Clonal T-cell Receptor Gene Rearrangements Research Articles

Use of multigeneration-family molecular dog leukocyte antigen typing to select a hematopoietic cell transplant donor for a dog with T-cell lymphoma

A 7-year-old Golden Retriever was examined because of anorexia, lethargy, vomiting, and gradual weight loss. Splenomegaly, pancytopenia, high serum calcium concentration, and high alkaline phosphatase activity were detected. Magnetic resonance imaging revealed an enlarged mesenteric lymph node and increased signals from the bone marrow of the ilium and vertebral bodies. Histologic examination and immunophenotyping of biopsy specimens confirmed a stage V (b) T-cell malignant lymphoma. Clinical remission was attained by use of 2 chemotherapy cycles, followed by an allogeneic hematopoietic cell transplant performed at 18 weeks after diagnosis. A donor was identified by molecular dog leukocyte antigen typing methods. The patient was conditioned with 2 fractions of 4 Gy total body irradiation delivered 3 hours apart at 7 cGy/min, followed by an IV infusion of recombinant canine granulocyte colony-stimulating factor mobilized leukapheresis product and postgrafting immunosuppression with cyclosporine. Chimerism analyses revealed full donor engraftment that has been maintained for at least 58 weeks after transplant. Remission has been confirmed by normal results of serum thymidine kinase assays and the absence of peripheral blood clonal T-cell receptor gene rearrangements. Systemic chemotherapy induces remissions; however, most dogs succumb to disease recurrence because of multidrug resistance. Outcome of allogeneic hematopoietic cell transplantation in dogs can be excellent because of improved donor-recipient selection by use of molecular dog leukocyte antigen typing, compared with early attempts, and better prevention of graft versus host disease, better supportive care, and substitution of peripheral blood mononuclear cells for bone marrow.

Transient clonal expansion of T-large granular lymphocytes during primary cytomegalovirus infection

Clonal immunoglobulin and T-cell receptor gene rearrangements are useful in distinguishing reactive lymphoproliferations from neoplastic processes. Here, we report a case of transient clonal expansion of CD8+ CD57- T-large granular lymphocytes (T-LGL) during primary cytomegalovirus infection. This case underlines that clonal expansion of T-LGL could be a reactive phenomenon related to an acute infectious disease and is not specific for lymphoid malignancy.

Tandem Application of Flow Cytometry and Polymerase Chain Reaction for Choice Targets of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia.

Objective In order to allow monitoring virtually all patients with childhood acute lymphoblastic leukemia for minimal residual disease in resource-poor countries or patient population. Methods Choice targets of minimal residual disease using tandem application of multi-parameter flow cytometry with various combinations of monoclonal antibodies for leukemia-associated immunophenotypes and polymerase chain reaction (PCR) of clonal T-cell receptor or immunoglobulin gene rearrangements was performed in 122 patients with newly diagnosis acute lymphoblastic leukemia on protocol ALL-XH-99. Results [circ1]The four-color antibody combinations consisted of CD10/CD34/CD19 plus another effective marker such as CD38, CD65, CD66c, and CD21. 106 cases of childhood B-cell precursor acute lymphoblastic leukemia were screening for antibodies combinations of interest and were identified in 95 of 106 cases (89.62%). The frequency of terminal deoxynucleotidyl transferase (TdT) in the immunophenotypic expression of leukemia cells is 59.43%, and then does CD38 and CD58. There is only one aberrant immunophenotype in 11 of 95 cases (11.58%) and most cases (88.42%) express at least two suitable combinations. [circ2] Due to lack of specimens in the leukemia cell bank, polymerase chain reaction of clonal T-cell receptor or immunoglobulin gene rearrangements was performed in 27 patients with newly diagnosis acute lymphoblastic leukemia including 7 cases with B-cell precursor ALL who were not detected targets of minimal residual disease by multi-parameter flow cytometry and 20 cases with T-lineage acute lymphoblastic leukemia (25 patients with T-ALL at the same time in our hospital). In 17 samples (65.38%), two or more monoclonal/bi-allelic gene rearrangements were identified, in 9 of 27 cases (34.62%), only one monoclonal rearrangement was detectable, and in one case no clonal T-cell receptor or immunoglobulin gene rearrangement could be identified. The vast majority (70%) of T-lineage ALL contain T-cell receptor VγI-Jγ1.3/2.3, and then T-cell receptor Vδ1-Jδ1, T-cell receptor VγIII-Jγ1.3/2.3. In B-cell precursor ALL, the high frequency of T-cell receptor VγI-Jγ1.3/2.3 emerged, and then the Kde rearrangements of IGK. Cross-lineage T-cell receptor rearrangements are found in many patients (57.14%) with B-lineage acute lymphoblastic leukemia. [circ3]Tandem application of multi-parameter flow cytometry with various combinations of monoclonal antibodies for leukemia-associated immunophenotypes and polymerase chain reaction (PCR) of clonal T-cell receptor or immunoglobulin gene rearrangements to detect MRD targets was investigated in 122 patients. Almost all patients except one case were detected suitable immunophenotypic abnormalities or antigen receptor gene rearrangements targets. Tandem application of two methods allows monitoring virtually all patients (99.18%) for MRD. Conclusion Choice targets of minimal residual disease using tandem application of flow cytometric detection of aberrant immunophenotypes, polymerase chain reaction (PCR) of clonal T-cell receptor or immunoglobulin gene rearrangements was to allow monitoring virtually in resource-poor countries or patient population for minimal residual disease.

Ehrlichiosis Mimicking T-Cell Lymphoma/Leukemia.

Immunophenotyping by flow cytometry has revolutinized the diagnosis of blood cell disorders such as leukemias and lymphomas and is now commonly used in diagnosis and prognosis of such patients. We describe a case of human ehrlichiosis mimicking T-cell lymphoma/leukemia based on flow cytometry of bone marrow cells and confirmed by T-cell receptor gene rearrangement (TCR) by polymerase chain reaction (PCR). Treatment with doxycycline reversed these findings. A 20-year-old, Amish female presented with fatigue, fever, chills, sweating, low back pain, and lower abdominal pain for 2 days. She admitted to multiple bites from ticks 2 weeks prior to presentation and also reported having numerous animals such as cats, dogs, cows, goats, horses at her farm where she lived. Clinical exam was significant for fever of 101.4 F, heart rate of 118/min, BP of 80/60 mm Hg and a distended urinary bladder which was treated by catheter drainage. Relevant laboratory tests are shown in table 1.Table 1Hemoglobin9.712–16 gm/dlWBC0.84–10.8 k/mm3Platelets16150–400 k/mm3Segments62%50–75%Lymphocytes15%20–40%Sodium140125–135 mmol/LAST1260–37 IU/LALT710–65 IU/LAlk. Phos.4950–136 IU/LLDH69191–190 IU/LChest radiograph, Ultrasound and Computed tomography scan of the abdomen were within normal limits. With a provisional diagnosis of septic shock and suspicion for Ehrlichiosis, therapy with intravenous(IV) fluids, vasopressors and doxycycline was initiated. Blood was cultured and a sample was forwarded to CDC for analysis of tick borne infections.In order to evaluate and exclude blood disorders like leukemia and lymphoma in a patient with fever and pancytopenia, a bone marrow aspiration and biopsy was performed. It showed cytologically abnormal-appearing, large sized lymphocyte population with irregular nuclear membranes. Flow cytometry of the bone marrow cells revealed 8–10% of phenotypically abnormal T-cells with abnormally weak intensity of membrane surface CD3, CD5, and CD7 expression and negativeCD4 and CD8 expression. These cells also expressed HLA-DR and CD38 at uncommonly bright intensity and there were no CD34 benign immature B-cells. Cytogenetics however was normal. Interestingly, PCR analysis was positive for clonal TCR gamma gene rearrangement. These results were reported as consistent with involvement of marrow by a peripheral T-cell lymphoma/leukemia [Display omitted] Since the patient was steadily improving with IV Doxycycline, we decided to wait and repeated the bone marrow aspiration a week later. This time the bone marrow exam was found to be normal morphologically, on flow cytometry and TCR gamma gene rearrangement by PCR.Patient was discharged on oral doxycycline after a stay of 13 days in the hospital. The blood test for ehrlichiosis from CDC was reported 3 weeks later as positive for Ehrlichia chaffeensis by PCR. Patient is doing well 6 months after the illness. This case illustrates that Ehrlichiosis can transiently cause T cell abnormalities resulting in false positive analysis on flow cytometry and TCR gamma gene rearrangement, thereby leading to false positive diagnosis of Ehrlichiosis. Reconfirmation with repeat studies need to be done before considering active treatment for lymphoma/leukemia.

Rapid Detection of Clonal T-Cell Receptor-β Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis

Various molecular methods have been developed to diagnose clonal T-cell receptor (TCR) gene rearrangements in clinical samples. Most polymerase chain reaction strategies for detecting clonal TCR gene rearrangements rely on either gel or capillary electrophoresis. However, a cumbersome manual transfer step separates amplification from analysis. Recently, we developed a novel polymerase chain reaction assay using the LightCycler system to detect clonal immunoglobulin heavy chain gene rearrangement. In the current study, we extend this work to include the TCR. We report that clonal TCR-beta (TCR-beta) gene rearrangements can be detected in less than 1 hour after preparing the DNA by measuring DNA melting immediately after amplification in a single closed capillary tube. We retrospectively studied 52 fresh-frozen tissue samples from patients clinically suspected of T-cell malignancy. A clonal TCR-beta gene rearrangement was detected in 14 samples by DNA melting curve analysis. When DNA melting was compared to the gold standard methods of Southern blot or denaturing gradient gel electrophoresis, it achieved a sensitivity equal to 71% and a specificity equal to 94%. We also compared melting curve analysis and polyacrylamide gel electrophoresis: melting curve analysis reached a sensitivity equal to 100% and a specificity equal to 97%. We conclude that DNA melting curve analysis in the LightCycler system has potential for clinical use as a new, ultra-fast method for the initial diagnosis of clonal TCR-beta gene rearrangements.

Acquired Pure Red Cell Aplasia Associated with Clonal T-Cell Receptor (TCR) Gene Rearrangements with and without Pathologic Features of T-Cell Large Granular Lymphocytic Leukemia (T-LGLL).

Acquired pure red cell aplasia (APRCA) is associated with a number of conditions including malignancies, infections and autoimmune disorders. APRCA also occurs in patients with T-LGLL. It is postulated that suppression of erythropoiesis in T-LGLL is related to the immune destruction of erythroid precursors by the LGL clone. We hypothesize that APRCA can be associated with clonal T-cell expansion without pathological evidence of T-LGLL.Methods: We identified patients who were diagnosed with APRCA and had clonal TCR gene rearrangements. Clinical course, bone marrow pathology and flow cytometry data were reviewed. Immunohistochemical studies were performed on available bone marrow biopsies using antibodies to CD3, CD8, CD20 and the cytotoxic granule proteins TIA-1 and granzyme B. Clonal TCR gene rearrangements were detected by PCR and Southern blot. The diagnosis of APRCA was based on the absence of erythroid progenitors, with intact granulo- and thrombopoiesis. A pathologic diagnosis of T-LGL leukemia was established by the presence of clonal TCR gene rearrangements and an immunophenotypically distinct CD8 positive T-cell population as previously described. The diagnostic flow cytometric features included abnormal patterns of T-cell antigen expression and aberrant co-expression of NK-cell antigens. Immunoperoxidase features supportive of a diagnosis of LGL were the presence of interstitial clusters or intravascular staining of cells positive for CD8 and TIA-1 and/or granzyme B.Results: We identified 28 patients with APRCA and the presence of clonal TCR gene rearrangements seen at Mayo Clinic Rochester between 7/1/1991 to 6/1/03. All patients presented with a transfusion dependent anemia (median hemoglobin 7.8 g/dL, range 3.6–10.3). Tissue was available for immunohistochemical studies for 21 cases. 13 of 21 patients (61%) had an immunophenotypically abnormal T-cell population which, in conjunction with the clonal TCR gene rearrangements were considered diagnostic of T-LGL. 8 patients (39%) had no pathological evidence of T-LGLL despite clonal TCR gene rearrangement studies. The most commonly used treatment consisted of oral cyclophosphamide which was used in 14 of 21 patients (8 and 6 in patients with and without pathological evidence of T-LGLL respectively). A normalization of hemoglobin was seen in 11 patients (6 and 5 in patients with and without evidence of T-LGLL respectively). Median duration of response was 30 months (range 22–120). None of 8 patients with APRCA and clonal TCR gene rearrangements without evidence of LGLL at the the time of initial diagnosis developed overt T-LGLL during follow up (median 64 months, range 30–118).Discussion: 39 % of patients with APRCA and evidence of clonal TCR gene rearrangements did not meet pathological criteria for diagnosis of T-LGLL. There was no apparent difference in disease presentation and response to therapy in patients with or without pathological evidence of LGLL. The significance of the presence of clonal TCR gene rearrangements in the absence of a detectable immunophenotypically abnormal T-cell population is unclear. It is possible that T-LGL was present which could not be detected by our analyses. Alternatively, the clonal TCR gene rearrangements could be indicative of a pertubation of the T-cell compartment related to an autoimmune disorder causing the APRCA. The fact that none of the patients developed progression to overt LGLL supports the latter possibility.

Prognostic Value of Early Treatment Response in Children with Acute Lymphoblastic Leukemia: A Single Institution Experience in Shanghai, China.

Early response to therapy is one of the most important prognostic factors in childhood ALL. In the early 1980s, assessment of early treatment response to therapy relied mainly on morphological examination of peripheral blood or bone marrow after single-agent or multi-agent remission induction therapy. More recently, measurement of minimal residual disease (MRD) by flow cytometric detection of aberrant immunophenotype or by polymerase chain reaction of clonal T-cell receptor or immunoglobulin gene rearrangement emerged as a more reliable prognostic indicator. We evaluated the prognostic significance of early treatment response in our recent clinical trial. Among the children with ALL who entered protocol of ALL-XH-99 from January 1998 to May 2003, 193 patients with newly diagnosed ALL comprise the basis for this report. We examined blast cell count in the bone marrow on day 19 of remission induction and at the end of 35 days of remission induction. Minimal residual disease was measured with the use of flow cytometry. Probability of event-free survival was estimated by Kaplan-Meier analysis and the distributions of pEFS were compared using the log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors. Univariate analysis indicated the probability of 4-year event-free survival (pEFS) was significantly worse for patients with ≥ 5% lymphoblasts in the bone marrow on day 19 as compared to those with less than 5% lymphoblasts on that date (42.59%±14.28% vs 74.24%±6.67%, P=0.0006). The probability of 4-year event-free survival (pEFS) was significantly worse for patients with any amount of lymphoblasts in the bone marrow on the remission date as compared to that of other patients with no morphologically identifiable blasts (63.47%±9.23% vs 76.41%±6.09%, P=0.0130). Patients who achieved a minimal residual level < 0.01%, fared significantly better than those with a higher level. (23.81%±20.26% vs 94.44%±5.4%, P=0.001). Early treatment response as assessed by morphologic examination or minimal residual leukemia determination by flow cytometry has important prognostic significance, and can be performed in a resource-poor patient population.

Sequence analysis of clonal immunoglobulin and T-cell receptor gene rearrangements in children with acute lymphoblastic leukemia at diagnosis and at relapse: implications for pathogenesis and for the clinical utility of PCR-based methods of minimal residual disease detection

AbstractImmunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements provide clonal markers useful for diagnosis and measurement of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We analyzed the sequences of Ig and TCR gene rearrangements obtained at presentation and relapse in 41 children with ALL to study clonal stability, which has important implications for monitoring MRD, during the course of the disease. In 42%, all original Ig and/or TCR sequences were conserved. In 24%, one original sequence was preserved but the other lost, and in 14% the original sequences were conserved with new sequences identified at relapse. In 20% only new sequences were found at relapse. Using primers designed from the novel relapse sequences, the relapse clone could be identified as subdominant clones in the diagnostic sample in 8 of 14 patients. Alteration of these clonal gene rearrangements is a common feature in childhood ALL. MRD detection should include multiple gene targets to minimize false-negative samples or include also multicolor flow cytometry. In some cases the leukemic progenitor cell might arise earlier in lineage before DHJHrecombination but retain the capacity to further differentiate into cells capable of altering the pattern of Ig and/or TCR rearrangements. (Blood. 2003;102:4520-4526)

Comparison between TaqMan and LightCycler technologies for quantification of minimal residual disease by using immunoglobulin and T-cell receptor genes consensus probes

Quantification of residual leukemic cells at early time points during therapy can reliably predict the outcome in children with acute lymphoblastic leukemia (ALL). Recently, semiquantitative minimal residual disease (MRD) detection assays such as dot-blot hybridization have been replaced by real-time quantitative PCR. We tested the flexibility of the two most used real-time PCR machines: the SDS 7700 or 'TaqMan' (TM) (Applied Biosystems) and the LightCycler (LC) (Roche) instruments. Clonal T-cell receptor and immunoglobulin gene rearrangements were used for MRD detection with germline hydrolyzation probes and clone-specific primers. Sensitivity tests for 65 clonal gene rearrangements and MRD quantification in 90 bone marrow samples during therapy of 49 children with ALL at diagnosis or relapse were performed with both machines. Both real-time PCR systems provided specific results for MRD quantification in all follow-up samples. In conclusion, we were able to demonstrate that TM and LC real-time PCR technologies produce similar MRD quantification results and that the quantification assays can be easily transferred from one detection system to the other. Using the same detection format, both techniques can be applied in combination in multicenter MRD studies.

Blastic natural killer cell and extranodal natural killer cell-like T-cell lymphoma presenting in the skin: report of six cases from the UK.

Some lymphomas express natural killer (NK)-cell markers such as the neural cell adhesion molecule, which is recognized by the CD56 antibody. These lymphomas may present in the skin, but do not represent a homogeneous group. The new World Health Organization classification of lymphoma/leukaemia recognizes several types of NK/T-cell neoplasm, including blastic NK-cell lymphoma, which characteristically presents with cutaneous lesions. To describe the clinical, pathological and molecular features in six cases of CD56+ lymphoma with cutaneous presentation. The clinical, histopathological and immunophenotypic features of six patients were reviewed. In addition, in situ hybridization (ISH) to identify Epstein-Barr virus (EBV) mRNA, and polymerase chain reaction analysis to identify the presence of a clonal population of T cells or B cells were performed on lesional skin. All patients presented with widespread nodules and plaques, which in five cases were a characteristic purple colour. Four patients developed disseminated disease, three with neurological involvement. These four patients died between 14 and 46 months following diagnosis (median 30 months). In four of six cases the histopathological and immunohistological features were in keeping with a blastic NK-cell lymphoma. No clonal immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangement was detected in the four cases consistent with an origin from NK cells. A further case fitted the criteria for an extranodal NK/T-cell lymphoma of nasal type and was also the only case to show evidence of EBV mRNA by ISH. A clonal T-cell population was identified in the final case. This patient also exhibited molecular evidence of a clonal B-cell population and a t(14;18) translocation confirmed by sequence analysis. Our data confirm that NK-cell lymphomas presenting in the skin are a heterogeneous group, and that in the U.K., blastic NK-cell lymphoma is more common than extranodal NK/T-cell lymphoma of nasal type. These lymphomas pursue an aggressive course, with rapid development of disseminated disease, and resistance to chemotherapy. Detailed immunophenotyping is needed to distinguish the different types. Our molecular data indicate that blastic NK-cell lymphoma cases lack clonal TCR/IgH gene rearrangements consistent with an NK-cell origin. Our ISH findings indicate that EBV plays a pathogenetic role only in extranodal NK/T-cell lymphoma of nasal type.

Primary follicular mucinosis: Long-term follow-up of patients younger than 40 years with and without clonal T-cell receptor gene rearrangement

Since the original descriptions of follicular mucinosis, accumulating experience shows that patient age, distribution of lesions, and duration or extent of disease do not reliably distinguish benign primary follicular mucinosis from secondary follicular mucinosis, associated with cutaneous lymphoma. More recently, it has been suggested that individuals with follicular mucinosis demonstrating a clonal T-cell receptor gene rearrangement may be at higher risk for the development of lymphoma. Long-term follow-up of 7 patients younger than 40 years with primary follicular mucinosis are reported. In all cases, there was no clinical or histologic evidence of associated dermatoses or lymphoma at the time of diagnosis. Five of the patients have clonal T-cell gene rearrangement as determined by Southern blot analysis. Clinically, at the time of diagnosis, lesions of primary follicular mucinosis ranged from papules confined to the face to widespread cutaneous plaques. After a mean follow-up of 10 years (range, 5-23 years) from the onset of disease, the majority of patients continue to have cutaneous manifestations of follicular mucinosis despite various treatments. There is no evidence of progression to cutaneous T-cell lymphoma in any patient despite the presence of a clonal T-cell receptor gene rearrangement. Continued prolonged follow-up of patients with clonal primary follicular mucinosis is necessary to determine the significance of infiltrates harboring a T-cell receptor gene rearrangement. However, in our experience with this group of selected patients, primary follicular mucinosis has been a clonal disorder with limited or “benign” cutaneous manifestations. (J Am Acad Dermatol 2002;47:856-62.)

Deletion 6q as a recurrent chromosomal aberration in T-cell large granular lymphocyte leukemia

T-cell large granular lymphocyte (T-LGL) leukemia is an uncommon disease, with limited information on karyotypic aberrations. No consistent chromosomal changes have thus far been described. We report two cases of T-LGL leukemia who presented with severe anemia. The LGL were CD3 +, CD4 −, CD8 +, CD56 −, and CD161 −, with variable expression of CD94, CD158a, and CD158b, and had clonal T-cell receptor gene rearrangement. A deletion of the long arm of chromosome 6 was the sole aberration in both cases. This is the first report of a recurrent chromosomal aberration in T-LGL leukemia.

Mucinosis folicular: nuevas entidades clínico-patológicas. Estudio clínico, histológico, inmunohistoquímico e inmunogenotípico de 23 pacientes

A clinical, histopathologic, inmunohistochemical and genotypic analysis review of twenty-three patients with follicular mucinosis (FM) is reported.Patients with primary follicular mucinosis (PFM) and secondary follicular mucinosis (SFM) associated with a systemic or cutaneous T-cell lymphoma were included in the study. Biopsy specimens of FM between the period 1975–2000 were selected based solely on the histopathologic finding of folicular mucin deposition from the dermatopathology files. Inmunostaining with monoclonal antibodies against pan T, pan B, T helper and T8 cells as performed in all biopsies. Detection of clonal T-cell receptor gene rearrangements was performed using a polymerase chain reaction (PCR) amplification of the T-cell ©gene from paraffin-embedded tissue in selected cases.Six cases with SFM (30,4%) were evaluated (all men; mean age: 54,1 y). The most frequent associated lymphoma was mycosis fungoides. Sixteen patients with PFM were evaluated (8 men and 8 women: 8 to 72 years). Four different clinical presentations of PFM could be identified after clinical review: localized PFM, diffuse PFM, acneiforme PFM and urticaria-like PFM.No histopathologic features separated clearly PFM from SFM. Only urticaria-like follicular mucinosis showed specific findigns, consisting in intense inflammatory infiltrate with abundant eosinophils. Inmunohistochemical findings showed predominance of T helper cells in all cases. Three cases of clonal PFM were observed. No differences could be detected between clonal cases and non-clonal cases of PFM. With long follow-up no evolution to cutaneous lymphoma was observed in clonal cases of PFM.Detection of PFM with clonal T-cell populations reinforces and expands the recently proposed concept of clonal benign dermatosis. Continued, long-term surveillance of patients with clonal PFM seems advisable.

Hypopigmented mycosis fungoides: frequent expression of a CD8+ T-cell phenotype.

Hypopigmented mycosis fungoides (MF) is a form of cutaneous T-cell lymphoma in which hypopigmentation occurs in the absence of classic lesions of MF. Hypopigmented MF predominantly affects people with dark complexions. The natural history of this variant of cutaneous T-cell lymphoma is similar to that of conventional MF, although the disease onset is usually in childhood or adolescence. In a retrospective study we evaluated the clinical, histopathologic, immunohistochemical, and molecular characteristics of hypopigmented MF in 15 patients. Similar to other reports, the disease onset occurred in childhood and adolescence in most of the cases. The survival rate was comparable with that of classic MF. We did not observe progression to systemic disease or lymph node involvement. Histopathologically hypopigmented lesions were indistinguishable from hyperpigmented or erythematous patches. On immunohistochemical analysis a predominantly CD8+ infiltrate was detected in the majority of cases (nine of 15 patients). To determine whether epidermotropic CD8+ T cells represent the malignant T-cell clone or whether these cells are innocent, tumor-infiltrating T lymphocytes, we performed microdissection of epidermotropic CD8+ T cells and analyzed T-cell receptor-gamma chain gene for rearrangements. The epidermotropic CD8+ T lymphocytes showed clonal T-cell receptor gene rearrangement and therefore represented the malignant T-cell clone. We conclude that hypopigmented MF tends to occur in young people and that it belongs to the group of CD8+ cutaneous T-cell lymphomas in the majority of cases.

Commentary on “Blastic Natural Killer Cell Leukemia/Lymphoma Presenting as Overt Leukemia”

B-cell and T-cell malignancies can be definitively diagnosed by demonstrating clonal rearrangements of their antigen-receptor genes. In contrast, natural killer (NK) cells do not have antigen-receptor genes, making unequivocal assignment of tumors to NK-cell lineage more problematic. Normal NK cells are classically defined functionally as a subset of receptornull lymphocytes that have cytotoxic activity in the absence of prior stimulation. CD56 and CD16 are expressed at variable levels by most normal NK cells and are two of the more commonly used diagnostic markers. However, CD16 is expressed on subsets of myeloid and T cells as well as some myeloid leukemias and T-cell tumors. CD56 expression is a feature of a subset of B-cell and T-cell lymphomas, monocytic leukemias, and approximately 10% of poorly differentiated myeloid leukemias. 1,2 Despite their lack of sensitivity and specificity, CD56 and CD16 remain the most commonly used diagnostic markers for NK-cell tumors. Although more specific markers of NK cells are now available, they are not yet in routine diagnostic use. 3 Recently, immunostaining for cytotoxic granule proteins (eg, TIA-1, perforin, and granzyme B) has become common. The diagnostic value of these markers is unclear given that many types of T-cell tumors (anaplastic large-cell lymphoma and mycosis fungoides) and some B-cell tumors show cytoplasmic immunoreactivity with these antibodies. In everyday practice, NK-cell lineage is commonly assigned to all lymphoid-appearing tumors that show expression of CD2 and/or cytoplasmic CD3 in the absence of T-cell–specific findings, such as surface expression of CD3 or clonal Tcell–receptor gene rearrangements. Using these criteria, a range of NK-cell tumors have been described that exhibit a wide spectrum in their clinical presentation and behavior. These tumors of proposed NKcell origin are summarized in Table 1. Traditionally, these tumors have been divided into lymphomas (ie, those presenting primarily in tissue sites such as the sinonasal area, skin, and gastrointestinal tract) and leukemias. However, many NK-cell lymphomas show a propensity for leukemic involvement and widespread extranodal dissemination leading to the designation of NK-cell lymphoma/leukemia.

Analysis of T-Cell Subpopulations in T-Cell Non-Hodgkin’s Lymphoma of Angioimmunoblastic Lymphadenopathy with Dysproteinemia Type by Single Target Gene Amplification of T Cell Receptor- β Gene Rearrangements

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin's lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4(+) and CD8(+) T cells and proliferating Ki67(+) cells of seven cases were micromanipulated from frozen tissue sections. TCRbeta gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4(+), and not among CD8(+) T cells, indicating that the tumor clones in AILD usually derive from CD4(+) T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRbeta chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.

T-Large Granular Lymphocyte Leukemia Accompanied by an Increase of Natural Killer Cells (CD3-) and Associated with Ulcerative Colitis and Autoimmune Hepatitis

Clonal expansion of large granular lymphocyte(LGL) have been classified into T-LGL and NK-LGL leukemia. T-LGL leukemia cells have a CD3+ phenotype and show clonal T-cell receptor(TCR) gene rearrangement. NK-LGL leukemia cells have a CD3-phenotype and no TCR gene rearrangement. We report a case of T-LGL leukemia accompanied by NK LGL expansions in a 65-year-old man who was observed 3 years earlier to have a LGL lymphocytosis in association with ulcerative colitis(UC) and autoimmune hepatitis (AIH). Phenotypic analysis of peripheral blood by flow cytometry disclosed an increase of both T-LGL(CD3+,CD56-,CD57+,and TCRαβ+) and NK-LGL (CD3-,CD16+,CD56+, and CD57+). Clonal rearrangement of the TCR β gene was detected. A diagnosis of UC and AIH was made on the basis of the X-ray and mucosal biopsy findings of the large intestine, and on the scoring system for diagnosis of AIH, respectively. The disease was nonprogressive, and mesalazine and prednisolone were successful for treatment of UC and AIH. Previously reported cases of T-LGL, NK-LGL leukemia, or NK cell lymphocytosis had no association with UC or AIH, and there have been no reports having both T-LGL leukemia with T-cell receptor gene rearrangement and chronic NK cell lymphocytosis co-existing in a single patient.

Clinicopathologic and genotypic study of extranodal nasal-type natural killer/T-cell lymphoma and natural killer precursor lymphoma among Koreans.

This study aimed to define genotypic profile and to describe the clinicopathologic features of nasal-type natural killer (NK)/T-cell lymphoma of nasal and extranasal origin and NK precursor lymphoma. NK/T-cell lymphomas from the upper aerodigestive tract (n = 45), skin (n = 2), gastrointestinal tract (n = 3), and soft tissue (n = 2) and NK precursor neoplasms (n = 3) were studied. Immunophenotype was analyzed by immunohistochemistry and flow cytometry. In situ hybridization with EBER 1/2 RNA probes was performed. T-Cell Receptor (TCR)-gamma gene rearrangement was analyzed by seminested polymerase chain reaction with heteroduplex analysis. Overall survival rate was correlated with clinicopathologic parameters and compared by Wilcoxon test. Clonal TCR-gamma gene rearrangement was detected in 3 of 31 upper aerodigestive and 1 of 2 skin tumors. When immunostained using paraffin embedded tissue, 6 upper aerodigestive lymphomas were negative for CD56 in which 4 cases lacked clonal TCR gene rearrangement. Epstein-Barr virus (EBV) mRNA was detected in 33 upper aerodigestive tumors including 26 of 29 nasal tumors (90%), and 7 of 10 extranasal tumors (70%). There was no histologic, immunophenotypic, or genotypic differences according to the lineage and EBV association in upper aerodigestive lymphomas. Among the patients with upper aerodigestive tumors, overall 1-year survival rate was 41%, and correlated well with the stage (P < 0.05) but not with the size of tumor cells, EBV status, and lineage (P > 0.05). Median survival rate of lymphomas from other sites excluding upper aerodigestive tract was not significantly different from that of upper aerodigestive lymphomas with same stage (P > 0.05). Unlike nasal-type NK/T-cell lymphomas, NK precursor lymphoma involved the bone marrow and lymph nodes at initial presentation or in the course of disease. Tumor cells were positive for TdT in all and myeloid markers in two. TCR gene rearrangement was germ line. Most upper aerodigestive nasal-type NK/T-cell lymphomas among Koreans are genotypically of NK derivation and few belong to T lineage. Presence or absence of EBV has no significant correlation with the histologic changes and the lineage of these lymphomas.

Frequent T and B Cell Oligoclones in Histologically and Immunophenotypically Characterized Angioimmunoblastic Lymphadenopathy

The identification of clonal rearrangements of T cell receptor (TCR) genes is central to the diagnosis of T cell lymphomas. However, in angioimmunoblastic lymphadenopathy (AILD), first described as a nonneoplastic proliferation associated with immunodeficiency, the heterogeneity of TCR and IgH gene rearrangements suggest that some cases may harbor multiple lymphoid clones. In this study we have isolated DNA from archival paraffin biopsy material from 22 cases of AILD identified on the basis of classical histological and immunohistochemical features with the aim of establishing the occurrence of clones and oligoclones, the frequency of TCR and immunoglobulin heavy chain (IgH) variable (v) gene use, and the relationship of these findings to the presence of Epstein-Barr virus. DNA extracted from the biopsies was amplified using the polymerase chain reaction (PCR) and sequenced to detect functional and nonfunctional gene rearrangements. Epstein-Barr virus-encoded short RNA species (EBERs) were detected using in situ hybridization combined with immunochemistry to identify the phenotype of the Epstein-Barr virus-infected cells. Fifty-seven clonal products were found in 20/22 patients: TCRgamma clonal products were identified in 16/22, TCRbeta clonal products in 16/22 and IgH clonal products in 6/22 cases. Oligoclonal PCR products were seen for TCR in 3/22 and for IgH in 3/22 cases. In one biopsy PCR products from all reactions were polyclonal. Sequence analysis revealed functional TCRgamma, TCRbeta, and IgH sequences in 6/12, 9/11, and 8/8 cases, respectively. Functional TCR and/or IgH oligoclones were detected in 6/20 (30%) cases. In addition, nonfunctional TCR and IgH sequences were found in 11 cases. EBERs were identified in 18/20 cases varying from occasional to 25 to 30% nuclei staining and were associated with both T and B cells, although the majority were of indeterminate phenotype. The presence of EBERs was not associated with all clonal IgH gene rearrangements but was associated with B cell oligoclones. Patterns of gene recombinations indicated that the majority of TCRgamma recombinations used GV1 and GJ1S3/2S3 genes. Six out of eleven cases used TCR BV4S1 or BV2S1 genes associated with various BJ and BD1/2 genes. No common IgH gene usage was identified, but 8 clones had varying degrees of replacement and silent mutations (0.6-10.1%), consistent with B cell clones having undergone somatic mutation in the germinal center, and 3 clones harbored unmutated V genes, consistent with naive B cells. Our data do not support the concept of AILD as a clearly defined peripheral T cell lymphoma (PTCL). Rather, they suggest that AILD as defined by histology and immunohistochemistry is either a heterogeneous entity or represents a lymphoproliferation associated with immunodeficiency in which clonal T cell or B cell proliferation may occur.

A long-term study of patients with chronic natural killer cell lymphocytosis.

Chronic natural killer cell lymphocytosis is a persistent state of natural killer (NK) cell (CD3-CD16/CD56+) excess in the peripheral blood that is not associated with clinical lymphoma. In 16 consecutive patients (median age 60.5 years, range 7-77), males were overrepresented (M:F 7:1) and the median absolute NK cell count was 4.09 x 10(9)/l (range 1.2-16.6). Bone marrow examination was performed in 14 patients and showed atypical granulomata in two; chromosome studies in seven patients were normal. Clonal T-cell receptor gene rearrangement was not found in any of 12 patients evaluated. At presentation, seven patients (44%) had no clinical symptoms or signs and the others had vasculitic skin lesions (three patients), non-neutropenic fever (three patients), recurrent neutropenic infection (two patients), musculoskeletal symptoms (two patients), peripheral neuropathy (two patients), aphthous ulcers (one patient), and splenomegaly (one patient). Five patients had anaemia, five had neutropenia, and two had thrombocytopenia. After a median follow-up of 5.1 years (range 0-10.2) from immunophenotypic diagnosis or 5.7 years (range 0.1-14.1) from documentation of absolute lymphocytosis, vasculitic glomerulonephritis developed in one patient, accelerated splenomegaly developed in a patient receiving myeloid growth factor treatment, and severe aplastic anaemia developed in one patient. Treatment with nonsteroidal anti-inflammatory drugs or immunosuppressive agents was variably successful.