Clonal T-cell Population Research Articles

Durable complete molecular remission of chronic myeloid leukemia following dasatinib cessation, despite adverse disease features

Patients with chronic myeloid leukemia, treated with imatinib, who have a durable complete molecular response, might remain in complete molecular response after stopping treatment. Previous reports of patients stopping treatment in complete molecular response have included only patients with a good response to imatinib. We describe 3 patients with stable complete molecular response on dasatinib treatment following imatinib failure. Two of the 3 patients remain in complete molecular response more than 12 months after stopping dasatinib. In these 2 patients we used highly sensitive patient-specific BCR-ABL1 DNA PCR to show that the leukemic clone remains detectable, as we have previously shown in imatinib-treated patients. Dasatinib-associated immunological phenomena, such as the emergence of clonal T-cell populations, were observed both in one patient who relapsed and in one patient in remission. Our results suggest that the characteristics of complete molecular response on dasatinib treatment may be similar to that achieved with imatinib, at least in patients with adverse disease features.

In immunocompromised patients, Epstein-Barr virus lymphadenitis can mimic angioimmunoblastic T-cell lymphoma morphologically, immunophenotypically, and genetically: a case report and review of the literature

The development of lymphomas and solid malignancies in association with immunosuppression is a well-documented occurrence in the medical literature. We report the case of a young man who developed progressive diffuse lymphadenopathy with associated extremely high levels of serum Epstein-Barr virus in the setting of chronic immunosuppressive treatment of glomerulonephritis. Excisional biopsy of a right inguinal node revealed a sclerosing process with the morphologic appearance of angioimmunoblastic T-cell lymphoma with a CD3(+), CD4(+) immunophenotype. In situ hybridization of Epstein-Barr virus-encoded RNA was positive. Molecular probe studies demonstrated a clonal T-cell population. Upon reduction of immunosuppression, the patient's lymphadenopathy and Epstein-Barr virus titer have resolved without recurrence over 2 years time. This case demonstrates that a benign Epstein-Barr virus-associated process can mimic angioimmunoblastic T-cell lymphoma and should be considered particularly in the setting of immunosuppression, emphasizing the need for close communication with the treating physician in the interpretation of lymph node biopsies.

Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL

Chronic lymphocytic leukemia (CLL) cells require complex microenvironmental and immunologic interactions to survive and proliferate. Such interactions might be best recreated in animal models; however, this needs extensive verification. We therefore investigated the composition of the T-cell compartment in the Eμ-TCL1 transgenic mouse, currently the most widely used murine model for CLL. Immunophenotyping and transplant approaches were used to define T-cell subsets at various stages of CLL. Analogous to human CLL, we observed a skewing of T-cell subsets from naive to antigen-experienced memory T cells that was more pronounced in lymph nodes than in blood. Transplantation of CLL into non-transgenic recipients was feasible without immunosuppression in a pure C57BL/6 background and resulted in the prominent skewing of the T cells of the recipient mice. Both in spontaneously developed CLL and in the transplantation setting, a loss in T-cell receptor diversity was observed, with a relevant number of clonal T-cell populations arising. This suggests that antigen-dependent differentiation toward the T memory pool is initiated by murine CLL cells. In summary, we validate the TCL1 transgenic mouse model for analysis of T-cell phenotypes and suggest a CLL-dependent antigen-driven skewing of T cells in these mice.

In‐vivo purging of the circulating clonal T‐cells with Alemtuzumab prior to autologous peripheral blood hematopoietic cell mobilization and transplantation

<i>In‐vivo</i> purging of the circulating clonal T‐cells with Alemtuzumab prior to autologous peripheral blood hematopoietic cell mobilization and transplantation

Molecular immunology lessons from therapeutic T-cell receptor gene transfer.

The T-cell receptor (TCR) is critical for T-cell lineage selection, antigen specificity, effector function and survival. Recently, TCR gene transfer has been developed as a reliable method to generate ex vivo large numbers of T cells of a given antigen-specificity and functional avidity. Such approaches have major applications for the adoptive cellular therapy of viral infectious diseases, virus-associated malignancies and cancer. TCR gene transfer utilizes retroviral or lentiviral constructs containing the gene sequences of the TCR-alpha and TCR-beta chains, which have been cloned from a clonal T-cell population of the desired antigen specificity. The TCR-encoding vector is then used to infect (transduce) primary T cells in vitro. To generate a transduced T cell with the desired functional specificity, the introduced TCR-alpha and TCR-beta chains must form a heterodimer and associate with the CD3 complex in order to be stably expressed at the T-cell surface. In order to optimize the function of TCR-transduced T cells, researchers in the field of TCR gene transfer have exploited many aspects of basic research in T-cell immunology relating to TCR structure, TCR-CD3 assembly, cell-surface TCR expression, TCR-peptide/major histocompatibility complex (MHC) affinity and TCR signalling. However, improving the introduction of exogenous TCRs into naturally occurring T cells has provided further insights into basic T-cell immunology. The aim of this review was to discuss the molecular immunology lessons learnt through therapeutic TCR transfer.

Collagenous Sprue

Collagenous sprue is a rare form of small bowel enteropathy characterized by chronic diarrhea and progressive malabsorption with little data available on its natural history. The pathologic lesion consists of subepithelial collagen deposition associated with variable alterations in villous architecture. The small bowel biopsies of 12 cases were reviewed. Clinical details, celiac serology, and T-cell receptor gene rearrangement study results, when available, were collated. There were 8 females and 4 males (age ranged from 41 to 84 y) who presented with chronic diarrhea and weight loss. Small intestinal biopsies showed subepithelial collagen deposition with varying degrees of villous atrophy and varying numbers of intraepithelial lymphocytes. Four patients had previous biopsies showing enteropathic changes without collagen deposition. Seven cases were associated with collagenous colitis and 1 also had features of lymphocytic colitis. Three patients also had collagen deposition in gastric biopsies. One case was associated with lymphocytic gastritis. Celiac disease (CD, gluten-sensitive enteropathy) was documented in 4 patients. Five patients made a clinical improvement with combinations of a gluten-free diet and immunosuppressive therapy. Two patients died of complications of malnutrition and 1 of another illness. Clonal T-cell populations were identified in 5 of 6 cases tested. Four of these patients improved clinically after treatment but 1 has died. Collagenous sprue evolved on a background of CD in 4 cases. There was no history of CD in others and these cases may be the result of a biologic insult other than gluten sensitivity. None has developed clinical evidence of lymphoma to date.

Detection of Clonal T Cells in the Circulation of Patients With Nephrogenic Systemic Fibrosis

Nephrogenic systemic fibrosis is a sclerodermalike disease in patients with acute or chronic renal insuffiency related to administration of gadolinium-containing contrast agents. Previous studies have demonstrated clonal T-cell populations in the blood of patients with systemic sclerosis, suggesting that these cells may be involved in the pathogenesis of the disease. Facing the clinical similarities of both diseases, we hypothesized that clonal expansion of T cells could be present in nephrogenic systemic fibrosis as well. Findings from polymerase chain reaction and high-resolution capillary electrophoresis for T-cell receptor gamma gene rearrangement analysis showed that all 6 prospectively evaluated patients (100%) with nephrogenic systemic fibrosis had detectable clonal T cells in their peripheral blood. In contrast, only 4 of the 15 control patients (27%) with chronic renal failure and none of the 12 healthy individuals analyzed in this study had evidence for T-cell clonality using the same type of examination. Clonal T-cell-positive patients with systemic sclerosis have previously been reported to better respond to extracorporeal photopheresis. However, this was not the case in 2 of our patients with nephrogenic systemic fibrosis. Conclusion As in systemic sclerosis, clonally expanded T-cell populations could play a critical role in the pathogenesis of nephrogenic systemic fibrosis, probably as an in vivo-activated inflammatory response to gadolinium exposure.

Lymphocytic vasculitis involving the central nervous system occurs in patients with X‐linked lymphoproliferative disease in the absence of Epstein–Barr virus infection

X-linked lymphoproliferative disease (XLP) is an immunodeficiency caused by defects in the adaptor molecule SAP. The manifestations of XLP generally occur following Epstein-Barr virus (EBV) infection and include fulminant mononucleosis, hypogammaglobulinemia and lymphoma. In this report, we describe two unrelated patients with fatal T-cell-mediated central nervous system vasculitis for whom repeated serologic and molecular testing for EBV was negative. In both patients, clonal T-cell populations were observed, but neither demonstrated evidence of lymphoma. Thus, loss of SAP function can lead to dysregulated immune responses characterized by the uncontrolled expansion and activation of T cells independent of EBV infection.

Aplastic anaemia: autoimmune sequel of thymoma

A 67-year-old male presenting with persistent cough was found to have a large anterior mediastinal tumour (top). He underwent mediastinotomy and resection of a thymic tumour, which was subsequently diagnosed as a benign thymoma (bottom left). Six months post-thymectomy he presented with symptomatic anaemia, with full blood examination revealing severe thrombocytopenia in addition to the anaemia: haemoglobin 66 g/l, white cell count 5AE2 · 10/l, platelet count 8 · 10/l, and neutrophils 1AE5 · 10/l. There had been no recent exposure to drugs or chemicals known to cause marrow aplasia and serology for parvovirus and hepatitis B and C were negative. Bone marrow trephine biopsy showed a markedly hypocellular marrow (cellularity c. 10%) with few haemopoietic elements present, consistent with evolving aplastic anaemia (bottom right); there was no clonal T-cell population or evidence of paroxysmal nocturnal haemoglobinuria detected by flow cytometry. A computed tomography scan showed no evidence of recurrent thymoma. The patient was treated with anti-thymocyte globulin and ciclosporin; the most recent blood tests showed a near complete response at 10 weeks. Aplastic anaemia is a rare complication of thymoma occurring concurrently in c. 1% of cases. Aplastic anaemia following thymectomy is exceedingly rare with only five other cases identified by us in the literature. Thymoma-related aplastic anaemia often fails to respond to thymectomy requiring immunosuppressive therapy for count recovery. Autoreactive T-cell clones generated by the thymoma are postulated to be the cause of aplastic anaemia and other autoimmune manifestations. The occurrence of aplastic anaemia as a late autoimmune complication following resection of thymoma indicates that autoreactive T-cell clones may persist post-thymectomy leading to late rather than concurrent autoimmune haematological manifestations.

Clonal T-cell populations are frequent in the skin and blood of patients with systemic sclerosis

Pilot studies were suggestive for a role of clonal T cells in the pathogenesis of systemic sclerosis (SSc). To investigate the presence of clonal T cells in both peripheral blood and skin of a large collection of patients with SSc. Polymerase chain reaction and high-resolution capillary electrophoresis for detecting T-cell clonality were performed in a series of 126 patients with SSc. Seventy-seven (61%) of 126 patients had clonal T cells in their peripheral blood. In contrast, a clonal T-cell population was present in only four of 29 (14%) age-matched healthy controls (P = 0.03). Older patients were more likely to have clonal T cells than younger patients with SSc (P < 0.0001). Clonal T cells were more commonly detected in the blood of patients with limited cutaneous SSc (48 of 65 patients, 74%) than in those with diffuse cutaneous disease (29 of 61, 48%; P = 0.0002). Lesional skin specimens from 20 of 44 patients (45%) had detectable clonal T-cell populations. There was no correlation between the presence of circulating clonal T cells and lesional clonal T cells, sex, disease duration, extent of skin sclerosis, digital ulcers, organ involvement (e.g. interstitial lung disease, kidney disease, oesophagus involvement), treatment of SSc, or autoantibody profile. Many patients with SSc have expanded clonal T cells in their peripheral blood and skin. These clonal T cells could play a critical role in the pathogenesis of SSc, especially in limited cutaneous disease.

Molecular identity of skin and blood T‐cell clones in cutaneous T‐cell lymphoma patients as determined from the migration pattern of the T‐cell receptor‐γ gene by capillary electrophoresis

The presence of a dominant clonal T-cell population in skin lesions is an important clue in the diagnosis of cutaneous T-cell lymphoma (CTCL). However, it has never been determined whether dominant T-cell receptor (TCR) rearrangements identified in skin lesions and blood from CTCL patients, displaying strictly identical migration patterns by capillary electrophoresis, actually correspond to identical clones. As this information has potential clinical relevance, TCR-gamma (TCRG) gene-derived amplified fragments from dominant blood and skin T-cell clones featuring either identical or slightly different capillary electrophoresis migration patterns were analyzed in eleven CTCL patients. In nine patients with identical electrophoretic migration patterns, sequence analyses revealed the dominant skin and blood T-cell clones to be identical. In contrast, in two patients displaying slight migration differences between skin and blood samples, the TCRG sequences were distinct. Additionally, capillary electrophoresis appears more sensitive and accurate than heteroduplex analysis and in silico analysis of samples of different origins is possible a posteriori. These results demonstrate the efficacy of capillary electrophoresis in assessing molecular identity and discrepancy of dominant T-cell populations obtained from different tissues or at different times, facilitating diagnosis and follow-up.

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.

Dominance of Nonmalignant T-Cell Clones and Distortion of the TCR Repertoire in the Peripheral Blood of Patients with Cutaneous CD30+ Lymphoproliferative Disorders

The CD30-positive cutaneous lymphoproliferative disorders (CLPD) include lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large T-cell lymphoma (cALCL). Despite the malign-appearing histology, an excellent prognosis and spontaneous regression of single lesions characterize LyP. Even after years of clinical remission newly erupting lesions often harbor a T-cell clone identical to the initial one. This fact raises the question whether the clonal T-cell population persists in the peripheral blood. Therefore we investigated genomic DNA of 126 samples of lesional skin and peripheral blood from 31 patients with CLPD, obtained during both active disease and clinical remission. We performed molecular genetic analysis by combining T-cell receptor (TCR)-gamma PCR with the GeneScan technique and assessed the TCR repertoire in selected blood samples by beta-variable complementarity-determining region 3 (CDR3) spectratyping qualitatively and quantitatively. We were able to detect a clonal T-cell population in 36/43 (84%) skin samples and in 35/83 (42%) blood samples. Comparison of the compartments in each patient demonstrated different T-cell clones in skin and blood, suggesting a reactive nature of the clonal T cells in the blood. Moreover, CDR3 spectratyping revealed a restricted T-cell repertoire in the blood, suggesting T-cell stimulation by an unknown antigen.

Case report of four patients with erythrodermic cutaneous T-cell lymphoma and severe photosensitivity mimicking chronic actinic dermatitis

The marked photosensitivity associated with chronic actinic dermatitis (CAD) is presumed to be due to a T cell-mediated response to ultraviolet (UV)-induced epidermal neoantigens. Photosensitivity is, however, a rare occurrence in cutaneous T-cell lymphoma (CTCL). We discuss a series of four patients with erythrodermic CTCL who exhibited marked photosensitivity mimicking CAD. Significantly, the tumour cells had a CD8 phenotype in half of these patients. All patients had T-cell clones in skin and also demonstrated identical peripheral T-cell clones in blood or lymph node involvement. Sézary cell counts ranged from 6% to 20%, CD4/CD8 ratios from 0.22 to 23.5. Clinical presentation was striking for a marked photosensitive distribution. Monochromator irradiation testing revealed reduced minimal erythema doses throughout UVB and UVA ranges, findings consistent with those seen in CAD. All patients subsequently died from systemic disease. These findings suggest that, rarely, malignant clonal T-cell populations may recognize a unique UV-induced neoantigen, resulting in the clinical features of severe photosensitivity mimicking those seen in CAD.

CD20+ T-cell Lymphoma

Rare cases of CD20+ T-cell lymphoma (TCL) have been reported, but the clinicopathologic spectrum of this disorder is not known. We identified 9 cases of CD20+ TCL diagnosed at our institution and 26 additional cases through a search of the English language literature. Among current cases, there were 7 men (ages 71 to 81, median 75 y) and 2 women (ages 36 and 37 y). Five patients presented with predominantly nodal disease (localized in 3 and widespread in 2 cases) and 4 patients presented with purely extranodal disease involving the parotid glands, skin, or small intestine. CD20 was uniformly and strongly expressed in 5 cases and dimly expressed or present on a subset of neoplastic cells in 4 cases. The proportion of CD20+ T cells changed over time in 3 cases. Three cases fulfilled diagnostic criteria for clinicopathologically defined subtypes of TCL (2 mycosis fungoides; 1 enteropathy-type TCL), whereas 6 were peripheral TCL unspecified with variable cytomorphology, T-cell immunophenotype, and sites of involvement. In 8 of 9 cases, a clonal T-cell population was identified by molecular genetic analysis. Among 8 cases with clinical follow-up, 5 behaved aggressively with death from disease within 3 years of diagnosis in 4 cases (median survival: 11 mo, range: 1 to 35 mo), and recurrent disease at 10 months in 1 case; 1 patient died of an EBV+ B-cell lymphoma (BCL) 66 months after the original diagnosis; in the remaining 2 cases, patients were alive and undergoing treatment (follow-up: 4 and 18 mo). Historical cases showed similar clinicopathologic variability. CD20+ TCL is rare, and clinically and pathologically heterogeneous. When CD20 expression is present in TCL, it may be dimmer than that of normal B cells, suggesting neoplastic transformation of a normal CD20dim+ T-cell subset. Cases of CD20+ TCL in which the proportion of CD20+ cells changes over time may reflect aberrant expression of CD20, possibly as an activation marker, by neoplastic T cells. CD20+ TCL may cause diagnostic difficulty, particularly in cases that clinically and pathologically mimic BCL. Knowledge of the unusual phenomenon of CD20 expression in TCL, in conjunction with careful morphologic analysis, the use of a panel of antibodies, and molecular genetic studies, is important in avoiding a misdiagnosis of BCL.

Relative quantification of TCR Vbeta-chain families by real time PCR for identification of clonal T-cell populations

BackgroundQuantification of T-cell receptor (TCR) chain families can be utilized for detection of clonal T-cell populations. Besides southern blotting and antibody-based approaches, quantitative real time PCR (qRT PCR) has been more widely applied in this context during the last years. Here, the heterogeneity of sequences within single families is the most challenging problem for exact quantification.MethodVβ-families were quantified using a universal reverse primer and family-specific forward primers with TaqMan technology on a light cycler instrument. Relative concentrations were calculated considering slopes and crossing points of each PCR reaction. Total expression of α/β TCR was assessed by quantification of the constant α-chain as a further control.ResultsThe method was tested by serial dilutions of clonal T-cells in mononuclear cells from healthy volunteers. Calculated percentages were in good correspondence with qRT PCR results demonstrating high reliability. Duplicates showed excellent technical reproducibility. We analyzed blood samples of 20 healthy volunteers for determination of mean and standard deviation for each family. The method was applied both to tissue and blood samples from patients with carcinomas and hematological disorders.ConclusionWe introduce a versatile method for the relative quantification of Vβ-families by real time PCR. The experimental strategy described allows the identification of alterations in the Vβ-family repertoire.

Regarding the algorithm for the diagnosis of early mycosis fungoides proposed by the International Society for Cutaneous Lymphomas: suggestions from routine histopathology practice

Routine clinicopathologic practice is expected to refine/validate the scoring system proposed in 2005 by the International Society for Cutaneous Lymphomas (ISCL) for the diagnosing early mycosis fungoides (eMF), classical type. An evaluation of 72 cases of erythematous and scaling dermatoses was employed with a partial implementation of the ISCL algorithm. The selected cases fulfilled the clinical criteria proposed by the ISCL; routine histopathology allowed to reach the ISCL minimum score for a diagnosis of eMF in 45 cases. A clonal T-cell population was found in 4 out of 12 cases tested with the polymerase chain reaction, three of which with an already established clinicopathologic diagnosis of eMF. An aberrant immunophenotype was found in 11 cases, all of which already labeled as eMF on the basis of clinical and histopathologic features. 6 out of 27 patients with inconclusive clinicopathologic data underwent a new skin biopsy, which allowed to reach a diagnosis of eMF in two cases. The diagnosis of eMF still rests upon clinical features and conventional histology; a new skin biopsy is recommended for cases with no clear-cut diagnostic features.

Primary Cutaneous Follicle Center Lymphoma Associated with Alopecia Areata

We report a patient who presented simultaneously with primary cutaneous follicle center lymphoma (PCFCL) of the face and scalp and alopecia areata of the scalp and beard bearing a clonal T-cell receptor gene rearrangement. To our knowledge, alopecia areata has not been previously reported in association with PCFCL. Both lesions regressed with topical imiquimod and systemic steroids, which suggests an inter-relationship in this case between the clonal B-cell and T-cell populations in driving outgrowth of these lesions.

TCR Gene Rearrangement in B-Cell Chronic Lymphocytic Leukaemia.

Background: Chronic lymphocytic leukaemia (B-CLL) is a heterogenous B cell disorder. However, clonal/oligoclonal T cell expansion has also been detected at a low frequency in B-CLL using a range of methodologies including Southern blotting and flow cytometric analysis. The underlying cause of this phenomenon is unclear and it has even been suggested that this clonal T cell receptor (TCR) gene rearrangement occurred in the leukaemic cells, leading to the concept of lineage infidelity. Other suggestions include that the observed T cell clonality may simply be related to the normal aging process or that it may result from the immune response to malignant cells or viral infection. During normal T cell maturation in the thymus, the TCR genes undergo a complex process of V(D)J segment rearrangement to produce a wide repertoire of antigen specificity. As it is an early event in T cell development, TCRγ gene rearrangement is commonly used as a marker of clonality. The TCRβ gene is also routinely investigated due to its greater combinatorial diversity. Lack of sufficiently sensitive detection methodology is often a limiting factor in the discrimination between clonal and polyclonal T-cell populations.Aims: The aim of this study was to use high resolution heteroduplex gel and fluorescence GeneScan analysis to screen for TCRγ and TCRβ gene rearrangements respectively in a cohort of B-CLL patients and to ascertain if the clonal rearrangement occurred in B cells or T cells.Methods: B and T cells were purified from peripheral blood from 34 B-CLL patients using a CD19+ or CD3+ magnetic bead system (autoMACS). DNA was extracted from purified B or T cells and the TCRγ and TCRβ genes were amplified by BIOMED-2 multiplex PCR assays (InVivoScribe Technologies). Heteroduplex and GeneScan analysis were then performed to detect clonal TCR gene rearrangments.Results: Clonal TCR gene rearrangements were detected in11/34 (32%) of cases in purified T cell fractions. No clonal TCR gene rearrangements were found in purified B cell fractions. Clonal TCRβ rearrangements were found in 10/34 (29%) and clonal TCRγ in 8/34 (23%). In most instances a weak clonal pattern was observed. Clonal TCR rearrangements were detected in 7 males and 4 females. Five patients had mutated IGHV genes, while the remaining 6 possessed unmutated IGHV genes. Seven patients presented with more advanced clinical stage (Binet B or C), and 8 patients received treatment.Summary/conclusions: These results are in agreement with previous work demonstrating that T-cell clonal/oligoclonal expansions occur frequently in B-CLL patients. However this is the first report to demonstrate that the clonal expansions occur in T cells and not in B cells. In most instances a weak clonal pattern was observed which was probably due to minor clonal T-cell populations. It is possible that T-cell clonality may be associated with a poorer prognosis in this disease category. Of interest, we found that 8/11(73%) patients in our B-CLL sub-group with T-cell expansion also had advanced stage disease and/or unfavourable molecular (IGHV gene usage/mutational status) markers. We are currently investigating the possibility of a specific antigenic stimulus resulting in this clonal T-cell expansion.

Budesonide in the Treatment of Refractory Celiac Disease

Corticosteroids are used in patients with refractory celiac disease. In order to minimize their systemic side effects, we assessed the role of a locally active sustained release corticosteroid with minimal systemic bioavailability in patients with refractory celiac disease in an open labeled noncontrolled study. Patients who received budesonide for refractory celiac disease were classified according to whether they were primarily or secondarily unresponsive to the diet, and whether they had a polyclonal (type I) or clonal (type II) expansion of intraepithelial lymphocytes. The response to budesonide was assessed globally and by reduction in bowel movements. Patients (N = 29, 72% female) received budesonide for a mean of 6.7 +/- 8.5 months, 5 patients (18%) had type II disease (clonal T-cell population); 76% responded to the medication, 55% completely. Response occurred when budesonide was used alone or with oral corticosteroids and/or azathioprine. There was an objective improvement in the number of bowel movements in those that responded. Response occurred in those with either primary or secondary refractory disease and in those with type II disease, irrespective of the presence of microscopic colitis (N = 7). There was no improvement in the duodenal biopsy over the study period and there were no side effects of budesonide. Budesonide may be of value in the management of refractory celiac disease.