Minimal residual disease in hypopigmented mycosis fungoides Pa-Fan Hsiao, MD, MMS,a,b Cheng-Hsiang Hsiao, MD,c Tsen-Fang Tsai, MD,d and Shiou-Hwa Jee, MD, PhDd Taipei, Taiwan We describe the case of a 13-year-old boy with stage I hypopigmented mycosis fungoides in whom minimal residual disease was detected with T-cell receptor g-polymerase chain reaction after the disease was in complete clinical remission. We further cloned and sequenced the T-cell receptor g-polymerase chain reaction product of the lesion in remission and found that the original T-cell clone still existed in decreased amounts. The patient was followed up for 3 1/2 years without any new lesions developing. The clinical significance of this residual malignant T-cell clone in mycosis fungoides remains to be elucidated. ( J Am Acad Dermatol 2006;54:S198-201.)
ypopigmented mycosis fungoides (MF) is an uncommon variant of MF, usually observed in dark-skinned individuals, especially in young patients.1-3 Although most of the reported cases have stage I disease, recurrences are common.1-4 Histopathologic examination is still the gold standard for diagnosis, but there are difficulties in interpreting post-treatment histology,5,6 and discrepancies frequently are found between clinical response and histologic response after treatment.5,7 Detecting monoclonal T cells by means of T-cell receptor gene rearrangement studies is helpful in establishing the diagnosis of MF, but residual T-cell clones can be detected even in lesions not diagnostic for MF after treatment. The persistence of a cutaneous T-cell clone in treated skin lesions of MF with complete clinical remission was defined as minimal residual disease (MRD) by Poszepczynska-Guigne.5 The precise location of these residual clonal T cells after treatment was unclear, and the prognostic value of MRD in MF remains to be clarified.
Supported by Stiefel Laboratories. From the Department of Dermatology, Mackay Memorial Hospitala; the Mackay Medicine, Nursing, and Management Collegeb; the Departments of Pathologyc and Dermatology,d National Taiwan University College of Medicine and National Taiwan University Hospital. Funding sources: None. Conflicts of interest: None identified. Reprint requests: Tsen-Fang Tsai, MD, Department of Dermatology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. E-mail: [email protected]
. 0190-9622/$32.00 ª 2006 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2005.08.044
Abbreviations used: PCR: MF: MRD: TCR:
polymerase chain reaction mycosis fungoides minimal residual disease T-cell receptor
We describe the case of a pediatric patient with hypopigmented MF, in whom MRD was detected in the dermis of the lesion after a complete clinical response to topical treatment with BCNU (carmustine).
CASE REPORT A 13-year-old boy was seen for an 8 cm 3 6 cm hypopigmented patch with focal central hyperpigmentation on his right forearm, and several hypopigmented macules on his left hand, thigh, knee, and popliteal fossa, which were present for more than 1 year. He had no previous significant medical history. Biopsy specimens were taken from his right forearm, left hand, and left popliteal fossa; histopathology of the 3 specimens showed focal lymphocytic infiltration along the basal epidermis accompanied by vacuolization (Fig 1, A). There also was dense perivascular lymphocytic infiltration in the dermis. Most lymphoid cells were CD3/CD8 positive cells admixed with some CD4-positive cells. Polymerase chain reaction (PCR) to detect T-cell receptor (TCR) g gene rearrangement showed a clonal T-cell population within the 3 specimens (Fig 2). Initial laboratory data, including complete blood cell count, and liver and renal function tests were within normal ranges. The patient had no
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Fig 2. Detection of TCR gene rearrangement by PCR amplification and 8% polyacrylamide gel electrophoresis. Lane P: positive control (T-cell lymphoma). Lane MW: molecular weight markers (Bioman). Lanes 1-3: skin biopsy specimens before treatment. Lane 4: a skin biopsy specimen in clinical remission. Lane N: negative control (reactive lymph node).
Fig 1. A, Single basal lymphocytes in linear arrangement along basal epidermis. B, Vacuolization of basal keratinocytes, and melanin incontinence. (Hematoxylin-eosin stain; original magnification: 3200.)
lymphadenopathy and no abnormality was found on physical examination. Chest and abdominal computed tomographic scans were normal. A bone marrow biopsy did not detect any malignant cells, and a whole body bone scan also was negative. After a complete staging evaluation, stage Ia hypopigmented MF was diagnosed. The patient was started on topical BCNU (carmustine) treatment (100 mg/100 mL); at follow-up, 6 months later, the lesions showed repigmentation and topical BCNU treatment was stopped. During follow-up, 1 month after treatment discontinuation, a biopsy was performed again from the right forearm adjacent to the site of initial biopsy. Histopathologic examination showed mild acanthosis and dyskeratosis in the epidermis. Vacuolization of basal keratinocytes and melanin incontinence were noted (Fig 1, B). No epidermotropism or single basal lymphocytes were found. The papillary dermis revealed mild interstitial lymphocytic infiltration. According to the clinical and histologic findings, complete clinical remission was impressed. Complete clinical remission was defined as complete disappearance of clinical lesions for at least 1 month.1 The patient subsequently was followed up for 3 1/2 years up to the present day; no new lesions have appeared.
Fig 3. DNA sequences of PCR products. 1-12: twelve clones selected from PCR product of skin biopsy specimen in clinical remission. K: the original clone from a skin biopsy specimen before treatment.
Gene analyses of the skin biopsy specimens were performed retrospectively by using paraffin-embedded sections. A clonal T-cell population was detected, with the use of TCRg- PCR, in the biopsy specimens before and after treatment (Fig 2). We also manually dissected the epidermis from the dermis, and the TCRg- PCR result was negative in the epidermal portion. To confirm whether the clonal T cells detected in skin sections before and after treatment were of the same malignant clone, we
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Fig 4. DNA sequences of PCR products. Clones 1, 2, 5, 9 and 12 have the same sequences as the original clone from a skin biopsy specimen before treatment.
further cloned the PCR products with pGEM-T Easy vector (Promega, Madison, WI, USA). The PCR products were ligated first with pGEM-T Easy vector and then transformed into colonies of Escherichia coli via heat shock. After incubation at 378C overnight, 12 colonies with color changes from blue to white (indicating successful transformation) were selected randomly. The plasmid DNA of E coli was extracted and digested with EcoRI to get the inserted PCR product after agarose gel electrophoresis. DNA sequences were identified with the use of an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Fremont, CA, USA) (Fig 3). Comparison of the DNA sequences of these 12 clones with the original T-cell clone detected in the first biopsy revealed that 5 of the 12 clones were the same as the original clone (Fig 4). We concluded that the original malignant T-cell clone persisted in the skin lesion after complete clinical remission, but was reduced in amount (5/12).
DISCUSSION Despite the increasing application of T-cell receptor gene rearrangement studies in MF, the existence of MRD in MF rarely has been addressed. MRD was detected in about 30% (4/13) to 38% (5/13) of MF patients in complete clinical remission.5,7 In Dereure’s study,7 all stage Ia patients displayed no dominant gene rearrangement after the treatment was completed, while a significant number of stage Ib patients had MRD in skin and/or blood. Our patient is an exception of MRD in stage Ia MF. To avoid detecting an insignificant rare amount of tumor cells (which occurs on rare occasions using more sensitive and expensive techniques), we used PCRpolyacrylamide gel electrophoresis, which is suitable for routine examination.8 There also are pitfalls in interpreting PCR results, but further cloning and sequencing can exclude possible false positivity. Furthermore, PCR results alone fail to quantify the
amount of residual T-cell clone. In our patient, we identified more clearly that the MRD detected by TCRg-PCR represented the persistence of the original clone in a reduced amount, rather than the emergence of a new clone. In previous studies, by separating epidermis from dermis in lesions of MF, different groups found that the clonal T cells resided mainly in the epidermis.9,10 Other studies using microdissection on patients with hypopigmented MF also confirmed this finding.3,4 No previous study focused on the location of the original clonal T cells in the MRD of MF. By manually separating epidermis from dermis in the treated lesion of this patient, we could not demonstrate the existence of the original T-cell clone in the epidermis. Since this patient received topical BCNU treatment, it was inferred that the clonal T cells located in the epidermis were eradicated more easily than those in the dermis. It was unclear whether MRD in MF has prognostic significance, which heralds higher relapse rates or has no relevance. Our patient remained disease-free for 3 1/2 years without further treatment. We inferred that the existence of the malignant T-cell clone might not be the only factor influencing the outcome of the disease. The immune status of the patient possibly played a role in controlling the outgrowth of malignant T-cell clones; a balanced immune status may thus have been achieved. In our patient, the residual clonal T cells confined to the dermis may be under the surveillance of the host immune system, rendering them nonepidermotropic. Considering the relative benign course of our patient, we do not suggest more aggressive therapy to obtain a negative PCR in skin lesion, as it might do harm to the immunity of the patient. At present, MRD detected by molecular methods should not replace pathologic examination in deciding on further treatment. Different study groups have suggested that the long-term outcome of MRD in MF requires a large-scale prospective study.5,7 Despite having MRD detected by TCRg-PCR, our patient had a good response to topical treatment alone. Molecular studies can aid in understanding MRD in an individual patient, but its clinical significance is still unclear. Long-term follow-up is mandatory in the future.
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