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R. Dobson et al.
(https://www.hmrn.org/ statistics/disorders/34). The poor clini- cal outcome is largely a consequence of the lack of under- standing of their molecular mechanism and targeted ther- apy.
The diagnosis of these lymphoma entities, particularly at an early stage, is a challenge because of the lack of spe- cific clinical and histological features, low tumor cell con- tent and the presence of prominent polymorphous inflam- matory infiltrates that often mask the neoplastic cells. To help the diagnosis, T-cell clonality analysis is commonly used, but this is frequently not helpful because of the paucity of malignant T cells. The diagnostic difficulty is further exacerbated by the increasing use of needle core biopsies to avoid more invasive surgical excision. When a lymphoproliferative lesion is suspected but uncertain on histological diagnosis, patients are commonly subjected to a “watch and wait” approach, and further biopsied when showing signs of disease progression.
There is a wide range of genetic changes in T-cell lym- phomas with TFH phenotype, including early TET2 and DNMT3A mutations, which occur in hematopoietic stem cells, and late genetic changes, which are specifically seen in lymphoma cells.1-4 Among the latter, RHOA mutation is the most frequent,2,5-9 occurring in 60-70% of cases, with Gly17Val (c.50G>T) accounting for 95% of the changes. Interestingly, studies of mouse models have sug- gested that RHOA Gly17Val mutation induces TFH cell differentiation and, together with loss-of-function TET2 mutations, can promote the development of AITL-like lymphomas.10-12 Importantly, RHOA mutation is preferen- tially associated with the above lymphoma entities and has, so far, been shown only in the neoplastic T-cell pop- ulation, but not in reactive B and T cells in these malignant conditions.2,5,13,14 Studies to date suggest that the RHOA Gly17Val mutation could be used as a marker for the diag- nosis of AITL and PTCL-TFH.6,15 However, it is unclear whether the mutation is present in tissue biopsies prior to the diagnosis of AITL and whether mutation analysis could help in the early detection of these lymphomas. To investigate these issues, we established a highly sensitive quantitative polymerase chain reaction (qPCR) assay to detect the RHOA mutation, and screened a large cohort (n=37 cases) of AITL and PTCL-TFH with multiple sequential biopsies together with 61 controls.
Methods
Tissue materials and DNA extraction
The use of archival tissues for research was approved by the ethics committees of the institutions involved. In total, 78 tissue specimens from 37 patients (multiple consecutive specimens in 29 cases) with AITL (n=35) or PTCL-TFH (n=2), together with 61 controls (13 with reactive lymph nodes with paracortical expan- sion of T cells, 10 with classic Hodgkin lymphoma and 1 with marginal zone lymphoma with a prominent background of T cells) and peripheral blood DNA samples from 16 individuals (TET2 and DNMT3A mutations in 2, TET2 mutation in 4, DNMT3A mutation in 5) with clonal hematopoiesis of indetermi- nate potential (CHIP) (1 individual classed as having high-risk CHIP) were successfully investigated. DNA samples were pre- pared from a whole tissue section of each specimen. Purified DNA was obtained using a QIAamp DNA-Micro kit (Qiagen), while crude DNA was obtained by digesting samples at 37°C overnight with proteinase K and NP-40 buffer. Purified DNA samples from
nine cases of AITL or PTCL-TFH with known RHOA mutations (Gly17Val, c.50G>T, n=6 with variant allele frequency [VAF] rang- ing from 2-32%; Gly17Leu, c.49-50GG>TT, n=2; Gly17Glu, c.50G>A, n=1; Ser26Arg, c.76A>C, n=1) were available from our previous study,2 while crude DNA samples were available from routine clonality analysis or similarly prepared. The histology of these specimens was reviewed by specialist hematopathologists.
Quantitative polymerase chain reaction with peptide nucleic acid clamp and locked nucleic acid probe
The qPCR with peptide nucleic acid (PNA) clamp and locked nucleic acid (LNA) probe was performed as previously described.16 The qPCR assay contained two probes and a PNA clamp (Figure 1A, Online Supplementary Table S1). The total probe served as an internal control to monitor the PCR performance. The PNA clamp specifically and strongly binds to the wild-type DNA sequence, resisting the 5’ nuclease activity of Taq DNA polymerase, and thus blocks the wild-type allele from PCR amplification. This results in preferential amplification of the mutant allele which is detected specifically by the LNA mutant probe.
PCR primers with various probes were first tested using puri- fied DNA samples, with the optimized conditions outlined below (Online Supplementary Table S1). Briefly, the PNA-LNA PCR was carried out in a 20 μL reaction containing 10 mL Premix Ex Taq (Probe qPCR) Master Mix (Takara, Shiga, Japan), 0.2 mM of each forward and reverse primer, 0.1 μM of each total and mutant probe, 0.05 mM of the PNA clamp probe, and 2 mL of crude DNA or 25 ng of purified DNA. Real-time PCR was carried out in tripli- cate using Quantstudio 6 (Thermo Fisher Scientific, Waltham, MA, USA) with denaturation at 95°C for 30 s followed by 45cyclesat95°Cfor3s,andat62°Cfor30s.
Targeted sequencing using the Fluidigm Access Array and Illumina MiSeq
Targeted sequencing was performed on a selected case with consecutive tissue biopsies as described in our recent study.2 Each of the DNA samples was investigated in duplicate for mutations in TET2, DNMT3A, IDH2, RHOA, PLCG1, CCND3, CD28 and TNFRSF21 by Fluidigm PCR and Illumina MiSeq sequencing. Sequence reads alignment, variant calling, filtering to eliminate false positive and benign changes were carried out according to our previously established protocols.2 Only the reproducible vari- ants that appeared in both replicates were regarded as true changes.
BaseScope in situ hybridization
This was performed on a selected case. The sequence of the
clonal TRB rearrangement in a case of AITL was available from a recent study.2 Based on the unique VDJ junctional sequence, unique BaseScope probes were designed and used to identify the lymphoma T cells by in situ hybridization. The BaseScope in situ hybridization was carried out according to the manufacturer's instructions (Advanced Cell Diagnostics, Newark, CA, USA),2 with the conditions optimized using the AITL specimen from which the clonal TRB rearrangement was sequenced. Tonsils were used as a negative control.
Results
Peptide nucleic acid – locked nucleic acid quantitative polymerase chain reaction is highly sensitive and specific for the detection of RHOA Gly17Val (c.50G>T)
Upon the optimization of PCR conditions for RHOA mutation detection, we first determined the sensitivity of
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haematologica | 2022; 107(2)