Early detection of T-cell lymphoma with T follicular helper phenotype by RHOA mutation analysis

Abstract

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma with T follicular helper phenotype (PTCL-TFH) are a group of complex clinicopathological entities that originate from T follicular helper cells and share a similar mutation profile. Their diagnosis is often a challenge, particularly at an early stage, because of a lack of specific histological and immunophenotypic features, paucity of neoplastic T cells and prominent polymorphous infiltrate. We investigated whether the lymphoma-associated RHOA Gly17Val (c.50G>T) mutation, occurring in 60% of cases, is present in the early "reactive" lesions, and whether mutation analysis could help to advance the early diagnosis of lymphoma. The RHOA mutation was detected by quantitative polymerase chain reaction with a locked nucleic acid probe specific to the mutation, and a further peptide nucleic acid clamp oligonucleotide to suppress the amplification of the wild-type allele. The quantitative polymerase chain reaction assay was highly sensitive and specific, detecting RHOA Gly17Val at an allele frequency of 0.03%, but not other changes in Gly17, nor in 61 controls. Among the 37 cases of AITL and PTCL-TFH investigated, RHOA Gly17Val was detected in 62.2% (23/37) of which 19 had multiple biopsies including preceding biopsies in ten and follow-up biopsies in 11 cases. RHOA Gly17Val was present in each of these preceding or follow-up biopsies including 18 specimens that showed no evidence of lymphoma by combined histological, immunophenotypic and clonality analyses. The mutation was seen in biopsies 0-26.5 months (mean 7.87 months) prior to the lymphoma diagnosis. Our results show that RHOA Gly17Val mutation analysis is valuable in the early detection of AITL and PTCL-TFH.

Figure 1.

Detection of RHOA p.Gly17Val (c.50G>T) by real time polymerase chain reaction with peptide nucleic acid/locked nucleic acid probes. (A) Schematic illustration of the real time polymerase chain reaction (PCR) design. The total probe is used as an internal control to monitor the PCR performance. The peptide nucleic acid (PNA) clamp binds to the wild-type sequence, resists 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 locked nucleic acid (LNA) mutant probe. (B) The PCR assay is highly sensitive, capable of detecting the RHOA mutation at a variant allele frequency (VAF) of 0.03% based on serial dilutions of an angioimmunoblastic T-cell lymphoma sample with known mutation allele frequency by next-generation sequencing (left panel). For simplicity, only the LNA mutant but not total probe signals are shown. The PCR assay shows a linear correlation among the serial dilutions (right panel).

Figure 2.

Analysis of RHOA (c.50G>T; p.Gly17Val) mutation by quantitative polymerase chain reaction in the RHOA-positive cases with longitudinal biopsies in patients with angioimmunoblastic T-cell lymphoma (only including cases with at least one biopsy classed as not diagnostic [“group B” or “group C”]). The diagnoses of these specimens were reviewed, and categorized into three groups: group A (filled circles): lymphoma diagnosed by histology and immunophenotype, further supported by clonal TCR gene rearrangement; group B (half-filled circles): lymphoma not diagnostic by histology and immunophenotype alone, but ascertained by clonal TCR gene rearrangement; group C (open circles): lymphoma not diagnosed by combined analyses. Red (regardless of the symbol: including outline only, half-filled, and completely filled symbols) indicates RHOA p.Gly17Val mutant positive biopsy. Gray indicates RHOA p.Gly17Val status unknown. E denotes extranodal biopsies. *denotes lymph node excision biopsies and all others are core biopsy specimens. An open square denotes a diagnosis of classic Hodgkin lymphoma with no apparent evidence of angioimmunoblastic T-cell lymphoma (AITL). Case 38 lacks a final diagnosis as the patient died, indicated by a dashed line. Cases with only group A multiple biopsies are not included in this figure.

Figure 3.

Histological and immunophenotypic findings in case 30. The first lymph node excision biopsy shows partial effacement of the lymph node architecture by polymorphous infiltrates, particularly B cells with plasmacytoid differentiation. A large proportion of B cells were EBER-positive and showed IG κ light chain restriction (not shown). The lymphoid follicles appear to be reactive and shows no apparent expansion of T follicular helper (TFH) cells, with only a few CD10-positive cells spilling out of the germinal center, consistent with pattern-1 histology of angioimmunoblastic T-cell lymphoma (AITL). The second lymph node biopsy shows effacement of the lymph node architecture by medium-sized atypical lymphoid cells with regressed follicles. There is a prominent proliferation of follicular dendritic cell meshworks and high endothelial venules with atypical lymphoid cells clustered in their vicinity. The atypical lymphoid cells are T cells expressing TFH markers, spilling out of the germinal center to the interfollicular region. EBER in situ hybridization shows only scattered positive cells.

Figure 4.

Histological and immunophenotypic findings in case 7. The first biopsy shows a subcutaneous perivascular infiltrate of CD3⁺ T cells with vasculitic features. The third biopsy shows cardinal features of angioimmunoblastic T-cell lymphoma (AITL) and also a prominent pleomorphic infiltrate including an EBER-positive B-cell population with Hodgkin and Reed/Sternberg (HRS)-like morphology and immunophenotype. The fifth biopsy shows no apparent evidence of AITL, but a polymorphous infiltrate with a more prominent EBER-positive B-cell population that has HRS cell morphology and immunophenotype, rosetting by CD4⁺ T cells.