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01.12.2013 | Research | Ausgabe 1/2013 Open Access

Journal of Hematology & Oncology 1/2013

Clinical significance of cytogenetic aberrations in bone marrow of patients with diffuse large B-cell lymphoma: prognostic significance and relevance to histologic involvement

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2013
Autoren:
Seon Young Kim, Hyo Jung Kim, Hye Jin Kang, Jin Seok Kim, Hyeon Seok Eom, Tae Min Kim, Sung-Soo Yoon, Cheolwon Suh, Dong Soon Lee, Korean Society of Hematology Lymphoma Working Party
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1756-8722-6-76) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

CS and DSL designed the study, SYK, HJK, HJK, JSK, HSE, TMK, SSY, CS, and DSL provided study materials or patients, collected and assembled data. SYK, CS, and DSL analyzed and interpreted data. SYK, HJK, HJK, JSK, HSE, TMK, SSY, CS, and DSL wrote the manuscript. All authors have read and approved the final manuscript.

Introduction

Bone marrow (BM) evaluations are an essential part of the routine staging of diffuse large B-cell lymphoma (DLBCL) [1]. DLBCL with BM involvement is rated as Ann Arbor stage IV, resulting in higher International Prognostic Index (IPI) scores and, thus, poor prognoses [2]. BM has traditionally been evaluated by morphological examination, which commonly includes immunohistochemical (IHC) staining. Histologic BM involvement has been reported in 10-30% of DLBCL cases [3, 4]. Recently, additional efforts have been made to detect even a minimal involvement of lymphoma cells using flow cytometry and molecular or cytogenetic techniques. With the application of these complementary tests, approximately 10-20% of cases that were initially classified as histologically negative have been reassessed as having BM involvement [57]. In a previous study in which BM was evaluated using flow cytometry and immunoglobulin gene rearrangement analysis, a change in IPI was noted in 11.5% on immunophenotyping alone, and 14.1% cases on immunophenotyping and molecular testing. The revised IPI model using immunophenotyping provided better differentiation between the IPI prognostic categories [6].
Classical cytogenetic studies of BM specimens play a pivotal role in the diagnosis and prognostic prediction of many hematologic malignancies. However, the cytogenetic data concerning DLBCL tissues are limited. DLBCL is a group of B-cell malignancies that are extremely heterogeneous histopathologically, biologically, and clinically. Consistent with this heterogeneity, various chromosomal abnormalities have been reported in patients with DLBCL [8]. Correlations between cytogenetic data and clinical outcomes have been attempted for DLBCL; however, controversy remains concerning the prognostic significance of these data, most of which were obtained before the initiation of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) therapy.
In Korea, cytogenetic studies of BM specimens using the G-banding technique have been a routine practice in many hospitals, primarily to aid in the detection of BM involvement when staging newly diagnosed DLBCL patients. Cytogenetic study of the BM can overcome the limitations of tumor tissue cytogenetics such as fuzzy chromosomes, failure in obtaining cells in metaphase, and contamination. The presence of chromosomal aberrations in the absence of histologic involvement of BM raises the question as to whether the abnormalities truly originate from BM involving-lymphoma cells or if the aberrations are just cytogenetic noise. In the present study, to investigate the characteristics of chromosomal aberrations in the BM of DLBCL patients and to determine their prognostic significance, we retrospectively analyzed cytogenetic data of BM specimens submitted for staging from a large series of DLBCL patients.

Materials and methods

Study population

A total of 1585 DLBCL cases were referred from six tertiary hospitals in Korea: Seoul National University Hospital (n = 646; 1996 to 2011); Asan Medical Center (n = 484; 2001 to 2009); National Cancer Center of Korea (n = 236; 2004 to 2009); Yonsei University Hospital (n = 118; 2004 to 2009); Hallym University Hospital (n = 57; 2004 to 2009); and Korea Cancer Center Hospital (n = 44; 2005 to 2009). The cases were selected on the basis of diagnoses established according to the 2008 World Health Organization (WHO) classification criteria for primary tissue biopsy specimens [9]. BM biopsies were conducted for staging purposes at the time of the initial diagnosis. The treatment protocols were heterogeneous but generally conformed to international standards, including combination chemotherapy using CHOP-like regimens for front-line therapy, as well as salvage chemotherapy followed by stem cell transplantation for refractory cases. The baseline patient characteristics are summarized in Table 1. All of the patients were Korean, and the median age was 57 years (range, 2–91 years). A total of 1128 patients (71.2%) received R-CHOP as the initial therapy, 380 patients (24.0%) received a therapy other than R-CHOP (157 CHOP and 223 other regimens), and 77 patients (4.9%) received an unknown treatment or no treatment. The median follow-up time was 25.7 months (range, 0.1-211.1 months).This study was reviewed and approved by the institutional review board of each hospital.
Table 1
The baseline characteristics of 1585 DLBCL patients and a comparison of the clinical features of the patients with histologic BM involvement (BMI histo  + ) and those without (BMI histo  – )
Characteristics
Total
BMIhisto+
BMIhisto
P value*
 
(n = 1585)
(n = 259)
(n = 1326)
 
Median age, years (range)
57.4 (1.9-90.9)
59.0 (1.9-86.4)
57.0 (5.3-90.9)
0.013
Age > 60 yr
679/1585 (42.8)
124/259 (47.9)
555/1326 (41.9)
0.073
Gender (male/female, %male)
881/704 (55.6)
132/127 (51.0)
749/577 (56.5)
0.102
B symptoms
394/1585 (24.9)
135/259 (52.1)
259/1326 (19.5)
<0.001
ECOG ≥ 2
226/1585 (14.3)
86/259 (33.2)
140/1326 (10.6)
<0.001
High serum LDH
975/1585 (61.5)
203/259 (78.4)
772/1326 (58.2)
<0.001
Stage 3 or 4
822/1585 (52.6)
259/259 (100)
574/1326 (43.3)
<0.001
Stage 3 or 4, excluding BM status
809/1585 (51.0)
237/259 (91.5)
572/1326 (43.1)
<0.001
Extranodal involvement ≥ 2 sites
497/1585 (31.4)
193/259 (74.5)
304/1326 (22.9)
<0.001
International Prognostic Index
    
  Low risk
392/1585 (24.7)
0/259 (0)
392/1326 (29.6)
<0.001
  Low/intermediate risk
462/1585 (29.2)
33/259 (12.7)
427/1326 (32.4)
 
  High/intermediate risk
467/1585 (29.5)
114/259 (44.0)
353/1326 (26.6)
 
  High risk
264/1585 (16.7)
112/259 (43.2)
152/1315 (11.5)
 
Non-GCB type
500/877 (57.0)
95/150 (63.3)
405/727 (55.7)
0.086
CD5-positive
33/341 (9.7)
7/57 (12.3)
26/284 (9.2)
0.466
Initial treatment
    
  R-CHOP
1128/1585 (71.2)
146/259 (56.4)
982/1326 (74.1)
<0.001
  CHOP
157/1585 (9.9)
30/259 (11.6)
127/1326 (9.6)
 
  Other treatment
223/1585 (14.1)
53/259 (20.5)
170/1326 (12.8)
 
  No therapy
77/1585 (4.9)
30/259 (11.6)
47/1326 (3.5)
 
Initial treatment response
    
  CR
1020/1394 (73.2)
118/207 (57.0)
902/1187 (76.0)
<0.001
  PR
268/1394 (19.2)
51/207 (24.6)
217/1187 (18.3)
 
  SD
33/1394 (2.4)
8/207 (3.9)
25/1187 (2.1)
 
  PD
73/1394 (5.2)
30/207 (14.5)
43/1187 (3.6)
 
Follow-up data
    
  Deaths
484/1585 (30.5)
134/259 (51.7)
350/1326 (26.4)
<0.001
  Median follow-up, months (range)
25.7 (0.1-211.1)
12.0 (0.1-141.4)
29.5 (0.1-211.1)
<0.001
*P value are based on the chi-square test for categorical variables and the Mann–Whitney U test for continuous variables.
The data represent the median (range) for continuous variables or the number (percentage) for categorical variables, unless otherwise indicated.
Abbreviations: BMI, bone marrow involvement; CR, complete remission; DLBCL, diffuse large B-cell lymphoma; ECOG, Eastern Cooperative Oncology Group; GCB, germinal center B-cell-like; LDH, lactic dehydrogenase; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone; PD, progressive disease; PR, partial remission; SD, stable disease.

Histopathology

A primary DLBCL diagnosis was established by examining hematoxylin and eosin (H&E)-stained sections of diagnostic biopsies from various tissues with IHC stains, including CD3, CD20, CD5, CD10, BCL2, BCL6, and IRF/MUM1, according to the diagnostic protocol of each institute. The type of DLBCL based on the cell of origin, either germinal center B-cell-like (GCB) or non-GCB, was defined in 877 cases using the algorithm of Hans et al. [10]. In general, the BM biopsies were performed bilaterally. Wright-stained BM smears and H&E-stained sections of BM biopsies were reviewed by hematopathologists at each institute. IHC staining was performed at the discretion of hematopathologists at each institute. The BM reports were reviewed centrally, and additional IHC staining targeting CD3, CD20, and CD79a was performed for cases with discrepant results between histologic examination and cytogenetic tests, to confirm the initial BM histologic diagnosis. The presence of benign lymphoid aggregates was distinguished from lymphoma involvement using previously described criteria [11]. Flow cytometric analysis was performed for some cases with diffuse infiltration of lymphoma cells (n = 46). According to the BM results, the cases were dichotomized into those with histological BM involvement (BMIhisto+) and those without (BMIhisto-).

Cytogenetic analysis of BM

Conventional cytogenetic tests using the G-banding technique were performed on BM aspirates from all the patients. The cytogenetic tests were performed locally, and the reports were reviewed centrally by two of the investigators (SYK and DSL). Cytogenetic studies using standard techniques were performed as a part of the diagnostic work-up at the time of initial diagnosis. Conventional G-banding cytogenetic analysis was performed using the short-term unstimulated culture (24–48 h) of BM cells. At least 20 metaphases were analyzed, whenever possible. Clonal abnormalities were defined as at least two cells with the same aberration if the aberration is a chromosome gain or a structural rearrangement, or 3 or more cells with the same chromosome missing. The karyotypes were recorded according to the International System for Human Cytogenetic Nomenclature (ISCN) [12]. We classified the karyotype results according to the complexity of the chromosomal abnormalities observed. A complex karyotype was defined as ≥ 3 chromosomal abnormalities, in accordance with previous studies [13, 14]. A monosomal karyotype was defined as either a single autosomal monosomy in the presence of one or more structural aberration or two or more distinct autosomal chromosome monosomies [15, 16]. In the interpretation of specific abnormalities, numerical aberrations included gains, losses of chromosomes (aneuploidy) and changes in ploidy. Structural aberrations included abnormalities such as deletions, translocations, isochromosomes, and duplications. Translocations included balanced reciprocal translocations and translocations with unknown partners (additions).
Fluorescence in situ hybridization (FISH) was performed in some cases (n = 235). The following target regions were investigated using the accompanying probes: 14q32/IGH using an IGH dual-color, break-apart rearrangement probe (n = 201); cMYC/IGH using a dual-color, dual-fusion probe (n = 11); BCL2/IGH using a dual-color, dual-fusion probe (n = 29); 9p21/p16 using a p16/CEP 9 dual-color probe (n = 178); 3q27/BCL6 using a BCL6 dual-color, break-apart rearrangement probe (n = 95); 8q24/cMYC using a cMYC break-apart probe (n = 39) or a cMYC/IGH using dual-color, dual-fusion probe (n = 11); 1q25 using a 1p32/1q25 probe (n = 49); 17p13/TP53 using a P53 probe (n = 34); and 18q23/BCL2 using a BCL2/IGH dual-color, dual-fusion probe (n = 29; all probes from Abbott/Vysis, Downers Grove, IL, USA). We analyzed interphase cells according to the manufacturer’s instruction and the ISCN criteria. At least 200 nuclei per sample were scored for normal or abnormal FISH signals. The normal cut-off values for translocation, deletion, or amplification were based on the mean (± 3SD) and the binomial distribution function [17] analyzed of 40 negative controls. The cut-off values for IGH break-apart probe was 2%, and cMYC/IGH probe was < 0.5%.

Statistical analyses

The data were compared using the Mann–Whitney and Kruskal-Wallis tests for continuous variables and χ2 test for categorical variables. Each numerical abnormality and the specific locus of each structural abnormality were dichotomized as present or absent, and hierarchical clustering was performed using Pearson correlation distance metrics and Wald linkage tests. The probabilities of overall survival (OS) and progression-free survival (PFS) [18] were plotted according to the Kaplan-Meier method, and the log-rank test with Bonferroni correction for multiple testing was used to compare the survival curves. A multivariate analysis was performed using the Cox regression method. The following parameters were analyzed for multivariate analysis: advanced age, gender, IPI risk groups, history of R-CHOP treatment, BMIhisto+ vs BMIhisto-, ≥ 2 abnormalities vs normal karyotype or 1 abnormality, and presence of several specific cytogenetic abnormalities abnormalities, which were associated with poor prognosis in univariate analysis and found in a minimum of 5 patients. Variables in the final model were selected using stepwise selection procedure with a threshold of P = 0.05. The statistical analyses were performed using SPSS version 15 (SPSS, Chicago, IL, USA) and the R statistical package (R Development Team 2012). A probability level of 0.05 was considered significant in the univariate analysis. When multiple hypothesis testing was performed, the P value was adjusted by Bonferroni correction.

Results

Comparison of histology and conventional cytogenetic tests for the detection of BM involvement

A total of 259 patients (16.3%) had BM involvement, as determined through histologic examinations. Among the 259 BMIhisto+ patients, 181 (69.9%) exhibited lymphoma cells on BM aspirate smears, and all 259 patients demonstrated lymphoma involvement in a BM biopsy. The median percentage of lymphoma cells in the BM aspirate smear was 6.2% (range, 0-98%). Compared with the BMIhisto group, the BMIhisto+ group had a poorer performance status, higher lactate dehydrogenase (LDH) levels, more advanced stage tumors, and more prevalent extranodal involvement; consequently, these patients had higher IPI scores (Table 1).
Chromosomal abnormalities were detected in 192/1585 patients (12.1%), of whom 124/192 (64.6%) were BMIhisto+ (Table 2). Among the 192 patients with chromosomal aberrations, 42 (21.9%) exhibited the following single numerical aberrations: 33 patients had Y chromosome loss; 7 patients had a loss or gain of other single chromosomes; and 2 patients had hyperdiploid clones (Additional file 1: Table S1). Among the 42 patients with the above single numerical aberrations, 4/42 (9.5%) were BMIhisto+, which was not significantly different from the proportion of BMIhisto+ patients with normal karyotypes (135/1393, 9.7%; P = 0.971). There were 12 patients with single structural abnormalities, of whom 4 patients exhibited single deletions [del(13q) in 2 patients and del(20)(q11.2) in 2 patients], and 1 patient had a duplication of Y chromosome; none were BMIhisto+. Seven patients exhibited translocations, including t(2;11)(p21;q23), t(4;10)(q28;p13), t(6;18)(p23;p11), t(10;11)(q22;q23), add(12)(q24), t(3;14)(q27;q32), and t(14;18)(q32;q21); the patients with the latter 3 types of abnormalities were BMIhisto+. The remaining 138/192 patients (71.9%) exhibited ≥ 2 aberrations. Ten of these patients exhibited 2 chromosomal abnormalities: 6 patients had 1 structural aberration and 1 numerical aberration, and 4 patients had 2 structural aberrations. Among the patients with 2 chromosomal abnormalities, 6/10 (60%) were BMIhisto+. A total of 128 patients exhibited complex karyotypes. Many of these patients had highly complex abnormalities, with a median of 9 total chromosomal abnormalities (range, 3–25) including many structural abnormalities (median, 7; range, 0–20). Among the patients with complex karyotypes, 111/128 were BMIhisto+ (86.7%). Consequently, 117/138 patients (84.8%) with ≥ 2 chromosomal aberrations were BMIhisto+. Among the patients with complex karyotypes, the percentages of abnormal metaphases were higher in the BMIhisto+ group compared with the BMIhisto- group (P = 0.024). The percentages of metaphases with Y chromosome losses were higher in the BMIhisto+ group (P = 0.021), however, there was no significant differences in the number of metaphases for patients with loss of single chromosomes other than Y, polyploidy or single structural abnormalities between the BMIhisto+ and BMIhisto- groups. Among the patients with complex karyotypes, only a weak correlation was found between the percentage of metaphases with aberrant karyotypes and the percentage of lymphoma cells in BM aspirates (r = 0.365). Our results indicate that a finding of BM cytogenetic aberrations involving single numerical abnormalities alone cannot be regarded as sufficient evidence of BM involvement of lymphoma cells, considering the low concordance between such findings and the histologic results. If multiple chromosomal abnormalities were considered as definite evidence of the presence of lymphoma cells, the 21/1585 (1.3%) patients with isolated cytogenetic aberrations without histologic involvement would be reassessed as having BM involvement.
Table 2
A comparison of the histologic and conventional cytogenetic analyses
Cytogenetic findings
No. of patients
No. of BMIhisto+cases
Percentage of abnormal metaphases
BM lymphoma cell percentage
BMIhisto+
BMIhisto-
P value*
Normal karyotype
1393 (87.9)
135/1393 (9.7)
NA
NA
NA
1 (0–98)
Abnormal karyotype
192 (12.1)
124/192 (64.6)
49 (8–100)
25 (5–100)
< 0.001
18 (0–95)
  Single abnormality
54 (3.4)
7/54 (13.0)
35 (8–100)
25 (9–100)
0.697
5 (0–86)
   Single numerical abnormality
42 (2.6)
4/42 (9.5)
60 (27–100)
21 (10–100)
0.038
3 (0–86)
    Loss of Y
33 (2.1)
3/33 (9.1)
70 (50–100)
20 (10–100)
0.021
6 (0–86)
    Loss of another single chromosome
7 (0.4)
1/7 (14.3)
27
22 (15–70)
0.617
0
    Polyploidy
2 (0.1)
0/2 (0)
NA
27 (22–31)
NA
NA
   Single structural abnormality
12 (0.8)
3/12 (25.0)
15 (8–35)
67 (9–100)
0.090
5 (0–29)
  Multiple abnormalities
138 (8.8)
117/138 (84.8)
50 (8–100)
25 (5–100)
0.009
19 (0–95)
   2 abnormalities
10 (0.7)
6/10 (60.0)
67 (10–100)
23 (15–58)
0.394
41 (12–73)
    1 structural and 1 numerical abnormality
6 (0.4)
4/6 (66.7)
67 (13–92)
39 (20–58)
0.355
27 (12–50)
    2 structural abnormalities
4 (0.3)
2/4 (50.0)
55 (10–100)
20 (15–25)
0.999
61 (49–73)
   ≥ 3 abnormalities
128 (8.1)
111/128 (86.7)
48 (8–100)
30 (5–100)
0.024
18 (0–95)
   (complex karyotype)
      
*P value by the Mann–Whitney U test comparing the percentage of abnormal metaphases between the BMIhisto+ and BMIhisto- groups.
Lymphoma cell percentage calculated from bone marrow aspirate smears.
This case showed diffuse DLBCL involvement in the bone marrow biopsy.
The data represent the median (range) for continuous variables or the number (percentage) for categorical variables, unless otherwise indicated.
Abbreviations: BMIhisto+, histologic bone marrow involvement; BMIhisto-, no evidence of histologic bone marrow involvement; NA, not applicable.

Chromosomal abnormalities and clinical and biological characteristics

We compared the clinical and biological characteristics of the BMIhisto+ patients with normal karyotypes to patients with abnormal karyotypes. Compared with the patients with normal karyotypes, the patients with a single numerical abnormality presented with less aggressive disease, as evidenced by their lower LDH levels, less involvement of extranodal sites, and lower IPI risk scores. In contrast, the patients with ≥ 2 chromosomal abnormalities had a poorer performance status, higher LDH levels, more advanced disease stages, more prevalent extranodal involvement, and thus, higher IPI scores (Table 3).
Table 3
A comparison of the clinical and laboratory characteristics of patients with histologic BMI (BMI histo + ) with normal karyotypes and patients with chromosomal abnormalities
Parameters
BMIhisto+and normal karyotype
Abnormal karyotypes
(n = 135)
1 numerical
1 structural
≥ 2 abnormalities
  
(n = 42)
(n = 12)
(n = 138)
Age (years)
    
  Median (range)
58 (2–86)
67 (11–83) *
56 (45–77)
60 (10–86)
  Age > 60 yr
64/135 (47.4)
34/42 (81.0) *
5/12 (41.7)
66/138 (47.8)
Gender (male/female)
59/76 (43.7)
35/7 (83.3) *
10/2 (83.3) *
76/62 (55.1)
ECOG ≥ 2
26/135 (19.3)
6/42 (14.3)
0/12 (0)
62/138 (44.9) *
High serum LDH
95/135 (70.4)
16/42 (38.1) *
6/12 (50.0)
123/138 (89.1) *
B symptoms
50/135 (37.0)
7/42 (16.7) *
6/12 (50.0)
98/138 (71.0) *
Stage 3 or 4,
123/135 (91.1)
18/42 (42.9) *
9/12 (75.0)
126/138 (91.3)
excluding BM status
    
Extranodal ≥ 2 sites
89/135 (65.9)
13/42 (31.0) *
5/12 (41.7)
112/138 (81.2) *
IPI risk
    
  Low
0/135 (0)
16/42 (38.1)
1/12 (8.3)
1/138 (0.7)
  Low/intermediate
28/135 (20.7)
10/42 (23.8)
5/12 (41.7)
4/138 (2.9)
  High/intermediate
65/135 (48.2)
10/42 (23.8)
6/12 (50.0)
58/138 (42.0)
  High
42/135 (31.1)
6/42 (14.3) *
0/12 (0) *
75/138 (54.4) *
Non-GCB type
46/73 (63.0)
17/26 (65.4)
3/8 (37.5)
53/88 (60.2)
CD5- positive
4/32 (12.5)
0/3 (0)
0/1 (0)
3/30 (10.0)
R-CHOP treatment
83/135 (61.5)
31/42 (73.8)
8/12 (66.7)
78/138 (56.5)
Death
53/135 (39.3)
10/42 (23.8)
2/12 (16.7)
90/138 (65.2) *
*Significant P value (< 0.05) for each subgroup compared with the group of BMIhisto+ patients with normal karyotypes.
The data represent the number (percentage) for categorical variables, unless otherwise indicated.
Abbreviations: DLBCL, diffuse large B-cell lymphoma; ECOG, Eastern Cooperative Oncology Group; GCB, germinal center B-cell-like; IPI, International Prognostic Index; LDH, lactic dehydrogenase; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone.

Comparison of BM histology and FISH results

The histologic examinations, conventional cytogenetics, and FISH results of a limited number of patients were compared (Table 4). Among the 235 patients for whom FISH studies were performed, there were 3 BMIhisto- patients with normal karyotypes and abnormal 14q32/IGH FISH results, although the frequencies of abnormal FISH signals were low (5%, 7%, and 8%). A single BMIhisto- patient exhibited cytogenetic abnormalities of the 8q24 locus despite normal FISH results using a specific probe.
Table 4
A comparison of conventional cytogenetic (CG) and fluorescence in situ hybridization (FISH) results
 
FISH positve
FISH negative
Probe
No. of patients
BMIhisto+/
BMIhisto+/
BMIhisto-/
BMIhisto-/
No. of patients
BMIhisto+/
BMIhisto+/
BMIhisto-/
BMIhisto-/
  
CGwhole+
CGwhole-
CGwhole+
CGwhole-
 
CGlocus+
CGlocus-
CGlocus+
CGlocus-
14q32/IGH
37/235 (15.7)
20 (54.1)
11 (29.7)
3 (8.1)
3 (8.1)
198/235 (84.3)
6 (3.0)
54 (27.3)
0 (0.0)
138 (69.7)
9p21/p16
17/178 (9.6)
14 (82.4)
3 (17.6)
0 (0)
0 (0)
161/178 (90.4)
3 (1.9)
50 (31.1)
0 (0)
108 (67.1)
3q27/BCL6
19/95 (20.0)
14 (73.7)
5 (26.3)
0 (0)
0 (0)
76/95 (80.0)
2 (2.6)
28 (36.8)
0 (0)
46 (60.5)
8q24/MYC
7/50 (14.0)
5 (71.4)
2 (28.6)
0 (0)
0 (0)
43/50 (86.0)
3 (7.0)
12 (27.9)
1 (2.3)
27 (67.8)
1p32/1q25
9/49 (18.4)
9 (100)
0 (0)
0 (0)
0 (0)
40/49 (81.6)
1 (2.5)
21 (52.5)
0 (0)
18 (45.0)
17p13/TP53
3/34 (8.8)
1 (33.3)
2 (66.7)
0 (0)
0 (0)
31/34 (91.2)
0 (0)
8 (25.8)
0 (0)
23 (74.2)
18q23/BCL2
3/29 (10.3)
0 (0)
2 (66.7)
1 (33.3)
0 (0)
26/29 (89.7)
2 (7.7)
7 (26.9)
0 (0)
17 (65.4)
The data represent the number (percentage) for categorical variables.
Abbreviations: FISH, fluorescence in situ hybridization; CGwhole+/CGwhole-, normal or abnormal results of conventional cytogenetic tests considering whole chromosomes; CGlocus+/CGlocus-, normal or abnormal results of conventional cytogenetic tests for the specific locus, which is targeted by FISH probes.

Characteristics of cytogenetic aberrations

We analyzed the frequencies of specific cytogenetic aberrations among the patients with chromosomal aberrations other than single numerical aberrations (n = 150) (Figure 1). The chromosomes most frequently involved were chromosome 1, 3, 6, 14, and 18. The most common numerical aberrations were trisomy 18, trisomy 7, trisomy 3, loss of Y, and loss of 13. The predominant structural aberrations involved the following loci: rearrangements involving 14q32, 19q13, 19p13, 1p32-p36, 3q27, 8q24, 18q21-q23, 1cen-1q12, 9p22-p24, 11q23-q25, 16q22-q24, and; deletions of 6q; and duplications of 1q. The well-known oncogenes and lymphoma-related genes that exhibited frequent breakpoints, including BCL6 (3q27), JAK2 (9p22), IGH (14q32), and BCL2 (18q21), as well as other possible oncogenes with breakpoints, are indicated in Figure 1D. Monosomal karyotypes were observed in 70 patients (46.7%), and hyperdiploidy was noted in 24 cases (16.0%). Reciprocal translocations were observed in 90 patients (60.0%); among them, 19 carried reciprocal translocations involving the 14q32/IGH region with defined partners. The translocation partners were the following: t(8;14)(q24;q32) in 5 cases; t(14;18)(q32;q21) in 4 cases; t(3;14)(q27;q32) in 3 cases; t(1;14)(q21;q32); t(1;14)(q25;q32); t(3;14)(p25;q32); t(6;14)(q25;q32); t(9;14)(p13;q32); t(9;14)(q13;q32); and t(14;19)(q32;q13).

Prognoses according to the chromosomal abnormalities and BM histology

We analyzed the prognostic impacts of chromosomal abnormalities according to the complexity of the chromosomal aberrations involved (Figure 2). There was a significant difference in prognosis between patients with ≥ 2 abnormalities and those with a normal karyotype or a single abnormality both in the BMIhisto+ group (5-year OS, 22.0% vs. 52.7%; P < 0.001) and BMIhisto- group (5-year OS, 31.8% vs. 66.5%; P < 0.001). In BMIhisto- groups, the patients with a single numerical abnormality and those with single structural abnormality did not exhibit significant differences in OS compared with those with normal karyotypes (P = 0.422 and 0.137, respectively). The number of BMIhisto+ patients with single abnormalities was too small to assess prognostic impact (Additional file 2: Figure S1). There was no significant difference in OS and PFS according to the number of abnormalities among the patients with ≥ 2 abnormalities (Additional file 3: Figure S2).
When patients were classified according to IPI risk groups, BMIhisto+ patients with ≥ 2 cytogenetic abnormalities presented significantly poorer prognosis compared with normal karyotypes in the high/intermediate-risk group (5-year OS, 35.4% vs. 69.4%, P < 0.001; Figure 3A). BMIhisto- patients with ≥ 2 abnormalities revealed significantly poorer prognosis in the high/intermediate and high risk groups (5-year OS, 31.8% vs. 58.4%, P = 0.027 for high/intermediate risk group; 0% vs. 40.6%, P < 0.001 for high risk group, respectively; Figure 3B).
When R-CHOP-treated patients were analyzed separately, ≥ 2 chromosomal abnormalities were associated with poorer prognoses compared with normal karyotypes or 1 abnormality among both the R-CHOP-treated BMIhisto+ patients (5-year OS, 33.9% vs. 70.9%; P < 0.001) xand the BMIhisto- patients (5-year OS, 39.5% vs. 72.2%; P = 0.003; Additional file 4: Figure S3).
The presence of a monosomal karyotype had no apparent prognostic value among patients with ≥ 2 abnormalities (5-year OS, 25.4% vs. 20.0%; P = 0.274). There was no significant prognostic value of hyperdiploidy among patients with ≥ 2 abnormalities (5-year OS, 24.7% vs. 23.1%; P = 0.413). Among the 877 patients for whom IHC information was available, the non-GCB group demonstrated a lower OS than the GCB group (5-year OS, 58.6% vs. 71.2%, respectively; P = 0.004). However, among patients with ≥ 2 abnormalities, there was no significant difference in OS between the non-GCB and GCB types (5-year OS, 25.4% vs. 23.4%, respectively; P = 0.467).

Prognoses according to specific chromosomal abnormalities

We investigated the prognostic impacts of the specific chromosomal abnormalities. Figure 4A presents hazard ratios (HRs) obtained by univariate Cox analysis for OS and PFS according to specific chromosomal abnormalities found in ≥ 8 patients using BMIhisto+ patients with normal karyotype as a reference group among 327 patients with either cytogenetic abnormalities or were BMIhisto+. The presence of structural abnormalities at 16q22-q24 was significantly associated with a higher risk for both OS (HR, 5.86, P < 0.001) and disease progression (HR, 4.05, P < 0.001). The abnormalities at 6p21 (HR for OS, 4.89, P < 0.001), 12q22-q24 (HR, 4.28, P < 0.001), and -17 (HR, 4.49, P < 0.001) were also significant association with adverse prognosis. Consistent results were observed in the R-CHOP-treated patients (Additional file 5: Figure S4). In addition, frequent loci of cytogenetic abnormalities, including 11q21-q23, 19q13, 18q21, 1q21-q23, 8q24, 19p13, 3q27, 6q, and 14q32 were also associated with adverse outcomes.
To identify the subgroups of BM-involved DLBCL that exhibited distinct cytogenetic aberrations, we performed cluster analyses using the loci associated with poor survival in the univariate analysis and frequent and characteristic breakpoints, including 14q32, 3q27, 8q24, 19p13, and 19p13. Based on the hierarchical cluster analysis, 138 patients with ≥ 2 chromosomal abnormalities were segregated into clusters with characteristic patterns of chromosomal abnormalities (Figure 4B). When we compared prognoses among the clusters, Cluster 7, which was composed of loci associated with poorer prognoses in the univariate analysis, revealed the poorest prognosis (Figure 4C).

Multivariate analysis of prognoses among patients with BM abnormalities diagnosed by either histologic examination or conventional cytogenetic testing

A multivariate analysis was performed for 327 patients who had either chromosomal abnormalities or were BMIhisto+ (Table 5). When the presence of ≥ 2 cytogenetic abnormalities was analyzed with other covariates, the presence of ≥ 2 cytogenetic abnormalities was also significantly associated with a worse OS (HR, 2.49; 95% CI, 1.75-3.54; P < 0.001). The high IPI score was strongly associated with a poor prognosis, whereas R-CHOP treatment was strongly associated with a better prognosis. When the specific chromosomal abnormalities which were associated with adverse prognosis in the univariate analysis were analyzed, the aberration at 19p13 was selected as an independent adverse prognostic factor (HR, 2.67; 95% CI, 1.50-4.76; P = 0.001), in addition to 7q22, 12q22-q24, 18q21, and 16q22-q24. When PFS was analyzed, aberrations at 19p13 and 8q24 emerged as factors independently associated with disease progression (HR, 3.02 and 2.61, respectively; P < 0.001 and P < 0.001, respectively). When 200 R-CHOP treated patients among 327 patients were analyzed separately, 19q13 (HR, 3.36, P = 0.003), 12q22-q24, 19p13, and 8q24 were independently predicted poor OS, and 19p13 and 8q24 were associated with disease progression (Table 5).
Table 5
Multivariate Cox analysis of the overall survival (OS) and progression-free survival (PFS) among 327 patients having either cytogenetic abnormalities (CAs) or histologic BM involvement (BMI histo + ) and among R-CHOP treated patients (n = 200) *
  
OS
PFS
Risk factors
No. of patients (%)
HR
95% CI
P
HR
95% CI
P
Total patients with CAs and/or BMI histo + (n = 327)
       
Presence of ≥ 2 cytogenetic abnormalities as a risk factor
       
  IPI risk group, high vs. low
123 (37.6)
6.46
1.56-26.72
0.010
7.15
1.73-29.46
0.007
  R-CHOP treatment, R-CHOP vs. others
200 (61.2)
0.26
0.18-0.35
<0.001
0.38
0.28-0.51
<0.001
  ≥ 2 cytogenetic abnormalities vs. 0 or 1 abnormality
138 (42.2)
2.49
1.75-3.54
<0.001
2.12
1.53-2.93
<0.001
Presence of specific cytogenetic abnormalities as risk factors
       
  IPI risk group, high vs. low
123 (37.6)
8.34
2.04-24.09
0.003
8.43
2.06-34.44
0.003
  R-CHOP treatment, R-CHOP vs. others
200 (61.2)
0.24
0.17-0.34
<0.001
0.32
0.24-0.44
<0.001
  19p13 abnormality present vs. absent
24 (7.3)
2.67
1.50-4.76
0.001
3.02
1.85-4.93
<0.001
  7q22 abnormality present vs. absent
11 (3.4)
3.03
1.51.-6.06
0.002
NS
NS
NS
  12q22-q24 abnormality present vs. absent
12 (3.7)
2.68
1.37.-5.26
0.004
NS
NS
NS
  18q21 abnormality present vs. absent
21 (6.4)
2.11
1.22.-3.64
0.007
NS
NS
NS
  16q22-q24 abnormality present vs. absent
14 (4.3)
2.27
1.23-4.18
0.009
NS
NS
NS
  11q23-q25 abnormality present vs. absent
19 (5.8)
1.81
1.03-3.15
0.038
NS
NS
NS
  8q24 abnormality present vs. absent
19 (5.8)
NS
NS
NS
2.61
1.54-4.43
<0.001
R-CHOP treated patients with CAs and/or BMI histo + (n = 200)
       
Presence of ≥ 2 cytogenetic abnormalities as a risk factor
       
  IPI risk group, high vs. low
67 (33.5)
5.29
1.23-22.86
0.026
5.55
1.32-23.37
0.020
  Male vs. female
112 (56.0)
1.71
1.01-2.91
0.046
NS
NS
NS
  ≥ 2 cytogenetic abnormalities vs. 0 or 1 abnormality
78 (39.0)
2.01
1.17-3.45
0.012
2.15
1.38-3.38
0.001
Presence of specific cytogenetic abnormalities as risk factors
       
  IPI risk group, high vs. low
67 (33.5)
5.92
1.40-25.04
0.016
7.61
1.82-31.92
0.006
  Male vs. female
112 (56.0)
2.00
1.15-3.47
0.014
1.71
1.09-2.69
0.021
  19q13 abnormality present vs. absent
13 (6.5)
3.36
1.50-7.54
0.003
NS
NS
NS
  12q22-q24 abnormality present vs. absent
6 (3.0)
3.65
1.42-9.44
0.007
NS
NS
NS
  19p13 abnormality present vs. absent
16 (8.0)
2.85
1.20-6.80
0.018
3.31
1.68-6.51
0.001
  8q24 abnormality present vs. absent
13 (6.5)
2.54
1.24-5.23
0.011
2.43
1.24-4.73
0.009
*The multivariate Cox regression models initially included age, gender, the International Prognostic Index (IPI) risk groups, history of R-CHOP treatment, presence of histologic bone marrow involvement, and either presence of ≥ 2 cytogenetic abnormalities, or presence of specific cytogenetic abnormalities found in minimum 5 R-CHOP-treated patients and associated with significant poor prosnosis in the univariate analysis (+3, -17, abnormalities at 14q32, 3q25-q27, 19p13, 8q24, 7q22, 1q21-q23, 18q21, 19q13, 9q34, 22q11, 11q23-q25, 12q22-q24, 16q22-q24, and deletion 6q). The stepwise selection procedure was used to select variables in the final models.
Abbreviations: CI, confidence interval; HR, hazard ratio; NS, not significant.

Discussion

With recent advances in genetic technology, the value of G-banding data is often underestimated compared with the data derived from higher-resolution techniques, such as FISH, array-CGH, and deep sequencing. Molecular testing for immunoglobulin gene rearrangements is also a sensitive test for detecting clonal cells within bone marrow, which detected BM positive cases in 16% of histologically negative cases in a previous study [6, 19]. Although conventional karyotyping has low resolution, this method remains the only technique used worldwide in many hospital laboratories; thus, it is readily applicable in routine practice. In addition, the advantages of conventional cytogenetic tests include their ability to detect abnormalities in proliferative clones; to provide information regarding whole chromosomes, including balanced translocations; and to distinguish between heterogeneous clones that coexist in a sample.
In fact, conventional cytogenetic testing is not as widely performed for DLBCL as for other hematologic malignancies, such as acute leukemia and multiple myeloma. The disadvantages of cytogenetic testing using tissue samples include the necessity of laborious tissue preparation, the presence of fuzzy chromosomes, a lack of metaphase cells, and contamination. Because BM studies are a standard process of DLBCL staging, the use of BM samples for additional conventional cytogenetic testing is easily applicable. There are two major possible advantages associated with the cytogenetic testing of BM samples in DLBCL patients: an increased sensitivity for detecting BM involvement in DLBCL and the information provided by the chromosomal abnormalities found in the BM. In this study, we analyzed retrospective data from a large series of patients to analyze both possible advantages.
One major difficulty in the decision about whether to perform cytogenetic testing on BM specimens is caused by the absence of BM involvement in many DLBCL patients. Even in patients with BM involvement, the lymphoma cells may be obscured by the more abundant normal hematopoietic cells [5, 6, 19, 20]. In our data, 8.8% of the total patients exhibited multiple chromosomal abnormalities, and 1.3% of the patients were diagnosed with BM involvement on the basis of cytogenetic results alone. Considering that 16.3% of the cases in our study were BMIhisto+, the detection rate of significant cytogenetic abnormalities in BM specimens was not negligible. Because karyotyping is based on metaphases, the number of metaphases with cytogenetic abnormalities does not necessarily represent the proportion of cells within BM cells [21]. Karyotyping may be a sensitive method, because a small clone with proliferative advantage over normal cells may be identified by karyotyping.
The interpretation of data from patients with a single abnormality can be difficult because the abnormality does not necessarily originate from lymphoma cells. In our study, single aneuploidies demonstrated only a 10% concordance rate with histologic examination, which indicates that a single aneuploidy cannot be the definite evidence for presence of lymphoma cells. In addition, patients with single aneuploidies had less aggressive disease than normal karyotype cases with histologic involvement and presented no significant differences in prognosis. Therefore, the presence of a single numerical abnormality cannot be an indicator of the advanced disease and a poor prognostic factor. In fact, loss of chromosome Y is a well-known normal age-related phenomenon in elderly males [22]. Although monosomy 21 can be detected as a sole cytogenetic abnormality in a variety of hematologic disorders, the random loss of chromosome as an artifact of cell culture or slide preparation can cause this abnormality [23]. Single structural abnormalities also presented lower concordance rate with histologic BM involvement. There were 2 patients with del(20q) without histologic evidence of BM involvement. Although these patients did not presented cytopenia or morphologic dysplasia, previous studies reported that myelodysplastic syndrome associated with isolated del(20q) can commonly present with minimal morphological dysplasia [24]. There were a patient with duplication of Y chromosome and patients with balanced translocation, which may be constitutional chromosomal aberrations [25, 26]. However, some single abnormalities can represent a primary event in lymphomagenesis. In this study, single structural abnormalities such as t(14;18)(q32;q21) or t(3;14)(q27;q32) can be reasonably regarded as originated from lymphoma cells considering their well-known chromosomal loci and their concordant histologic BM results. Because of the small number of analyzed cases, it was hard to make convincing conclusion about the significance and prognostic impact of these single abnormalities in this study. When these abnormalities are detected, cautious BM examination with other ancillary tests may be needed. Further studies will be needed for more clear conclusion and to make a guideline on the appropriate interpretation of ambiguous cytogenetic abnormalities.
Most importantly, this study demonstrated that BM cytogenetic results have a powerful prognostic significance that is independent of BM histology. The presence of ≥ 2 chromosomal abnormalities was associated with very poor prognosis among both patients with and without histologic BM involvement. This finding confirms that a BM cytogenetic result is a stronger prognostic predictor than histologic BM involvement. The significant prognostic effect of cytogenetic abnormalities was observed in patients that had been stratified according to their IPI risk scores, especially for patients with high intermediate risks.
The chromosomal abnormalities observed in this study were nonrandom and recurrent, implying that they may belong to the recurrent lymphomagenesis pathway [27]. There have been previous reports of high frequencies of many of these abnormalities, including rearrangements at 14q32, 1p, 3q27, 8q24, 11q23-q25, and 18q21-q23; duplications at 1q; and deletions at 6q. The most common numerical abnormality in DLBCL, trisomy 18, has also been frequently reported in many other B-cell neoplasms [28, 29]. The 14q32/IGH rearrangement, which was the single most common abnormality in the present study, is frequently observed in all B-cell neoplasia [3036]. Because 14q32/IGH rearrangements mainly involve balanced translocations, these rearrangements can be detected only by G-banding, not through array methods [3741].
In our data from patients with multiple abnormalities, which specific loci presented abnormalities was a more significant prognostic factor than the number of abnormalities. Several abnormalities, including aberrations at 16q22-q24, 6p21-p25, 12q22-q24, 11q23-q25, 19q13, 1q21-q23, 8q24, and 19p13, and -17 appeared to be associated with a worse prognosis in the univariate analysis. The multivariate analysis demonstrated that several chromosomal abnormalities including aberrations at 19p13, 7q22, 12q22-q24, 18q21, and 16q22-q24 are independent adverse prognostic factors for survival among DLBCL patients with BM involvement. In addition, abnormalities at 19q13, 12q22-q24, 19p13, and 8q24 were associated with poor prognoses among R-CHOP treated patients with BM abnormalities. Because of the small number of patients having each specific cytogenetic abnormality, and the high variability of the cytogenetic abnormalities, to make clear conclusion about the prognostic importance of each specific chromosomal loci might be difficult. However, we considered a recurrent cytogenetic abnormality with a high statistical significance, such as abnormalities at 19p13 can be a potentially important target for further investigation. Aberrations in chromosome 19 have been reported in several previous series of DLBCL cases, although their prognostic impact was not explored [30, 33, 35, 36, 4042]. The identity of the specific gene on 19p13 that is associated with DLBCL is not yet known; however, TCF3 has been reported to be associated with acute lymphoblastic leukemia [43], and microRNA (miRNA) genes have been implicated in mature B-cell neoplasia [44]. In addition, a recent study using array-CGH and SNP-chip analyses reported that recurrent deletions of the tumor suppressor genes, TNFSF7 and TNFSF9 at the 19p13.3 region were observed in DLBCL and Burkitt lymphomas [45]. These genes may play an important role in the pathogenesis of DLBCL leading to disease progression and BM involvement. The MYC/8q24 rearrangement, which has been reported to be associated with a poor prognosis when detected in tissues using FISH at the time of diagnosis [4649], was independently associated with disease progression in this study. The nonrandom nature of the observed chromosomal abnormalities and their association with prognosis suggest that investigations into the clonal evolution of DLBCL could provide useful insights into the pathogenesis of this disease.
Our study has several limitations. First, this study was performed retrospectively, and the patients had undergone heterogeneous therapies. Second, the evaluations of the cell of origin and CD5 positivity were limited. Third, several BM-related prognostic factors, including the extent and histologic characteristics of the BM involvement, could not be investigated [3, 4, 50, 51]. However, considering the comparable results of the BMIhisto+ and BMIhisto- cases, it could be suggested that cytogenetic aberrations are a stronger prognostic factor than other BM-related factors.
The results of this study may be representative of the major characteristics of the BM chromosomal abnormalities in the Korean DLBCL population, considering the large size of our data series. A larger series of international investigations may be needed to characterize and confirm the prognostic significance of the BM cytogenetic aberrations in DLBCL patients from other ethnic groups, considering the variety of external genotoxic agents that can cause chromosomal changes and the diversity of host susceptibility factors to chromosomal breaks. Results from classic cytogenetics can guide the design of additional studies using other techniques, such as FISH or arrays.
In conclusion, the conventional cytogenetic testing of BM may provide essential information for newly diagnosed DLBCL patients. We suggest that cytogenetic testing of BM samples should be integrated with DLBCL staging apart from histologic examination, and more effective therapeutic strategies should be developed. In addition, frequently affected cytogenetic regions, such as 19p13, must be intensively investigated to characterize the underlying molecular pathogenesis of DLBCL.

Acknowledgments

This work was supported by (1) the Basic Science Research Program through the National Research Foundation of Korea (NRF) Funded by the Ministry of Education, Science and Technology (2012–0002257), (2) a grant (10172KFDA993) from Korea Food & Drug Administration, (3) a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120216).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

CS and DSL designed the study, SYK, HJK, HJK, JSK, HSE, TMK, SSY, CS, and DSL provided study materials or patients, collected and assembled data. SYK, CS, and DSL analyzed and interpreted data. SYK, HJK, HJK, JSK, HSE, TMK, SSY, CS, and DSL wrote the manuscript. All authors have read and approved the final manuscript.

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Zusatzmaterial
Additional file 1: Table S1: Detailed karyotypes of 192 patients with chromosomal abnormalities. Karyotype results were classified according to the complexity of chromosomal abnormalities observed. (XLSX 29 KB)
13045_2013_345_MOESM1_ESM.xlsx
Additional file 2: Figure S1: Kaplan-Meier survival plots of OS according to histologic bone marrow involvement (BMI) and chromosomal abnormalities found in bone marrow cells in DLBCL patients. (A) The patients with a single abnormality did not exhibit significant differences in OS in patients without bone marrow involvement (BMIhisto-). (B) The patients with complex karyotypes (≥3 abnormaliteis) exhibited significantly worse survival in both the BMIhisto+ and BMIhisto- groups. (PDF 391 KB)
13045_2013_345_MOESM2_ESM.pdf
Additional file 3: Figure S2: Survival according to the total number of chromosomal abnormalities in 1585 DLBCL patients. (A) overall survival (OS). (B) progression-free survival (PFS). The patients with ≥ 2 abnormalities exhibited significantly worse OS and PFS; however, there was no significant difference according to the number of abnormalities among patients with ≥ 2 abnormalities. (PDF 357 KB)
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Additional file 4: Figure S3: Kaplan-Meier survival plots of overall survival (OS) of R-CHOP treated DLBCL patients according to chromosomal abnormalities (A) in patients with histologic bone marrow involvement (BMIhisto+) and (B) in patients without histologic bone marrow involvement (BMIhisto-). (C and D) OS according to risk stratification by international prognostic index (IPI) scores and chromosomal abnormalities (C) in R-CHOP-treated BMIhisto+ patients and (D) in R-CHOP-treated BMIhisto- patients. (PDF 446 KB)
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Additional file 5: Figure S4: Forest plots showing hazard ratios (HRs) obtained by univariate Cox analysis for (A) overall survival (OS) and (B) progression-free survival (PFS) according to the presence of specific chromosomal abnormalities using patients with lymphoma bone marrow involvement and normal karyotype as a reference group in R-CHOP-treated patients having either abnormal karyotypes or histologic BM involvement (n = 200). (PDF 474 KB)
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Authors’ original file for figure 3
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