Page 210 - Haematologica May 2020

F. Schieppati et al.
DLBCL studied by interphase fluorescence in situ hybridization (FISH), both before11 and after12,13 the intro- duction of rituximab. Overall, MYC genetic rearrange- ments have been described in 5-10% DLBCL at diagno- sis.10,11 The presence of dual translocations involving both MYC and BCL2 (“double-hit”), associated or not to the translocation of BCL6 (“triple-hit”), have shown a dismal clinical course.14 The importance of the molecular study of MYC, BCL2 and BCL6 status is highlighted by the updat- ed World Health Organization (WHO) classification of 2016, which identifies a specific diagnostic category called “High Grade Lymphoma with MYC and BCL2 with or without BCL6 translocation”, irrespective of the morpho- logical subtype of DLBCL. In addition, preliminary studies suggest that the partner gene in the MYC translocation may also influence tumor behavior.15 In particular, MYC rearrangements with immunoglobulin genes, but not with other partner genes, seem to have a negative prognostic impact on patients with DLBCL treated with immuno- chemotherapy.16,17
Recent studies have revealed that also numerical alter- ations of MYC gene detected by FISH can occur in DLBCL.18 In the same way as chromosome translocations juxtapose oncogenes to the promoter of genes that are constitutively expressed, a gain of gene copy-number or the amplification of MYC may cause its over-transcription and protein over-expression leading to uncontrolled prolif- eration.19 Different studies have shown that numerical alterations of MYC may influence the outcome of patients with DLBCL, but their incidence and prognostic relevance is still controversial.19,20 Moreover, the definition of MYC copy number changes is not homogeneous across studies, where the terms “gain” and “amplification” are used to define different conditions. In the present study, we ana- lyzed the frequency and the clinical outcome of patients with MYC numerical aberrations in the setting of DLBCL with particular emphasis on the number of MYC extra- copies, on their frequency, and on their correlation with the clinical outcome in a consecutive series of DLBCL patients.
Methods
Study design and participants
In this retrospective, observational study, participants were enrolled between January 2011 and June 2016. Eligible patients were consecutive adults receiving a diagnosis of DLBCL during the study period. FISH study at diagnosis was performed on patients considered fit for treatment with curative intent. Tumors were classified according to the 2008 WHO Classification. No immunodeficiency-associated lymphomas were included. Disease burden was assessed by Ann-Arbor staging and IPI classification.2 All patients signed informed consent to provide material for bio- logical studies. The study was conducted in accordance with good clinical practice guidelines and approved by the institutional ethi- cal committee. All patients with DLBCL were treated with ritux- imab-containing immuno-chemotherapy programs. The follow- ing were considered standard dose regimens: CHOP, and COMP (cyclophosphamide, vincristine, liposomal doxorubicin, and pred- nisone), whereas the following were considered intensified regi- mens: GMALL B-ALL/NHL 2002 protocol,21 and DA-EPOCH (dose adjusted etoposide, doxorubicin, cyclophosphamide vin- cristine, and prednisone). Autologous stem cell transplantation (ASCT) was used in approximately 25% of cases as intensification
of first-line treatment, using BEAM/FEAM as conditioning regi- mens [carmustine (BCNU) or fotemustine, etoposide, cytarabine, melphalan followed by autologous stem cell infusion]. ASCT as intensification of a first-line treatment with R-CHOP was consid- ered an intensified regimen.
Interphase fluorescence in situ hybridization analysis Fluorescence in situ hybridization analysis was performed on 4-mm sections of formalin-fixed paraffin-embedded (FFPE) tissue using break-apart DNA probes (Dako, Glostrup, Denmark) for c- MYC (8q24), BCL2 (18q21) and BCL6 (3q27). FISH was carried out according to the manufacturer’s guideline. FISH images were cap- tured at x100 magnification and elaborated using the Genikon software (Nikon Instruments S.p.A., Italy). The presence of three or more red/green signals of MYC, BCL-2 or BCL-6 was consid- ered to indicate an increased copy number of these genes (namely MYC-ICN, BCL2-ICN, and BCL6-ICN).20 A “cloud-like” FISH pat- tern due to countless copies of MYC was defined as “amplifica- tion” (MYC-AMP) (Figure 1). We did not regularly use a chromo- some 8 centromeric probe in this study. However, in 11 cases with MYC-ICN, single centromeric chromosome 8 probe (CEP8 SpectrumGreen, Abbott Molecular Inc., USA) was also used in
order to exclude polysomy as cause of MYC-ICN.
Immunohistochemistry
Four-micron thick tissue sections were used for immunohisto- chemical staining for c-MYC (clone Y69, -Abcam; dilution 1:75), which was performed on a Bond III automated immunostainer (Leica Microsystem, Bannockburn, IL, USA) using controls in par- allel. Diaminobenzidine was used to reveal the in situ hybridiza- tion (ISH) reaction and sections were counterstained with hema- toxylin. A cut-off of >40% was used for positive MYC expression by immunohistochemistry (IHC).
Response criteria and statistical analysis
Standard definitions of complete response (CR), progression- free survival (PFS), and overall survival (OS) were used.22 Categorical data were compared using Fisher’s exact test, whereas the Mann-Whitney test was used for continuous parameters. OS was measured from date of diagnosis to death from any cause, and PFS from the date of treatment start to the date of disease pro- gression, relapse or death. The actuarial survival analysis was car- ried out according to the method described by Kaplan and Meier and the curves compared by the log-rank test with 95% confi- dence intervals (CI).23 Differences between the results of compar- ative tests were considered significant at two-sided P<0.05.
Results
General clinical characteristics and outcome of the study population
Of 504 patients diagnosed with DLBCL at our Institution, FISH was performed on 385 consecutive patients considered fit for treatment with curative intent. Tumors were classified according to the WHO 2008 Classification of Tumours of Haematopoietic and Lymphoid Tissues, as follows: 365 DLBCL not otherwise specified (NOS) (95%), and 20 B-cell lymphoma, unclassi- fiable (BCLU), with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5%). Thirty-four (8.3%) cases were transformed from a low- grade lymphoma.
Ninety-five patients of the whole cohort of DLBCL had a structural or numerical aberration of MYC at FISH (25%),
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