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RHOA mutation analysis in early lymphoma diagnosis
Discussion
By using a highly sensitive qPCR assay, we confirmed that RHOA Gly17Val (c.50G>T) mutation is specifically associated with AITL or PTCL-TFH, but is not found in other lymphoproliferative conditions including those with florid infiltration of T-helper cells. More importantly, detection of RHOA Gly17Val (c.50G>T) mutation could help early detection of AITL and PTCL-TFH, and also diagnosis of their extranodal involvement.
The difficulty in making a histological diagnosis of AITL or PTCL-TFH is well recognized, particularly when the neoplastic cell content is low, inconspicuous by histologi- cal and immunophenotypic assessment and undetectable by analysis of TCR gene rearrangements. Apart from the paucity of neoplastic T cells, the polymorphous infiltrate, particularly the presence of numerous EBV-positive B cells, including HRS-like cells (EBV-positive or -negative), may lead to diagnostic consideration of an EBV-driven B-cell proliferation.19-21 Such misleading diagnostic features may also be reinforced by the frequent demonstration of clonal IG gene rearrangement. As shown in this study, EBV-dri- ven B-cell proliferation was considered as a diagnosis in four of 11 of the initial biopsies. Indeed, it has been report- ed by several independent studies that EBV-associated lymphoproliferation is a frequent pitfall in the diagnosis of AITL.19-22
In the present study, the majority of the initial speci- mens that were not diagnostic were core biopsies. It is possible that these core biopsies were not totally represen- tative, with characteristic lymphoma components missed because of sampling errors. Nevertheless, two initial non- diagnostic biopsies were excisional lymph node speci- mens, indicating that an absence of diagnostic features of AITL/PTCL-TFH in the initial biopsies was also a real issue. Interestingly, the time interval between the initial non-diagnostic biopsies and the follow-up biopsies that established the diagnosis varied considerably, ranging from 0 to 26.5 months. In the cases with a long interval, it is likely that the initial biopsy represented an early pre- malignant lesion, while the follow-up biopsy reflected more progressed disease, thus having more cardinal fea- tures for making the AITL/PTCL-TFH diagnosis. While in the cases with a short interval, it is possible that the enlarged lymph nodes were variably involved by AITL/PTCL-TFH and the initial non-diagnostic biopsies represented early involvement by the lymphoma.
Both the above possibilities may exist, without exclud- ing the other. It is impossible to distinguish the two sce- narios based on the analysis of a single biopsy, including RHOA mutation analysis. Nonetheless, detection of RHOA Gly17Val (c.50G>T) mutation can certainly raise the alarm to perform more in-depth histological and immunophenotypic investigations, for example a more careful search for evidence of TFH cell expansion, as shown in the first biopsy in case 30. It is important to emphasize that detection of a RHOA mutation is not equivalent to a diagnosis of lymphoma because mutation- al analysis by qPCR or targeted sequencing is highly sen- sitive, and the mutation is seen in biopsies without histo- logical evidence of AITL. As discussed above, the RHOA mutation-positive non-diagnostic biopsy may represent a premalignant lesion or early involvement by the lym- phoma. Therefore, RHOA mutation analysis should be used as an auxiliary tool with the results interpreted in the
context of histological and immunophenotypic findings. If a histological diagnosis of AITL/PTCL-TFH cannot be made, an excision biopsy or a low threshold for an early follow up biopsy, when appropriate, is indicated.
In a few case studies, RHOA mutation was detected in circulating free DNA or peripheral blood lymphocytes from patients with AITL/PTCL-TFH,15 and the mutation burden appeared to correlate with the treatment outcome.23,24 It remains to be investigated whether the mutation is detectable in circulating free DNA or periph- eral blood lymphocytes at the time of initial non-diagnos- tic biopsies. Given that the RHOA mutation burden in cir- culating free DNA and peripheral blood lymphocytes reflects, at least theoretically, the overall lymphoma load, simultaneously analyzing blood samples, in addition to tissue biopsy, could add further value in lymphoma diag- nosis, particularly when a specimen is not representative.
Apart from the initial biopsies, the diagnosis of extra- nodal involvement by AITL/PTCL-TFH such as skin and bone marrow is also difficult for reasons similar to those discussed above, including low tumor cell content and polymorphous infiltrate.25 In particular, extranodal infil- trates may not harbor the cardinal features of AITL such as TFH marker expression in the neoplastic T cells and follicular dendritic cell meshworks. The DNA sample from these extranodal sites is often not informative for clonality analysis because of insufficient lymphoid cells and/or poor DNA quality. As shown in our study, RHOA mutation analysis is highly valuable for confirming AITL/PTCL-TFH involvement in follow-up biopsies. Similarly, RHOA mutation analysis is valuable in the diagnosis of AITL/PTCL-TFH from cytological speci- mens.9
Aside from the RHOA mutation, there are several other genetic changes including IDH2, CD28, PLCG1, VAV1 and TNFRSF21 mutations, and VAV1-STAP2, CTLA4-CD28 and ITK-SYK fusions, which occur at variable frequencies in AITL and PTCL-TFH.2,26-28 Of note, mutations in VAV1, a signaling molecule downstream of TCR, occurs in 8.2% of AITL and appears to be mutually exclusive of the RHOA mutation.28 Detection of these additional lym- phoma-associated genetic changes could also help the early detection of AITL/PTCL-TFH, together with RHOA mutation detection, potentially being valuable for diagno- sis in up to 90% of these T-cell lymphomas. These diverse genetic changes could be readily investigated by targeted sequencing either alone or as part of a comprehensive lymphoma panel.
The detailed analyses of multiple biopsies in case 7 also revealed the mutation burden in non-malignant T cells. The fifth lymph node biopsy showed little involvement by AITL as the lymphoma clone was undetectable by BaseScope in situ hybridization and the VAF of the RHOA mutation was only 1%, but the specimen had a high TET2 mutation burden (20% VAF). This implies that the TET2 mutation must have been present in a large proportion of reactive B and T cells,1,5,29-31 probably up to 40% of the total cell population, which is well above the EBER-posi- tive cell fraction (~5%). Hence, this enlarged lymph node was essentially caused by lymphoid proliferation driven by the TET2 mutation and/or EBV infection. These find- ings further highlight the markedly variable histological presentation of enlarged lymph nodes in patients with AITL and, hence, the danger of potential sampling errors in the diagnosis of AITL. In this context, it is pertinent to
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