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R. Dobson et al.
(EBER) in situ hybridization was carried out in eight cases during the routine diagnostic workup, and four showed variable positivity from a few scattered to numerous EBER-positive cells, of which three displayed clonal IG gene rearrangement. In four cases, EBV-driven prolifera- tion or classic Hodgkin lymphoma were considered in the initial diagnosis.
Six follow-up biopsies were not diagnostic of involve- ment by AITL or PTCL-TFH by routine diagnostic inves- tigations; these included two bone marrow and two skin specimens. In each specimen, a CD4+ T-cell infiltrate was noted, but a definite aberrant immunophenotype and expression of TFH markers could not be ascertained. T- cell clonality analysis showed weak polyclonality in two and a weak clonal or oligoclonal pattern in two.
Representative cases
Case 30. A 79-year-old man presented with bilateral tender neck lymph nodes, and had mild sweats, but no weight loss or fever. Clinical examination revealed multi- ple bilaterally enlarged neck and groin lymph nodes (up to 1.5 cm diameter), and palpable liver and spleen. Right level II neck lymph node excision biopsy showed partial effacement of the lymph node architecture and expansion of the interfollicular area by a polymorphous population of lymphoid cells, including B cells with plasmacytoid dif- ferentiation, and scattered large cells (Figure 3). These B cells expressed pan B-cell markers (CD20, CD79a, CD19), and MUM1, but were negative for CD10 and BCL6 (data not shown). A high proportion of the B cells were EBER- positive and showed IG k light chain restriction. The lym- phoid follicles appeared to be reactive and showed no apparent expansion of TFH cells, with only a few CD10- positive cells spilling out of the germinal centers (Figure 3). BIOMED-2 clonality analyses showed clonal IGH and IGK gene rearrangements, but a weak oligoclonal pattern with TRG and TRB. The histological diagnosis was uncertain: a clonal EBV-positive polymorphous lympho- proliferation was considered. Close follow-up with a low threshold for re-biopsy was recommended.
Two months later, the patient presented with increasing fatigue, fever, maculopapular chest rash and increased size of peripheral lymphadenopathy. Positron-emission tomography (PET) scan revealed extensive bilateral cervi- cal, mediastinal, bilateral iliac and groin lymphadenopa- thy. Left level V neck lymph node excision biopsy showed partial effacement of the lymph node architecture by an infiltrate of medium-sized atypical lymphoid cells with regressed follicles (Figure 3). There was hyperplasia of fol- licular dendritic cell meshworks and high endothelial venules with the atypical lymphoid cells clustered in their vicinity. The atypical lymphoid cells were positive for CD3, CD5 and TFH markers (PD1, CD10, ICOS, BCL6) (Figure 3). EBER in situ hybridization revealed only scat- tered positive cells. BIOMED-2 clonality analyses showed clonal TRB and TRG gene rearrangements, and also weak clonal IGH and IGK gene rearrangements which were dif- ferent from those of the previous biopsy in the size of their amplified IG products. A diagnosis of AITL was made. The patient was initially treated with six cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), then avelumab (cycle 12 at the most recent follow-up) under the AVAIL-T trial, and was well, show- ing no constitutional symptoms or palpable cervical lymph nodes, 20 months following the AITL diagnosis.
Retrospective analysis showed the presence of RHOA Gly17Val mutation in the above two specimens by qPCR and targeted sequencing with 3% and 23% VAF in the first and second biopsy, respectively. The targeted sequencing also revealed a pathogenic nonsense substitu- tion in DNMT3A (c.2311C>T, p.R771*) in the first and second biopsy with 17% and 26% VAF, respectively.
Case 7. An 82-year-old man, with remission of a previ- ous mantle cell lymphoma, presented with a skin rash on the right calf. A punch biopsy showed a mild perivascu- lar infiltrate by CD3+ T cells, with a histological diagnosis of panniculitis (Figure 4, Table 1). To investigate potential lymphoma relapse, lymph node core biopsies were taken at 11 and 12 months of follow-up: neither specimen showed evidence of mantle cell lymphoma, but both revealed cardinal features of AITL and also a prominent pleomorphic infiltrate including an EBER-positive B-cell population with Hodgkin and Reed/Sternberg (HRS)-like morphology and immunophenotype (Figure 4, Table 1). T-cell clonality analysis demonstrated clonal rearrange- ments by TRG-A and TRB-A with identical sized ampli- fied products between the two biopsies, and an addition- al clonal rearrangement by TRB-C in the second biopsy. Further follow-up biopsies were taken at 25 and 26 months, and neither showed apparent evidence of AITL, but both had polymorphous infiltrates with a more prominent EBER-positive B-cell population that had HRS- cell morphology and immunophenotype (Figure 4, Table 1). Neither specimen showed any evidence of the clonal TRG/TRB rearrangements seen in the early lymph node biopsies, although a fifth biopsy displayed an isolated clonal rearrangement by TRB-C. B-cell clonality analyses demonstrated polyclonal IG gene rearrangements in both specimens (Figure 4, Table 1). A classic Hodgkin lym- phoma arising from the EBV-positive B-cell component of the AITL was considered.
In a retrospective study, the TRB-A and B PCR products from the second biopsy were sequenced using an Illumina MiSeq platform and a dominant TRBV5-J2 rearrangement (86%) was identified.2 Based on the unique VDJ junctional sequence, we designed unique BaseScope probes to iden- tify the lymphoma T cells by in situ hybridization (Online Supplementary Figure S2). As expected, both the second and third biopsies with an AITL diagnosis showed diffuse positivity with the lymphoma clone-specific probe. Interestingly, the initial skin biopsy also displayed isolated positive cells, while the fifth biopsy with a diagnosis of EBV lymphoproliferative disease (LPD) gave a negative result. Both the second (AITL) and fifth (EBV-LPD) biop- sies were investigated by panel sequencing for recurrent somatic mutations, and this identified five shared muta- tions – one DNMT3A, three TET2 and one RHOA changes – between the two specimens, and one further TET2 mutation only in the second specimen (Table 1). In gener- al, the mutation load in each of the above shared changes was much higher in the second biopsy (AITL) than in the fifth biopsy (EBV-LPD). This was particularly striking for the RHOA Gly17Val mutation, with a VAF of 20% in the second biopsy but of only 1% in the fifth biopsy. As expected, qPCR for the RHOA mutation demonstrated its strong positivity in both the second and third biopsies showing AITL, but a weak positive signal in the initial skin biopsy, and the fourth and fifth biopsies displaying classic Hodgkin lymphoma-like EBV-LPD (Figure 4, Online Supplementary Figure S2).
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haematologica | 2022; 107(2)