Prevalence and clinical implications of cyclin D1 expression in diffuse large B-cell lymphoma (DLBCL) treated with immunochemotherapy: A report from the International DLBCL Rituximab-CHOP Consortium Program

Authors


Abstract

BACKGROUND

Cyclin D1 expression has been reported in a subset of patients with diffuse large B-cell leukemia (DLBCL), but studies have been few and generally small, and they have demonstrated no obvious clinical implications attributable to cyclin D1 expression.

METHODS

The authors reviewed 1435 patients who were diagnosed with DLBCL as part of the International DLBCL rituximab with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP) Consortium Program and performed clinical, immunohistochemical, and genetic analyses with a focus on cyclin D1. All patients who were cyclin D1-positive according to immunohistochemistry were also assessed for rearrangements of the cyclin D1 gene (CCND1) using fluorescence in situ hybridization. Gene expression profiling was performed to compare patients who had DLBCL with and without cyclin D1 expression.

RESULTS

In total, 30 patients (2.1%) who had DLBCL that expressed cyclin D1 and lacked CCND1 gene rearrangements were identified. Patients with cyclin D1-positive DLBCL had a median age of 57 years (range, 16.0-82.6 years). There were 23 males and 7 females. Twelve patients (40%) had bulky disease. None of them expressed CD5. Two patients expressed cyclin D2. Gene expression profiling indicated that 17 tumors were of the germinal center type, and 13 were of the activated B-cell type. Genetic aberrations of B-cell leukemia/lymphoma 2 (BCL2), BCL6, v-myc avian myelocytomatosis viral oncogene homolog (MYC), mouse double minute 2 oncogene E3 ubiquitin protein ligase (MDM2), MDM4, and tumor protein 53 (TP53) were rare or absent. Gene expression profiling did not reveal any striking differences with respect to cyclin D1 in DLBCL.

CONCLUSIONS

Compared with patients who had cyclin D1-negative DLBCL, men were more commonly affected with cyclin D1-positive DLBCL, and they were significantly younger. There were no other significant differences in clinical presentation, pathologic features, overall survival, or progression-free survival between these two subgroups of patients with DLBCL. Cancer 2014;120:1818–1829. © 2014 American Cancer Society.

INTRODUCTION

Diffuse large B-cell lymphoma (DLBCL) is the most common type of B-cell lymphoma in the world.[1] DLBCL, as currently defined, is a heterogeneous group of diseases with many morphologic and immunophenotypic variants and molecular subtypes.[2] The standard therapy for patients with DLBCL is rituximab with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP); and the 10-year overall survival (OS) and disease-free survival (DFS) rates are 43.5% and 36.5%, respectively.[3]

Mantle cell lymphoma (MCL) represents 4% to 10% of B-cell lymphomas worldwide and is characterized by t(11;14)(q13;q32) resulting in immunoglobulin heavy locus/cyclin D1 (IGH/CCND1) fusion, which results in marked over-expression of the cyclin D1 protein.[4] MCL is an aggressive lymphoma, and affected patients respond poorly to conventional chemotherapy. MCL can exhibit a wide range of morphologic appearances. A blastoid form occurs in up to 10% of patients, and another histological form, the pleomorphic variant, exhibits morphologic overlap with DLBCL. Distinguishing pleomorphic variant MCL from DLBCL is important because R-CHOP therapy is considered inadequate for patients with MCL; and, in some institutions (including ours), patients with MCL most often receive treatment with a dose-intensified regimen: rituximab combined with fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (R-hyper-CVAD) alternating with rituximab in combination with high-dose methotrexate-cytarabine (R-MA).[5]

Previous studies have demonstrated that a subset of patients with DLBCL overexpress cyclin D1 at the immunohistochemical level, and this expression occurs independently of the t(11;14).[6-11] There have been only few case reports and case series of cyclin D1 expression in DLBCL. The objectives of the current study were: 1) to determine the frequency of cyclin D1 expression in a large cohort of patients with DLBCL; 2) to comprehensively characterize patients who have DLBCL with cyclin D1 expression; and 3) to estimate of the frequency with which pleomorphic MCL might be misdiagnosed as DLBCL.

MATERIALS AND METHODS

Patients

In total, 1435 patients with de novo DLBCL were evaluated. All patients were a part of the International DLBCL Rituximab-CHOP Consortium Program[12-15] and were diagnosed according to World Health Organization (WHO) classification criteria. Exclusion criteria were transformation from low-grade B-cell lymphoma and immunodeficiency-associated cases, especially human immunodeficiency virus infection. This study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review boards of all participating institutions. The overall collaborative study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center.[12-15]

Tissue Microarray and Immunohistochemistry

Hematoxylin-eosin stained slides from each of the 1435 patients with DLBCL were reviewed, and tumor-rich areas were selected. Tissue microarrays (TMAs) were constructed using a tissue microarrayer (Beecher Instrument, Silver Spring, Md). Immunohistochemical analyses were performed on 4-μm TMA sections using a streptavidin-biotin complex technique with antibodies that were reactive for the following antigens: cluster of differentiation 5 (CD5; a type I transmembrane protein), CD10 (a type II transmembrane protein), CD30 (a type I transmembrane protein), B-cell leukemia/lymphoma 2 (BCL-2), BCL-6, cyclin D1, cyclin D2, cyclin D3, forkhead box P1 (FOXP1), germinal center B-cell–expressed transcript 1 (GCET1), multiple myeloma antigen 1 (MUM1), and nuclear factor-κB (NF-κB) pathway components protein 65 (p65), p50, p52, and c-Rel (a protein encoded by the REL gene); and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in all cases. Because of tissue exhaustion, staining was not always available for each marker. Antigen expression was scored in 10% increments by assessing the percentage of immunoreactive tumor cells. A cutoff value for each marker was established using an analysis of receiver operating characteristic (ROC) curves and/or X-Tile analysis to achieve maximum specificity and sensitivity as described previously.[13] The cutoff scores for these markers were established as follows: 20% for CD30, 30% for CD10 and BCL-6; 50% for cyclin D2 and cyclin D3; 60% for GCET1, MUM1, and FOXP1; and 70% for BCL-2. When an optimal cutoff value could not be determined by ROC curve and/or X-Tile analyses, cutoff values were established based on a literature review, which indicate cutoff scores of 20% for CD5[16] and 30% for pSTAT3.[17] Any nuclear expression of cyclin D1 and each NF-κB component was considered positive. We also performed cyclin D1 and CD20 double staining in selected cases.

Fluorescence in Situ Hybridization for CCND1, BCL2, BCL6, MYC, MDM2, MDM4, and TP53 Sequencing

Fluorescence in situ hybridization (FISH) was performed on paraffin-embedded tissue sections with a locus-specific CCND1 dual-color, break-apart probe (Vysis, Downers Grove, Ill); BCL2 and BCL6 dual-color, break-apart probes (Vysis); v-myc avian myelocytomatosis viral oncogene homolog (MYC) locus-specific IGH/MYC/chromosome enumeration probe 8 (CEP8) tricolor, dual-fusion probes; a locus-specific MYC dual-color, break-apart probe (Vysis); and mouse double minute 2 oncogene E3 ubiquitin protein ligase (MDM2) and MDM4 probes as described previously.[13, 15] Tissues on the TMA were considered for evaluation if at least 200 tumor cell nuclei per core displayed positive signals. Abnormal FISH signals were recorded as the percentage of cells that had an abnormality. Tumor protein 53 (TP53) exon sequencing with a p53 AmpliChip (Roche Molecular Systems, Pleasanton, Calif) was performed in 618 patients as previously described.[12]

Cell-of-Origin Classification

Cell-of-origin (COO) classification was established by combining data from gene expression profiling (GEP), which is considered the “gold standard,” and immunohistochemistry (IHC) data. RNA was extracted from each of 583 sections using the HighPure RNA Extraction Kit (Roche Applied Science, Indianapolis, Ind) and was subjected to GEP as previously described.[12, 13, 15] COO classification could be determined in 528 cases by GEP. When COO was not classifiable with GEP, it was determined by IHC according to the Visco-Young and Choi algorithms.[13, 18] The correlation between GEP and IHC for COO classification was 86% overall.

Response Definitions and Statistical Analysis

Clinical and laboratory features were compared using the Fisher exact test for categorical variables and the Mann-Whitney U test for continuous variables. OS and PFS were calculated from the date of diagnosis to the date of last follow-up or death and from the date of diagnosis to the date of progression or death, respectively. Kaplan-Meier survival curves were used to estimate OS, and the log-rank test was used to assess differences in survival between groups. All differences with P values < .05 were considered statistically significant.

RESULTS

Patients' Characteristics

There were 821 males and 614 females (male to female ratio, 1.3:1), and the median patient age was 62.9 years (range, 3.4-96.0 years). B-symptoms were present in 35.6% of patients. Approximately half of the patients had advanced Ann Arbor stage disease (51.3%), or had an elevated serum lactate dehydrogenase level (49.4%), or had both. The Eastern Cooperative Oncology Group performance status was considered good (performance status <2) in 78.1% of patients. The International Prognostic Index was low or low-intermediate (<3) in 63% of patients. Two or more extranodal sites were involved in 23.2% of patients, and bulky tumor (≥6 cm) was identified in 44.4% of patients. In total, 964 patients received R-CHOP or an R-CHOP-like regimen, and 348 patients received CHOP or a CHOP-like regimen. Twenty-one patients received other regimens, such as doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) or fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-CVAD). No information regarding treatment was available for 102 patients. Overall, 750 (52.3%) and 645 (44.9%) DLBCLs were classified as the germinal center B-cell (GCB) and activated B-cell (ABC) subtypes, respectively. Forty tumors (2.8%) did not have sufficient data for classification either because GEP was not performed or because of tissue exhaustion.

Cyclin D1 Is Expressed in a Subset of DLBCL

Cyclin D1 protein was expressed in 30 patients (2.1%). These patients had a median age of 57 years (range, 16.0-82.6 years) (Table 1). There were 23 males and 7 females. Lymph nodes were involved in 17 patients, the spleen was involved in 1 patient, and various extranodal sites were involved in 12 patients, including small intestine, humerus, vertebra, submandibular glands, and breast. Ten patients had small disease (<6 cm), and 12 patients had bulky disease (≥6 cm). Size was unknown in 8 patients. Histologically, 23 DLBCLs were the centroblastic variant, 3 were immunoblastic, and 4 were anaplastic (Table 2, Fig. 1). None of these tumors were positive for CD5. Two tumors were positive for cyclin D2, and all tumors were negative for cyclin D3. Eleven tumors (36.7%) were positive for p50, 8 tumors (26.7%) were positive for p52, 7 tumors (23.3%) were positive for pSTAT3, 4 tumors (13.3%) were positive for p65, and 3 tumors (10%) were positive for c-Rel.

Table 1. Clinical Findings in Patients With Cyclin D1-Positive Diffuse Large B-Cell Lymphoma
No.Age, yrSexTissue TypeTreatmentResponseOS, moStatusStageLDHECOG PS≥2 Extranodal InvolvementsSize ≥ 6 cmB-SymptomsIPI Score
  1. Abbreviations: CHOP, cyclophosphamide, daunorubicin, vincristine, and prednisone; CR, complete remission; ECOG PS, Eastern Cooperative Oncology Group performance status; F, female; IPI, International Prognostic Index; LDH, lactate dehydrogenase; M, male; NA, nonapplicable; OS, overall survival; PD, progressive disease; PR, partial remission; R-CHOP, rituximab with cyclophosphamide, daunorubicin, vincristine, and prednisone; SD, stable disease.

166.9MShoulderCHOPCR149.8AliveIVNormal1NoYesNo2
269.0MSpleenNACR88.8AliveNANormalNAYesYesNoNA
327.0MLymph nodeR-CHOPCR41.0AliveIVHigh0YesNoYes3
457.0FLymph nodeR-CHOPPD7.7DeadIVNormal1YesNoYes2
569.0MLymph nodeCHOPPD17.4DeadIIHigh1NoNoNo2
640.8MSmall intestineR-CHOPPD2.2DeadIIHigh1NoYesNo1
739.8MSmall intestineCHOPCR15.9DeadIIHigh1NoYesNo1
851.3MLymph nodeCHOPCR24.4DeadIHigh1NoYesNo1
955.8MLymph nodeCHOPCR99.5DeadIVHigh2YesYesNo4
1038.9MSkinCHOPCR120.9DeadIHigh3NoNoNo2
1157.0MLymph nodeR-CHOPPR15.0DeadIINANANoNoYesNA
1237.0MLymph nodeR-CHOPCR74.5AliveIIIHigh0NoNANA2
1359.0MLymph nodeR-CHOPSD11.8DeadIIIHigh1NoNANA2
1457.0MLymph nodeR-CHOPPR38.4DeadIVHigh3YesNANA3
1577.0FLymph nodeR-CHOPCR75.7AliveIIHigh1NoNANA2
1616.0MSubmandibular glandR-CHOPCR68.8AliveIHighNANoNoNoNA
1730.0MLymph nodeR-CHOPPR5.5AliveNAHighNANoNoNoNA
1855.0MBreastR-CHOPCR60.4AliveINormal0NoNANo1
1976.0MLymph nodeR-CHOPCR75.7AliveIHigh1NoYesNo2
2058.0FNeckR-CHOPCR38.1AliveIIIHighNAYesYesYesNA
2159.0FHumerusR-CHOPPR22.2AliveNANormalNANANANANA
2259.0MLymph nodeR-CHOPPD5.1DeadIVHigh2YesNoYes4
2382.6FLymph nodeR-CHOPPR14.2NAIIINormalNANANAYesNA
2474.0FVertebral bodyCHOPPR17.2AliveIIINormal3NANANoNA
2522.0MMediastinumR-CHOPCR41.6AliveINormal0NoYesNA1
2658.0MLymph nodeR-CHOPNA50.9AliveIVNormal1NoYesNo1
2773.0MLymph nodeR-CHOPNA28.5AliveINormal1NoNoNo1
2868.0MLymph nodeR-CHOPNANAAliveIVNormalNANoYesNANA
2933.4MBone marrowCHOPPD5.4DeadIVHigh1YesNoNo2
3037.8FParotid glandCHOPCR3.5DeadNAHigh2NoYesNoNA
Table 2. Molecular and Phenotypic Findings in Patients With Cyclin D1-Positive Diffuse Large B-Cell Lymphoma
No.COOHistoD1 (%)CCND1 FISHD2D3CD5Ki-67, %p50p52p65c-RelpSTAT3BCL2BCL6MYCMDM2MDM4TP53
  1. Abbreviations: −, negative; +, positive; A, amplification; ABC, activated B-cell type; Ana, anaplastic variant; BCL2, B-cell leukemia/lymphoma 2; BCL6, B-cell leukemia/lymphoma 6; CB, centroblastic variant; CCDN1, cyclin D1 gene; CD5, cluster of differentiation 5; COO, cell of origin; D1, cyclin D1 immunohistochemistry; D2, cyclin D2 immunohistochemistry; D3, cyclin D3 immunohistochemistry; F, fluorescence in situ hybridization failed; FISH, fluorescence in situ hybridization; GCB, germinal center B-cell type; Histo, histology; IB, immunoblastic variant; MDM2, mouse double minute 2 oncogene E3 ubiquitin protein ligase; MDM4, mouse double minute 4 oncogene E3 ubiquitin protein ligase; MYC, v-myc avian myelocytomatosis viral oncogene homolog; N, normal; NA, nonapplicable; pSTAT3, phosphorylated signal transducer and activator of transcription 3; R, rearranged; T, translocation; TP53, tumor protein p53; WT, wild-type.

  2. a

    Patients 1, 2, and 3 were negative for the CCND1 gene rearrangement but had amplification of CCND1.

1GCBCB30a80NNTNNNA
2GCBCB40a40+++NNNFNWT
3GCBCB40aNA80++NRNNNWT
4ABCAna5080+++NRNNNWT
5GCBCB4050RNTNNM246V
6GCBIB80NANA70NANANANANNNNNG245S
7ABCCB2040NNNNFNA
8ABCCB30+50FNANANNNA
9ABCCB2070++NNANNNNA
10ABCCB20+40FNANANNNA
11GCBCB5030NNNANFWT
12ABCCB3080NNNNANAWT
13ABCCB20NANA60NANANANFNANANAWT
14GCBCB3050+++NNNANANAWT
15ABCCB4090NRNNANAWT
16GCBCB4090NNNNNWT
17GCBCB2095++NRNNNNA
18ABCIB20NANA50+++NAFFNANFNA
19GCBCB5090NFNANANAWT
20ABCCB3080+NNNNFWT
21GCBCB30NANA95NA+NANANNNNFWT
22ABCCB2050+NFNANNWT
23ABCCB30NAANNNANANA
24ABCIB70NA+FNNNANANA
25GCBAna30NA+++NNNNANANA
26GCBCB20NA+NNNNANANA
27GCBCB30NA++NNNNANANA
28GCBAna60NA++NNNNANANA
29GCBAna50No tissueNANA90+NANA+NANNNNNWT
30GCBCB20No tissueNANA30+FNNNNNA
Figure 1.

Photomicrographs of diffuse large B-cell lymphoma with cyclin D1 expression show (A,D) a centroblastic variant of diffuse large B-cell lymphoma with cyclin D1 expression from patient 12 (H&E staining, original magnification ×400); (B,E) an immunoblastic variant of diffuse large B-cell lymphoma with cyclin D1 expression from patient 6 (H&E staining, original magnification ×400); and (C,F) an anaplastic variant of diffuse large B-cell lymphoma with cyclin D1 expression from patient 29 (H&E staining, original magnification ×400). (G-I) Cells with cyclin D1 expression (nuclear; brown) that also expressed CD20 (membrane; magenta) were observed in (G) patient 3, (H) patient 16, and (I) patient 17. These patients were negative for CCND1 rearrangements by fluorescence in situ hybridization (results not shown).

GEP demonstrated that 17 tumors were GCB and 13 were ABC. FISH detected CCND1 amplification in 3 tumors. CCND1 abnormalities were not detected by FISH in 25 tumors. The remaining 2 tumors did not have a sufficient amount of tissue for analysis. FISH for other genes identified 1 of 30 tumors with BCL2 rearrangement, 4 of 27 tumors with BCL6 rearrangement, 2 of 22 tumors with MYC translocations, and no abnormalities involving MDM2 (n = 19) or MDM4 (n = 18). Two tumors had TP53 mutation: 1 had an A to G substitution at codon 246 in exon 7, resulting in a methionine to valine replacement; and the other had a G to A substitution at codon 245 in exon 7 of the TP53 gene, resulting in glycine to serine replacement in the protein. Both mutations were missense mutations, resulting in loss of function of the p53 protein. One case with TP53 mutation also had an MYC translocation. Treatment data were available for 29 patients with cyclin D1-positive DLBCL (Table 1). Twenty of these patients received R-CHOP and the other 9 patients received CHOP. In the former group, 6 patients died, 13 patients remained alive, and 1 patient was lost to follow-up. Seven patients were dead and 2 patients remained were alive in the latter group. The 5-year OS rate was 63.2% and 40% in patients who received treatment with R-CHOP and CHOP, respectively (P = .335).

FISH-Identified Rare, Probable, Pleomorphic MCL

From the original study group (n = 1435), 6 patients had cyclin D1 expression, and FISH revealed CCND1 rearrangements, supporting reclassification as MCL of the pleomorphic variant. The median age of these 6 patients was 66.4 years (range, 53.0-85.0 years), and they included 4 women and 2 men. Three patients had involved lymph nodes, and 2 had involved extranodal sites. The disease site was not available for 1 patient. Two patients had bulky disease (≥6 cm), 2 patients had small lymph nodes (< 6 cm), and size was not available for 2 patients.

Histologically, the pleomorphic MCLs could not be distinguished from the DLBCLs. All tumors had diffuse proliferation of large, atypical, lymphoid cells with round-to-oval nuclear contours, vesicular chromatin, 2 or 3 membrane-bound nucleoli, and a moderate amount of cytoplasm intermixed with scattered immunoblasts, mimicking the centroblastic variant of DLBCL (Fig. 2). All pleomorphic MCLs overexpressed cyclin D1 in at least 60% of tumor cells, in which cyclin D2 and cyclin D3 were negative. All tumors were positive for CD5. Two tumors were positive for p52; and p50, p65, c-Rel, and pSTAT3 were positive in 1 tumor each. There were no abnormalities in BCL2 (n = 6), BCL6 (n = 6), MYC (n = 5), MDM2 (n = 5), or MDM4 (n = 5). TP53 mutation analysis was performed in 4 tumors, and 1 tumor had a nonsense mutation. The C to T substitution at codon 192 in exon 6 (Q192X) resulted in a stop codon.

Figure 2.

These are photomicrographs of two cases of pleomorphic mantle cell lymphoma. (A,D) Diffuse sheets of large lymphoid cells mimic the centroblastic variant of diffuse large B-cell lymphoma (H&E staining, magnification ×1400). (B,E). Cyclin D1 is expressed in the majority of lymphoma cells (magnification ×1400). (C,F) Fluorescence in situ hybridization for CCND1 reveals fused normal alleles (yellow arrows) and separated red and green signals corresponding to the rearranged alleles (original magnification ×1000).

Four patients received treatment with R-CHOP, and 2 received treatment with CHOP. Five patients passed away and 1 patient remained alive at the last follow-up.

Cyclin D1-Positive DLBCL Versus Cyclin D1-Negative DLBCL

We compared the patients who had cyclin D1-positive DLBCL with those who had cyclin D1-negative DLBCL (Table 3). When age was analyzed as a continuous variable, patients with cyclin D1-positive DLBCL were significantly younger than those with cyclin D1-negative DLBCL (median age, 57 years vs 63 years, respectively; P = .022). This trend was reproduced when age was analyzed as a categorical variable (age ≥60 years vs <60 years; P = .004). Men were more commonly affected by cyclin D1-positive DLBCL (P = .039). All other clinical findings, including COO classification, CD5 expression, NF-κB components, and pSTAT3 status, were similar in the 2 groups. GEP identified 22 genes, including CCND1, that were up-regulated in patients who had DLBCL with cyclin D1 expression compared with those who had DLBCL without cyclin D1 expression (Fig. 3A). Some of the other substantially up-regulated genes were solute family carrier 2A member 4 regulator (SLC2A4RG), zinc finger protein 319 (ZNF319), ZNF444, ZNF519, and MYC-associated zinc finger protein (purine-binding) (MAZ) transcription factors; Fas apoptotic inhibitory molecule 3 (FAIM3) (anti-apoptotic function); and ATP-binding cassette, subfamily C, member 5 (ABCC5) (ATP-binding cassette transporter). However, striking differences in GEP between the 2 groups were not observed. Compared with patients who had DLBCL without cyclin D1 expression, patients who had cyclin D1-positive DLBCL had similar OS and PFS (P = .788 and P = .847, respectively) (Fig. 4). Separating out the GCB type from the ABC type, patients with cyclin D1-positive DLBCL and cyclin D1-negative DLBCL had similar OS and PFS in the GCB group (P = .969 and P = .999, respectively) and the ABC group (P = .788 and P = .914), respectively.

Table 3. Comparison Between Diffuse Large B-Cell Lymphoma Without Cyclin D1 Expression and Diffuse Large B-Cell Lymphoma With Cyclin D1 Expression
 No. of Patients (%) 
VariableCyclin D1-Negative DLBCLCyclin D1-Positive DLBCLTwo-sided P
  1. Abbreviations: ABC, activated B-cell type; CD5, cluster of differentiation 5 (a type I transmembrane protein); c-REL, protein encoded by the REL gene; DLBCL, diffuse large B-cell lymphoma; ECOG PS, Eastern Cooperative Oncology Group performance status; GCB, germinal center B-cell type; IPI, International Prognostic Index; LDH, lactate dehydrogenase; MCL, mantle cell lymphoma; pSTAT3, phosphorylated signal transducer and activator of transcription 3.

  2. a

    Two-tailed P values were determined with the Mann-Whitney U test. Values in boldface indicate a statistically significant difference.

Age, y   
Median [range]63.0 [3.4-96.0]57.0 [16.0-82.6].022a
≥60718 (57.6)9 (30)-
<60528 (42.4)21 (70).004
Sex   
Male717 (57.5)23 (76.7)-
Female530 (42.5)7 (23.3).039
Stage   
I-II573 (49.1)12 (46.2)-
III-IV595 (50.9)14 (53.8).844
LDH   
Normal576 (53.3)11 (37.9)-
High504 (46.7)18 (62.1).131
ECOG PS   
<2816 (79.1)16 (72.7)-
≥2216 (20.9)6 (27.3).436
Extranodal involvement   
<2898 (77.3)19 (70.4)-
≥2263 (22.7)8 (29.6).362
B-symptoms   
No707 (64.4)17 (73.9)-
Yes391 (35.6)6 (26.1).388
IPI score   
0-2665 (63.6)16 (80)-
3-5381 (36.4)4 (20).161
Tumor size, cm   
<6518 (57)10 (45.5)-
≥6391 (43)12 (54.5).286
Cell of origin   
GCB651 (53.2)17 (56.7)-
ABC572 (46.8)13 (43.3).853
CD5   
Negative1155 (94.9)29 (100)-
Positive62 (5.1)0 (0).395
p50   
Negative682 (63.1)17 (60.7)-
Positive398 (36.9)11 (39.3).844
p52   
Negative812 (76)20 (71.4)-
Positive256 (24)8 (28.6).654
p65   
Negative784 (70.4)24 (85.7)-
Positive330 (29.6)4 (14.3).093
c-Rel   
Negative770 (74)24 (88.9)-
Positive270 (26)3 (11.1).115
pSTAT3   
Negative695 (67.3)18 (72)-
Positive338 (32.7)7 (28).829
Figure 3.

Gene expression profiling is shown. (A) Diffuse large B-cell lymphoma (DLBCL) is shown with and without cyclin D1 expression. (B) Pleomorphic mantle cell lymphoma (MCL) and DLBCL are shown with cyclin D1 expression.

Figure 4.

Overall survival (OS) and progression-free survival (PFS) are compared between patients who had diffuse large B-cell lymphoma with and without cyclin D1 expression. The top row illustrates the survival of all patients with diffuse large B-cell lymphoma. The middle and bottom rows denote the survival of patients who had the germinal center B-cell (GCB) and activated B-cell (ABC) subtypes of diffuse large B-cell lymphoma, respectively.

Cyclin D1-Positive DLBCL Versus Pleomorphic MCL

When we compared patients who had cyclin D1-positive DLBCL with patients who had pleomorphic MCL, all clinical characteristics, COO classification, the Ki-67 proliferation rate, the expression of NF-κB components, and pSTAT3 expression did not differ significantly (P > .05) (Table 4). However, CD5 was expressed in all patients with pleomorphic MCL but not in patients with cyclin D1-positive DLBCL (P < .001). The median proportion of cyclin D1-stained cells also was much higher in patients who had pleomorphic MCL versus those with cyclin D1-positive DLBCL (85% vs 30%, respectively; P < .001). OS did not differ significantly between the 2 groups after separating the subgroups who received R-CHOP and CHOP (P = .464 and P = .778, respectively). Genetic aberrations regarding BCL2, BCL6, MYC, MDM2, and MDM4 were rare in both groups. GEP revealed a striking distinction between DLBCL with cyclin D1 expression and pleomorphic MCL (Fig. 3B). In total, 66 genes were differently expressed between the 2 groups: 19 genes were up-regulated and 47 genes were down-regulated in DLBCL with cyclin D1 expression compared with pleomorphic MCL. Most down-regulated genes in DLBCL with cyclin D1 expression were genes related to ubiquitination (ubiquitin-protein ligase E3C [UBE3C], DNA excision repair cross-complementing gene 8 [ERCC8], ubiquitin-conjugating enzyme E2K [UBE2K], ubiquitin-like modifier-activating enzyme 5 [UBA5], UBA6, mex-3 RNA binding family member C [MEX3C], HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 [HERC1], and tripartite motif containing 33 [TRIM33]) and transcription factors (ZNF770, ZNF616, ZNF85, activity-dependent neuroprotective protein [ADNP], mediator complex subunit 13-like [MED13L], and solute family carrier 30 [zinc transporter] member 9 [SLC30A9]).

Table 4. Comparison Between Diffuse Large B-Cell Lymphoma With Cyclin D1 Expression and Pleomorphic Mantle Cell Lymphoma
 No. of Patients (%) 
VariableCyclin D1-Positive DLBCLPleomorphic MCLTwo-Sided P
  1. Abbreviations: ABC, activated B-cell type; c-REL, protein encoded by the REL gene; DLBCL, diffuse large B-cell lymphoma; ECOG PS, Eastern Cooperative Oncology Group performance status; GCB, germinal center B-cell type; IPI, International Prognostic Index; LDH, lactate dehydrogenase; MCL, mantle cell lymphoma; pSTAT3, phosphorylated signal transducer and activator of transcription 3.

  2. a

    Two-tailed P values were determined with the Mann-Whitney U test. Values in boldface indicate a statistically significant difference.

Age, y   
Median [range]57.0 [16.0-82.6]66.4 [53.0-85.0].053a
≥609 (30)4 (66.7)-
<6021 (70)2 (33.3).161
Sex   
Male23 (76.7)2 (33.3)-
Female7 (23.3)4 (66.7).057
Stage   
I-II12 (46.2)1 (25)-
III-IV14 (53.8)3 (75).613
LDH   
Normal11 (37.9)1 (20)-
High18 (62.1)4 (80).635
ECOG PS   
<216 (72.7)3 (60)-
≥26 (27.3)2 (40).616
Extranodal involvement   
<219 (70.4)5 (100)-
≥28 (29.6)0 (0).296
B-symptoms   
No17 (73.9)2 (50)-
Yes6 (26.1)2 (50)0.558
IPI score   
0-216 (80)2 (50)-
3-54 (20)2 (50).251
Tumor size, cm   
<610 (45.5)2 (50)-
≥612 (54.5)2 (50)1.000
Cell of origin   
GCB17 (56.7)4 (66.7)-
ABC13 (43.3)2 (33.3)1.000
CD5   
Negative29 (100)0 (0)-
Positive0 (0)6 (100)<.001
p50   
Negative17 (60.7)5 (83.3)-
Positive11 (39.3)1 (16.7).389
p52   
Negative20 (71.4)4 (66.7)-
Positive8 (28.6)2 (33.3)1.000
p65   
Negative24 (85.7)5 (83.3)-
Positive4 (14.3)1 (16.7)1.000
c-Rel   
Negative24 (88.9)5 (83.3)-
Positive3 (11.1)1 (16.7)1.000
pSTAT3   
Negative18 (72)4 (80)-
Positive7 (28)1 (20)1.000
Cyclin D1: Median [range], %30 [20-80]85 [60-90]<.001a
Ki-67: Median [range], %70 [30-95]52.5 [30-75].166a

Mutations in TP53 were rare in both groups. The nonsense mutation identified in 1 pleomorphic MCL was a very rare event in hematolymphoid neoplasms. Three patients with pQ192* mutations in TP53 have been documented in the International Agency for Research on Cancer (IARC) TP53 Database, comprising 0.18% (3 of 1677 tumors) of all deposited hematopoietic neoplasms. Functional information (sorting intolerant from tolerant [SIFT] class) is not available for this mutation. According to the IARC TP53 Database, the 2 missense mutations in DLBCL with cyclin D1 expression also are rarely observed in hematolymphoid neoplasms, comprising 25 of 1677 (1.5%) G245S mutations and 12 of 1677 (0.72%) M246V mutations. However, both mutations are functionally deleterious according to their SIFT class.

DISCUSSION

Although the overexpression of cyclin D1 is characteristically observed at high levels in MCL, cyclin D1 expression can also be observed in a subset of other lymphoid tumors, including hairy cell leukemia (approximately 50%) and plasma cell myeloma (approximately 33%), and uncommonly in DLBCL.[19, 20] Other investigators have reported cyclin D1-positive DLBCL mostly as part of case reports and small case series.[6-8, 10, 21, 22] The reported frequency in most studies has ranged from 1.5% to 4.3%. One study reported that 15% of DLBCLs expressed cyclin D1, but that study seems to be an outlier.[6] In the current study, in accordance with earlier studies, 30 of 1429 (2.1%) de novo DLBCLs overexpressed cyclin D1.

Prior data on DLBCL with cyclin D1 expression indicated that these tumors are more often of a non-GCB type based on the Hans algorithm.[6, 8, 23] Our data contradict this previous conclusion, because more tumors (17 of 30 DLBCLs; 56.7%) were of the GCB type. One explanation for the contradiction may be that GEP was used for COO classification in our study, in contrast to previous reports in which the Hans algorithm was used. It is noteworthy that the Hans algorithm has reported 71% and 88% concordance with the GCB and ABC types, respectively, by GEP classification of DLBCL.[20] Our cohort was far larger (n = 1429) than those from prior studies by Ehinger et al (n = 231)[8] and Vela-Chavez et al (n = 66)[6] and, thus, also may be more representative.

Three patients with cyclin D1-positive DLBCL had CCND1 amplification, which is the likely explanation for cyclin D1 overexpression in these 3 tumors. This is similar to the situation in plasma cell myeloma, in which extra copies of chromosome 11 explain cyclin D1 expression in a subset of DLBCLs.[20] The explanation for cyclin D1 overexpression in the remaining 26 cyclin D1-positive DLBCLs is unclear. Except for the occurrence in relatively younger men, cyclin D1 expression in DLBCL does not confer difference in clinical manifestations, immunophenotype, genetic aberrations, or survival compared with DLBCL without cyclin D1 expression. The reason for the difference in age and sex in patients who have DLBCL with cyclin D1 expression is not clear.

It is known that NF-κB, a signaling pathway involved in cell growth and development, is constitutively activated in ABC-type DLBCL as well as MCL.[24, 25] STAT3 regulates many cell functions, including proliferation, differentiation, survival, and angiogenesis. Constitutive activation of STAT3 also has been observed in ABC-type DLBCL and in 70% of leukemic MCL.[26, 27] Recently, inhibitors of NF-κB and STAT3 have been actively studied as a new approach to cancer therapy. For these reasons, we evaluated the expression of NF-κB components (p50, p52, p65, and c-Rel) and pSTAT3 in patients who had DLBCL with cyclin D1 expression. The expression of NF-κB components was observed more commonly in DLBCL with cyclin D1 expression versus pleomorphic MCL. It is interesting that the expression of pSTAT3 was detectable in 7 of 25 (28%) cyclin D1-positive DLBCLs, and 5 of those tumors were of the GCB type. The reason for pSTAT3 expression in GCB-type DLBCL is unclear, and further studies are warranted for clarification.

We identified 6 tumors originally classified as DLBCL that were cyclin D1-positive and carried CCND1 rearrangement as part of the current study. These tumors were reclassified as probable pleomorphic MCL according to the current WHO classification. It is noteworthy that GEP of pleomorphic MCL in our study differed somewhat from that of classic MCL reported by Fu et al,[28] who demonstrated that 28 genes were significantly up-regulated in classic MCL. We included 24 of the 28 genes reported by Fu et al in our GEP study. Compared with DLBCL, only 1 gene, arachidonate 5-lipoxygenase (ALOX5), was significantly up-regulated; 17 genes were relatively up-regulated without statistical significance; 2 genes had inconsistent regulation patterns; and 4 genes were down-regulated in pleomorphic MCL. The discrepancy in GEP raises the issue of exact classification when FISH is used to separate MCL from DLBCL. Juskevicius et al, in a recently published case report of DLBCL with CCND1 rearrangements, reported that the exact classification may be a matter of debate.[29] However, according to the current WHO classification, these tumors are better designated as bona fide MCL.

Our data suggest that pleomorphic MCL is misdiagnosed as DLBCL in a small subset of patients, representing 6 of 1435 patients (0.42%) in the current study. The clinical characteristics of affected patients and morphologic features of the neoplasms were very similar between the cyclin D1-positive DLBCL and pleomorphic MCL groups. One helpful finding was CD5 expression, which was observed in all 6 patients with pleomorphic MCL. In addition, cyclin D1 expression was observed in higher proportions of tumor cells in pleomorphic MCL (median, 85%) than in DLBCL (median, 30%). Very little genetic information (other than conventional cytogenetic data) is available for pleomorphic MCL. Our study demonstrated that genetic aberrations involving BCL2, BCL6, MYC, MDM2, and MDM4 are rare in pleomorphic MCL. The TP53 mutation (Q192*) is also rarely observed in any hematolymphoid neoplasm, and this mutation appears to be a random event.

In summary, a small subset (2.1%) of patients with DLBCL overexpresses cyclin D1. Patients with cyclin D1-positive DLBCL tend to be younger men but otherwise are not clinically significantly different from patients with cyclin D1-negative DLBCL. Unlike earlier studies, over half of the cyclin D1-positive DLBCL patients in the current study had the GCB type. FISH studies revealed a low frequency or absence of translocations involving BCL2, BCL6, MYC, MDM2, and MDM4 as well as a low frequency of TP53 mutations in patients with cyclin D1-positive DLBCL. The distinction of cyclin D1-positive DLBCL from MCL is critical because of important differences in prognosis and therapy.

FUNDING SUPPORT

This study was partially supported by the National Cancer Institute/National Institutes of Health (grants R01CA138688, 1RC1CA146299, P50CA136411, and P50CA142509 to K.H.Y.). Dr. Ok is the recipient of a Hematopathology Fellowship Award. Dr. Xu-Monette is the recipient of the Shannon Timmins Leukemia Fellowship Award at The University of Texas MD Anderson Cancer Center. Dr. Young is supported by The University of Texas MD Anderson Cancer Center Institutional Research and Development Fund, an Institutional Research Grant Award, an MD Anderson Cancer Center Lymphoma Specialized Programs on Research Excellence (SPORE) Research Development Program Award, an MD Anderson Cancer Center Myeloma SPORE Research Development Program Award, a Gundersen Lutheran Medical Foundation Award, and MD Anderson Cancer Center Collaborative Funds with Roche Molecular System and HTG Molecular Diagnostics. The Consortium Program acknowledges the support from Drs. April Chiu, William W. L. Choi, Weiyun Ai, Xiaoying Zhao, Jooryung Huh and Andres J. M. Ferreri during this collaboration and apologizes for not being included into the authorship due to limit of author numbers from the journal.

CONFLICT OF INTEREST DISCLOSURES

Dr. O'Malley reports a salary from Clarient/GE Healthcare.

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