Page 241 - Haematologica March 2020

SOX11 and TP53 status in MCL
ditionally considered an aggressive and incurable disease, an indolent variant has been recognized that presents with leukemic nnMCL, and treatment is usually deferred for months or years.14,15 Interestingly, nnMCL, in contrast to cMCL, is characterized by a lack of SOX11 expression, the presence of IGHV mutations and usually low genomic complexity, indicating that these two variants follow differ- ent pathways of lymphomagenesis.15-17 The lack of SOX11 expression in nnMCL is believed to play an important role for the indolent behavior of the disease.11,14,16,18-20 SOX11 is a transcription factor that belongs to the SOX gene family and is widely expressed in embryogenesis, but not in nor-
21
mal hematopoiesis. In MCL, it is controversial whether
SOX11 acts as an oncogene or as a tumor suppressor gene. SOX11 has been proposed to act as an oncogene inducing cell proliferation, enforcing PAX5 expression and inhibiting terminal B-cell differentiation into plasma cells via PRDM1 and BCL6.16,22 Additionally, it has been shown to promote tumor angiogenesis through the PDGFA axis.23 More recent- ly, in a murine model, it was demonstrated that SOX11 overexpression in B-cells promotes B-cell receptor signaling and results in a disease phenotype similar to MCL.24 In con- trast, it has been shown that SOX11-binding targets could repress proliferation, and therefore, it has been suggested that SOX11 in MCL may act as a tumor suppressor gene.25 The prognostic relevance of SOX11 in MCL is equally con- troversially discussed. Although originally it was thought that SOX11 negative cases carried an indolent clinical course, other studies have shown that high SOX11 expres- sion is associated with improved survival in a subset of MCL patients, and low or negative SOX11 expression with poor overall survival.25-28 Further work identified a group of MCL lacking SOX11 expression with a dismal prognosis.11 Interestingly, many of these cases were associated with TP53 mutations. TP53 mutations are preferentially associat- ed with blastoid morphology (up to 30%),29 but also occur in both cMCL with classic morphology and nnMCL, and associated with a poor prognosis.30-32
SOX11 expression is usually investigated by immunohis- tochemistry (IHC) in tissue specimens; however, IHC is not a quantitative technique. RNAscope is a relatively new in situ hybridization (ISH) technique that allows a highly sen- sitive visualization of molecular markers in the morpholog- ical context by target-specific amplification of signals with suppression of the background.33 In recent years, some stud- ies have analyzed this technology in different tissues and found it to be a method comparable to immunohistochem- istry and RT-qPCR.34-36
By integrating TP53 status and SOX11 expression in the diagnostic workup of MCL the risk stratification could be improved. Given the lack of reliable quantification of IHC and the conflicting results concerning the prognostic role of SOX11, we aimed to establish a sensitive ISH analysis of SOX11 mRNA allowing its quantification within the histopathological context and to compare it with IHC and RT-qPCR analyses. Furthermore, the TP53 status was inves- tigated by IHC and next-generation sequencing (NGS) and correlated with SOX11 mRNA expression levels.
Methods
Patient selection
Sixty-six cases with the diagnosis of MCL were selected from the files of the Institute of Pathology, University of Tübingen. In
addition, 12 cases with the diagnosis of small cell B-cell lym- phoma (chronic lymphocytic leukemia (CLL), follicular lym- phoma (FL) and marginal zone B-cell lymphoma (MZL), four cases each) were used as controls. The study was approved by the local ethics committee (TÜ-730/2018BO2).
Histology and immunohistochemistry
Hematoxylin and Eosin (H&E) sections and all immunohisto- chemical stains were reviewed. BM trephines were formalin- fixed, decalcified in EDTA, and paraffin-embedded (FFPE). Immunohistochemical stains were performed using an automat- ed immunostainer (Ventana Medical Systems, Tuscon, USA), according to the manufacturer´s protocol. The following anti- bodies were used: SOX11 (MRQ-58) (Medac Diagnostika, Wedel, Germany) and p53 (DO-7) (Novocastra Liquid, Leica Biosystems, Newcastle, UK). SOX11 stain was scored as nega- tive (0%), low (1-10% positive cells) and positive (>10% posi- tive cells). p53 was considered positive when >20% cells were strongly positive.
RNAscope
The mRNA ISH was performed using the RNAscope 2.5 VS reagent kit-RED with custom designed SOX11-RNA, as target (Advanced Cell Diagnostics, ACD, Hayward, CA), according to the manufacturer`s protocol, as previously described33 (Online Supplementary Material and Methods). The RNAscope procedure was performed in the Ventana Discovery XT autostainer for open procedures. Tissue mRNA preservation was assessed by performing RNAscope analysis of mRNA of the housekeeping gene peptidylpropyl isomerase B (PPIB) (Online Supplementary Figure S1).
RNAscope scores
The results were scored according to the guidelines described in the manufacturer´s protocol. Briefly, staining score 0 was defined as: no staining or less than 1 dot to every 10 cells (40x magnification), score 1: 1-3 dots/cell (visible at 20-40x magnifi- cation), score 2: 4-9 dots/cell, and very few dot clusters (visible at 20-40x magnification), score 3: 10-15 dots/cell, less than 10% positive cells have dot clusters (visible at 20x magnification), and score 4: >15 dots/cell, more than 10% positive cells have dot clusters (visible at 20x magnification). Score 0-1 was considered negative/low, score 2 intermediate, and score 3-4 high SOX11 mRNA expression.
RNA isolation and Real-time quantitative PCR (RT-qPCR)
RNA was isolated using the Maxwell® 16 LEV RNA FFPE Purification Kit and the Maxwell® 16 Instrument (Promega, Madison, WI, USA). RT-qPCR was performed to quantify SOX11 mRNA levels (see Online Supplementary Material and Methods). Data were analyzed using the 2-ΔΔCp method and the mean of the SOX11 negative cases was defined as calibrator.
Next generation sequencing of TP53 gene
NGS was performed using the Ion AmpliSeqTM TP53 Panel
(Life Technologies) on the Ion GeneStudioTM S5 Prime System according to the manufacturer`s protocol (see Online Supplementary Material and Methods).
Statistical analysis
Statistical analysis was performed with JMP software version 10 (SAS Institute GmbH) using Fisher exact or χ2 test for com- parison of nominal data. Statistical significance was concluded for values of P<0.05.
haematologica | 2020; 105(3)
755