T-cell Clonality Analysis Research Articles

Clonal Hematopoiesis and Bone Marrow Infiltration in Patients With Follicular Helper T-Cell Lymphoma of Angioimmunoblastic Type

Follicular helper T-cell (TFH) lymphoma harbors recurrent mutations of RHOAG17V, IDH2R172, TET2, and DNMT3A. TET2 and DNMT3A mutations are the most frequently affected genes in clonal hematopoiesis (CH). The aim of our study was to investigate the frequency of CH in bone marrow biopsies (BMB) of TFH/angioimmunoblastic T-cell lymphoma (TFH-AITL) patients and its association with myeloid neoplasms. A total of 29 BMB from 22 patients with a diagnosis of TFH–AITL were analyzed by next-generation sequencing (NGS) with a custom panel. Morphologically, 5 BMB revealed that TFH–AITL infiltrates of >5% of bone marrow (BM) cellularity confirmed in 4 cases by NGS-based T-cell clonality. IDH2R172 was demonstrated only in 1 (3%) of 29, and RHOAG17V in 2 (7%) of 29 samples. TET2 and DNMT3A were identified in 24 (83%) of 29 and 17 (59%) of 29 BMB, respectively. In the parallel lymph node the frequencies of mutations were 27% (IDH2R172), 64% (RHOAG17V), 86% (TET2), and 50% (DNMT3A). TET2 and/or DNMT3A mutations identical in lymph node and BMB were present in 18 (82%) of 22 patients, regardless of BM infiltration. In 3 cases the CH mutations were detected 13, 41, and 145 months before TFH–AITL diagnosis. Cases with TET2/DNMT3A mutations and BM variant allele frequencies >40% (7/18, 39%) showed lower blood counts. However, only low platelet count was statistically significant (P = .024). Myeloid neoplasms and/or myelodysplastic syndrome-related mutations were identified in 4 cases (4/22; 18%); all with high TET2 variant allele frequencies (>40%; P = .0114). In conclusion, CH is present in 82% of TFH–AITL and can be demonstrated up to 145 months before TFH–AITL diagnosis. NGS T-cell clonality analysis is an excellent tool to confirm TFH–AITL BM infiltration. Concurrent myeloid neoplasms were identified in 18% of the cases and were associated with TET2 mutations with high allelic burden (>40%). We demonstrated that myeloid neoplasms might occur simultaneously or precede the diagnosis of TFH lymphoma.

OA14 Subcutaneous panniculitis-like T-cell lymphoma: a mimic of lupus erythematous panniculitis

Abstract Background/Aims Lupus erythematosus panniculitis (LEP) is a rare variant of cutaneous lupus erythematosus characterised by tender, erythematous subcutaneous nodules or plaques on the face, trunk and proximal extremities. Without treatment, profound lipoatrophy frequently occurs, which often has a devastating psychosocial impact. Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare form of cutaneous T-cell lymphoma, which may appear in a similar manner. SPTCL initially preferentially infiltrates the subcutaneous adipose tissue and symptoms vary widely depending on the stage of the disease. Early-stage disease presents as painless self-resolving subcutaneous nodules on limbs and trunk. Methods We present the case of a young woman who presented with progressively developing, widespread painful cutaneous lesions, pyrexia of unknown origin and general malaise for one month. She had a history of hurthle cell neoplasm and a strong family history of malignancy. Initial examination revealed 1-2 cm firm, skin-coloured subcutaneous nodules over her lower back, chest and thigh, with groin and axillary lymphadenopathy. Further lesions progressively developed over a three-month period, with lesions turning increasingly erythematous and with associated ulceration. Of note, a significant periorbital lesion appeared, associated with an ipsilateral facial palsy. Results Initial bloods revealed an elevated LDH with cholestatic liver function tests. PET-CT imaging showed an enlarged axillary node with extensive pathology throughout the subcutaneous fat of unknown aetiology. Due to the family history, underlying haematological malignancy and atypical Sweet’s were considered for this unusual presentation. Seropositivity for anti-Ro and ENA, alongside raised angiotensin converting enzyme, broadened the differentials to include LEP, mixed atypical connective tissue disease and cutaneous sarcoid. Notably, complement, myositis panel and creatine kinase were normal. Lupus vulgaris was considered due to high local prevalence and excluded with T-SPOT testing. Histopathological analysis of lesional skin from the upper arm found predominantly subcutaneous infiltrates of atypical lymphoid cells with irregular hyperchromatic nuclei and rimming of individual fat cells. T-cell clonality analysis detected clonal T-cell receptor beta (V-J and D-J) gene rearrangements consistent with SPTCL. Her orbital and cutaneous lesions responded well to six cycles of CHOEP (cyclophosphamide, hydroxydaunorubicin, oncovin, etoposide, prednisone) and the patient returned to functional baseline. Conclusion This case demonstrates the diagnostic difficulties when managing a patient with relapsing remitting panniculitis. Whilst the initial presentation of SPTCL here was classical, with widespread multifocal subcutaneous nodules, the appearance of facial symptoms in SPTCL is rare. Diagnostic uncertainty is frequent in both LEP and SPTCL due to the initial non-specific cutaneous presentation. This case emphasises the importance of clinicopathological correlation for accurate diagnosis. Interestingly, up to 20% of SPTCL patients have concomitant autoimmune diseases, and there is evidence suggesting a potential association between LEP and subcutaneous SPTCL beyond chance, leading some researchers to propose that these two entities exist along a spectrum. Disclosure D. Li: None. L. Spencer: None.

BioIns-PO-08 - Use of the T cell receptor B-chain constant domains 1 and 2 (TCRBC1 and TCRBC2) studies for the analysis of T-cell clonality

BioIns-PO-08 - Use of the T cell receptor B-chain constant domains 1 and 2 (TCRBC1 and TCRBC2) studies for the analysis of T-cell clonality

The combination of clinical parameters and immunophenotyping of intraepithelial lymphocytes allows to assess disease severity in refractory celiac disease

BackgroundFlow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis. AimsTo investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease. MethodsFlow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data. ResultsFlow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients’ allocation (sensitivity 100%, specificity 96%). ConclusionImmunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy.

Bio-O1-01 - Diagnostic performance of high throughput sequencing of the T-cell receptor beta gene for the diagnosis of cutaneous T-cell lymphoma

Bio-O1-01 - Diagnostic performance of high throughput sequencing of the T-cell receptor beta gene for the diagnosis of cutaneous T-cell lymphoma

Abstract PS17-07: Impact of chemotherapy (chemo) on t-cell maturation and clonal proliferation in early-stage and metastatic breast cancer

Abstract Background Adaptive immunity is initiated by T-cell receptor (TCR) engagement with cognate tumor antigen, leading to T-cell maturation (i.e. conversion from naïve to effector state), clonal proliferation, and potentially tumor elimination. Therefore, broad T-cell repertoire diversity could be an important determinant of anti-tumor immunity. In breast cancer, chemo may facilitate adaptive immunity, but conversely may also be lymphotoxic. We characterize the impacts of curative-intent chemo and palliative combination chemo/immune checkpoint inhibition (ICI) on lymphocyte quantity, maturation, and clonal proliferation. Methods Peripheral blood mononuclear cells (PBMCs) were collected at baseline and serially in stage I-III subjects receiving curative-intent chemo (dose-dense doxorubicin, cyclophosphamide, paclitaxel, n=20), and in stage IV subjects receiving palliative ICI (pembrolizumab) plus chemo (paclitaxel, n=15; or capecitabine, n=14). Flow cytometry was conducted on fresh PBMCs to minimize cellular losses related to cryopreservation. DNA was extracted for T-cell clonality analysis using the immunoSEQ Assay (Adaptive Biotechnologies) at deep resolution. T-cell richness, calculated using the iChao1 richness estimator was used to assess overall T-cell diversity. Results Both dose-dense chemo and palliative chemo/ICI were lymphodepleting, however dose-dense chemo was associated with greater reductions in CD4+ naïve T cell count (week 8 counts 34% vs. 96% of baseline, p<.001) and T-cell richness (week 8 richness 57% vs 90% of baseline, p<.001). These effects were durable, with reductions in lymphocyte subsets (ALC, CD4, CD8, naïve, effector, and central memory subsets) and T-cell richness (estimated rearrangements 4.37 vs 3.29 x 105/sample) sustained at metastatic relapse. T-cell richness correlated with CD4+ naïve cell proportion (R2=0.52), suggesting a major contribution of CD4+ naïve cells to overall T-cell diversity. In this dataset, acute memory T-cell expansions were observed following initiation of chemo, but only among younger patients (age<60). Conclusions Curative-intent cytotoxic chemotherapy durably alters the quantity and diversity of peripheral T-cells, particularly of naïve cells, contributing to lymphopenia and reduced T-cell richness at metastatic relapse. Acute expansions of EM subsets were also observed but only in younger individuals. These contrasting immunologic effects highlight the potential importance of sequencing of ICI relative to chemo, and raise concerns regarding age-related thymic involution and immunosenescence. Ongoing research is warranted to investigate the predictive/prognostic utility of T-cell diversity/quantity in breast cancer, and whether thymic T-cell regeneration can be targeted therapeutically to enhance immunotherapy response, for example with cytokine therapies (IL-12 or IL-7, NCT04095689) or sex steroid inhibition (NCT03650894). Citation Format: Brie M Chun, Joanna Pucilowska, Valerie Conrad, Mark Schmidt, Isaac Kim, Paul Fields, Alison Conlin, Heather McArthur, David B Page. Impact of chemotherapy (chemo) on t-cell maturation and clonal proliferation in early-stage and metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-07.

Clinical Experience With Modified, Single-Tube T-Cell Receptor Vβ Flow Cytometry Analysis for T-Cell Clonality.

Analysis of T-cell clonality using the IO Test Beta Mark (Beckman Coulter, Fullerton, CA) flow cytometry assay is cumbersome owing to the required eight-part analyses that limit its clinical utility. Here, we describe a simple modification that may improve its clinical utility. By simply combining all T-cell receptor (TCR) Vβ antibodies into one reagent tube, we show that T-cell repertoire analysis can be efficiently performed with an increased potential to closely link the aberrant immunophenotype of a neoplastic T-cell clone to its apparent clonal TCR Vβ restriction. We review our clinical experience and include flow cytometry sorting studies in conjunction with TRG gene rearrangement analysis to demonstrate the feasibility of this approach. We show in representative cases that this modified approach permits evaluation of TCR Vβ clonality in T-cell neoplasms without a priori knowledge of the TCR Vβ isoform restriction and that this approach is suitable for samples containing a minor population of neoplastic T cells, hypocellular samples such as skin biopsy specimens and body fluids, and fine-needle aspirations. We estimate a false-positive rate for identifying a clonal population for this approach to be less than 6.2%. Our modified approach of TCR Vβ analysis could improve routine clinical evaluation for abnormal T-cell populations by flow cytometry.

Expansion of Memory-Type CD8+ T Cells Correlates With the Failure of Early Immunosuppression Withdrawal After Cadaver Liver Transplantation Using High-Dose ATG Induction and Rapamycin

We report on a pilot study investigating the feasibility of early immunosuppression withdrawal after liver transplantation (LT) using antithymocyte globulin (ATG) induction and rapamycin. LT recipients received 3.75 mg/kg per day ATG from days 0 to 5 followed by rapamycin-based immunosuppression. In the absence of acute rejection (AR), rapamycin was withdrawn after month 4. Immunomonitoring included analysis of peripheral T-cell phenotypes and clonality, cytokine production in mixed lymphocyte reaction, and characterization of intragraft infiltrating cells. Ten patients were enrolled between October 2009 and July 2010. In the first three patients, complete withdrawal of immunosuppression after month 4 led to AR. No further withdrawals of immunosuppressive were attempted. Two AR occurred in the remaining seven patients. ATG induced profound T-cell depletion followed by CD8(+) T-cell reexpansion exhibiting memory/effector-like phenotype associated with progressive oligoclonal T-cell expansion (Vβ/HPRT ratio) and gradually enhanced anti-cytomegalovirus and anti-Epstein-Barr virus T-cell frequencies. Patients developing AR were characterized by decreased TCAIM expression. AR were associated with increased donor-specific production of interferon (IFN)-γ and interleukin (IL)-17, increased intragraft expression of IFN-γ mRNA, and significant CD8(+) T-cell infiltrates colocalizing with IL-17(+) cells. High-dose ATG followed by short-term rapamycin treatment failed to promote early operational tolerance to LT. AR correlates with expansion of memory-type CD8(+) T cells and increased levels of IFN-γ and IL-17 in mixed lymphocyte reaction and in the graft. This suggests that resistance and preferential expansion of effector memory T-cell in lymphopenic environment could represent the major barrier for establishment of tolerance to LT in approaches using T-cell-depleting induction.

Clonality in Sarcoidosis, Granuloma Annulare, and Granulomatous Mycosis Fungoides

The histological discrimination of granulomatous cutaneous T-cell lymphomas (CTCLs) from reactive granulomatous disorders such as sarcoidosis and granuloma annulare (GA) may be difficult due to overlapping histological features. We analyzed the T-cell receptor gene rearrangement in sarcoidosis and GA to investigate the value of the detection of clonal T cells as an adjunctive diagnostic marker in the differentiation between sarcoidosis and GA versus granulomatous CTCLs. Rearrangement of T-cell receptor γ genes was examined by the use of automated high-resolution polymerase chain reaction fragment analysis in 35 cases of sarcoidosis and 15 cases of GA and compared with a series of 19 cases of granulomatous CTCLs. A monoclonal T-cell population was found in none of the cases of sarcoidosis and in 2 of 15 cases of GA (13%). In granulomatous CTCLs, a neoplastic T-cell clone was detected in 94%. Presence of clonal T cells argues in favour of a granulomatous CTCL, while a polyclonal T-cell population makes the presence of a sarcoidosis or a GA more likely. The analysis of T-cell clonality is a useful diagnostic adjunct in the differentiation between sarcoidosis and GA from granulomatous CTCLs.

The frequency of dual TCR-PCR clonality in granulomatous disorders

A granulomatous infiltrate in association with cutaneous T-cell lymphoma is uncommon. The diagnosis of mycosis fungoides can be difficult in the setting of an exuberant granulomatous infiltrate that obscures the neoplastic lymphoid infiltrate, thereby mimicking a granulomatous dermatitis. Therefore, the clinical context and supplemental molecular analysis, such as the demonstration of a monoclonal T-cell population, may assist in diagnosis. Monoclonal T-cell populations have been reported in association with inflammatory conditions and serve as a diagnostic pitfall. The frequency of T-cell clonality in association with granulomatous dermatitides has not yet been established. We identified 29 patients with granulomatous dermatitis who had biopsies at two distinct body sites. Results were correlated with clinical follow up and with clonal T-cell receptor-gamma chain rearrangement as detected by polymerase chain reaction-based analysis (dual TCR-PCR). Clinical follow up was obtained in 17 of 29 cases (58.6%). Twenty-five of 29 cases of granulomatous dermatitis lacked T-cell monoclonality. Three cases of granuloma annulare contained a T-cell clone in one of the two biopsies. One case of necrobiotic xanthogranuloma showed an identical T-cell clone in multiple biopsies. The use of dual TCR-PCR analysis, that is, T-cell clonality analysis in biopsy specimens from two different sites, serves as an adjunct to assist in distinguishing granulomatous inflammatory reactions from granulomatous T-cell lymphoma, including granulomatous mycosis fungoides. The occasional finding of a T-cell clone in a granulomatous dermatitis underscores the importance of clinicopathological correlation in daily diagnosis.

M1189 Collagenous Sprue: Clinical Characteristics, Response to Steroids, and Prognosis

Background: Collagenous sprue (CS) is a very rare cause of severe malabsorption with an enteropathy that is characterized by villous atrophy and a distinctive band of subepithelial collagen deposition. A poor outcome has been reported historically. Aim: To describe the clinical characteristics, treatment, and prognosis in a contemporary cohort of patients with CS. Methods: We included cases with biopsy-proven CS evaluated at the Mayo Clinic Results: 15 patients (80% females) with a median age of 67 years (range, 53-83) were included. The clinical manifestations were diarrhea (100%), loss of weight (93%), and abdominal pain (14%). Hospitalizationwas required because severe diarrhea in 9 (64%) patients. Hypoalbuminemia and anemia were evident in 9 (64%) and 7 (50%) patients, respectively. Humanleukocyte antigen genes DQ2 or DQ8 were present in 12 (80%) patients (DQ2+ single dose [n=8], DQ2+ double dose [n=3], and DQ2+/DQ8+ [n=1]). Celiac antibodies were present in only 1 patient. A prior diagnosis of celiac disease was evident in 7 (47%) patients. Histologically, all cases had both the abnormal collagen deposition and some degree of villous atrophy. Aberrant (clonal) intraepithelial lymphocytes were detected in 1 (of 12) patients tested by both immunostaining and T-cell clonality analysis. Associated disorders were collagenous gastritis (n=4), collagenous colitis (n=4), small-intestine bacterial overgrowth (n=4), lymphocytic colitis (n=2), lymphocytic gastritis (n=1), autoimmune hepatitis (n=1), and hyposplenism (n=1). All patients received treatment with a combination of a strict gluten-free diet and steroids such as budesonide 9mg/day (n=12), prednisone 30-40 mg/day (n=2) and dexamethasone 8 mg/day (n=1). Parenteral nutrition was necessary in 11 (79%) patients. One patient developed enteropathy-type T-cell lymphoma during followup and received a combination of nitrogen mustard and methylprednisolone. Clinical followup after treatment was available in 12 (92%) patients (median time = 16 months; range, 172), disappearance of diarrhea was observed in 11 with complete histologic remission documented in one case. Three patients died during follow-up because of emaciation, enteropathy-type T-cell lymphoma, and aspiration pneumonia, respectively. Conclusions: We report the largest case series of CS. Treatment with a combination of gluten-free diet and steroids (especially budesonide) was useful to control symptoms in most patients; however, length of follow-up was limited so far to a few months in the majority of our patients. CS was associated with collagen deposition in other organs, autoimmune conditions, and lymphoma.

M1190 Natural History of Eosinophilic Gastroenteritis

Background: Collagenous sprue (CS) is a very rare cause of severe malabsorption with an enteropathy that is characterized by villous atrophy and a distinctive band of subepithelial collagen deposition. A poor outcome has been reported historically. Aim: To describe the clinical characteristics, treatment, and prognosis in a contemporary cohort of patients with CS. Methods: We included cases with biopsy-proven CS evaluated at the Mayo Clinic Results: 15 patients (80% females) with a median age of 67 years (range, 53-83) were included. The clinical manifestations were diarrhea (100%), loss of weight (93%), and abdominal pain (14%). Hospitalizationwas required because severe diarrhea in 9 (64%) patients. Hypoalbuminemia and anemia were evident in 9 (64%) and 7 (50%) patients, respectively. Humanleukocyte antigen genes DQ2 or DQ8 were present in 12 (80%) patients (DQ2+ single dose [n=8], DQ2+ double dose [n=3], and DQ2+/DQ8+ [n=1]). Celiac antibodies were present in only 1 patient. A prior diagnosis of celiac disease was evident in 7 (47%) patients. Histologically, all cases had both the abnormal collagen deposition and some degree of villous atrophy. Aberrant (clonal) intraepithelial lymphocytes were detected in 1 (of 12) patients tested by both immunostaining and T-cell clonality analysis. Associated disorders were collagenous gastritis (n=4), collagenous colitis (n=4), small-intestine bacterial overgrowth (n=4), lymphocytic colitis (n=2), lymphocytic gastritis (n=1), autoimmune hepatitis (n=1), and hyposplenism (n=1). All patients received treatment with a combination of a strict gluten-free diet and steroids such as budesonide 9mg/day (n=12), prednisone 30-40 mg/day (n=2) and dexamethasone 8 mg/day (n=1). Parenteral nutrition was necessary in 11 (79%) patients. One patient developed enteropathy-type T-cell lymphoma during followup and received a combination of nitrogen mustard and methylprednisolone. Clinical followup after treatment was available in 12 (92%) patients (median time = 16 months; range, 172), disappearance of diarrhea was observed in 11 with complete histologic remission documented in one case. Three patients died during follow-up because of emaciation, enteropathy-type T-cell lymphoma, and aspiration pneumonia, respectively. Conclusions: We report the largest case series of CS. Treatment with a combination of gluten-free diet and steroids (especially budesonide) was useful to control symptoms in most patients; however, length of follow-up was limited so far to a few months in the majority of our patients. CS was associated with collagen deposition in other organs, autoimmune conditions, and lymphoma.

Refractory sprue – precursor lesion of enteropathy type T-cell lymphoma – A clinicopathological case report

Refractory sprue is characterised by distinctive morphologic alterations and the emergence of clonal intraepithelial lymphocytes. In this case report the authors emphasize the importance of histopathology in the diagnosis of refractory sprue. The sequential biopsies from this patient have been investigated with routine histology, immunohistochemistry and molecular genetics for T-cell clonality analysis. The severely cachectic patient presenting with malabsorption syndrome has been diagnosed with celiac disease through a duodenal biopsy, and the CD8 negativity of the intraepithelial lymphocytes suggested the possible diagnosis of refractory sprue. Azathioprine and glucocorticoid therapy was administered due to the failed jejunal feeding and gluten-free diet, resulting in clinically complete, morphologically partial remission. Intestinal T-cell lymphoma developed in the ileocecal region within two years after the first clinical presentation. Refractory sprue and the enteropathy-type T-cell lymphoma constitute a disease spectrum. The reported case shows how a simple method can provide crucial information in the diagnosis of refractory sprue.

T-Cell Clonality Assays: How Do They Compare?

The distinction between certain benign cutaneous lymphocytic infiltrates and cutaneous T-cell lymphoma (CTCL) can be a challenging problem in clinical practice. An imperfect but useful tool in this circumstance is the analysis of T-cell clonality or monoclonality by assessing T-cell receptor (TCR) gene rearrangements. "Monoclonality" describes the origin of a specific human malignant tumor from one single cell from which the entire tumor is derived. The term is used to describe the early steps in tumorigenesis; in later stages of tumor growth and progression, monoclonal tumor cells may acquire a mutator phenotype, rendering the genome unstable. The resulting genetic heterogeneity may ultimately lead to subclones in the formerly monoclonal population of tumor cells.

Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease

Background: Refractory celiac disease (RCD) patients with aberrant, often clonal, intraepithelial T-cells are at high risk for development of enteropathy associated T-cell lymphoma (EATL). Early detection of those patients that actually develop EATL is of utmost importance for curative intervention. Aim: First, to establish an optimal cut-off value for the percentage of aberrant lymphocytes, previously determined based on clinical observations, via reference ranges for aberrant T-cells in the duodenal mucosa of celiac disease patient and control groups. Secondly, to compare aberrancy with intestinal T-cell clonality as a prognostic parameter for EATL development in RCD. Methods: Immunophenotyping using flow cytometry was performed on small intestinal biopsy-derived lymphocytes, obtained from distinct celiac disease (CD) patient and control groups ( N = 167 in total). T-cell clonality in duodenal biopsy specimens was assessed by PCR in RCD, ulcerative jejunitis and EATL patients ( N = 31 in total). Results: In 95% of non-refractory CD patients, the highest percentage aberrant T-cells was 20%. Using this cut-off value, EATL development was exclusively seen in RCD with more than 20% aberrant T-cells (median 52% aberrant T-cells, range 27–94%). When compared with T-cell clonality analysis, > 20% aberrancy showed a much higher negative predictive value and sensitivity (both 100%) for EATL development in RCD patients than T-cell clonality analysis (respectively 75% and 78%). Conclusions: Quantification of aberrant T-cells by flow cytometry is preferable to T-cell clonality analysis for identification of RCD patients at risk for EATL development. A cut-off value of 20% is of use in risk stratification, therapeutic options and subsequent follow-up of RCD patients.

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.

T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides

The diagnosis of mycosis fungoides (MF) is often difficult because of significant clinical and histopathologic overlap with inflammatory dermatoses. T-cell receptor (TCR)gamma chain rearrangement by polymerase chain reaction (PCR) (TCR-PCR) is a helpful adjuvant tool in this setting, but several of the inflammatory dermatoses in the differential diagnosis of MF may contain a clonal T-cell proliferation. We examined whether analysis for T-cell clonality and comparison of the clones with the standardized BIOMED-2 PCR multiplex primers for the TCRgamma chain from two anatomically distinct skin sites improves diagnostic accuracy. We examined two biopsy specimens each from 10 patients with unequivocal MF, from 18 patients with inflammatory dermatoses, and from 18 patients who could initially not be definitively given a diagnosis based on clinical and histopathologic criteria. Eight of 10 patients with unequivocal MF had an identical clone in both biopsy specimens. Two of 18 patients with inflammatory dermatoses were found to have a clone in one of the biopsy specimens. On further follow-up of the 18 patients with morphologically nondiagnostic biopsy specimens, 13 of 18 were later confirmed to have MF and 5 of 18 had inflammatory dermatoses. Eleven of 13 patients with MF had an identical clone in both biopsy specimens; two of 13 had a polyclonal amplification pattern in both biopsy specimens. Four of 5 patients with inflammatory dermatoses had no clone in either biopsy specimen. One patient with an inflammatory dermatosis had an identical clone in both specimens. The sensitivity of TCR-PCR analysis to evaluate for an identical clone at different anatomic skin sites (dual TCR-PCR) is 82.6% and the specificity is 95.7%. The number of patients in the study group was limited. These data suggest that dual TCR-PCR is a very promising technique with high specificity in distinguishing MF from inflammatory dermatoses.

A practical strategy for the routine use of BIOMED‐2 PCR assays for detection of B‐ and T‐cell clonality in diagnostic haematopathology

BIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first-line assays for routine B- and T-cell clonality analysis in diagnostic haematopathology.

Distinguishing angioimmunoblastic T‐cell lymphoma from peripheral T‐cell lymphoma, unspecified, using morphology, immunophenotype and molecular genetics

To identify distinguishing histological, immunophenotypic and molecular genetic features between angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma (PTL). Nodal T-cell lymphomas examined (n =137), included AITL (n = 89), PTL (n = 22), anaplastic large cell lymphoma (n = 16) and 'AITL/PTL indeterminate' (n = 10) with overlapping features between AITL and PTL, showing morphology typical of AITL but lacking follicular dendritic cell expansion. Immunohistochemistry for CD3, CD20, CD21 and CD10, in situ hybridization for Epstein-Barr virus encoded RNA (EBER) and polymerase chain reaction for T-cell and B-cell clonality analysis were performed. Of the AITLs, 74/89 showed typical morphology, whereas 15/89 showed hyperplastic follicles. AITL and 'AITL/PTL indeterminate' showed a polymorphous infiltrate and prominent vascularity in all cases. In both groups, CD10 was present in the majority and clear cells and EBER positivity were specific (but not universal) features lacking in PTL. Detection of T-cell clonality was significantly higher in AITL (90%) compared with PTLu (59%). Clear cells and EBV infection (when present) are useful distinguishing features and CD10 a sensitive and specific marker of AITL. Hyperplastic follicles are present in a significant minority of AITL. AITL/PTL indeterminate probably falls within the spectrum of AITL rather than PTL.

Usefulness of Cutaneous T-Cell Clonality Analysis for the Diagnosis of Cutaneous T-Cell Lymphoma in Patients With Erythroderma

Demonstration of a dominant T-cell clone in skin biopsy specimens by a molecular assay constitutes an additional diagnostic criterion to differentiate cutaneous T-cell lymphomas (CTCLs) from inflammatory dermatoses. To determine which patients, depending on their clinical presentations, could most benefit from a cutaneous T-cell clonality analysis in addition to histopathologic analysis for the diagnosis of CTCL. Comparison of sensitivity and specificity of histopathologic analysis and a combination of this method and the detection of a T-cell receptor gamma chain gene rearrangement by polymerase chain reaction denaturing gradient gel electrophoresis performed on skin biopsy specimens obtained at initial presentation. One hundred forty consecutive patients were classified into 4 groups, depending on their clinical presentation: (1) eczematous patches suggestive of early-stage mycosis fungoides (MF) (IA and IB of the TNM classification) (n = 42); (2) plaques, nodules, or tumors that arise on or are associated with plaques suggestive of late-stage MF (IIB and III of the TNM classification) (n = 16); (3) erythroderma (n = 50); and (4) nodules or tumors that arise in normal skin, suggestive of non-MF CTCL (n = 32). When compared with histopathologic examination, the addition of clonality analysis increased the sensitivity of CTCL diagnosis in all groups of patients except those with cutaneous lesions suggestive of late-stage MF, because the diagnosis was made based on histopathologic analysis alone in 100% of these cases. The main increase in sensitivity of CTCL diagnosis was observed in patients with erythroderma: 62% with histopathologic analysis alone to 87% with the combination of both methods (P = .04). Diagnostic specificity of molecular assays decreased from 100% to 76% (P = .01) in patients with patch lesions and from 100% to 70% (P = .04) in patients with nodules that occurred in normal skin due to the detection of a T-cell clone in 6 patients with follicular mucinosis without a histologic pattern of MF and in 5 of 20 cases of T-cell pseudolymphoma (25%), respectively. In contrast, a T-cell clone was not detected in the 34 patients with erythroderma of inflammatory origin. Polymerase chain reaction analysis of cutaneous T-cell clonality could be useful for the diagnosis of CTCL in patients who present with erythroderma.