Page 146 - Haematologica May 2022

A. Dodero et al.
amycin, vincristine, prednisone), leading to a long-lasting complete remission in approximately 60% of cases.1,2 For these patients, the International Prognostic Index (IPI), is an easy and valid tool for prognostic stratification.3
In 2000 Alizadeh et al. used gene expression profiling (GEP) to identify the cell of origin (COO) and described prognostic subgroups according to diversity in gene expression patterns indicative of different stages of B-cell differentiation.4 Immunohistochemical algorithms, used as surrogates of GEP for the identification of COO sub- groups, have been extensively used but have a limited concordance with GEP when applied to patients with DLBCL treated with R-CHOP.5
Among other possible prognostic indicators for patients with DLBCL, tumor protein p53 (TP53) mutations seem to represent simple and attractive biomarkers to be used in the daily routine clinical practice. The TP53 gene is involved in maintaining genomic stability in response to DNA damage by activating DNA repair programs and by triggering cell cycle arrest. The loss of TP53 is associated with lymphomagenesis and resistance to chemotherapy.6 TP53 mutations are present in 10% of DLBCL patients and confer a poor prognosis in patients treated with R- CHOP.7,8 Recently, Chapuy et al. performed whole genomic sequencing in 304 primary DLBCL patients and identified genetically different subgroups among germi- nal center B-cell like (GCB) and activated B-cell (ABC) lymphoma patients. Patients with TP53 mutations were described as a distinct cluster with a very poor prognosis.9
MYC and BCL2 represent other possible poor prognos- tic markers when rearranged or overexpressed in DLBCL and lymphomas harboring the double or triple transloca- tion (DH or TH) represent new entities in the recently updated 2016 World Health Organization Lymphoma Classification. Most DH lymphomas are in the GCB cat- egory, they present with advanced stages and have a very poor prognosis when treated with the R-CHOP regi- men.10-12 Also, the BCL2 translocation alone has been shown to play a significant prognostic role in GCB DLBCL patients treated with R-CHOP.13,14 Additionally, the presence of MYC rearrangements seems to be a poor prognostic marker, although its role in the absence of either BCL2 or p53 alterations remains controversial.15 The concomitant overexpression of MYC and BCL2 on the tumor cell surface observed in double expressor lym- phomas (DEL), could be the result of other mechanisms, different from translocation such as copy gain/amplifica- tion. The prognosis of DEL patients treated with standard R-CHOP is worse than that of non-DEL patients,16,17 and these individuals have a risk of central nervous system (CNS) relapse of 9.7% at 2 years.18 Moreover, a consensus on the optimal treatment for these patients has yet to be established.
Since 2013 at our Institution, DEL patients have been treated with the intensive chemotherapy regimen dose- adjusted EPOCH and rituximab (DA-EPOCH-R), achiev- ing a promising 2-year progression-free survival (PFS) and overall survival (OS) of 62% and 85%, respectively. These survival rates were better than those reported with R-CHOP in a historical cohort.19
The aim of the present study was to assess the inci- dence and prognostic role of TP53 mutations and BCL2, MYC translocations in a large cohort of DEL patients con- secutively treated with DA-EPOCH-R. The analysis of
the cumulative risk of CNS relapse as well as the effect of the different CNS prophylaxis applied in this selected cohort of DEL patients was also assessed.
Methods
Patients
All consecutive patients with a diagnosis of DEL treated since 2013 with DA-EPOCH-R at four hematological divisions in northern Italy were retrospectively identified. For inclusion in this observational study, the following inclusion criteria were required: i) histologically proven diagnosis of DEL with an age ≥18 years; ii) availability of formalin-fixed, paraffin-embedded (FFPE) samples; iii) no exposure to previous therapy except for the first cycle of R-CHOP that was allowed while waiting for immunohistochemistry (IHC) results and cytogenetic character- ization. Exclusion criteria were HIV positivity, CNS involvement at diagnosis, and histology other than DEL. The ethics commit- tees of the participating centers approved the study (INT 35/17). Written informed consent was obtained from all patients.
Immunohistochemistry and fluorescence in situ hybridization analysis
FFPE tissue samples were sectioned at 3-μm thickness. IHC was performed using the EnVision FLEX+, mouse, high pH method (Dako Denmark A/S Produktionsvej 42 DK-2600 Glostrup, Denmark) and a Dako Autostainer Link48 (Dako, Italia, SPA, Milano, Italy). Slides were stained with monoclonal antibodies against CD19, CD20, CD10, BCL2, BCL6, MUM1, MYC, and Ki67.
Fluorescence in situ hybridization (FISH) analyses for BCL2, BCL6, and MYC rearrangements were performed in all patients using “LSI BCL2 LSI BCL6 (ABR), C-MYC “break apart” probes (Vysis/Abbott Molecular, Illinois, USA) according to the manu- facturer’s instructions (detailed in the Online Supplementary Appendix).
TP53mutations
TP53 is located on chromosome 17p13.1 and consists of 14
exons (1-11), 10 of which are coding sequences for the p53 pro- tein.20,21 TP53 mutations were assessed according to Institutional practice, Sanger sequencing was used for samples collected from patients at Humanitas Cancer Center while next-generation sequencing followed by direct sequencing was used for all other samples (procedures described in the Online Supplementary Appendix).
Treatment and central nervous system prophylaxis
Patients received the DA-EPOCH-R regimen therapy every 21 days for six cycles. In all patients dose adjustment based on cell counts between cycles according to the NCI algorithm was applied.22 At diagnosis, all the patients performed lumbar punc- ture for cytology and flow cytometry analyses of cerebrospinal fluid (CSF). During this first procedure, all patients received intrathecal chemotherapy with methotrexate (MTX) and cytara- bine. Neuro-imaging was considered only in case of the pres- ence of neurological signs or symptoms. In absence of definitive clinical guidelines on CNS prophylaxis, the choice was deter- mined by the treating physician. High-dose MTX (HD-MTX: 3 g/m2) was used as prophylaxis in 66 patients (for the majority at the end of the treatment). Intrathecal chemotherapy at day 1 of every cycle, including MTX/cytarabine/dexamethasone, was given to 40 patients.
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