T-cell Receptor Gamma Gene Research Articles

Clinicopathological features and T-cell receptor gene rearrangement findings of mycosis fungoides in patients younger than age 20 years

Mycosis fungoides (MF) is the most common form of cutaneous T-cell lymphoma that usually arises in middle-aged or older people. The incidence of childhood MF is low, but studies in childhood MF suggests that the incidence is rising. The number of studies in childhood MF are few and there are currently no studies about childhood MF in Asians. We investigated the clinicopathological features, T-cell receptor (TCR) gene rearrangement findings, treatment and follow up in childhood MF. The retrospective study was performed on a population consisting of 23 MF patients aged 4-19 years. The duration of the disease ranged from 1 month to 10 years (mean, 2.7 years). In Tumor-Node-Metastasis classifications, all cases were confined to stage IA or IB. Histopathological findings revealed epidermotropism and perivascular infiltrates, epidermotropism in the infundibulum, Pautrier's microabscess, haloed lymphocytes, epidermal lymphocytes larger than dermal lymphocytes, atypical cells with hyperchromatic nuclei and wiry bundles of collagen. TCRgamma gene rearrangement was performed except for four patients, and monoclonality was detected in 16 of 19 cases (84%). Treatment was done with psoralen and ultraviolet (UV)A, UVA1, narrow-band UVB, topical steroid, retinoic acid and calcipotriol. Most patients showed a good response. At a mean follow up of 90.4 months, no patient had either an exacerbation of the disease or extracutaneous involvement. Compared with adult-onset MF, MF in children may show a variety of clinical features. It is considered to have a good prognosis. Moreover, histopathological study and TCR gene rearrangement study can help in the diagnosis of MF in children.

Primary Cutaneous CD4+ Small-/Medium-Sized Pleomorphic T-Cell Lymphoma: A Cutaneous Nodular Proliferation of Pleomorphic T Lymphocytes of Undetermined Significance? A Study of 136 Cases

Patients with skin nodules characterized by the infiltrate of pleomorphic small/medium T lymphocytes are currently classified as "primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma" (SMPTCL) or as T-cell pseudolymphoma. The distinction is often arbitrary, and patients with similar clinicopathologic features have been included in both groups. We studied 136 patients (male:female = 1:1; median age: 53 years, age range: 3-90 years) with cutaneous lesions that could be classified as small-/medium-sized pleomorphic T-cell lymphoma according to current diagnostic criteria. All but 3 patients presented with solitary nodules located mostly on the head and neck area (75%). Histopathologic features were characterized by nonepidermotropic, nodular, or diffuse infiltrates of small- to medium-sized pleomorphic T lymphocytes. A monoclonal rearrangement of the T-cell receptor-gamma gene was found in 60% of tested cases. Follow-up data available for 45 patients revealed that 41 of them were alive without lymphoma after a median time of 63 months (range: 1-357 months), whereas 4 were alive with cutaneous disease (range: 2-16 months). The incongruity between the indolent clinical course and the worrying histopathologic and molecular features poses difficulties in classifying these cases unambiguously as benign or malignant, and it may be better to refer to them with a descriptive term such as "cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance," rather than forcing them into one or the other category. On the other hand, irrespective of the name given to these equivocal cutaneous lymphoid proliferations, published data support a nonaggressive therapeutic strategy, particularly for patients presenting with solitary lesions.

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.

CD3-positive Large B-cell Lymphoma

It is not uncommon for some B-lineage non-Hodgkin lymphomas (NHLs) to aberrantly coexpress T-cell markers, particularly CD5, as well as CD7, CD2, CD4, and/or CD8 in rare cases. Cases of CD3-positive B-cell NHL, however, have not previously been described in the literature. We present 4 cases of large B-cell lymphoma aberrantly coexpressing T-cell marker CD3 and B-lineage markers as well as demonstrating clonal rearrangement of the immunoglobulin genes but not the gamma T-cell receptor gene. To our knowledge, this represents the first series report of B-cell NHL coexpressing T-lineage-specific marker CD3. The identification of such cases indicates that the use of CD3 antibody alone in paraffin sections may lead to an incorrect determination of cell lineage in some B-cell NHL. Immunohistochemistry using additional cell lineage specific markers or molecular analysis for antigen receptor gene rearrangements are necessary for correct determination of the cell lineage in such cases.

Application of BIOMED-2 primers in analysis of T-cell receptor gamma gene rearrangements in paraffin-embedded tissue specimens of T-cell lymphoma

To evaluate the practical values of PCR detectable T-cell receptor (TCR) gene rearrangement in paraffin embedded tissue samples in the diagnosis of T-cell malignancies using BIOMED-2 PCR multiplex tubes TCRgamma(A+B). Traditional phenol-chloroform method was used to extract DNA from 55 cases of archival paraffin embedded tissues samples of T-cell malignancies and the DNA quality was evaluated by PCR-based amplification of housekeeping gene beta-globin. The selected BIOMED-2 PCR multiplex tubes TCRgamma(A+B) were used to detect TCR gene rearrangement and comparison with the results of universal TCR primers (T(VG)/T(JX)) was performed. Positive detection rates by the BIOMED-2 multiplex tubes TCRgamma(A+B) and the universal primers (T(VG)/T(JX)) were 76.4% and 60.0%, respectively. There were not statistical difference between the methods (P > 0.05). BIOMED-2 multiplex tubes TCRgamma(A+B) is suitable for detection of clonal rearrangements of TCR genes in current archival paraffin embedded tissue samples of T-cell malignancies.

Flow cytometric assessment of TCR-VBETA expression in the evaluation of peripheral blood involvement by mature T-cell leukaemias

Background Mature T-cell leukaemias have always been considered difficult and challenging to diagnose by flow cytometry, owing to a paucity of immunophenotyping markers that are sensitive for detection of a clonal T-cell population. Demonstration of the neoplastic nature of T-cell proliferations traditionally has relied on morphological criteria and molecular tests. TCR gamma gene rearrangement studies have been used as the only reliable test to prove clonal T-cell receptor. This notion is changing now. Aim To validate the current commercially available kit IOTest TCR Vβ Repertoire Kit (#IM3497, Beckman Coulter) for routine use in the clinical flow cytometry laboratory. Methods EDTA antigoagulated blood specimens: 10 controls and 10 patients with known or suspected T-cell lymphoproliferative disorders were tested for the expression of 24 TCR Vβ antigens with TCR Vβ Repertoire Kit. All specimens were subjected to four-colour immunophenotyping on FC 500 cytometer. Correlation was performed with TCR gene rearrangement by PCR using Cohen’s Unweighted Kappa score. Results All 13 patients had clonal T-cell population detected by flow cytometry. PCR confirmed T-cell clonality in all 13 cases studied– Kappa score 1. No false positive or negative results were observed in our settings. Conclusion Detection of T-cell clonality is no longer restricted to molecular techniques or research laboratories. It is now possible to prove T-cell clonality by means of flow cytometry, based on abnormal distribution of TCR Vβ antigens. Apart from the very high cost, the assay is easy to perform and interpret, with a turnaround time of approximately 2 hours. This test represents a powerful tool for rapid diagnosis of peripheral T-cell lymphoma before the disease becomes a medical emergency and, in some instances, might eliminate the need for further genetic testing.

Analysis of Clonal T-Cell Receptor Gamma Gene Rearrangements, NOTCH1, FBW7 and PTEN Mutations in Paired Pediatric T-Cell Acute Lymphoblastic Leukemia Samples at Diagnosis and Relapse Reveals Evidence of Relapse in New Leukemic Clones.

Relapse following remission induction chemotherapy remains the major challenge in the successful treatment of childhood T cell acute lymphoblastic leukemia (T-ALL). Relapse often results from the outgrowth of residual leukemia cells that are present below the limit of detection or involves a new therapy-related secondary leukemia. Individualization of treatment might improve the outcome and long-term quality of life for T-ALL patients. Molecular genetic markers represent clinically useful factors which predict responses to therapy. T-cell receptor gamma (TCRG) gene rearrangements occur in more than 90% of T-ALL and provide markers of lymphoblast clonality. Determining rearrangements in the TCRG could be critical to the diagnosis and treatment of T-ALL in children and adults. Mutations in the NOTCH1, FBW7, and PTEN genes have been identified at high frequencies in pediatric T-ALL cases. Activating NOTCH1 mutations have been found in more than 50% of ALL patients, resulting in constitutive NOTCH1 signalling, whereas PTEN mutations are inactivating, resulting in increased PI3K/AKT signalling. FBW7 has been identified as an important tumor suppressor. Several studies reported that frequent mutations in the substrate binding domain (e.g. Arg465, Arg479, Arg505) for FBW7 in T-ALL cell lines and primary T-ALL specimens result in sustained NOTCH1 levels and downstream signalling and gamma secretase inhibitor resistance, suggesting an alternate mechanism for NOTCH1 deregulation. To investigate the mechanism of T-ALL relapse, we analyzed the TCRG gene rearrangements and mutational status of the NOTCH1, FBW7, and PTEN genes by comparing sequences in paired diagnostic and relapsed T-ALL samples from 11 children to evaluate their stabilities throughout disease progression and association with treatment failure. The age distribution of 11 patients ranged from four years to fifteen years. Original TCRG sequence (a measure of leukemia clonality) was fully preserved at relapse in 3 (27.3%) patients. Clonal evolution was identified in 8 (72.7%) patients, reflected in changes in TCRG sequence. In 3 patients at diagnosis, NOTCH1 mutations were detected. At relapse, the major leukemia clones exhibited different NOTCH1 mutations. For another patient, a NOTCH1 mutation was detected at relapse but not at diagnosis. No FBW7 mutations were detected either at diagnosis or relapse. In 5 patients at diagnosis, PTEN mutations were detected and at relapse, 2 preserved the same mutation and 2 lost their mutations, while the additional sample harbored a different PTEN mutation. Our comparative sequence analysis of pediatric T-ALL samples provided detailed insight in the stabilities and changes of TCRG rearrangements and NOTCH1, FBW7 and PTEN mutation status during disease development. Re-emergence of the initial ALL clone or the occurrence of a secondary ALL clone may be clinically important to guide subsequent therapy. Collectively, our results suggest that for the majority of cases, relapse is associated with appearance of a new leukemic clone. For a subset of these cases, this is accompanied by a distinct subset of NOTCH1 mutations and, to a lesser extent, PTEN mutations. FBW7 mutations are rare. Better understanding of the changes in oncogenes and tumor suppressor genes with progression of T-ALL may identify new targets for therapy and facilitate the design of individualized therapy for this disease. Further study is needed to determine whether the newly identified relapse ALL clones were present at diagnosis as minor subclinical populations.

Incidence ofTCRandTCL1Gene Translocations and Isochromosome 7q in Peripheral T-Cell Lymphomas Using Fluorescence In Situ Hybridization

Translocations involving the T-cell receptor (TCR) and TCL1 genes occur in T-cell precursor lymphoblastic leukemia/lymphoma and prolymphocytic leukemia; isochromosome 7q has been associated with hepatosplenic T-cell lymphoma. However, the incidence of these abnormalities in peripheral T-cell lymphomas (PTCLs) as a whole has not been well defined. We studied genetic abnormalities in 124 PTCLs seen at the Mayo Clinic, Rochester, MN, between 1987 and 2007. Tissue microarrays were screened using 2-color break-apart fluorescence in situ hybridization probes flanking the TCRalpha (TCRA, 14q11), TCRbeta (TCRB, 7q35), and TCRgamma (TCRG, 7p15) genes and the TCL1 gene (14q32). Isochromosome 7q was analyzed by using a 2-color probe to 7p and 7q32.1. Translocations involved TCRA in 3 (2.9%) of 102 cases and TCRB in 1 (1%) of 88. Isochromosome 7q was detected in 2 cases of extranodal NK/T-cell lymphoma, nasal type, and 2 cases of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. One of the latter cases also had a translocation of TCRA, and further studies confirmed a novel t(5;14) translocation.

Mucosal Intraepithelial T-lymphocytes in Refractory Celiac Disease: A Neoplastic Population With a Variable CD8 Phenotype

Celiac disease (CD) is characterized by villous atrophy and an increase in intraepithelial lymphocytes (IEL). The IEL usually exhibit a suppressor/cytotoxic phenotype (CD3 and CD8) and display a polyclonal profile for T-cell receptor (TCR) rearrangement as opposed to the monoclonality of refractory CD (RCD) with CD8 IEL. A complication of CD is the loss of response to a gluten-free diet called RCD that may progress to an enteropathy-associated T-cell lymphoma. We reviewed 20 uncomplicated CD and 23 complicated CD (19 RCD and 4 diagnosed at the same time as enteropathy-associated T-cell lymphoma). In complicated CD, the IEL phenotype was CD8 in 9 cases and CD8 in 14 cases. In 100% of cases, IEL showed a monoclonal TCR rearrangement. All the 9 CD8 complicated CD exhibited a monoclonal TCR rearrangement and 3 of them were associated with a T-cell lymphoma (2 at the same time as CD and 1 after 43-mo follow-up) and bore the same monoclonal rearrangement in IEL and in lymphoma. Interestingly, the 13 cases (100%) of CD with a CD8 phenotype were also found monoclonal and 2 of them were associated with a T-cell lymphoma diagnosed at the same time as CD and exhibiting the same rearrangement in IEL and in lymphoma. An aberrant CD3 CD8 IEL phenotype is a good criterion for RCD diagnosis. However, cases with a normal CD3 CD8 IEL phenotype may correspond to RCD. In such cases, we suggest that molecular analysis of TCR-gamma genes is a useful method for identifying cases with RCD.

A case of pericoronary pseudotumor due to localized Castleman's disease

A 59-year-old male had a latent epicardial mass discovered at cardiovascular imaging during the assessment of an aortic murmur. The resected mass surrounded the left anterior descending coronary artery. It was a well-limited pericoronary cellular lesion. It was made of a mixture of polytypic plasma cells, lymphocytes with lymphoid follicles, hyaline vascular hyperplasia, and focal eosinophils. No immunoglobulin and TCR-gamma gene rearrangements were detected. In this immunocompetent patient, HHV-8 was negative. The pattern was consistent with a pericoronary localized Castleman's disease of composite histologic subtype.

TCRγ-Chain Gene Rearrangement by PCR-Based GeneScan: Diagnostic Accuracy Improvement and Clonal Heterogeneity Analysis in Multiple Cutaneous T-Cell Lymphoma Samples

Cutaneous T-cell lymphomas are a heterogeneous group of lymphomas where the tumor population emerges within a multiple subclone pattern ("clonal heterogeneity"). PCR analysis has been shown to be useful in the diagnosis of mycosis fungoides (MF) and Sézary Syndrome (SS). Focusing the attention on clonal heterogeneity, the efficacy of the multiplex/heteroduplex (HD) PCR and the GeneScan (GS) capillary electrophoresis analysis was compared in the early diagnosis of MF/SS, using a multiple sample approach. Indeed, GS demonstrated TCRgamma gene rearrangement (GR) in all the 57 SS (100%) and in 123/146 (84%) of the MF samples, whereas the multiplex/HD PCR was less sensitive. An increase in clonality was observed in connection with both a worsening of the cutaneous disease (79% T1/T2; 100% T3/T4) and an increase in the histopathological score (HS < 5, 76%; HS > or = 5, 94%). Clonal heterogeneity with adjunctive reproducible skin TCRgamma-GRs was also observed. "Clonal instability," with different GRs, was present in a small percentage of patients. Therefore, it can be concluded that GS analysis in TCRgamma-GR is able to improve diagnosis in MF/SS patients and the multiple sample approach is helpful for a correct interpretation of clonal patterns in skin lesions, especially in early-stage MF and in SS skin/blood samples.

Mycosis fungoides mimicking erysipelas

Mycosis fungoides may rarely simulate facial erysipelas. In a patient with that clinical presentation, a biopsy specimen revealed a diffuse dermal infiltrate with numerous neutrophils that also mimicked erysipelas histopathologically. The diagnosis of mycosis fungoides was made on the basis of atypical lymphocytes with focal epidermo- and folliculotropism. It was confirmed by typical findings of mycosis fungoides in a second biopsy from a clinically inconspicuous patch. An identical T-cell receptor gamma-gene rearrangement was detected in lymphocytes of both biopsy specimens.

Nodal T/NK‐cell lymphoma of nasal type: a clinicopathological study of six cases

To investigate the clinicopathological features of six unusual cases of nodal CD56+ and Epstein-Barr virus (EBV)+ T/natural killer (NK)-cell lymphoma, a putative nodal counterpart of nasal NK/T-cell lymphoma (nodal T/NK-cell lymphoma of nasal type) in comparison with nasal NK/T-cell lymphoma with secondary lymph node involvement (n = 24) and peripheral T-cell lymphoma (PTCL) of cytotoxic molecule (CTM)+ and EBV+ type (n = 21). All cases of nodal T/NK-cell lymphoma of nasal type exhibited diffuse infiltration of pleomorphic medium-sized to large tumour cells, reminiscent of those in CTM+ EBV+ PTCL. The tumour cells had a typical phenotype of nasal NK/T-cell lymphoma: CD2+, CD3epsilon+, CD4-, CD5-, CD56+, T-cell intracellular antigen-1+, granzyme B+, perforin+ and EBV+. However, four of six cases demonstrated clonal T-cell receptor gamma-gene rearrangement on polymerase chain reaction analysis, unlike nasal NK/T-cell lymphoma. Comparison of clinical parameters and overall survival among the three groups demonstrated only minor differences. Nodal T/NK-cell lymphoma may occupy the grey zone between extranodal nasal-type NK/T-cell lymphoma and nodal CTM+ PTCL in a spectrum of NK to T-cell lymphomas that are EBV+. The close relationship between NK/T-cell lymphomas and cytotoxic T-cell lymphomas was also substantiated.

Primary T-cell lymphoma of the thyroid: case report and review of the literature

Here we report a rare case of primary T-cell lymphoma of the thyroid gland. A 32-year-old Chinese man was admitted to our hospital because of marked goiter and subcutaneous nodules. Computed tomography (CT) scan of the neck disclosed diffuse enlargement of the thyroid gland. A diagnosis of peripheral T-cell lymphoma was made according to cytopathological findings of fine-needle aspiration (FNA) of the thyroid gland and skin biopsy. Immunohistochemistry stain showed that the tumor was positive for CD3 and negative for CD20. Clonal T-cell receptor (TCR-gamma) gene rearrangements were demonstrated by polymerase chain reaction. After six cycles of combination chemotherapy (bleomycin, cyclophosphamide, doxorubicin, vincristine, and prednisone), the thyroid retracted to normal size gradually.

Clinical, Histopathological, Immunophenotypic and Molecular Analysis of 60 Patients with Cutaneous T-cell Infiltrates with Follow up of Indeterminate Cases to Identify T-cell Lymphoma

Diagnosis of primary cutaneous T-cell lymphomas, especially of mycosis fungoides could be difficult in early stage due to clinical and histopathological similarity to reactive inflammatory dermatoses. To assess diagnostic value of complex histological, immunophenotypic and T-cell receptor gamma gene rearrangement analysis, skin biopsy specimen and peripheral blood samples of 60 patients with suspected cutaneous T-cell lymphoma were analyzed. Our results indicate clear distinction between reactive dermatoses (benign cases, n = 31) and cutaneous T-cell lymphomas (lymphoma cases, n = 17). As definite diagnosis was not obtained in a smaller group of patients (indeterminate cases, n = 12), these patients were followed up. Repeated skin biopsy confirmed mycosis fungoides in 6/12 cases, however in 6/12 patients the diagnosis remained indeterminate. We concluded that careful and complex clinical follow up and repeated histopathological, immunophenotypic and molecular analysis is needed for an appropriate diagnosis in the assessment of early stage mycosis fungoides and uncertain clinical cases.

CNS T-cell lymphoma: an under-recognized entity?

The incidence of CNS lymphoma has increased significantly in the past 30 years, primarily in the elderly and immunocompromised. While T-cell lymphomas comprise 15-20% of systemic lymphomas, they comprise less than 4% of primary CNS lymphomas, suggesting that they may be under-recognized compared to their systemic counterparts. To investigate this, we studied brain biopsies from three patients who were diagnosed with T-cell lymphoma confined to the brain. They had enhancing lesions by MRI, arising in the cerebellum and brainstem in one and temporal lobe in two. We compared these to biopsies from three patients who had reactive lymphoid infiltrates and who had clinical signs/symptoms and radiographic findings that were indistinguishable from the lymphoma group. Biopsies from both the lymphoma group and reactive group showed considerable cytomorphologic heterogeneity. Although one lymphoma case contained large atypical cells, the other two contained small, mature lymphocytes within a heterogeneous infiltrate of neoplastic and reactive inflammatory cells. Surface marker aberrancies were present in two lymphoma cases, but this alone could not reliably diagnose T-cell lymphoma. The proliferation index was not useful for differentiating lymphoma from reactive infiltrates. In five of the six cases the diagnosis was most influenced by clonality studies for T-cell receptor-gamma gene rearrangements. We conclude that because of the high degree of overlap in cytomorphologic and immunophenotypic features between T-cell lymphoma and reactive infiltrates, T-cell lymphoma may not be recognized unless studies for T-cell receptor gene rearrangements are performed for CNS lesions composed of a polymorphous but predominantly T-cell infiltrate.

A novel nude mice model of human extranodal nasal type NK/T-cell lymphoma

A novel nude mice model of human extranodal nasal type NK/T-cell lymphoma was established by subcutaneously implanting the sample taken from the patient with secondary extranodal nasal type NK/T-cell lymphoma of the stomach into the right axillary region of a BALB/c (nu/nu) nude mouse. This model had been successfully transplanted in vivo for thirty-two generations with a stable growth cycle. The survival rates of both resuscitation and transplantation were 100%. Histologically, the tumor cells were medium to large size and arranged in sheets, with a little mesenchyma, and disseminated almost in all passages of the lymphoma-bearing nude mice. Immunologically, the tumor cells were positive for CD56, cytoplasmic CD3, granzyme B or TIA-1 and LMP1, sometimes for CD8 but negative for surface CD3, CD7, CD20 and CD1a. EBER1/2 was found. No T-cell receptor gamma gene rearrangement was detected in the transplanted tumors. Furthermore, both human sequencing-tagged sites SY14 and Y chromosome were detected by PCR or fluorescent in situ hybridization, respectively, in the transplanted tumor. The transplanted tumor in this novel nude mice model maintained the essential features of human extranodal nasal type NK/T-cell lymphoma, and it would be an ideal tool in vivo for further research of the tumor.

Pulmonary manifestations of peripheral T‐cell lymphoma: case report and review of the literature

Peripheral T-cell lymphomas (PTCL) represent approximately 10% of non-Hodgkin's lymphomas. Pulmonary involvement is an uncommon manifestation of this heterogeneous group of malignancies. Report of a case. This case report describes a 75-year-old man with fever, weight loss, anemia, enlargement of spleen and liver, atypical lymphocytes and pulmonary nodules. Lung biopsy showed lymphocytic infiltration of the lung parenchyma. T-cell receptor gamma gene rearrangement by polymerase chain reaction confirmed the diagnosis of peripheral T-cell lymphoma. Unfortunately, the patient died because of refractory and aggressive disease. Pulmonary and pleural involvement are seen in patients with PTCL and usually carry a poor prognosis. The subject of pulmonary involvement in peripheral T-cell lymphoma is discussed.

Optimization of PCR amplification for B-and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples

In many cases, particularly in retrospective studies, only formalin-fixed and paraffin-embedded (FFPE) tissue samples are available for molecular studies. DNA recovered from FFPE tissues generally consists of fragmented small target sequences with chemical alterations. Clonality analysis is not easy on FFPE samples, in fact, it requires even more experience than that of performed on fresh samples or is more complicated than most genomic PCR amplifications for somatic genes. In our study, we have performed a multi-parameter PCR evaluation investigating immunoglobulin heavy chain gene (IgH) and T-cell receptor gamma gene (TCRgamma) rearrangements on non-purified crude lysates of FFPE samples, in order to establish the significance of different variables on performance of PCR amplification. The results showed that a slight decrease in the concentration of primers in combination with a slight increase in MgCl2 and Taq polymerase concentrations, as well as the use diluted crude template and a standard amount of dNTPs can be the modifications of choice while adjusting IgH and TCRgamma clonality tests on poor quality DNA FFPE samples. Using our improved protocol, 74% (17/23) of the tested B-cell lymphomas and 68% (31/46) of the tested T-cell lymphomas demonstrated monoclonal PCR product, proving the applicability of our optimized method. Our experience may be of help during the optimization process in technically difficult cases as well as to determine which parameters and how should be changed to minimize false-negative and false-positive results.

Analysis of T-Cell Clonality Using Laser Capture Microdissection and High-Resolution Microcapillary Electrophoresis

Identification of clonal lymphocytic populations by polymerase chain reaction may be difficult in cases with scant cellular infiltrates or those with a heterogeneous population of cells. Here, we assessed the diagnostic utility of laser capture microdissection (LCM) and high-resolution microcapillary electrophoresis in the analysis of clonality of small biopsy specimens. Clonality was determined in 24 cases: five reactive tonsils, five reactive lymph nodes, six inflammatory skin lesions, and eight T-cell lymphomas. CD3-positive T lymphocytes were captured by LCM from paraffinized immunohistochemically stained sections. Genomic DNA was analyzed for T-cell receptor-gamma gene rearrangement by polymerase chain reaction followed by high-resolution microcapillary electrophoresis with the DNA 500 LabChip and the Agilent Bioanalyzer. In the reactive specimens, T-cell receptor-gamma polymerase chain reaction revealed monoclonal bands when 10 to 1000 cells were captured. This pattern changed to polyclonal when higher numbers of cells were microdissected (2000 to 10,000 cells). In contrast, lymphoma cells were consistently monoclonal whether low or high numbers were microdissected. Microcapillary electrophoresis coupled with LCM facilitated clonality analysis in equivocal cases. In two of eight lymphoma cases, LCM revealed diagnostic monoclonal bands, whereas routine T-cell receptor-gamma assessment of whole tissue sections with 10% polyacrylamide gel electrophoresis demonstrated only minor clonal bands. We conclude that clonality determined by LCM is cell number-dependent. Biopsy specimens containing low numbers of reactive polyclonal T cells may produce pseudomonoclonal bands and therefore should be interpreted with great caution.