T-Cell Clonality Assays: How Do They Compare?

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See related article on pg 1030

T-Cell Clonality Assays: How Do They Compare? Antonio Cozzio1 and Lars E. French1 The distinction between certain benign cutaneous lymphocytic infiltrates and cutaneous T-cell lymphoma (CTCL) can be a challenging problem in clinical practice. An imperfect but useful tool in this circumstance is the analysis of T-cell clonality or monoclonality by assessing T-cell receptor (TCR) gene rearrangements. “Monoclonality” describes the origin of a specific human malignant tumor from one single cell from which the entire tumor is derived. The term is used to describe the early steps in tumorigenesis; in later stages of tumor growth and progression, monoclonal tumor cells may acquire a mutator phenotype, rendering the genome unstable. The resulting genetic heterogeneity may ultimately lead to subclones in the formerly monoclonal population of tumor cells. Journal of Investigative Dermatology (2008), 128, 771–773. doi:10.1038/jid.2008.49

Mycosis fungoides is the most common form of primary CTCL. In advanced stages of disease, it is readily diagnosed by the outgrowth of skin tumors. In its early stages, however, it can be difficult to distinguish from benign inflammatory dermatoses such as eczema. Furthermore, long-term follow-up studies and clinical practice suggest a clinical spectrum that ranges from a “purely” inflammatory disease (parapsoriasis en petites plaques), with no or an extremely low tendency to develop into CTCL, to inflammatory diseases with a well-demonstrated

propensity to develop CTCL (parapsoriasis en grandes plaques) and, finally, at the far end of the spectrum, to lymphoproliferative and malignant disorders such as mycosis fungoides and Sézary syndrome. There is a need for validated diagnostic tools that are helpful in the early distinction of these diseases. In recent years, Southern blot analyses on the T-cell receptor β locus (TCRβ), which require relatively large amounts of starting materials, and polymerase chain reaction (PCR) analyses of the TCRγ locus have been proposed,

TCRGAMMA: Chromosome 7p15 Vl

ψV1 V2

V3

V4

V5

Vll

ψV5 ψV6 ψV7 V8

Vlll

VlV

ψVA V9 ψV10 ψVB ψV11

J1

1 23

C1

J2 C2

13

Figure 1. The TCRγ� chain gene contains 14 Vγ, 2 Cγ� and 5 Jγ �gene segments. The Vγ� gene segments are named and grouped into four families (V I to V IV) according to their order in the genomic organization, and to their sequence homologies. The expressed Vγ repertoire includes only six Vγ genes (Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, and Vγ9), but rearrangement also occurs in the pseudogenes ψVγ7, ψVγ10, and ψVγ11. Rearrangement of ψVB (also known as ψVγ12) is very rare. Family I of Vγ �gene can be amplified using a consensus primer, as it consists of seven Vγ gene segments that are highly homologous. Families II, III, and IV each consist of a single variable gene segment and do not share similar homology. The five Jγ �gene segments are divided into two groups comprised of Jγ1.1, Jγ1.2, Jγ1.3, located upstream of Cγ1, and, Jγ2.1, Jγ2.3, located upstream of Cγ2. PCR analysis of the TCRγ gene rearrangement generally is based on primers targeting one of the four families of Vγ �gene segments and one of the five Jγ gene segments. Department of Dermatology, Zurich University Hospital, Zurich, Switzerland

1

Correspondence: Lars French, Department of Dermatology, Zurich University Hospital, Gloriastrasse 31, Geneva CH-8091, Switzerland. E-mail: [email protected]

© 2008 The Society for Investigative Dermatology

among others, as tools to differentiate neoplastic from reactive lymphoproliferation. PCR clonality analysis of the TCRγ locus is the most widely used technique to date owing to (1) the restricted repertoire of V and J segments (14 V segments, 2 duplicated J-C segments; Figure 1) that can fairly easily be covered by a limited number of PCR primers and (2) the existence of TCRγ recombinations in both TCRαβ and TCRγδ clonal T-cell proliferations (as a result of the chronological order in TCR gene recombinations (γ → α and β → δ) in T-cell progenitors). Various detection systems have been developed for TCR-PCR fragment analysis, including denaturing gradient gel electrophoresis (DGGE) (Wood et al., 1994), singlestrand conformation polymorphism (SSCP) (Yamamoto et al., 1996), heteroduplex analysis (HD) (Marguerie et al., 1992), fluorescent gene scanning (Assaf et al., 2000), and cloning

|

Methods of detection are similiar but not equal.

and sequencing (Wilson et al., 1993). Oligonucleotide microchip analysis was recently introduced as a new tool to determine T-cell clonality (Gra et al., 2007). Whereas DGGE, heteroduplex analysis, and SSCP depend on the conformation of the nucleic acids in a test tube, gene scanning analysis (GS) allows discrimination by size and V family usage. GS thus allows a more accurate comparison of amplicons from different tissue samples in the same patient. Cloning and sequencing of the CDR3 region of the TCR represents the gold standard but is costly and time-consuming (Table 1). In this issue, Ponti and colleagues (2008) report on the analysis of 270 skin samples from 196 patients by comparative use of two TCR-PCR assays: multiplex/heteroduplex (multiplex/HD) www.jidonline.org

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Table 1. PCR-based clonality assays for T-cell lymphoproliferative disorders Assay

Characteristics

Denaturing gradient gel electrophoresis; single-strand conformation polymorphism

Sequence-/conformation-based separation of DNA Technically more demanding than PAGE

Multiplex/heteroduplex PCR analysis on PAGE

Length-/composition-dependent separation of DNA Standardization efforts (Biomed 2)a Rapid, simple, inexpensive

Fluorescent GeneScanning (capillary electrophoresis)

Length-dependent separation of DNA Clear cutoff values for signal relevance (2× background signal strength) Standardization efforts (Biomed 2) Rapid, simple Uses expensive equipment More sensitive than heteroduplex analysis Precise determination of the size of the PCR product can be used for monitoring the clonal proliferation during follow-up of the patient

Cloning and sequencing

Time-consuming, more expensive

Oligonucleotide microchip analysis

Sequence-dependent hybridization signal. Little experience

Comparison of specificity and sensitivity of the PCR-based clonality assays of T-cell lymphoproliferative disorders is not feasible, as the various techniques have not been tested against one another on standard sets of histologically/clinically correlated and well-defined cases of CTCL. In addition, primer sets vary among the various PCR assays. PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction. avan Dongen et al., 2003.

PCR and multiple-sample GS. They show that for three diagnostic classes—Sézary syndrome, mycosis fungoides, and inflammatory diseases—GS analysis proved to be more specific and more sensitive than multiplex/HD PCR. Sensitivity for both techniques depended on the histological score for CTCL (0 to 7) according to the proposal of Guitart et al. (2001) and was lowest for patients at stage T1/T2, in which the histologic grade was lower than 5 (on a scale of 7, meaning that mycosis fungoides cannot be excluded). More interestingly, for GS, the positive predictive value (PPV) of clonality was high in the group of histologically nondiagnostic samples (the PPV of a test is the probability that a positive test reflects the condition being tested for). Specifically, 20 of 33 of these samples revealed T-cell clonality, and in 18 of 20 of the latter, clinical follow-up confirmed a diagnosis of CTCL, yielding a PPV of 90%. For multiplex/HD PCR, the corresponding numbers were 14 of 33, of which 9 were later diagnosed as CTCL (PPV of 64%). By analogy, in the same group of histologically nondiagnostic samples, the negative predictive value (NPV)—the proportion of patients with negative test results who were correctly diagnosed—was 100% for GS (13 of 33 patients, no falsenegative results) and 53% for multiplex/ HD PCR (19 of 33 negative, 9 falsenegative results). Thus, both sensitivity 772

and specificity were higher for GS than for multiplex/HD in this group of patients, a group that poses significant diagnostic problems. PPV = Number of true positives/ number of true positives + number of false positives NPV = Number of true negatives/ number of true negatives + number of false negatives Ponti and colleagues also confirm what has been pointed out by others (Klemke et al., 2006; Vega et al., 2002), that the concepts of clonal heterogeneity and pseudomonoclonality are important to consider when dealing with the molecular diagnosis of CTCL. Clonal heterogeneity describes the coexistence of two different T-cell clones in distinct samples from the same patient and may reflect a mixture of neoplastic and reactive T-cell clones or the selection of a newly arising clone. Distinction of clonal heterogeneity from homogeneity in multiple samples from the same patients may be important, because it has been proposed that clonal homogeneity correlates with faster clinical progression of CTCL (Vega et al., 2002). On the other hand, inflammatory (but not neoplastic) T-cell proliferations frequently show pseudomonoclonality, i.e., dominant PCR amplicons that vary in repeated independent PCR analyses

Journal of Investigative Dermatology (2008), Volume 128

of the same samples. Thus, ruling out pseudomonoclonality by assay repetition prevents overdiagnosis of CTCL and should be a standard procedure. Taken together, GS as performed by Ponti et al. appears to be superior to multiplex/HD for the early detection of clonal T-cell neoplasms. It should be remembered, however, that certain CTCL subtypes may represent “molecular pitfalls.” This is the case for T cell lymphones, with loss of TCR expression, NK-cell lymphomas, or T-cell-rich B-cell lymphomas, in which a negative TCR assay may be misleading if not followed by additional immunohistochemical or molecular studies. These limitations, together with persisting sensitivity and specificity issues of T-cell clonality analyses for the diagnosis of CTCL, illustrate the need for additional diagnostic tools. Because the border between inflammation and cancer appears somewhat difficult to identify with respect to lymphocytic clonality, we believe that early differentiation of neoplastic infiltrates from inflammatory ones—and, equally important, differentiation of early-progressing from non- or late-progressing CTCL—will ultimately require concurrent analyses by immunohistochemistry, clonality assays, and even additional assessments such as expression analysis of the specific lymphoproliferative T-cell population at the nucleic acid and/or protein levels.

COMMENTARY

CONFLICT OF INTEREST

The author states no conflict of interest.

heterogeneity analysis in multiple cutaneous T-cell lymphoma samples. J Invest Dermatol 128:1030–1038

REFERENCES

van Dongen JJ, Langerak AW, Bruggeman M, Evans PA, Hummel M, Lavender FL, et al. (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17:2257–317

Gra OA, Sidorova JV, Nikitin EA, Turygin AY, Surzhikov SA, Melikyan AL et al. (2007) Analysis of T-cell receptor-gamma gene rearrangements using oligonucleotide microchip: a novel approach for the determination of T-cell clonality. J Mol Diagn 9:249–57

Vega F, Luthra R, Medeiros LJ, Dunmire V, Lee SJ, Duvic M et al. (2002) Clonal heterogeneity in mycosis fungoides and its relationship to clinical course. Blood 100:3369–73

Assaf C, Hummel M, Dippel E, Goerdt S, Müller HH, Anagnostopoulos I et al. (2000) High detection rate of T-cell receptor beta chain rearrangements in T-cell lymphoproliferations by family specific polymerase chain reaction in combination with the GeneScan technique and DNA sequencing. Blood 96:640–6

Guitart J, Kennedy J, Ronan S, Chmiel JS, Hsiegh YC, Variakojis D (2001) Histologic criteria for the diagnosis of mycosis fungoides: proposal for a grading system to standardize pathology reporting. J Cutan Pathol 28:174–83 Klemke CD, Poenitz N, Dippel E, Hummel M, Stein H, Goerdt S (2006) T-cell clonality of undetermined significance. Arch Dermatol 142:393–4 Marguerie C, Lunardi C, So A (1992) PCR-based analysis of the TCR repertoire in human autoimmune diseases. Immunol Today 13: 336–8 Ponti R, Fierro MT, Quaglino P, Lisa B, di Celle Paola F, Michela O et al. (2008) TCRγ-chain gene rearrangement by PCR-based GeneScan: diagnostic accuracy improvement and clonal

Wilson KB, Quayle AJ, Suleyman S, Kjeldsen-Kragh J, Førre O, Natvig JB et al. (1993) Heterogeneity of the TCR repertoire in synovial fluid T lymphocytes responding to BCG in a patient with early rheumatoid arthritis. Scand J Immunol 38:102–12 Wood GS, Tung RM, Heaffner AC, Crooks CF, Liao S, Orozco R et al. (1994) Detection of clonal T-cell receptor γ gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE). J Invest Dermatol 103:34–41 Yamamoto K, Masuko-Hongo K, Tanaka A, Kurokawa M, Hoeger T, Nishioka K et al. (1996) Establishment and application of a novel T cell clonality analysis using single-strand conformation polymorphism of T cell receptor messenger signals. Hum Immunol 48:23–31

See related article on pg 816

Vitamin D Regulation of Cathelicidin in the Skin: Toward a Renaissance of Vitamin D in Dermatology? Siegfried Segaert1 1,25-Dihydroxyvitamin D3, the active form of vitamin D, is a major regulator of the expression of the cationic antimicrobial peptide cathelicidin, not only in monocytes but also in epidermal keratinocytes. The involvement of cathelicidin in wound healing and skin diseases as diverse as psoriasis, rosacea, and atopic dermatitis may create new opportunities for the use of vitamin D in dermatology. Journal of Investigative Dermatology (2008), 128, 773–775. doi:10.1038/jid.2008.35

With epidermal photosynthesis of vitamin D3 as its main source, vitamin D is not a true vitamin for humans. Vitamin D3 is biologically inert and needs successive hydroxylation by 25-hydroxy1

lase (CYP27A1) in the liver and 1αhydroxylase (CYP27B1) in the kidney to yield 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], also known as calcitriol. In vitamin D target cells, calcitriol

Department of Dermatology, University Hospital Leuven, Belgium

Correspondence: Dr Siegfried Segaert, UZ Sint-Rafaël, Kapucijnenvoer 33, B-3000 Leuven, Belgium. E-mail: [email protected]

subsequently activates the vitamin D receptor (VDR), resulting in altered expression of genes involved in calcium metabolism, proliferation, differentiation, apoptosis, and adaptive immunity (Reichrath, 2007). Epidermal keratinocytes occupy a unique position within the vitamin D system because they not only possess the full machinery for ultraviolet B-dependent photoproduction of 1,25(OH)2D3 (Vantieghem et al., 2006) but also contain VDR and respond to 1,25(OH)2D3 with growth arrest, differentiation, and changes in cytokine expression (Segaert et al., 1997). Almost two decades ago, this property resulted in the successful introduction of topical vitamin D analogs totreatpsoriasis (Reichrath, 2007). However, other dermatologic indications for vitamin D derivatives remained largely unexplored. The recent identification of the cationic antimicrobial peptide cathelicidin as a vitamin D target gene (Gombart et al., 2005) and of CYP27B1 and VDR upregulation in monocytes as the link between Toll-like receptor2 (TLR-2) activation on the one hand and cathelicidin production and intracellular mycobacteria killing on the other hand (Liu et al., 2006) created a previously unknown and unexpected link between innate immunity and the vitamin D system. Vitamin D status, as determined by its cutaneous photosynthesis, promptly became a plausible explanation for increased susceptibility of African-American individuals to tuberculosis, seasonal peaking of viral infections in winter, and the therapeutic effect of phototherapy in lupus vulgaris, for which Niels Ryberg Finsen received the Nobel prize more than a century ago (Liu et al., 2006). Whether cutaneous photosynthesis of 1,25(OH)2D3 (Vantieghem et al., 2006) directly enhances innate immunity in the skin by induction of cathelicidin in keratinocytes (Schauber et al., 2007) or whether skin-photoproduced vitamin D3 acts via hepatic conversion to 25-hydroxyvitamin D3 to reach target cells, including monocytes and keratinocytes, remains to be determined (Segaert and Simonart, 2008). In addition, the mechanism of action of Finsen’s phototherapy is an www.jidonline.org

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