MYC & BCL2 or BCL6 co-expression in DLBCL Zawam et al.
85
analysis to recognize DH/TH category of diffuse large
B-cell lymphoma, NOS (Swerdlow, 2014).
It is the resolve of this study to record cases of double/
triple-expressor DLBCL in our institution, to appraise
their potential use as prognostic markers and finally to
help single out the subset of patients who require further
gene rearrangement detection by FISH.
Patients and methods
This study was conducted after protocol approval by
both the Ethical Committee at Oncology and Pathology
Department at Faculty of Medicine, Cairo University.
It included 24 patients with primary DLBCL presented
to Kasr El-Aini Hospital, Faculty of Medicine, Cairo
University, in the period from August 2015 until
December 2017. Inclusion criteria for selection were
availability of tissue paraffin blocks at the pathology
department, full clinical data and uniform treatment with
R-CHOP. Exclusion criteria comprised paucity of tissue
in paraffin blocks, HIV positivity, primary central
nervous system DLBCL and treatment regimens other
than R-CHOP. The patients’ medical records were
retrieved from the oncology department. All parameters
of international prognostic index were available, and the
revised international prognostic index (R-IPI) was
calculated for each patient (Sehn et al., 2007).
At least two pathologists reviewed and reassessed all the
archived slides of the cohort included in the study,
hematoxylin and eosin-stained sections, CD20, and CD3
to confirm the primary diagnosis of DLBCL according to
2016 WHO classification (Swerdlow et al., 2016).
Clinical staging according to modified Ann Arbor staging
system was done (Cheson et al., 2014). PET scan was used
for evaluation of treatment response with the exception of
two patients, where computed tomography or MRI was used.
Response to treatment criteria and evaluation
All patients received R-CHOP as the first-line chemo-
therapy; the median number of cycles was six cycles
(range: 3–7). The response to treatment was evaluated
according to Deauville five-point scoring system
(Barrington et al., 2010). All data for overall survival
(OS) had been analyzed by 31 December 2017. OS was
calculated from the date of initial diagnosis until the date
of death from any cause or the date of last follow-up.
Progression-free survival (PFS) was defined as the time
from initial diagnosis until the date of disease progression
or death from any cause. The median follow-up time was
also calculated from the date of beginning of treatment.
Immunohistochemical staining
Multiple sections of 5-µm thickness were cut from paraffin
block of all cases. In summary, the steps of deparaffinization,
antigen retrieval, endogenous peroxidase activity block,
antibody dispense and incubation were all done on fully
automated Ventana medical system (BenchMark XT auto-
mated staining system; Ventana, Tucson, Arizona, USA)
following the manufacturer’s instructions. The following
antibodies were applied: C-MYC Ab-2 (Thermo Scientific,
Fremont, California, USA; cat. no. MS-1756-P0, clone 9E
10.3), BCL2 (clone 124; Dako, Glostrup, Denmark), BCL6
(clone LN22; Leica Microsystems, Ernst Leitz Strasse,
Wetzlar, Germany), and Ki-67 (clone GM001, Genemed,
Torrance, California, USA). All markers (C-MYC, BCL2,
BCl6 and Ki-67) were pre-diluted by the manufacturer and
supplied ready to use with 32 min incubation for each.
Appropriate positive controls were included in each run:
breast invasive duct carcinoma for C-MYC and reactive tonsil
for BCL2, BCL6 and Ki-67. Omission of the primary
antibodies was performed as negative control.
The cutoff points for evaluation of marker positivity were
at least 40% nuclear staining for C-MYC; at least 50%
cytoplasmic staining for BCL2; and at least 50% nuclear
staining for BCL6. Ki-67 proliferative activity was
reported as low as 90% or high of at least 90% (Swerdlow,
2014; Yan et al., 2014).
Double-hit score
Using cutoff points for C-MYC, BCL2 and BCL6
(previously mentioned), the double-hit score (DHS)
was calculated for all patients that ranged from 0 to 2.
Each patient was given one point for each of the markers
expressed at or above the cutoff points. DHS ranged
from 0 to 2, and no cases showed expression of all three
markers. Score of 0–1 is considered as low DHS, whereas
DHS of 2 is considered as a high score (Yan et al., 2014).
Statistical analysis
Patients’ clinicopathologic characteristics were tabulated,
and SPSS software version 17 (IBM corporation, New
York, New York, USA) was used for analysis of data
correlation. χ2-Test was performed.
Kaplan–Meier estimates were used to predict the OS and
PFS, as compared using log-rank tests. All calculated
P values were two-pronged, with P values of less than
0.05 being recorded as statistically significant.
Results
Patients
The patients’ clinicopathological characteristics are listed
in Table 1. Most of our cases were females (15 females
vs. nine males), with median age of 46 years (range:
22–71). The median follow-up period was 10 months
(range: 3–28). Thirteen patients had low IPI score (≤ 2),
whereas 11 patients had high IPI score (3–5). After first-
line treatment with R-CHOP, complete response (CR)
was observed in 13/24 patients, partial response in 3/24
patients and progressive course in 8/24 patients. High
R-IPI was significantly associated with high DHS
(P = 0.03) (Table 2). Moreover, high R-IPI was asso-
ciated with significantly shorter PFS and OS (P = 0.035
and 0.04, respectively) (Fig. 1).
Morphology and immunohistochemistry
Conventional hematoxylin and eosin-stained slides of the
studied cohort were reviewed by at least two pathologists. All
cases displayed the morphological picture of classical DLBCL
including CD20 + , CD3 − immunophenotyping, associated
with high Ki-67 proliferation index at least 90% (Fig. 2) or