Clonal TCR Gamma Gene Rearrangement Research Articles

Focal Myositis with CD8+T-Cell predominance: an Inflammatory Myositis Mimicking a Soft Tissue Neoplasm

Abstract Introduction/Objective FM is a rare self-limiting T-cell rich lesion arising within muscles of young adults as a solitary lesion and can be confused with a variety of neoplastic/inflammatory conditions or lymphoma. Here we describe a rare T-cell rich variant of FM with CD8 predominance. Methods/Case Report A 51-year-old female presented with two-month history of left trapezius swelling. MRI showed an enhancing tumor within the muscle, suspicious of sarcoma, less likely myeloma. Results (if a Case Study enter NA) Biopsy showed diffuse infiltration of small lymphocytes in a fibrotic background and atrophic skeletal muscle. These lymphocytes stained positive for CD2, CD3, CD7, CD8, CD5 (partial), TIA1 (partial), CD43, CD57 and negative for CD4, CD30, CD56, CD57, granzyme B, perforin, BCL-6, BCL-2, Pax5, TCL1, and CD56. Ki-67 was (<10%) with few background CD20+ B cells. By flow cytometry, the CD8+ T-cells co-express CD2, CD3, CD7, with dim CD5 expression. TCR gene rearrangement study by PCR showed no clonal TCR Gamma gene rearrangement. Conclusion FM is extremely rare with only 22 cases well-described in the literature, all predominantly composed of CD4+ T-cells, clinically concerning for low-grade sarcomas, as inflammatory myofibroblastic tumor, inflammatory leiomyosarcoma, or liposarcoma. Careful analysis is essential for correct diagnosis. Mimics include other causes of myositis, such as polymyositis and inclusion body myositis. Depending on the extent of lymphocytic infiltrate, gene rearrangement studies might be necessary to rule out clonality. Recognition of this rare entity with excellent prognosis is crucial to provide appropriate management and avoid unnecessary, aggressive procedures.

Composite angioimmunoblastic T-cell and diffuse large B-cell lymphoma

A58-year-old female smoker with a history of hypertension presented to the emergency department with a recurrent episode of worsening shortness of breath over five days. Her exam was significant for sinus tachycardia, cervical lymphadenopathy, and a firm left supraclavicular node. Laboratory values demonstrated a normal white cell count and differential, normocytic anemia, thrombocytopenia, and a lactate dehydrogenase of 803 IU/L. She was HIV negative. Computerized tomography (CT) scan of the chest/abdomen/pelvis demonstrated extensive bilateral cervical, supraclavicular and axillary adenopathy, mediastinal and hilar lymphadenopathy with infiltrate and consolidation of the right lower and posterior upper lobe and encasement of the right pulmonary artery, as well as splenomegaly with paraaortic and retroperitoneal adenopathy. An echocardiogram revealed a normal left ventricle and a severe pericardial effusion. She underwent a pericardial window and an excisional supraclavicular lymph node biopsy, which revealed T-cells with features of angioimmunoblastic T-cell lymphoma (AITL) and large atypical B-cells consistent with diffuse large Bcell lymphoma (DLBCL) (Figure 1A). Destruction of the lymph node architecture was noted, with many medium-sized atypical lymphocytes with irregular nuclei and moderate amounts of pale cytoplasm. In addition, scattered and focally increased large atypical lymphocytes with prominent nucleoli were seen. Blood vessels were increased. Immunostains demonstrated atypical T-cells expressing CD3 (Figure 1B), CD4 (Figure 1C), CD5, CD2, CD7, CD4, CD10, PD-1 (Figure 1D), as well as clustered large B-cells positive for expression of CD20, Bcl-6 and BOB-1 (Figure 1E). Polymerase chain reaction (PCR) testing revealed a clonal TCR gamma gene rearrangement, as well as a clonal IgH gene rearrangement. Epstein-Barr virus-encoded RNA (EBER) was negative by in situ hybridization (ISH). Bone marrow biopsy and CSF analysis were negative for lymphoid or plasma cell infiltrates. The patient was initiated on R-CHOEP (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone) and completed three cycles of chemotherapy. Her CT scan after three cycles was consistent with a partial response, with significantly diminished bilateral axillary lymphadenopathy, improved mediastinal, perihilar and right lower lobe infiltrates, decreased splenomegaly, and upper abdominal and retroperitoneal adenopathy. The patient eventually had progression of her disease during the course of subsequent R-CHOEP cycles. She then progressed through two cycles of gemcitabine-carboplatin and one course of romidepsin. Her performance status deteriorated, not enabling any further treatment and the patient finally expired, approximately 24 months after initial diagnosis. This case illustrates a rare hematologic malignancy presenting with a dual primary lymphoma population. The treatment of composite lymphoma with simultaneous Band T-cell components is challenging and the less favorable prognosis component determines

Genomic Characterization Of Histiocytic Lesions Following Pediatric T-Cell Acute Lymphoblastic Leukemia

IntroductionThe development of histiocytic lesions after acute lymphoblastic leukemia (ALL) is rare, occurring in 6 of 971 patients enrolled on BFM treatment regimens for T-ALL (Trebo 2005). Few cases of histiocytosis arising after a history of T-ALL have been characterized at the molecular level for genomic alterations. One case of fatal Langerhans cell histiocytosis (LCH) following treatment of T-ALL revealed activating mutations in NOTCH1; in contrast, no NOTCH1 mutations were identified in 24 other cases of LCH or Rosai-Dorfman disease without a previous history of T-ALL (Rodig 2008). In patients without prior leukemia, BRAF V600 mutations have been identified in a significant proportion of patients with LCH or Erdheim-Chester Disease but not in other histiocytoses (Haroche 2012). Methodology and Principal FindingsNext generation focused exomic sequencing of 236 genes and 47 Introns was conducted on samples of histiocytic lesions from two patients with a previous history of T-ALL. Case 1A 2 year-old male presented with marked adenopathy, mediastinal mass and white blood cell count 67,000 cells/uL with 30% blasts. The blasts expressed CD45, CD2, CD3 (surface/cyto), CD5, CD7, CD38, CD45 (bright), TCR gamma-delta, but were negative for CD4, CD8, and TdT. Karyotype was 46,XY,t(8;14)(q24;q11.2),der(12)t(12;20)(q11;q13.3),der(20)t(12;20)(q21;q13.3) with rearrangement of MYC (8q24) confirmed by FISH. He was treated for T-ALL per Children's Oncology Group (COG) protocol AALL0434 and had 5% residual bone marrow blasts at day 29 of induction. MRD-negative remission was ultimately achieved with high-dose cytarabine and methotrexate followed by consolidation with nelarabine. He underwent matched unrelated cord blood transplant following conditioning with cyclophosphamide and total body irradiation with cranial boost, and engrafted at day +14. Surveillance bone marrow at day +110 revealed systemic juvenile xanthogranuloma (JXG) without T-ALL. PET/CT revealed FDG-uptake in the diffusely enlarged spleen and throughout the skeleton. Due to progressive cytopenias, therapy was initiated with vinblastine and prednisone as per LCHIII. However, refractory cytopenias exacerbated by splenic sequestration developed following induction, and he was then treated with thalidomide and splenectomy. The spleen weighed 404 grams (expected 40 grams) and was diffusely infiltrated with JXG. The cytopenias dramatically improved and he continued thalidomide for 2 months until PET scan demonstrated progression. Genomic analysis of the JXG lesion revealed NRAS G13D mutation, and FISH demonstrated MYC rearrangement identical to the initial T-ALL sample. Case 2A 12-year-old male presented with WBC 142,700 cells/uL and CNS leukemia. Flow cytometry showed T-ALL with CD2, surface CD3, CD4, CD5, CD7, CD8, CD24 (subset), CD71, HLA-DR (subset) and TdT (partial). There was a clonal TCR gamma gene rearrangement and a biallelic CDKN2A (p16) deletion by FISH. He was enrolled on COG AALL0434 and had a rapid response with remission in both CNS and marrow at induction day 29. Following completion of high-dose methotrexate interim maintenance he developed hepatosplenomegaly, pancytopenia and elevated serum bilirubin, ferritin, and triglycerides. Bone marrow aspirate showed rare hemophagocytosis but no evidence of T-ALL. He was treated with dexamethasone and etoposide with no response. Follow-up bone marrow revealed brisk hemophagocytosis and a diffuse histiocytic neoplasm. Karyotype was 48,XY,+7,+11[2] /49,idem,+18[3] /46,XY[14]. PET/CT showed hepatosplenomegaly with FDG uptake in anterior mediastinum, hepatic nodules, spleen, and bone marrow. He was treated with Campath and then with intensive chemotherapy with fludarabine, cytarabine, and liposomal daunorubicin with no response and ultimately succumbed to disease. Genomic analysis of the clonal histiocytic infiltrate revealed KRAS G12C, BRAF G469V, NOTCH1 Q2440, and CCND2 G268R mutations, and FISH positive for biallelic CDKN2A (p16) deletion similar to original T-ALL. ConclusionsOur extensive genomic characterization suggests a unique molecular pathogenesis for histiocytic disorders arising after T-cell ALL and identified RAS signaling pathway and NOTCH1 mutations. Furthermore, these findings strongly indicate a potential derivation or trans differentiation from the malignant leukemic stem cell clone. Disclosures:No relevant conflicts of interest to declare.

Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selection of T lymphocytes

Background:The aim of this study was to investigate the prognostic effect of tumour-infiltrating lymphocytes (TILs) in serous stage III ovarian carcinoma to determine TIL clonality and to correlate this to Her2/neu expression.Methods:Formalin-fixed and paraffin-embedded ovarian carcinomas were examined for CD20-, CD3-, CD4- and CD8-positive lymphocytes (n=100), and for Her2/neu-positive tumour cells (n=55/100) by immunohistochemistry. Clonality analysis was carried out by T-cell receptor γ (TCRγ) gene rearrangements (n=93/100). Statistical analyses included experimental and clinico-pathological variables, as well as disease-free (DFS) and overall (OS) survival.Results:CD20-positive B lymphocytes were present in 57.7% (stromal)/33.0% (intraepithelial) and CD3-positive T lymphocytes in 99.0% (stromal)/90.2% (intraepithelial) of ovarian carcinomas. Intraepithelial CD3-positive T lymphocytes were correlated with improved DFS in optimally debulked patients (P=0.0402). Intraepithelial CD8-positive T lymphocytes were correlated with improved OS in all optimally debulked patients (P=0.0201) and in those undergoing paclitaxel/carboplatin therapy (P=0.0092). Finally, rarified and clonal TCRγ gene rearrangements were detected in 37 out of 93 (39.8%) and 15 out of 93 (16.1%) cases, respectively. This was marginally associated with improved DFS (P=0.0873). Despite a significant correlation of HER2/neu status and intraepithelial CD8-positive lymphocytes (P=0.0264), this was non-directional (R=−0.257; P=0.0626).Conclusion:Improved survival of ovarian cancer patients is related to the infiltration, clonal selection and intraepithelial persistence of T lymphocytes.

Small cell variant of peripheral T-cell lymphoma, not otherwise specified: a clinicopathologic and immunophenotypic analysis

To study the clinicopathologic features and differential diagnosis of small cell variant of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). The clinicopathologic features of 5 cases of small cell variant of PTCL, NOS were retrospectively reviewed, with immunohistochemical study, T-cell receptor (TCR) gene rearrangement analysis and evaluation for Epstein-Barr virus (EBV) status. All the 5 patients were males. The mean age was 52.6 years. The median duration before diagnosis was 1 month. Clinically, 3 patients presented in stage IV and 2 in stage III. Four of them had generalized lymphadenopathy and splenomegaly. Hepatomegaly and massive effusion were found in 1 and 2 cases, respectively. Marrow involvement was detected in 3 of the 4 patients with bone marrow biopsy performed and one of them also accompanied by lymphocytosis. Histologically, the involved lymph nodes showed partial or complete effacement of nodal architecture and replacement by a monomorphous population of small lymphoid cells. Scanty large lymphoid cells were also identified in 4 cases. Increase in number of blood vessels was noticed in two of them as well. Immunohistochemically, the lymphoma cells in all cases expressed two or more of the T-cell markers and CD43. The staining for CD20, TdT, CD56 and granzyme B was negative. CD99 expression was noted in 3 of the 4 cases. The Ki-67 index ranged from 5% to 15%. Clonal TCRgamma gene rearrangement was detected in the 4 cases studied and one of them also showed TCRbeta gene rearrangement. In-situ hybridization for EBV-encoded RNA was negative in the 4 cases studied. Follow up information was available in 3 of the 5 cases. All of the 3 patients died of the disease, with an average survival of 21.7 months. Small cell variant of PTCL, NOS represents a rare disease entity which often presents in advanced tumor stage and carries a poor prognosis.

T-cell lymphoproliferative disorder of hand-mirror cell morphology presenting in an eosinophilic loculated peritoneal effusion, with omental "caking"

BackgroundCells with "hand mirror" morphology have not, to the best of our knowledge, been described in a primary effusion sample. This paper describes a case of T-cell lymphoma with eosinophilia in a patient with suspected peritoneal carcinomatosis. Rarely, a T-cell lymphoproliferative process may mimic primary peritoneal carcinomatosis, clinically suggested by a presentation in CT imaging of omental caking with bilateral massive loculated effusions in a patient without lymphadenopathy or splenomegaly.MethodsA 60 year old caucasian male presented with vague abdominal discomfort and increasing abdominal girth. Computed tomography showed a two centimeter thick omental cake and a small loculated effusion. The clinical presentation and imaging findings were most consistent with peritoneal carcinomatosis. Cytologic evaluation of the effusion was undertaken for diagnostic study.ResultsRapid intraprocedural interpretation of the effusion sample showed a monomorphic population of cells with "hand-mirror" cell morphology exhibiting cytoplasmic extensions (uropodia) with 3–5 course dark cytoplasmic granules and a rim of vacuolated cytoplasm capping the opposing "mirror head" side. These cells were seen within a background of mature eosinophils. Flow cytometric evaluation of the ascites fluid demonstrated an atypical T-cell population with the following immunophenotype: CD2-, CD3+, CD4-, CD5-, CD7-, CD8+, CD56+. T-cell receptor (TCR) gene rearrangement was positive for clonal TCR-gamma gene rearrangement, supporting the diagnosis of a T-lymphoprolifereative disorder.ConclusionA T-cell lymphoproliferative process may present with "hand mirror" morphology in an effusion sample. These cells may show polar cytoplasmic vacuolization and 3–5 course granules within the "handle" of these unique cells. Cytoplasm shows peripheral constriction around the nucleus.

Biphenotypic Gamma-Delta T-Cell Leukemia/Lymphoma with Coexpression of Pax-5 and Other B-Cell Specific Antigens.

Lineage commitment of B- and T-lymphocytes occurs in early stages of normal differentiation. Pax-5 promotes commitment of B-cell and blocks early development of T-cell. Under neoplastic conditions, aberrant expression of less specific T-cell antigens (CD2, CD5 and CD8) has been detected in certain B-cell lymphomas, and B-cell antigens CD20 and CD79a have been seen in rare cases of T-cell lymphomas. However, coexpression of true lineage specific B-cell antigens Pax-5 and CD19 in T-cells with surface CD3 and T-cell receptors has not been reported in either normal or neoplastic T-cells. We report 4 cases of aggressive T-cell lymphoma/leukemia with a consistent but unusual immunophenotype, which was determined by immunohistochemistry (IHC) in all four cases with paraffin embedded tissue and by flow cytometry (FCM) in 2/4 cases. Molecular and cytogenetic analyses were also attempted in two patients and clinical information will be provided. All patients were male aged 21-79 years. Three patients, including 2 cases of T-ALL, presented with generalized lymphadenopathy and bone marrow involvement (clinical stage IV); 1/3 also had splenic involvement. A distinct mediastinal mass was not identified in any of the patients. Two patients undergoing therapy received the hyper-CVAD regimen and had a poor response to early treatment. One of the two was subsequently treated with Campath without success. The 4th patient presented with an isolated chest wall mass. Detailed clinical information is currently unavailable for 2 patients. In all cases, the neoplastic cells were characterized by blastic morphology and coexpression of bilineage lymphoid antigens. The neoplastic cells in 3 cases with systemic disease were immunoreactive with T-cell antigens CD3, CD5, CD7, CD43, and B-cell antigens CD79a and Pax-5; 2/3 cases that had FCM data both showed expression of CD19 and surface γ/δ-TCR, as well as myeloid antigens CD11c and CD33. The case without FCM data lacked βF1 (α/β-TCR) expression by IHC. In addition, 2/3 cases also had immunophenotypic features of typical T-lymphoblastic leukemia or lymphoma (T-ALL) (CD34+ and TdT+). All three cases lacked NK cell antigen CD56 and T-cell antigen CD2, and were double negative for CD4 and CD8. The tumor cells from the 4th case were positive for all pan-T cell antigens (CD2, CD3, CD7, CD43) except CD5 by IHC, and were double negative for CD4 and CD8. These features were phenotypically consistent with a γ/δ-T cell lymphoma, which was also supported by lack of detectable βF1 expression. Similar to other cases, however, these cells were also positive for B cell antigens Pax-5 and CD79a, as well as CD20. The only patient who had successful molecular and cytogenetic analyses had isochromosome 9 and clonal TCR gamma gene rearrangement. In summary, T-cell lymphoma or leukemia with bi-lineage lymphoid antigens is associated with Pax-5 expression, which may account for the upregulation of CD19. It appears to be a rare disease of γ/δ-T-cell origin with a unique phenotypic profile. Further study is needed to determine if the observed i(9) involves disruption of the Pax-5 locus at 9q13.

Detection of TCR-gamma gene rearrangements in early mycosis fungoides by non-radioactive PCR-SSCP.

Early mycosis fungoides (MF) can mimic numerous benign inflammatory dermatoses on routine histological examination. In this study, a recently developed non-radioactive polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique was used to assess T-cell clonality in paraffin-embedded skin biopsies clinically and pathologically suspicious for early MF. Non-radioactive PCR-SSCP is a simple, sensitive, reproducible and rapid procedure requiring minimal instrumentation. DNA was extracted from 22 skin biopsies of 20 patients with suspected patch stage MF and 15 skin biopsies of inflammatory dermatoses. V gamma1-8, V gamma9, V gamma10, V gamma11 and J gamma1/J gamma2 consensus primers were used for T-cell receptor (TCR)-gamma gene rearrangement amplification. PCR products were analyzed by non-radioactive SSCP. Clonal TCR-gamma gene rearrangements were detected in 17 of 22 (77%) suspected MF specimens. Clonal SSCP banding patterns were different among individual patients. In addition, identical banded patterns were demonstrated in serial skin biopsies from the same patient. No dominant T-cell clones were found in the inflammatory dermatoses studied. Therefore, non-radioactive PCR-SSCP is a useful molecular diagnostic tool for assessment of T-cell clonality in paraffin-embedded specimens suspicious for early MF. The SSCP imprint of PCR products is specific for each TCR-gamma gene rearrangement, and may be used to evaluate concurrent/recurrent disease in individual patients.

Primary gastric apoptosis-rich T-cell lymphoma co-expressing CD4, CD8, and cytotoxic molecules

In contrast to primary gastric lymphomas of B-cell type, little is known about primary gastric T-cell lymphomas. We describe three cases with remarkably similar features: diffuse growth, epitheliotropism, medium too large cell size, high apoptotic rates, and a CD3+, CD4+, CD8+, CD45RO+ immunophenotype. Clonal TCRgamma gene rearrangement was shown in two cases. Epstein-Barr virus infection was excluded in two cases. Taking advantage of fresh-frozen material, we analyzed two cases further, revealing CD5-, CD16+, CD56-, CD57-, CD25+, CD30+, CD103 (alphaEbeta7)+, bcl-2 protein+, CD95+, CD95 ligand(L)-. CD95L, however, was detected in histiocytic and fibroblastoid by stander cells. The lymphomas expressed granzyme B, perforin, and the TIA-1 antigen in various combinations. All three cases had a very unfavorable clinical course characterized by local recurrence and/or dissemination to other epithelial sites, leading to death within 6-12 months after the initial diagnosis despite surgery and aggressive antineoplastic treatment. These data suggest a novel variant of peripheral T-cell lymphoma operationally characterized as primary gastric, apoptosis-rich, CD103+, EBV-, T-cell lymphoma co-expressing CD4, CD8, CD16 and cytotoxic molecules.

Analysis of T-cell antigen receptor gamma chain gene rearrangement by polymerase chain reaction in combination with denaturing gradient gel electrophoresis in the differential diagnosis of cutaneous T-lymphoproliferative diseases.

A polymerase chain reaction (PCR)-based strategy has been developed for analysis of clonal rearrangement of the T-cell receptor gamma gene (TCR gamma) and was shown to be useful for detection of clonal T-cell populations. In this study, we performed PCR combined with denaturing gradient gel electrophoresis (DGGE) on fresh frozen biopsy samples from 16 patients with cutaneous T-lymphoproliferative diseases in whom a definite diagnosis was difficult to make on morphological and immunohistochemical grounds alone. Ages of the patients at biopsy ranged from 28 to 81 (median 62) years, and the subjects consisted of 8 men and 8 women. They presented with erythema on the extremities in 5 cases, trunk in 7, buttock in 2, and papules on the trunk and face in one case each. Clonal rearrangement of TCR gamma was observed in 3 of 16 cases. Clinical diagnoses of these three cases were mycosis fungoides, cutaneous invasion of adult T-cell leukemia (ATL), and large granular lymphocytic leukemia (LGL) of T-cell type, respectively, but they were histologically difficult to differentiate from reactive cutaneous T-cell proliferation. The skin lesions of the LGL case worsened, and this patient died two years after biopsy. Another patient with suspected mycosis fungoides in the plaque stage died due to dissemination of tumors 22 months after biopsy. The remaining one patient with ATL survived with cutaneous lesions for over four years. Clonality was not demonstrated in the remaining 13 cases, and their clinical courses were favorable. These findings showed that demonstration of clonal TCR gamma gene rearrangement using the PCR-DGGE method is very helpful for diagnosis of cutaneous T-cell neoplasms.

Polymerase chain reaction analysis of T-cell receptor gamma gene rearrangements in cutaneous B-cell lymphomas.

PCR analyses of T-cell receptor (TCR) gamma gene rearrangements in B-lymphoid neoplasms have shown lineage infidelity and double rearrangements involving both immunoglobulin heavy chain (igH) and TCRgamma genes. In order to investigate if this event is also a feature of cutaneous B-cell malignancies, we tested for clonal TCRgamma rearrangements a panel of immunophenotypically and genotypically well characterized cutaneous B cell lymphomas (CBCL). Fifteen samples of frozen CBCL biopsies were selected for the study. Diagnoses were established by routine histology and immunohistochemistry. Each of these cases displayed clonal igH gene rearrangement. Polymerase chain reaction (PCR) analysis of the TCRgamma rearrangements followed by high-resolution polyacrylamide gel electrophoresis was utilized for detection of clonal TCRgamma rearrangements. In our investigation, none of the cases of CBCL investigated by PCR showed the presence of clonal TCRgamma gene rearrangements. These data indicate that double rearrangements involving both igH and TCRgamma genes are not a feature of CBCL and confirm the B cell lineage specificity of this group of cutaneous lymphoid neoplasms.

Sensitive non-radioactive PCR detection of clonal T-cell receptor-gamma gene rearrangements in childhood acute lymphoblastic leukaemia.

Rearrangement of the T-cell receptor gamma (TCRgamma) gene potentially provides a valuable target for monitoring minimal residual leukaemia by the Polymerase Chain Reaction (PCR). However, existing strategies directed at this locus frequently lack sensitivity or specificity. We describe a novel PCR strategy for improved detection of clonal TCRgamma gene rearrangements based on the design of two overlapping clone-specific reverse PCR primers spanning the TCRgamma junctional region, which are used sequentially in conjunction with forward V-region specific primers. Unlike other strategies, specificity is generated from the initial and subsequent round of PCR amplification. This non-radioactive technique is highly specific and sensitive, and should prove effective for monitoring minimal residual leukaemia.

PCR DETECTION OF CLONAL TCR GAMMA-GENE REARRANGEMENTS IN A GROUP OF CUTANEOUS T-CELL LYMPHOMAS INCLUDING A CASE OF LOCALIZED PAGETOID RETICULOSIS EXPRESSING THE GAMMA-DELTA TCR

In this report, we describe the analysis of clonality performed using a PCR-based approach in a group of cutaneous T-cell lymphomas including a case of localised pagetoid reticulosis expressing the gamma delta T-cell receptor. For our PCR analysis DNA was extracted from frozen tissue of 10 cases of cutaneous T-cell lymphomas and subjected to polymerase chain reaction amplification using primers recognizing conserved sequences of the variable and joining regions of the T-cell receptor gamma chain gene. PCR products were then analyzed by high resolution non-denaturing polyacrylamide gel electrophoresis. Clonal TCR gamma gene rearrangements were detected in all the cases investigated including the case of localised pagetoid reticulosis. The PCR-based approach used in this study may provide an alternative to conventional Southern blot analysis for the analysis of cutaneous T-cell lymphomas as it is easy to perform and requires only minimal amounts of DNA. The findings in localized pagetoid reticulosis are consistent with the proliferation of monoclonal T-cells in lesional skin of this condition and provide evidence for the lymphoproliferative nature of this clinically benign disorder.

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)

We used a gene amplification strategy to analyze T-cell receptor (TCR) gene rearrangements in 185 specimens, including mycosis fungoides/Sezary syndrome (MF/SS), other cutaneous neoplasms, inflammatory dermatoses, reactive lymphoid tissues, and normal skin. Genomic DNA was extracted from lesional tissues and rearrangements of the TCR-gamma chain gene were amplified using the polymerase chain reaction (PCR) with primers specific for rearrangements involving V gamma 1-8 or V gamma 9 gene segments. The resulting PCR products were then separated according to their nucleotide sequence as well as size by denaturing gradient gel electrophoresis (DGGE). Dominant clonal TCR-gamma gene rearrangements were detected in 61 of 68 MF/SS cases by PCR/DGGE. This sensitivity of 90% compared to a sensitivity of only 59% when dominant clonality was sought in 17 of these same cases by Southern blot analysis of TCR-beta gene rearrangements. This difference in sensitivity was greatest in early, minimally infiltrated skin lesions. PCR/DGGE was also more sensitive than Southern blot analysis for detecting peripheral blood involvement in two cases of early MF. Among 12 additional specimens of suspected MF/SS, nine (75%) showed clonal TCR-gamma gene rearrangements by PCR/DGGE including six of eight cases with a previously confirmed diagnosis of MF/SS and three of four cases without prior known MF/SS. Among 105 non-MF/SS specimens, dominant TCR-gamma gene rearrangements were detected in only six cases (6%). Four were diagnosed as chronic dermatitis and two were diagnosed as cutaneous lymphoid hyperplasia. We conclude that the large majority of MF/SS cases, including patch phase disease, possess dominant clonal TCR-gamma gene rearrangements. PCR/DGGE is more sensitive than Southern blot analysis for detecting dominant clonality and staging disease in patients with a confirmed diagnosis of MF/SS. However, because PCR/DGGE is sensitive enough to detect dominant TCR-gamma gene rearrangements in a subset of patients with chronic dermatitis, it cannot be used as the sole criterion for establishing a diagnosis of T-cell lymphoma. As with other molecular biologic clonality assays, clinicopathologic correlation is essential. Nevertheless, the detection of dominant clonality in some cases of histologically nonspecific dermatitis allows the identification of a previously unrecognized subset of patients, i.e., those with "clonal dermatitis." It will be important to determine the long-term risk of MF/SS among these patients because our study indicated that MF/SS can sometimes present with lesions indistinguishable from clonal dermatitis.