Keywords
cutaneous T-cell lymphoma - mycosis fongoides - clonality - TCRG - BIOMED-2 protocol
- histology
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.[1]
[3] 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.
DNA Isolation
Genomic DNA was extracted using the QIAamp DNA formalin-fixed paraffin-embedded tissue
kit, following the manufacturer's instructions (Qiagen, Hilden, Germany). In cases
with insufficient DNA, a vacuum centrifugal concentration was carried out using an
Eppendorf Vacufuge plus Vacuum Concentrator (ID: 22820109). In all cases, before proceeding
to PCR, the DNA concentration and purity were assessed to ensure high-quality genomic
DNA in the samples.
Clonality Assessment via Analysis of TCRG Gene Rearrangements
TCRG gene rearrangements were assessed using the standardized BIOMED-2 multiplex PCR
clonality assays (InVivoScribe Technologies, San Diego, California, United States).
Genomic DNA was amplified with a single master mix that contains primers that target
the Vγ2, 3, 4, 5, 8, 9, 10, and 11 and Jγ1/Jγ2, JγP, and JγP1/JγP2 regions, generating
PCR amplicons with an expected size range between 159 and 207 nucleotides. The reaction
included both a clonal and a polyclonal control. The PCR components and standard program
were set up to follow the provider's protocols. After TCR amplification, 8 µL of PCR
products was loaded on 1% agarose gels to check whether PCR products had been formed.
PCR amplification was followed by a capillary electrophoresis, fluorescence-labeled
PCR products were run on an ABI Prism 3500 Genetic Analyzer, and the resulting data
were analyzed using the GeneMapper V 5.0 software (Applied Biosystems, Foster City,
California, United States).
Statistical Analysis
Statistical analysis was performed using IBM SPSS (v. 18.00; IBM Corp., Armonk, New
York, United States). Data were summarized using frequencies, percentages, medians,
and ranges. Fisher's exact test was used to determine whether a statistically significant
relationship exists between TCRG monoclonality and confirmation of the preliminary
diagnosis. The cutoff level for statistical significance was set at 0.05. Concordance
was analyzed by the use of kappa coefficient. It was valued between 0 and 1 (0.93–1.00:
excellent; 0.81–0.92: very well; 0.61–0.80: well; 0.41–0.60: moderate; 0.21–0.40:
below the middle; and 0.01–0.20: poor concordance).
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.
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.
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.
Fig. 2 Number of clonal peaks for confirmed and doubtful CTCL cases in the monoclonal group.
CTCL, cutaneous T-cell lymphoma.
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-Value[a]
|
|
TCRG monoclonality
|
1
|
11
|
0.038
|
|
TCRG polyclonality
|
4
|
3
|
|
Abbreviation: TCRG, T-cell receptor gamma.
a 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.[6]
[7]
[8] 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.[9]
[10]
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.[13]
[14] 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.[11]
[13]
[15] 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.
