Introduction

Cutaneous T-cell lymphomas (CTCLs) demonstrate a variety of clinical, histological, and molecular features and can follow an indolent or very aggressive course.1 While the term CTCL includes a number of rare disease entities, the most common subtypes are mycosis fungoides (MF) and Sézary's syndrome (SS), accounting for approximately 70 to 75% of all cases.2 However, the underlying pathogenetic mechanisms of CTCL are not fully understood.13 Therefore, accurate evaluation in each individual case is crucial for an adequate, stage-adapted therapeutic approach.4 In a significant number of suspected T-cell lymphoproliferative disorders, regarded as doubtful cases, a definitive diagnosis of malignancy cannot be made without additional evidence of a clonal process.4 Clonality may establish a form of molecular confirmation in instances in these doubtful cases, in which the malignity of a lymphoid lesion is not certain based on histopathological or immunophenotypic assessment. T-cell receptor gamma (TCRG) assays provide molecular evidence of clonality in malignant or doubtful lymphoproliferative disorders and can facilitate minimal residual disease assessment.5 This study aimed to explore the concordance in terms of the diagnostic value of BIOMED-2 TCRG polymerase chain reaction (PCR) protocol with the histological diagnosis.

Methods

Doubtful and confirmed CTCLs were included in this descriptive cross-sectional study performed in the Habib Thameur Hospital in 2021. These cases were followed in the department of dermatology from 2012 to 2021. Patients were excluded if there was lack of information on clinical data, or if the block was exhausted. In all cases, histopathological and immunophenotypic studies were performed in the pathology department. Molecular clonality analysis was done in the Molecular Biology Laboratory of the Pathology Department and the Human Genetics Laboratory of the Faculty of Medicine of Tunis. Demographic data, number of lesions per patient, and histopathological and immunophenotypical findings were collected. We grouped the lesions into MF, other CTCL, and doubtful cases with uncertain clinical presentation, noncontributory histology, and nondemonstrative immunohistochemistry (IHC) of the T-cell infiltrate.

Results

In total, 113 skin biopsies from 54 patients were included in this study. The lesions were solitary in 35 cases, and multiple lesions were observed in 19 cases. Thirty-seven patients were male, with a median age at diagnosis of 56.5 years (18–73 years). Based on clinical, histological, and immunohistochemical (CD3, CD4, CD8, CD2, CD5, CD7) criteria, cases were divided into (35/54, 65%) confirmed CTCL cases with MF (28/35, 80%) and other CTCL (7/35, 20%) and (19/54; 35%) doubtful CTCL cases including 10 doubtful MF, 4 eczematous dermatoses, and 1 angioimmunoblastic T-cell lymphoma (AITL).

TCRG Clonality Interpretation

Results were interpreted according to the latest EuroClonality/BIOMED-2 guidelines for interpretation and reporting of TCR clonality testing in suspected lymphoproliferations. Fig. 1 shows the presence of a clear monoclonal TCRG rearrangement.

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Fig. 1: T-cell receptor gamma gene rearrangement in an angioimmunoblastic T-cell lymphoma case showing a monoclonal peak with a size of 185 bp in a polyclonal background.

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Among the 54 cases, 30 (56%) were monoclonal for TCRG rearrangement, and 24 (44%) were polyclonal; monoclonality was found in 18 of 35 (51%) cases of confirmed CTCL, in 16 of 28 MF (57%), in 2 of 7 other CTCL, and in 12 of 19 (63%) doubtful cases. Fig. 2 shows the number of clonal peaks that were detected. Among the cases with monoclonality results, 9/30 cases (30%) had two clonal peaks, and 1 case (3%) had three peaks.

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Fig. 2: Number of clonal peaks for confirmed and doubtful CTCL cases in the monoclonal group. CTCL, cutaneous T-cell lymphoma.

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Among the 19 doubtful cases, 12 were converted to malignant diagnosis (11 MF and 1 AITL) and 7/19 remained doubtful (Table 1). Analysis of TCRG clonality as a discriminator between the confirmation and the rejection of the preliminary diagnosis was performed using Fisher's exact test. There was a significant correlation (p = 0.038) between TCRG monoclonality and confirmation of the preliminary histopathological diagnosis (Table 2).

Table 1

Preliminary and final diagnoses of TCRG monoclonal cases among the doubtful cases

Preliminary diagnosis	Monoclonal TCRG rearrangement	Polyclonal TCRG rearrangement	Final diagnosis
Doubtful MF cases (n = 14)	10	4	MF (n = 10)
Eczematous dermatoses (n = 4)	1	3	MF (n = 1)
Suggestive of AITL (n = 1)	1	0	AITL (n = 1)

Abbreviations: AITL, angioimmunoblastic T-cell lymphoma; MF, mycosis fungoides; TCRG, T-cell receptor gamma.

Table 2

Correlation between the preliminary diagnosis and clonality among doubtful cases

TCRG clonality	Change of diagnosis (N = 7)	No change of diagnosis (N = 12)	p-Valuea
TCRG monoclonality	1	11	0.038
TCRG polyclonality	4	3

Abbreviation: TCRG, T-cell receptor gamma.

Significant at p ≤ 0.05.

The results of TCRG clonality assay and the histology diagnosis were concordant in 14 cases (74%) and discordant in 5 cases (26%). A moderate concordance was found between histology and molecular clonality (kappa = 0.41).

Discussion

Our results showed a concordance in terms of the diagnostic value of BIOMED-2 TCRG PCR protocol with the histological diagnosis. In order to unify results and overcome technical problems, the BIOMED-2 protocol is considered the gold standard for clonality assessment in suspected lymphocytic infiltrations.678 Another recent development in the area of T- and B-cell clonality testing by PCR is the use of capillary electrophoresis, which has significantly improved the resolution and quantitation of PCR products.910

Of the 54 patients included in this study, 30 (56%) were monoclonal for TCRG rearrangement. Multipeak monoclonality was observed in 10 cases, and the explanation for double-peak cases is either the presence of two dominant clones in the same sample or a clone with TCRG biallelic rearrangements. Three peaks in cases could indicate the presence of a minor tumor clone.11 We detected monoclonality in 16/28 (57%) patients with confirmed MF. Similar detection rates were found in a large study by Massone et al.12 Also, these findings are in concordance with many other studies, which demonstrated that in well-established CTCL cases, TCR clonality has been shown to detect T-cell monoclonality in 52 to 90% of established CTCL biopsy specimens.13 Kirsch et al revealed superior frequencies using high-throughput sequencing of established CTCL cases.3

While MF and SS are the most common CTCLs, there are limited data about the other CTCLs. In the present study, among all the seven cases of other CTCL, two were monoclonal for a TCRG rearrangement. Analysis of clonal TCRG gene rearrangement plays an important role in the diagnosis of lymphoproliferative disorders. The identification of T-cell monoclonality within the lymphoid infiltrate can provide strong evidence of the diagnosis of CTCL.1314 Combining this approach with histopathologic and immunophenotypic examinations may aid in the diagnosis of doubtful cases. Twelve of 19 (64%) of doubtful cases revealed monoclonal TCRG gene rearrangement. Our finding is comparable with the results of many studies.111315 In a series of borderline lesions from 22 patients who subsequently developed MF and 32 newly suspected CTCL patients, TCRG monoclonality was detected in 50% (10/22) and 19% (6/32) of cases, respectively.15 Regarding our study, the positive rate of TCRG gene rearrangement in suggestive MF cases was 71% (10/14). Similar rates were found by Hsiao et al.16 However, different detection rates were found in other studies.17 Several factors accounting for the variability include different sensitivity of PCR detection methods, different stages and duration of early lesions, the inclusion of nonspecific lesions, the subjectivity of interpretation by pathologists, and the protocol followed in IHC.

In 74% of doubtful cases (12/19), TCRG molecular analysis allowed the confirmation of the malignant nature of lymphoid proliferation. We demonstrated concordant TCRG molecular analysis and histology results, with moderate kappa score (kappa = 0.41). Eleven (58%) doubtful cases: 10 MF and 1 AITL demonstrated TCRG monoclonality, confirming their clonal origin and thus the diagnosis of CTCL. We have shown that the results of TCRG clonality demonstrated significant correlation with histopathologic diagnoses (p = 0.038). Similar results were found by Sandberg et al; in 17 samples, the molecular findings correlated with the histopathologic diagnosis of malignant T-cell proliferations.18 In contrast, a previous study reported that differences in histologic parameters between PCR positive and PCR negative were not statistically significant. A polyclonal proliferation is not equivalent to benign lesions. This was justified by the fact that molecular studies are not very useful when the histopathologic findings show a more severe inflammation because inflammation dilutes the malignant T-cell clones. Even if there is a negative PCR result, it is important to consider the dilution of clonal T cells by reactive lymphocytes (in the case of a small clonal T-cell population) and more similar lesions need to be included.16

Conclusion

Detection of monoclonal TCRG gene rearrangement is a very powerful supplement to the diagnosis of CTCL. It is especially useful in the diagnosis of doubtful cases with atypical histopathologic features. BIOMED-2 PCR TCR clonality protocol has wide availability and produces satisfactory results, and thus, it is a useful tool in routine clinical practice. Results of molecular clonality testing in this study emphasize the importance of interpreting data in association with morphologic features as well as immunophenotypic characteristics of the lesions. New multicenter prospective studies are mandated to incorporate new possible markers related to given treatments.