Figure 1 Morphologic and immunohistochemical features characteristic of DLBL, BCLU and BL. A-C: Low power (200x, A) and high power (400x, B) views of DLBL stained with H&E shows characteristic nuclear pleomorphism, prominent nucleoli and abundant cytoplasm. This example of DLBL shows strong immunohistochemical staining for BCL2 (Dakocytomaton) and weak staining for CD10 (Novacastra) and BCL6 (Biocare Medical) and has a Ki67 (Dakocytomaton) proliferation index of approximately 50-60% (C). D-F: Low (D) and high power (E) views of BCLU stained with H&E show intermediate sized cells with abundant tingible-body macrophages giving a low power "starry sky" appearance (D). The cells show some nuclear pleomorphism with only a portion of cells containing prominent nucleoli (E). This case of BCLU shows staining for BCL2, BCL6 and CD10, and a Ki67 proliferation index of approximately 80-90% (F). G-I: Low (G) and high power (H) view of BL stained with H&E shows the classic "starry sky" appearance of BL composed of intermediate sized cells with monotonous nuclei containing finely clumped chromatin and no prominent nucleoli. There is abundant apoptosis and necrosis (H). This case of BL shows strong staining for BCL6 and CD10, negative staining for BCL2 and has a Ki67 proliferation index approaching 100% (I). Low power views were taken at 200x magnification and high power views were taken at 400x magnification. Images were taken using an Olympus BX41 microscope with Olympus 20x/0.40 and 40x/0.65 lenses with an Olympus DP70 digital camera and recorded with scale bars embedded into representative images using Olympus DP Controller Version 3.1.1.267 imaging software. Images cropped from their original size have accompanying scale bars. All scale bars represent 50 µm. DLBL, Diffuse large B-cell lymphoma; BCLU, Bcell lymphoma unclassifiable with features intermediate between DLBL and BL; BL, Burkitt lymphoma; H&E, Hemetoxolin and Eosin.

Figure 2 Overall and progression free survival curves of patents with BCLU and DLBL. (A) Overall survival of BCLU ( ) and DLBL ( ) patients. X-axis represents time (days); y-axis represents the cumulative survival. (B) Progression free survival of BCLU and DLBL patients. X-axis represents time (days); y-axis represents the cumulative survival. Analysis was performed using multiple Cox regression analysis adjusting for IPI and treatment regimen. Hazard Raito was 2.46 (95% CI 1.16-5.19) for overall survival (A) and 1.96 (95% CI 1.01-3.93) for progression free survival (B).

Figure 3Response to treatment and response duration of treatment responders in BCLU and DLBL patients. (A) Histogram showing the percentage (yaxis) of BCLU and DLBL patients who were classified as showing a complete response (Complete), a partial response (Partial), no significant change in tumor burden (Stable) or progression of disease (Progression) after first-line chemotherapy. (B) Response duration, defined as the time when criteria for complete or partial response were met to the first documentation of relapse or disease progression, of BCLU ( ) and DLBL ( ) patients. X-axis represents time (days); y-axis represents the cumulative survival. Analysis was performed using multiple Cox regression analysis adjusting for IPI and treatment regimen. Hazard Raito was 0.9, 95% CI 0.29-2.74 (B).

Figure 4 Overall survival curves of patents with BCLU according to MYC/ BCL2 status and of BCLU patients without double-hit MYC/BCL2 compared to DLBL patients. (A) Overall survival of BCLU patients with double-hit MYC/BCL2 alterations (DH, ) and without double-hit MYC/BCL2 (non-DH, ). (B) Overall survival of BCLU without double-hit MYC/BCL2 alterations (BCLU non-DH, ) compared to DLBL ( ) patients. X-axis represents time (days); y-axis represents the cumulative survival. Analysis was performed using multiple Cox regression analysis adjusting for IPI and treatment regimen. Hazard Raito was 13.8 (95% CI 2.3-83.6) for DH compared to non-DH (A) and 2.75 (95% CI 0.89-8.52) for BCLU non-DH compared to DLBL (B).

Table 1. Immunohistochemical Panel

Antibody

Manufacturer

Dilution

Antigen Retrieval

Clone

CD20

Dakocytomaton

1/100

none

L26

CD10

Novacastra

1/25

Citrate, HIER

56C6

BCL2

Dakocytomaton

1/25

Citrate, HIER

124

BCL6

Biocare Medical

1/75

BORG, HIER

PG-B6p

Ki-67

Dakocytomaton

1/1000

Citrate, HIER

MIB-1

TdT

Novacastra

1/25

Dako lo, HIER

SEN28

CD34

Novacastra

1/50

none

QBend10

CD3

Cell Marque

1/500

Citrate, HIER

Polyclonal

HIER, Heat induced epitope retrieval; Citrate, 0.01M citrate buffer, pH 6.0; BORG and Dako lo, pre-treatment reagents supplied by the manufacturer

Table 2. Demographic and clinical characteristics at diagnosis and treatment regimens.

 

 

Variable

DLBL
(n=97)

  n     (%)

BCLU
(n=34)

   n    (%)

 

 

P-value

Male Gender

45

(46)

18

(53)

0.511

B symptoms

37

(41)

12

(40)

0.949

Positive BM

21

(26)

5

(19)

0.453

CNS involved

4

(5)

6

(20)

0.009

Bulky disease   

18

(20)

12

(40)

0.031

Extranodal disease

35

(39)

14

(47)

0.427

IPI Score

0
1
2
3
4
5

 

6
16
19
18
11
14

 

(7)
(19)
(23)
(21)
(13)
(17)

 

3
5
8
6
7
0

 

(10)
(17)
(28)
(21)
(24)
(0)

0.219

EGOC

0
1
2
3
4

 

31
27
12
15
6

 

(34)
(30)
(13)
(17)
(7)

 

11
10
6
1
2

 

(37)
(33)
(20)
(3)
(7)

0.434

Ann Arbour stage

1  
2
3
4

 

18
18
14
41

 

(20)
(20)
(15)
(45)

 

8
5
8
9

 

(27)
(17)
(27)
(30)

0.330

Treatment regimen

BL-like
DLBL-like
Palliative
No treatment

 

4
74
13
6

 

(4)
(76)
(13)
(6)

 

4
24
1
5

 

(12)
(71)
(3)
(15)

 

 

 

 

 

 

 

DLBL, Diffuse large B-cell lymphoma; BCLU, B-cell lymphoma unclassifiable with features intermediate between DLBL and BL; BL, Burkitt lymphoma; IPI, International Prognostic Index; ECOG, Eastern Cooperative Oncology Group Performance Status; BL-like refers to French LMB, CODOX-M or Hyper-CVAD protocols with or without rituximab; DLBL-like refers to CHOP, GDP or DHAP protocols with or without rituximab.