p< Months 20 DH (n=10) p< Months FIGURE 4. Prognostic significance of traditional MYC/BCL2 double-hit (DH) lymphoma and immunohistochemistry defined MYC/BCL expression lymphoma (DP). A, MYC/BCL2 protein expression, overall survival (OS); (B) MYC/BCL2 protein expression, progression-free survival (PFS); (C) MYC/BCL2 translocation, OS; (D) MYC/BCL2 translocation, PFS. DHL. 26 In a follow-up abstract, 20 atypical MYC/BCL2 DHL were compared with 48 cases of classic MYC/BCL2 DHL. 41 The clinical, morphologic, immunophenotypic features including proliferation rate, karyotypic complexity, and prognosis of patients with atypical MYC/BCL2 DHL appear to be very similar to patients with traditional MYC/ BCL2 DHL. The only statistically significant differences are that cases of atypical MYC/BCL2 DHL more often resemble DLBCL and express CD10 less frequently. Although information about atypical MYC/BCL2 DHL cases is sparse in the literature, the results from Li et al 26,41 and the few cases reported by others 15,20,25 suggest that these cases appear to fall within the biological spectrum of MYC/BCL2 DHL (Table 3). Indirect evidence to support this concept can be found in a study by Yoon et al 61 who studied 154 DLBCL cases. They showed that 14 patients who had tumors had MYC rearrangement and 11 patients who had tumors with increased MYC copy number had a similarly poor prognosis as compared with patients with DLBCL without MYC abnormalities. Therefore, it seems likely that genetic abnormalities other than translocations that involve MYC and BCL2 result in upregulation and fit within the MYC/ BCL2 DHL category. However, more studies are required to definitively address this issue. B-CELL LYMPHOMAS WITH CONCURRENT MYC AND BCL2 EXPRESSION SHOWN BY IMMUNOHISTOCHEMISTRY Extending the logic described above, recent studies have used immunohistochemical methods to demonstrate MYC and BCL2 overexpression in a subset of DLBCL cases. In a series of DLBCL patients treated with R-CHOP, Green et al 39 showed that high-level expression of MYC and BCL2 was associated with poorer overall and progression-free survival (Fig. 4). Green and colleagues used cutoffs of Z40% and Z70%, for MYC and BCL2, respectively. Coexpression of MYC and BCL2 was also associated with concurrent MYC and BCL2 rearrangements, but the frequency of concurrent MYC and BCL2 expression, 29%, was substantially higher than concurrent MYC and BCL2 rearrangements, 6% (Fig. 4). In similar studies, Johnson et al 38 and Hu et al 62 assessed large numbers of patients with DLBCL treated with R-CHOP and showed concurrent MYC and BCL2 expression in 20% to 30% of all cases of DLBCL, whereas concurrent MYC and BCL2 rearrangements occurred less frequently, approximately 5% of patients. Clearly, there are multiple mechanisms that can upregulate MYC or BCL2 expression in DLBCL (Fig. 3) r 2013 Lippincott Williams & Wilkins

9 Adv Anat Pathol Volume 20, Number 5, September 2013 MYC/BCL2 Double-Hit Lymphoma TABLE 4. Treatment and Prognosis of Patients With MYC/BCL2 Double-Hit Lymphoma References Total DHL Cases Treatment Median OS (y) Prognostic Factors Macpherson 13 CHOP-variant or cyclophosphamide + MTX 0.21 NA et al 20 (6); HDC ± SCT (3); P (4) Kanungo et al CT-NOS (11); R(1); CT and BMT (1); CT, <1 NA BMT, and RT (1) Le Gouill et al CEEP/COPADM + Auto-SCT/BEAM (1); 0.42 NA CHOP/IVAM (1); COPADM/CYVE (3); COPADM (1); COPADM + Auto-SCT/ BEAM (1); COPADM + Allo-SCT/Bu/Cy (1) CEEP/DHAP + Auto-SCT/BEAM (1); R-CHOP (4); CHOP (1); steroids (1); R-CEEP + Allo-SCT/TBI/Cy (1) Bertrand et al NA < 1 NA Johnson et al R-CHOP (11/54);HDC ± SCT (6/54); CHOP (23/54); P (14/54) Niitsu et al CyclOBEAP (6); CHOP + HD MTX (3); CHOP (4); R-CHOP (3), R-CyclOBEA (3) Tomita et al CHOP or CODOX-M/IVAC or Hyper- CVAD (R, n = 14; -R, n = 8) Snuderl et al R-ICE + MTX/ASCT (1); CHOP (1); R-CHOP (3); R-CHOP + MTX (6); R-CHOP + MTX + ASCT (1); R- EPOCH + MTX (3); CODOX-MTX/ R-IVAC (3); P (1); NK(1) Li et al R-CHOP (19), R-Hyper-CVAD + MTX and Cytarabine (28), CODOX (1), R + fludarabine + mitoxantrone + dexamethasone (1), + intrathecal MTX (13), + SCT (11) Pedersen et al R-CHOP (11); R-CVP (3), R-ICE/R-DHAP (2); R-CHOEP/R-COPE (1), + ASCT (1); + radiotherapy (3), + HD MTX (1); + intrathecal (3) HDC 0.26; R-CHOP 1.40; CHOP like 0.42, P = 0.07 IPI, BM status, DLBCL/ BCLU morphology, IG/non- IG partner, BCL2 protein negative 1.5 NA 0.5 NA 0.38 ECOG 1.55 IPI, LDH, stage, BM status, CNS status, extranodal site >1 Bold indicates multivariant analysis still significant. ASCT indicates autologous stem cell transplantation; BCLU, B cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma; BEAM, carmustine, etoposide, cytarabine, and melphalan; BMT, bone marrow transplantation; Bu, busulfan; CEEP, cyclophosphamide, etoposide, epidoxorubicin, and cisplatin; CODOX, cyclophosphamide, vincristine, and doxorubicin; COPADM, cyclophosphamide, vincristine, prednisone, doxorubicin, and high-dose methotrexate; CT-NOS, intensive combination chemotherapy, not otherwise specified; CYVE, cytarabine and etoposide; Cy, cyclophosphamide; CyclOBEAP, cyclophosphamide, vincristine, bleomycin, etoposide, doxorubicin, prednisone; DHAP, dexamethason, high-dose cytarabine, and cisplatin; DHL, double-hit lymphoma; DLBCL, diffuse large B-cell lymphoma; EPOCH, rituximab plus etoposide, doxorubicin, vincristine, prednisone, and cyclophosphamide; HDC, high-dose chemotherapy; HD, high dosage; Hyper-CVAD, hyperfractionated cyclophosphamide, vincristine, adriamycin, dexamethasone; IPI, International Prognostic Index; IVAC, ifosfamide, etoposide, and high-dose cytarabine; IVAM, ifosfamide, etoposide, cytarabine, and methotrexate; LDH, lactate dehydrogenase; MTX, methotrexate; NA, not available; NK, not known; OS, overall survival; P, palliative; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; R, rituximab; R-ICE, rutuximab plus ifosfamide, carboplatin, and etoposide plus rituximab; RT, radiotherapy; SCT, stem cell transplantation; TBI, total body irradiation. NA NA These findings also raise 2 questions. First, and perhaps most important, is there any prognostic difference between patients with traditionally defined MYC/BCL2 DHL (with MYC and BCL2 translocations) versus patients with high-grade B-cell lymphoma with immunohistochemistry defined MYC and BCL2 overexpression (without MYC and BCL2 translocations)? Although the studies by others did not directly compare overall or progression-free survival between patients with classic MYC/BCL2 DHL versus tumors with immunohistochemistry proven MYC and BCL2 expression, Johnson and colleagues demonstrated that the median overall survival of patients with traditionally defined MYC/BCL2 DHL was significantly shorter than that of patients with immunohistochemistry defined MYC/BCL2 DLBCL. 38 Second, are the levels of MYC and BCL2 expression similar or different when comparing traditionally defined MYC/BCL2 DHL versus immunohistochemistry defined MYC/BCL2 DLBCL? Although it would be more convenient to substitute immunohistochemistry for conventional cytogenetics or FISH in the workup of high-grade B-cell lymphomas, reducing expense and improving turnaround time, we suggest that immunohistochemical assessment of MYC and BCL2 expression is not a simple surrogate for cytogenetic analysis and therefore it is premature to use immunohistochemistry to replace genetic analysis. RECOMMENDATIONS FOR WORKUP In the past it seems clear that the frequency of MYC/ BCL2 DHL was underestimated, in large part because many pathology laboratories did not routinely study highgrade B-cell lymphoma cases using conventional cytogenetic or FISH methods. We suggest that all biopsy specimens of high-grade B-cell lymphoma at first diagnosis should be analyzed using conventional cytogenetic r 2013 Lippincott Williams & Wilkins 323

10 Li et al Adv Anat Pathol Volume 20, Number 5, September 2013 methods. We acknowledge, however, that an argument can be made that it is not cost effective to perform conventional cytogenetics on every lymphoma case. It is also convenient that FISH can be performed on fixed, paraffin-embedded tissue sections, 10,63 and that MYC and BCL2 rearrangements can be detected with commercially available probes. A dual color breakapart probe is most useful for MYC rearrangement assessment. Dual-color dual-fusion BCL2 and IGH FISH probes are also available for detecting t(14;18)(q32;q21)/igh@bcl2. If FISH is used after the initial diagnosis of high-grade B-cell lymphoma is established by routine morphologic assessment and immunophenotyping, clinicopathologic and immunohistochemical findings can be used to choose which cases are most appropriate for FISH testing. We suggest that FISH testing for MYC and BCL2 abnormalities is most likely to be positive in the following scenarios. B-cell lymphoma with aggressive clinical features, including advanced stage disease, extranodal involvement, or high level of serum LDH. B-cell lymphoma with a starry-sky pattern, high mitotic and apoptotic activity, or high proliferation rate as shown by using immunohistochemistry for Ki-67. A patient with a history of follicular lymphoma in whom histologic transformation is suspected. Follicular lymphoma with blastoid cytologic features, starry-sky pattern, or high proliferation rate. Recommendations for using immunohistochemistry to assess overexpression of MYC and BCL2 are less clear, as cases of DLBCL that overexpress MYC and BCL2 only recently have been recognized. Given the simplicity and rapid turnaround time of immunohistochemistry to assess for MYC and BCL2 expression, it seems reasonable to assess all cases of high-grade B-cell lymphoma and DLBCL. Although MYC immunostaining has not been routinely available in many pathology laboratories, commercial antibodies specific for MYC that are effective in the analysis of routinely processed tissue sections are available. Appropriate cutoffs of MYC and BCL2 expression should be standardized using receiver operator curves, in correlation with clinical outcome, to achieve optimal sensitivity and specificity. CONCLUSIONS In summary, MYC/BCL2 DHL is defined as a highgrade B-cell lymphoma that harbors both MYC/8q24 rearrangement and IGH@BCL2/t(14;18)(q32;q21). The frequency of these tumors is approximately 10%. Patients with MYC/BCL2 DHL have clinically aggressive disease that often presents with advanced stage, extranodal involvement, and high serum LDH levels. Central nervous system and bone marrow involvement are common and the IPI score is often high-intermediate or high. Most cases of MYC/BCL2 DHL have morphologic features of BCLU or DLBCL. Essentially all cases have a germinal center B-cell immunophenotype and a high proliferation rate. Despite a variety of approaches that have been used to date, patients with MYC/BCL2 DHL have a very poor prognosis with median overall survival of <2 years. Novel therapeutic approaches are needed for these patients. More recent data suggest that MYC/BCL2 DHL can be expanded to include tumors in which cytogenetic abnormalities of MYC and BCL2 coexist, even if translocations involving these loci are absent, designated here as atypical MYC/BCL2 DHL. In addition, the recent demonstration of MYC and BCL2 coexpression using immunohistochemistry in 20% to 30% of DLBCL cases may further expand the spectrum of these tumors, although tumors with concurrent MYC and BCL2 expression may not be biologically equivalent to MYC/BCL2 DHL and the poor prognosis of each patient subset may be attributable to different biological signatures. 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