To the Editor:
In cutaneous T-cell lymphomas/lymphoproliferative disorders (CTLs) the T-cells are usually monoclonal, whereas in reactive infiltrates they are most often polyclonal.1 Analysis of the T-cell receptor (TCR) using multiplex polymerase chain reaction (PCR) is standard for clonality testing, however this method is time-consuming, expensive, and technically limited.2
A monoclonal antibody (mAb) against the C1 constant region of the TCRβ chain (TRBC1) has advanced clonality testing in flow cytometric analysis of αβ T-cell lymphomas3 including CTLs.4 During TCR gene rearrangement, one of two mutually exclusive TCR constant β chains (TRBC1 or TRBC2) is randomly selected. Normal αβ T-cells comprise a mixture of TRBC1-positive and TRBC1-negative (presumptively TRBC2-positive) cells, whereas neoplastic T-cell populations arise from a single clone and exclusively express either TRBC1 or TRBC2. Restricted expression of TRBC1 can thus serve as a marker for clonality.5 Here, we report our results using a TRBC1 mAB in immunohistochemistry (IHC) on FFPE specimens from 30 αβCTLs and 9 rashes as a surrogate for molecular testing.
We performed a retrospective review of clinicopathologic data from patients biopsied for possible CTL in 2022. Cases needed to have warranted IHC and molecular analysis for diagnosis and were TCRαβ+. Hematoxylin and eosin (H&E)-stained sections and CD3 IHC were reviewed by light microscopy (blinded to diagnosis) for atypical lymphoid infiltrates. >/=90% of morphologically atypical cells needed to show CD3 for baseline T-cell distribution. TRBC1 (E6Z3S; 1:600; 79485, Cell Signaling Technology) immunostaining was assessed as a percentage of total atypical CD3+ T-cell infiltrates. Cases were assigned a predictive category of monoclonal (PM) or polyclonal (PP) based on percent atypical T-cells labeled by TRBC1 with percentages of ≥70% or ≤25% indicating TRBC1-restriction/predicted monoclonal. We then compared TRBC1 predictions to clonality interpretations by PCR and clinical data.
Twenty-six (68%) samples were PM and twelve (32%) were PP by TRBC1. Overall concordance with PCR was observed in 21/26 (81%) PM and 9/12 (75%) PP, with TRBC1 PM showing 88% agreement for monoclonality with fragment analysis (21/24 PCR-clonal cases). Agreement improved to 23/26 when PCR results were re-evaluated, resulting in changed interpretation as monoclonal in two cases.
TRBC1 PM was more sensitive for CTLs (24/29, 83%) vs. PCR (22/29, 76%), although the specificity was lower with PM rendering a PPD of 89% compared to 96% with PCR. Both techniques showed clonality in all high grade CTLs. Neither technique was strong at confirming reactive disease (NPD 50% for TRBC1, and 53% for PCR). Qualitatively, TRBC1, when restricted positive, aided in diagnosis of strictly intraepidermal CTL.
Our findings indicate that using a single anti-TRBC1 antibody with an appropriate pan-T-cell marker allows rapid and low-cost assessment of clonality in archival tissues. Problems with visual “gating” by morphology and emphasis on CD3+ αβCTLs limit the generalizability of the results. Inflamed or polymorphous infiltrates are challenging with single IHC techniques. The small sample size and few reactive cases warrant caution in attributing statistical significance to comparisons with PCR. Cutoff values for TRBC1 monotypia require more stringent analysis. As with PCR, false positives are possible due to oligoclonal reactive processes.
Supplementary Material
Figure 1.
Skin biopsy specimen from a patient with mycosis fungoides which shows an atypical lymphocytic infiltrate predominantly located in the epidermis. The atypical lymphocytes are diffusely positive for CD3 (A, x400) and negative for TRBC1 (B, x400), consistent with a monotypic-negative TRBC1 expression pattern. The underlying dermal reactive immune infiltrate shows a TRBC1 polytypic expression pattern.
Funding Sources:
Research reported in this publication was supported in part by the Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute under award number P30CA008748.
ABBREVIATIONS & ACRONYMS:
- CTL
Cutaneous T-cell lymphoma/lymphoproliferative disorder
- TRBC1
T-cell receptor β constant region 1
- TCR
T-cell receptor
- IHC
immunohistochemistry
- FFPE
formalin-fixed paraffin-embedded
- PCR
polymerase chain reaction
- mAb
monoclonal antibody
- H&E
hematoxylin and eosin
- NGS
next generation sequencing
- PM
predicted monoclonal
- PP
predicted polyclonal
- PPD
positive predictive value
- NPD
negative predictive value
Footnotes
Conflicts of Interest: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.
IRB Approval Status: Reviewed and approved by Memorial Sloan Kettering Cancer Center (Derm 16–1613)
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