ORIGINAL STUDY
Multiple Distinct T-Cell Clones in Folliculotropic Mycosis Fungoides Panagiota Mantaka, MD,*† Agnieszka Malecka, MSc,‡ Gunhild Trøen, PhD,‡ Per Helsing, MD,* Petter Gjersvik, MD, PhD,*† and Jan Delabie, MD, PhD†‡
Abstract: Multiple distinct T-cell clones have been demonstrated in a subset of mycosis fungoides (MF), but have so far not been documented in folliculotropic MF, a clinical and histological variant of MF. We analyzed T-cell receptor (TCR) gene rearrangements in 8 patients with folliculotropic MF with multiple biopsies (range, 2–5) taken during the course of disease. Two patients had disease stage IA–IIA, 5 stage IIB–IVA, whereas data were not available for 1 patient. TCR b and g gene rearrangements were analyzed according to the BIOMED-2 PCR protocol. Multiple clonal TCR gene rearrangements indicating more than 1 T-cell clone were found in 5 patients. Although the number of patients is small, the finding of multiple distinct T-cell clones in 5 out of 8 patients suggests that chronic T-cell stimulation contributes to the development of folliculotropic MF. Key Words: folliculotropic mycosis fungoides, T-cell receptor gene rearrangements, T-cell clones
been shown to correlate significantly with clinical progression by Vega et al6,8 but needs confirmation in larger patient populations. Folliculotropic mycosis fungoides (FMF) is a variant of MF with distinct clinical and histopathological features.1 FMF shows a more aggressive clinical course and is more difficult to treat compared with conventional MF as demonstrated in several case series.11–13 Folliculotropism of the neoplastic T cells is a histological hallmark of the disease but the homing of T cells to follicles is as yet unexplained. Whether specific homing receptors or specific antigen stimulation within the follicular epithelium plays a role is not known. The purpose of this study was to evaluate clonal T-cell proliferation by analyzing T-cell receptor (TCR) gene rearrangements in skin biopsies from different anatomic sites at time of diagnosis or during the course of the disease in patients with FMF. The clinical data of the patients were also reviewed.
(Am J Dermatopathol 2014;36:972–976)
MATERIALS AND METHODS
INTRODUCTION Primary cutaneous T-cell lymphomas (CTCL) are clonal lymphoproliferative disorders characterized by, most frequently CD4+, neoplastic T cells that home to the skin.1 T cells develop in the thymus where TCR genes are rearranged early in the T-cell maturation process to produce antigen-specific T cells.2 Consequently, T-cell malignancies derived from mature T cells such as cutaneous T-cell lymphoma show clonally rearranged TCR genes, the analysis of which is useful for diagnosis.3–5 Multiple T-cell clones showing different TCR gene rearrangements have previously been described in up to 30% of cases of mycosis fungoides (MF) and Sézary syndrome.6–10 Of interest, a high prevalence of oligoclonal TCR gene rearrangements is reported in early stage CTCL, which suggests that antigen stimulation might play a role in the early stages of the disease.4,8 The persistence of the same clone over time has From the *Department of Dermatology, Oslo University Hospital, Rikshospitalet, Oslo, Norway; †Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; and ‡Department of Pathology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway. The authors declare no conflicts of interest. Supported by the Norwegian Cancer Society. Reprints: Panagiota Mantaka, MD, Department of Dermatology, Oslo University Hospital, Rikshospitalet, Postboks 4950 Nydalen, Oslo N-0424, Norway (e-mail: [email protected]). © 2014 Lippincott Williams & Wilkins
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Patients Patients were identified in the cutaneous T-cell lymphoma database at Oslo University Hospital, comprising all patients diagnosed in the period 1997–2012 (n = 95). Twentytwo patients with FMF were registered in the database. Of these, 8 patients had undergone multiple skin biopsies at the time of diagnosis and during the course of disease. Second biopsies were taken from 3 months to 4 years after the first biopsy. All biopsies were histologically reviewed, and the diagnosis of FMF was confirmed. The diagnosis of FMF and the staging of the patients were according to the WHO/EORTC classification for CTCL,1 based on clinical examination, skin biopsy, blood cell counts, and serum chemistry. Computed tomography of thoracic and abdominal regions and in some patients also of cervical and pelvic regions was performed upon clinical indication. Flow cytometry with quantitation of lymphoma cells in the blood was performed in 5 patients according to EORTC recommendations for staging of MF/Sézary syndrome.14 Disease progression was defined by one of the following criteria: (1) progression from patch/plaque to tumor or erythroderma, (2) histologically confirmed nodal involvement in patients with disease that was previously limited to the skin, (3) the development of visceral involvement in patients with previously only skin or lymph node involvement, or (4) Am J Dermatopathol Volume 36, Number 12, December 2014
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dissemination to the blood or death due to lymphoma. Clinical details of the patients have previously been published by our group.13 The study was approved by the Institutional Review Board of Oslo University Hospital and by the Regional Ethics Committee.
TCR Gene Rearrangement Studies TCR b and g gene rearrangements were studied in a total of 22 biopsies obtained from the 8 patients. DNA was isolated from paraffin-embedded or snap-frozen tissue, using the EZ1 tissue kit (Qiagen, Hilden, Germany), according to the instructions of the manufacturer. DNA quality was verified by PCR amplification of the albumin gene. TCR b and g gene rearrangements were detected by multiplex PCR according to the BIOMED-2 protocol.15 An in-house test was used for the latter, whereas a kit was used for the former (InVivoScribe Technologies, San Diego, CA). PCR products were size-fractionated on a 3130 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany) and analyzed using Gene Mapper version 4.1 software (Applied Biosystems).
Multiple T-Cell Clones in FMF
All analyses were performed in duplicate. The interpretation of the results was performed as recommended by Langerak et al.16 In short, a clonal TCR gene rearrangement was diagnosed when the height of one peak on the electropherogram was at least 3 times higher than that of other peaks resulting from the same competitive PCR product. Given the fact that both TCR gene alleles can be rearranged, the presence of multiple clones was defined when more than 2 clonal TCR g or more than 2 clonal TCR b gene rearrangements were observed in the same biopsy, when novel clonal TCR g or clonal TCR b PCR fragments were identified in different biopsies from the same patient or when one of the clonal TCR gene fragments was lost while other were retained.
RESULTS Clinical Findings Five patients were male, 3 were female. Median age was 54 years (range, 28–73 years). Patients presented with
TABLE 1. TCR Gene Rearrangements and Clinical Information in FMF Skin TCR g Gene TCR b Gene No. Patient Biopsies Time
Fragment (bp)
Clones* 1
Plaques and infiltrating tumors
IIB–IVA
3 mo
2
Erythematous and ulcerating plaques
IB–IIB
1 yr
3
Multiple erythematous plaques
NA
NA
2
Diffuse facial erythema, leonine facies, lichenoid dermatitis of upper body, ulcerating tumors
IVA
4 yrs
2
Erythematous plaques over eyebrows, arms, knees, milia-like lesions in the periorbital region, and infiltrating plaque/tumor on the extremities
IB–IIB
12 yrs
3
Infiltrating plaques and tumors on the face
IIB
2 yrs
1
Multiple erythematous plaques and tumors; infiltrating tumors on the trunk and extremities
IIB
1 yr
1
Multiple acne- and comedo-like lesions and infiltrating plaques and tumors on the head, neck, trunk, and extremities
IIB
3 mo
4
A B C A B A B A
2005 2007 2008 2003 2007 2005 2005 2009
104A, 165B 104A, 165B 104A, 165B 249A, 252A 249A, 252A 250A, 252A 207A, 230A 182B, 210A
280A 280A 280A No distinct tops 269B 271B 271B Not performed
5
B C D E A
2010 2010 2010 2011 2003
182B, 210A 159A, 182B, 210A 182B, 210A 182B, 210A 160A, 165A
262A 262A Not performed Not performed No distinct tops
7
B C A B A
2006 2006 2010 2011 2012
160A, 160A, 189A, 176B 221A,
8
B C A
2012 2012 2012
221A, 239A 221A, 239A 208A
266B, 277A 266B, 277A 252A, 261B
B
2012
208A
252A, 261B
1
2 3
6
165A 255A A 165 255A A A 206 , 218 250B, 252A 265B 239A 266B, 277A
Clinical Presentation
Stage at Diagnosis Follow-Up and Last Stage Time
Fragment (bp)
A, tube A primers (InVivoScribe Technologies); B: tube B primers (In VivoScribe Technologies). *A diagnosis of multiple clones was defined when more than 2 clonal TCR g or clonal TCR b gene rearrangements were observed in the same biopsy; when novel clonal TCR g or clonal TCR b PCR fragments were identified in different biopsies from the same patient or when one of the clonal TCR gene fragments was lost while other were retained.
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FIGURE 1. The left panel illustrates plaques and ulcerations seen in a patient with FMF. The right panel shows the typical histology of FMF with prominent infiltration of the hair follicle by malignant lymphoid cells (H&E–stained slide, magnification, ·40).
clinically diverse skin lesions, including infiltrated erythematous plaques, tumors, diffuse facial and eyebrow involvement, milia, acneiform or comedo-like lesions, and lichenoid dermatitis of the upper body (Table 1). Two patients had early stage disease with patch or plaque lesions (TNM stage IA–IIA), and 5 patients had an intermediate/advanced stage (TNM stage IIB–IVA) at the time of diagnosis. Detailed clinical data were not available for 1 patient. During follow-up, 3 patients showed progressive and 4 stable disease. Two patients progressed from stage IB to IIB disease, and 1 progressed from stage IIB to IVA disease. The remaining 4 patients had stable disease in stage IIB or IVA. Median follow-up time was 1 year (range, 3 months–12 years). One patient (patient 4) had advanced stage (IVA) disease at time of diagnosis and developed Hodgkin disease 3 years later. The patient died due to sepsis while under chemotherapy (Table 1, Fig. 1).
TCR Gene Rearrangement The results are summarized in Table 1. Five out of 8 patients (14 samples) showed multiple clonal TCR gene rearrangements, which is consistent with the presence of more than 1 clone (Table 1). In these patients, biopsies had been taken with intervals ranging from 3 months to 4 years after time of diagnosis. Loss (patients 3 and 6) and gain (patients 2, 3, 4, 5, and 6) of clonal TCR gene rearrangements was seen indicating loss and gain of a T-cell clone, respectively (see example in Fig. 2). Patients 1, 7, and 8 showed evidence of only 1 T-cell clone, using a strict interpretation of results.
DISCUSSION Gain or loss of clonal TCR gene rearrangements in addition to persistent rearrangements were seen in this series of FMF patients. The results indicate that some FMF patients may have multiple T-cell clones at diagnosis or in successive skin biopsies taken during the course of disease. It is striking that evidence for multiple clones were found in 5 of 8 patients with FMF indicating that the presence of multiple T-cell
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clones is common in FMF. This has not been demonstrated before in FMF. We did not find any striking correlation with disease stage or progression. However, the number of patients is too small for such an analysis. Investigation for multiple T-cell clones has also been performed in conventional MF.5–9 Multiple distinct T-cell clones were only found in 2 of the studies, with one reporting multiple clones in 23% of patients8 and the other in 48% of patients.9 The use of different PCR techniques may possibly account for this discrepancy. In our study, we used the BIOMED-2 primers and applied a strict interpretation of the results as previously published.15,16 For the interpretation of the number of clones, we used a strict definition, assuming that 1 clone shows at most 2 TCR g and 2 TCR b gene rearrangements in the case of biallelic arrangement of the 2 genes. However, this may not always have been the case, and the number of patients with multiple T-cell clones may therefore have been underestimated. Clonal heterogeneity could also be explained by the evolution of distinct clones from the dominant T-cell clone by subsequent rearrangements or deletions at the TCR locus.17,18 However, this hypothesis is considered less likely because FMF is a malignant lymphoma of mature T cells without on-going TCR rearrangements. The occurrence of multiple distinct T-cell clones in FMF is intriguing and needs further investigation. It has been previously suggested that pre-existing chronic T-cell stimulation due to chronic inflammatory skin disease, such as psoriasis or eczema, may lead to the development of 1 or more malignant clones in MF. Also, superantigens, such as bacterial proteins, or therapeutic interventions have been proposed to contribute to clonal evolution by TCR stimulation. Perhaps, this is also the case for FMF. The hair follicle is a major entry point for infectious agents and serves as a reservoir for many pathogens, including, for example, papillomaviruses. The microflora of the hair follicle, including Staphylococcus aureus, might also contribute to chronic stimulation of T cells.19 Multiple T-cell clones have been reported in both early and late stages of MF.4,5,8,9 This indicates that selection of 2014 Lippincott Williams & Wilkins
Am J Dermatopathol Volume 36, Number 12, December 2014
Multiple T-Cell Clones in FMF
FIGURE 2. Electropherograms of patient 4 demonstrating multiple T-cell clones during disease course. A, TCR gamma tube A (2009), peak shown at 210 bp; B, TCR gamma tube A (2010), peak shown at 159 and 210 bp; C, TCR gamma tube B (2009), peak shown at 182 bp; and D, TCR gamma tube B (2010) peak shown at 182 bp.
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a dominant clonal population may occur also in late stage of MF.8,9 It is possible that different treatment modalities could give rise to novel T-cell clones by allowing minor resistant T-cell clones to dominate the treatment-sensitive original clone.8 In FMF patients, multiple T-cell clones seem to arise independent of disease stage. Multiple T-cell clones may have prognostic importance in MF. Vega et al.8 have demonstrated that MF patients with a common clone identified in multiple concurrent skin samples were more likely to have a clinically progressive disease course versus those with more than 1 clone, but this finding has not yet been replicated in other studies. The number of patients in our study is too small to study the prognostic importance of multiple T-cell clones. However, we found no correlation between clonal heterogeneity and disease stage. In conclusion, multiple T-cell clones can be found in patients with FMF. This finding indicates that chronic T-cell stimulation is part of the pathogenesis of FMF. This is an interesting finding that needs further investigation. Whether multiple distinct T-cell clones are more frequent in FMF than in conventional MF and whether multiple clones have prognostic impact needs to be demonstrated in studies with more patients. REFERENCES 1. Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classification for cutaneous lymphomas. Blood. 2005;105:3768–3785. 2. Hayday AC, Saito H, Gillies SD, et al. Structure, organization, and somatic rearrangement of T cell gamma genes. Cell. 1985;40:259. 3. Dippel E, Assaf C, Hummel M, et al. Clonal T-cell receptor gammachain gene rearrangement by PCR based GeneScan analysis in advanced cutaneous T-cell lymphomas: a critical evolution. J Pathol. 1999;188: 146–154. 4. Klemke CD, Dippel E, Dembinski A, et al. Clonal T cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in the skin and blood of patients with parapsoriasis and early-stage mycosis fungoides. J Pathol. 2002;197:348–354. 5. Thurber SE, Zhang B, Kim YH, et al. T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides. J Am Acad Dermatol. 2007;57:782–790.
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6. Liebmann RD, Anderson B, McCarthy KP, et al. The polymerase chain reaction in the diagnosis of early mycosis fungoides. J Pathol. 1997;182: 282–287. 7. Delfau-Larue MH, Petrella T, Lahet C, et al. Value of clonality studies of cutaneous T lymphocytes in the diagnosis and follow-up of patients with mycosis fungoides. J Pathol. 1998;184:185–190. 8. Vega F, Luthra R, Medeiros LJ, et al. Clonal heterogeneity in mycosis fungoides and its relationship to clinical course. Blood. 2002;100: 3369–3373. 9. Ponti R, Fierro MT, Quaglino P, et al. TCRg-chain gene rearrangement by PCR-based GeneScan: diagnostic accuracy improvement and clonal heterogeinity analysis in multiple cutaneous T-cell lymphoma samples. J Invest Dermatol. 2008;128:1030–1038. 10. Cozzio A, French LE. T-cell clonality assays: how do they compare? J Invest Dermatol. 2008;128:771–773. 11. van Doorn R, Scheffer E, Willemze R. Follicular mycosis fungoides, a distinct disease entity with or without associated follicular mucinosis: a clinico-pathologic and follow-up study of 51 patients. Arch Dermatol. 2002;138:191–198. 12. Gerami P, Guitart J. The spectrum of histopathologic and immunohistochemical findings in folliculotropic mycosis fungoides. Am J Surg Pathol. 2007;31:1430–1438. 13. Mantaka P, Helsing P, Gjersvik P, et al. Clinical and histopathological features of folliculotropic mycosis fungoides: a Norwegian patient series. Acta Derm Venereol. 2012;93:325–329. 14. Vonderheid EC, Bernengo MG. The Sézary syndrome: hematologic criteria. Hematol Oncol Clin North Am. 2003;17:1367–1389. 15. van Dongen JJ, Langerak AW, Brüggemann M, et al. 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. 2003;17:2257–2317. 16. Langerak AW, Groenen PJ, Brüggemann M, et al. EuroClonality/ BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia. 2012;26: 2159–2171. 17. Rowen L, Koop BF, Hood L. The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. PCR-based clonality studies in lymphoproliferations. Science. 1996;272:1755–1762. 18. Rübben A, Kempf W, Kadin ME, et al. Multilineage progression of genetically unstable tumor subclones in cutaneous T-cell lymphoma. Exp Dermatol. 2004;13:472–483. 19. Bologna JL. Hair shaft—immunology. In: Bologna JL, Jorizzo JL, Rapini RP, eds. Dermatology. Philadelphia, PA: Lippincott Williams & Wikins; 2008:972–978.
2014 Lippincott Williams & Wilkins
Multiple Distinct T-Cell Clones in Folliculotropic Mycosis Fungoides Panagiota Mantaka, MD,*† Agnieszka Malecka, MSc,‡ Gunhild Trøen, PhD,‡ Per Helsing, MD,* Petter Gjersvik, MD, PhD,*† and Jan Delabie, MD, PhD†‡
Abstract: Multiple distinct T-cell clones have been demonstrated in a subset of mycosis fungoides (MF), but have so far not been documented in folliculotropic MF, a clinical and histological variant of MF. We analyzed T-cell receptor (TCR) gene rearrangements in 8 patients with folliculotropic MF with multiple biopsies (range, 2–5) taken during the course of disease. Two patients had disease stage IA–IIA, 5 stage IIB–IVA, whereas data were not available for 1 patient. TCR b and g gene rearrangements were analyzed according to the BIOMED-2 PCR protocol. Multiple clonal TCR gene rearrangements indicating more than 1 T-cell clone were found in 5 patients. Although the number of patients is small, the finding of multiple distinct T-cell clones in 5 out of 8 patients suggests that chronic T-cell stimulation contributes to the development of folliculotropic MF. Key Words: folliculotropic mycosis fungoides, T-cell receptor gene rearrangements, T-cell clones
been shown to correlate significantly with clinical progression by Vega et al6,8 but needs confirmation in larger patient populations. Folliculotropic mycosis fungoides (FMF) is a variant of MF with distinct clinical and histopathological features.1 FMF shows a more aggressive clinical course and is more difficult to treat compared with conventional MF as demonstrated in several case series.11–13 Folliculotropism of the neoplastic T cells is a histological hallmark of the disease but the homing of T cells to follicles is as yet unexplained. Whether specific homing receptors or specific antigen stimulation within the follicular epithelium plays a role is not known. The purpose of this study was to evaluate clonal T-cell proliferation by analyzing T-cell receptor (TCR) gene rearrangements in skin biopsies from different anatomic sites at time of diagnosis or during the course of the disease in patients with FMF. The clinical data of the patients were also reviewed.
(Am J Dermatopathol 2014;36:972–976)
MATERIALS AND METHODS
INTRODUCTION Primary cutaneous T-cell lymphomas (CTCL) are clonal lymphoproliferative disorders characterized by, most frequently CD4+, neoplastic T cells that home to the skin.1 T cells develop in the thymus where TCR genes are rearranged early in the T-cell maturation process to produce antigen-specific T cells.2 Consequently, T-cell malignancies derived from mature T cells such as cutaneous T-cell lymphoma show clonally rearranged TCR genes, the analysis of which is useful for diagnosis.3–5 Multiple T-cell clones showing different TCR gene rearrangements have previously been described in up to 30% of cases of mycosis fungoides (MF) and Sézary syndrome.6–10 Of interest, a high prevalence of oligoclonal TCR gene rearrangements is reported in early stage CTCL, which suggests that antigen stimulation might play a role in the early stages of the disease.4,8 The persistence of the same clone over time has From the *Department of Dermatology, Oslo University Hospital, Rikshospitalet, Oslo, Norway; †Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; and ‡Department of Pathology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway. The authors declare no conflicts of interest. Supported by the Norwegian Cancer Society. Reprints: Panagiota Mantaka, MD, Department of Dermatology, Oslo University Hospital, Rikshospitalet, Postboks 4950 Nydalen, Oslo N-0424, Norway (e-mail: [email protected]). © 2014 Lippincott Williams & Wilkins
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Patients Patients were identified in the cutaneous T-cell lymphoma database at Oslo University Hospital, comprising all patients diagnosed in the period 1997–2012 (n = 95). Twentytwo patients with FMF were registered in the database. Of these, 8 patients had undergone multiple skin biopsies at the time of diagnosis and during the course of disease. Second biopsies were taken from 3 months to 4 years after the first biopsy. All biopsies were histologically reviewed, and the diagnosis of FMF was confirmed. The diagnosis of FMF and the staging of the patients were according to the WHO/EORTC classification for CTCL,1 based on clinical examination, skin biopsy, blood cell counts, and serum chemistry. Computed tomography of thoracic and abdominal regions and in some patients also of cervical and pelvic regions was performed upon clinical indication. Flow cytometry with quantitation of lymphoma cells in the blood was performed in 5 patients according to EORTC recommendations for staging of MF/Sézary syndrome.14 Disease progression was defined by one of the following criteria: (1) progression from patch/plaque to tumor or erythroderma, (2) histologically confirmed nodal involvement in patients with disease that was previously limited to the skin, (3) the development of visceral involvement in patients with previously only skin or lymph node involvement, or (4) Am J Dermatopathol Volume 36, Number 12, December 2014
Am J Dermatopathol Volume 36, Number 12, December 2014
dissemination to the blood or death due to lymphoma. Clinical details of the patients have previously been published by our group.13 The study was approved by the Institutional Review Board of Oslo University Hospital and by the Regional Ethics Committee.
TCR Gene Rearrangement Studies TCR b and g gene rearrangements were studied in a total of 22 biopsies obtained from the 8 patients. DNA was isolated from paraffin-embedded or snap-frozen tissue, using the EZ1 tissue kit (Qiagen, Hilden, Germany), according to the instructions of the manufacturer. DNA quality was verified by PCR amplification of the albumin gene. TCR b and g gene rearrangements were detected by multiplex PCR according to the BIOMED-2 protocol.15 An in-house test was used for the latter, whereas a kit was used for the former (InVivoScribe Technologies, San Diego, CA). PCR products were size-fractionated on a 3130 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany) and analyzed using Gene Mapper version 4.1 software (Applied Biosystems).
Multiple T-Cell Clones in FMF
All analyses were performed in duplicate. The interpretation of the results was performed as recommended by Langerak et al.16 In short, a clonal TCR gene rearrangement was diagnosed when the height of one peak on the electropherogram was at least 3 times higher than that of other peaks resulting from the same competitive PCR product. Given the fact that both TCR gene alleles can be rearranged, the presence of multiple clones was defined when more than 2 clonal TCR g or more than 2 clonal TCR b gene rearrangements were observed in the same biopsy, when novel clonal TCR g or clonal TCR b PCR fragments were identified in different biopsies from the same patient or when one of the clonal TCR gene fragments was lost while other were retained.
RESULTS Clinical Findings Five patients were male, 3 were female. Median age was 54 years (range, 28–73 years). Patients presented with
TABLE 1. TCR Gene Rearrangements and Clinical Information in FMF Skin TCR g Gene TCR b Gene No. Patient Biopsies Time
Fragment (bp)
Clones* 1
Plaques and infiltrating tumors
IIB–IVA
3 mo
2
Erythematous and ulcerating plaques
IB–IIB
1 yr
3
Multiple erythematous plaques
NA
NA
2
Diffuse facial erythema, leonine facies, lichenoid dermatitis of upper body, ulcerating tumors
IVA
4 yrs
2
Erythematous plaques over eyebrows, arms, knees, milia-like lesions in the periorbital region, and infiltrating plaque/tumor on the extremities
IB–IIB
12 yrs
3
Infiltrating plaques and tumors on the face
IIB
2 yrs
1
Multiple erythematous plaques and tumors; infiltrating tumors on the trunk and extremities
IIB
1 yr
1
Multiple acne- and comedo-like lesions and infiltrating plaques and tumors on the head, neck, trunk, and extremities
IIB
3 mo
4
A B C A B A B A
2005 2007 2008 2003 2007 2005 2005 2009
104A, 165B 104A, 165B 104A, 165B 249A, 252A 249A, 252A 250A, 252A 207A, 230A 182B, 210A
280A 280A 280A No distinct tops 269B 271B 271B Not performed
5
B C D E A
2010 2010 2010 2011 2003
182B, 210A 159A, 182B, 210A 182B, 210A 182B, 210A 160A, 165A
262A 262A Not performed Not performed No distinct tops
7
B C A B A
2006 2006 2010 2011 2012
160A, 160A, 189A, 176B 221A,
8
B C A
2012 2012 2012
221A, 239A 221A, 239A 208A
266B, 277A 266B, 277A 252A, 261B
B
2012
208A
252A, 261B
1
2 3
6
165A 255A A 165 255A A A 206 , 218 250B, 252A 265B 239A 266B, 277A
Clinical Presentation
Stage at Diagnosis Follow-Up and Last Stage Time
Fragment (bp)
A, tube A primers (InVivoScribe Technologies); B: tube B primers (In VivoScribe Technologies). *A diagnosis of multiple clones was defined when more than 2 clonal TCR g or clonal TCR b gene rearrangements were observed in the same biopsy; when novel clonal TCR g or clonal TCR b PCR fragments were identified in different biopsies from the same patient or when one of the clonal TCR gene fragments was lost while other were retained.
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FIGURE 1. The left panel illustrates plaques and ulcerations seen in a patient with FMF. The right panel shows the typical histology of FMF with prominent infiltration of the hair follicle by malignant lymphoid cells (H&E–stained slide, magnification, ·40).
clinically diverse skin lesions, including infiltrated erythematous plaques, tumors, diffuse facial and eyebrow involvement, milia, acneiform or comedo-like lesions, and lichenoid dermatitis of the upper body (Table 1). Two patients had early stage disease with patch or plaque lesions (TNM stage IA–IIA), and 5 patients had an intermediate/advanced stage (TNM stage IIB–IVA) at the time of diagnosis. Detailed clinical data were not available for 1 patient. During follow-up, 3 patients showed progressive and 4 stable disease. Two patients progressed from stage IB to IIB disease, and 1 progressed from stage IIB to IVA disease. The remaining 4 patients had stable disease in stage IIB or IVA. Median follow-up time was 1 year (range, 3 months–12 years). One patient (patient 4) had advanced stage (IVA) disease at time of diagnosis and developed Hodgkin disease 3 years later. The patient died due to sepsis while under chemotherapy (Table 1, Fig. 1).
TCR Gene Rearrangement The results are summarized in Table 1. Five out of 8 patients (14 samples) showed multiple clonal TCR gene rearrangements, which is consistent with the presence of more than 1 clone (Table 1). In these patients, biopsies had been taken with intervals ranging from 3 months to 4 years after time of diagnosis. Loss (patients 3 and 6) and gain (patients 2, 3, 4, 5, and 6) of clonal TCR gene rearrangements was seen indicating loss and gain of a T-cell clone, respectively (see example in Fig. 2). Patients 1, 7, and 8 showed evidence of only 1 T-cell clone, using a strict interpretation of results.
DISCUSSION Gain or loss of clonal TCR gene rearrangements in addition to persistent rearrangements were seen in this series of FMF patients. The results indicate that some FMF patients may have multiple T-cell clones at diagnosis or in successive skin biopsies taken during the course of disease. It is striking that evidence for multiple clones were found in 5 of 8 patients with FMF indicating that the presence of multiple T-cell
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clones is common in FMF. This has not been demonstrated before in FMF. We did not find any striking correlation with disease stage or progression. However, the number of patients is too small for such an analysis. Investigation for multiple T-cell clones has also been performed in conventional MF.5–9 Multiple distinct T-cell clones were only found in 2 of the studies, with one reporting multiple clones in 23% of patients8 and the other in 48% of patients.9 The use of different PCR techniques may possibly account for this discrepancy. In our study, we used the BIOMED-2 primers and applied a strict interpretation of the results as previously published.15,16 For the interpretation of the number of clones, we used a strict definition, assuming that 1 clone shows at most 2 TCR g and 2 TCR b gene rearrangements in the case of biallelic arrangement of the 2 genes. However, this may not always have been the case, and the number of patients with multiple T-cell clones may therefore have been underestimated. Clonal heterogeneity could also be explained by the evolution of distinct clones from the dominant T-cell clone by subsequent rearrangements or deletions at the TCR locus.17,18 However, this hypothesis is considered less likely because FMF is a malignant lymphoma of mature T cells without on-going TCR rearrangements. The occurrence of multiple distinct T-cell clones in FMF is intriguing and needs further investigation. It has been previously suggested that pre-existing chronic T-cell stimulation due to chronic inflammatory skin disease, such as psoriasis or eczema, may lead to the development of 1 or more malignant clones in MF. Also, superantigens, such as bacterial proteins, or therapeutic interventions have been proposed to contribute to clonal evolution by TCR stimulation. Perhaps, this is also the case for FMF. The hair follicle is a major entry point for infectious agents and serves as a reservoir for many pathogens, including, for example, papillomaviruses. The microflora of the hair follicle, including Staphylococcus aureus, might also contribute to chronic stimulation of T cells.19 Multiple T-cell clones have been reported in both early and late stages of MF.4,5,8,9 This indicates that selection of 2014 Lippincott Williams & Wilkins
Am J Dermatopathol Volume 36, Number 12, December 2014
Multiple T-Cell Clones in FMF
FIGURE 2. Electropherograms of patient 4 demonstrating multiple T-cell clones during disease course. A, TCR gamma tube A (2009), peak shown at 210 bp; B, TCR gamma tube A (2010), peak shown at 159 and 210 bp; C, TCR gamma tube B (2009), peak shown at 182 bp; and D, TCR gamma tube B (2010) peak shown at 182 bp.
2014 Lippincott Williams & Wilkins
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Am J Dermatopathol Volume 36, Number 12, December 2014
Mantaka et al
a dominant clonal population may occur also in late stage of MF.8,9 It is possible that different treatment modalities could give rise to novel T-cell clones by allowing minor resistant T-cell clones to dominate the treatment-sensitive original clone.8 In FMF patients, multiple T-cell clones seem to arise independent of disease stage. Multiple T-cell clones may have prognostic importance in MF. Vega et al.8 have demonstrated that MF patients with a common clone identified in multiple concurrent skin samples were more likely to have a clinically progressive disease course versus those with more than 1 clone, but this finding has not yet been replicated in other studies. The number of patients in our study is too small to study the prognostic importance of multiple T-cell clones. However, we found no correlation between clonal heterogeneity and disease stage. In conclusion, multiple T-cell clones can be found in patients with FMF. This finding indicates that chronic T-cell stimulation is part of the pathogenesis of FMF. This is an interesting finding that needs further investigation. Whether multiple distinct T-cell clones are more frequent in FMF than in conventional MF and whether multiple clones have prognostic impact needs to be demonstrated in studies with more patients. REFERENCES 1. Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classification for cutaneous lymphomas. Blood. 2005;105:3768–3785. 2. Hayday AC, Saito H, Gillies SD, et al. Structure, organization, and somatic rearrangement of T cell gamma genes. Cell. 1985;40:259. 3. Dippel E, Assaf C, Hummel M, et al. Clonal T-cell receptor gammachain gene rearrangement by PCR based GeneScan analysis in advanced cutaneous T-cell lymphomas: a critical evolution. J Pathol. 1999;188: 146–154. 4. Klemke CD, Dippel E, Dembinski A, et al. Clonal T cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in the skin and blood of patients with parapsoriasis and early-stage mycosis fungoides. J Pathol. 2002;197:348–354. 5. Thurber SE, Zhang B, Kim YH, et al. T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides. J Am Acad Dermatol. 2007;57:782–790.
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