T-cell Receptor Polymerase Chain Reaction Research Articles

Blinatumomab for Acute Lymphoblastic Leukemia: The First Bispecific T-Cell Engager Antibody to Be Approved by the EMA for Minimal Residual Disease.

On November 15, 2018, the Committee for Medicinal Products for Human Use (CHMP) recommended the extension of indication for blinatumomab to include the treatment of adults with minimal residual disease (MRD) positive B-cell precursor acute lymphoblastic leukemia (ALL). Blinatumomab was authorized to treat relapsed or refractory B-precursor ALL, and the change concerned an extension of use. On March 29, 2018, the U.S. Food and Drug Administration (FDA) granted accelerated approval to blinatumomab to treat both adults and children with B-cell precursor ALL who are in remission but still have MRD. On July 26, 2018, the CHMP had originally adopted a negative opinion on the extension. The reason for the initial refusal was that although blinatumomab helped to reduce the amount of residual cancer cells in many patients, there was no strong evidence that it led to improved survival. During the re-examination, the CHMP consulted the scientific advisory group. The CHMP agreed with the expert group's conclusion that, although there was no strong evidence of patients living longer, the available data from the main study (MT103-203) indicated a good durable response to blinatumomab, with an overall complete response rate for the primary endpoint full analysis set (defined as all subjects with an Ig or T-cell receptor polymerase chain reaction MRD assay with the minimum required sensitivity of 1 × 10-4 at central lab established at baseline [n = 113]) as 79.6% (90/113; 95% confidence interval, 71.0-86.6), with a median time to complete MRD response of 29.0 days (range, 5-71). Therefore, the CHMP concluded that the benefits of blinatumomab outweigh its risks and recommended granting the change to the marketing authorization. The Committee for Orphan Medicinal Products, following reassessment, considered that significant benefit continued to be met and recommended maintaining the orphan designation and thus 10 years market exclusivity (the Orphan Designation is a legal procedure that allows for the designation of a medicinal substance with therapeutic potential for a rare disease, before its first administration in humans or during its clinical development). The marketing authorization holder for this medicinal product is Amgen Europe B.V. IMPLICATIONS FOR PRACTICE: Immunotherapy with blinatumomab has excellent and sustainable results, offering new hope for patients with minimal residual disease-positive acute lymphoblastic leukemia, a disease with poor prognosis. New recommendations and change of practice for treatment of this patient group are detailed.

Deep Sequencing of the T-cell Receptor Repertoire Demonstrates Polyclonal T-cell Infiltrates in Psoriasis

It is well known that infiltration of pathogenic T-cells plays an important role in psoriasis pathogenesis. However, the antigen specificity of these activated T-cells is relatively unknown. Previous studies using T-cell receptor polymerase chain reaction technology (TCR-PCR) have suggested there are expanded T-cell receptor (TCR) clones in psoriatic skin, suggesting a response to an unknown psoriatic antigen. Here we describe the results of high-throughput deep sequencing of the entire αβ- and γδ- TCR repertoire in normal healthy skin and psoriatic lesional and non-lesional skin. From this study, we were able to determine that there is a significant increase in the abundance of unique β- and γ- TCR sequences in psoriatic lesional skin compared to non-lesional and normal skin, and that the entire T-cell repertoire in psoriasis is polyclonal, with similar diversity to normal and non-lesional skin. Comparison of the αβ- and γδ- TCR repertoire in paired non-lesional and lesional samples showed many common clones within a patient, and these close were often equally abundant in non-lesional and lesional skin, again suggesting a diverse T-cell repertoire. Although there were similar (and low) amounts of shared β-chain sequences between different patient samples, there was significantly increased sequence sharing of the γ-chain in psoriatic skin from different individuals compared to those without psoriasis. This suggests that although the T-cell response in psoriasis is highly polyclonal, particular γδ- T-cell subsets may be associated with this disease. Overall, our findings present the feasibility of this technology to determine the entire αβ- and γδ- T-cell repertoire in skin, and that psoriasis contains polyclonal and diverse αβ- and γδ- T-cell populations.

Deep Sequencing of the T-cell Receptor Repertoire Demonstrates Polyclonal T-cell Infiltrates in Psoriasis.

It is well known that infiltration of pathogenic T-cells plays an important role in psoriasis pathogenesis. However, the antigen specificity of these activated T-cells is relatively unknown. Previous studies using T-cell receptor polymerase chain reaction technology (TCR-PCR) have suggested there are expanded T-cell receptor (TCR) clones in psoriatic skin, suggesting a response to an unknown psoriatic antigen. Here we describe the results of high-throughput deep sequencing of the entire αβ- and γδ- TCR repertoire in normal healthy skin and psoriatic lesional and non-lesional skin. From this study, we were able to determine that there is a significant increase in the abundance of unique β- and γ- TCR sequences in psoriatic lesional skin compared to non-lesional and normal skin, and that the entire T-cell repertoire in psoriasis is polyclonal, with similar diversity to normal and non-lesional skin. Comparison of the αβ- and γδ- TCR repertoire in paired non-lesional and lesional samples showed many common clones within a patient, and these close were often equally abundant in non-lesional and lesional skin, again suggesting a diverse T-cell repertoire. Although there were similar (and low) amounts of shared β-chain sequences between different patient samples, there was significantly increased sequence sharing of the γ-chain in psoriatic skin from different individuals compared to those without psoriasis. This suggests that although the T-cell response in psoriasis is highly polyclonal, particular γδ- T-cell subsets may be associated with this disease. Overall, our findings present the feasibility of this technology to determine the entire αβ- and γδ- T-cell repertoire in skin, and that psoriasis contains polyclonal and diverse αβ- and γδ- T-cell populations.

Mesalamine for Refractory Celiac Disease

Mesalamine for Refractory Celiac Disease

Evaluation of T-cell Clonality in Archival Skin Biopsy Samples of Cutaneous T-cell Lymphomas Using the Biomed-2 PCR Protocol

Recently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) gamma and TCRbeta PCR as well as an in-house TCRgamma PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRgamma PCR revealed 81% clonality, the in-house TCRgamma method revealed 86% clonality, and the Biomed-2 TCRbeta revealed 78% clonality in CTCL samples generating at least the 300 bp fragment in the Biomed-2 control PCR. We found clonal TCRbeta rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRgamma PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRgamma assay verifies our previously published findings on clonally expanded T-cells in CTCL.

Febrile ulceronecrotic Mucha–Habermann disease associated with herpes simplex virus type 2

Febrile ulceronecrotic Mucha-Habermann disease is a rare severe variant of pityriasis lichenoides et varioliformis acuta, a disease within the pityriasis lichenoides spectrum of disorders. It remains uncertain whether these disorders are preneoplastic or reactive against infectious or other antigenic stimuli. Febrile ulceronecrotic Mucha-Habermann disease varies in severity and may be accompanied by a range of systemic symptoms. We present a case associated with herpes simplex virus infection. In this case, no significant T cell clone was identified. Clonality may be a prognostic marker, but reports with T-cell receptor polymerase chain reaction results are limited to eight previous cases, and further reports are required. Awareness of the condition is important because of its fulminant potential.

A Comparison of Morphologic Features, Flow Cytometry, TCR-VβAnalysis, and TCR-PCR in Qualitative and Quantitative Assessment of Peripheral Blood Involvement by Sézary Syndrome

The strengths and weaknesses of various laboratory methods for peripheral blood (PB) Sézary cell quantitation have not been compared rigorously. In this study, manual Sézary cell counting, qualitative and quantitative flow cytometry, T-cell receptor (TCR) Vbeta flow cytometry, and TCR polymerase chain reaction were performed on PB specimens from 11 patients with Sézary syndrome (SS), 9 with reactive erythroderma, 6 with mycosis fungoides, and 11 healthy control subjects. These methods identified neoplastic cells in more than 90% of SS cases. The diagnostic specificities of these tests varied; they were enhanced by applying criteria proposed by the International Society for Cutaneous Lymphoma. Comparison of sequentially analyzed specimens from 6 patients with SS revealed that although the absolute number of clonal cells was reduced, in some cases, these cells still constituted the vast majority of the CD4+ T-cell subset, suggesting that quantitative subset analysis might be sufficient to monitor changes in the PB tumor burden.

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.

The T-cell receptor Vβ repertoire in naive and human immunodeficiency virus-1 (HIV-1) infected chimpanzees

Using a panel of human T-cell receptor (TCR) variable region beta chain (V beta) polymerase chain reaction (PCR) primers, we performed cross-sectional and longitudinal analyses of the TCR V beta repertoire in naive and HIV-1 infected chimpanzees. We demonstrate that our TCR PCR primer panel will support amplification of chimpanzee cDNA from most of the TCR V beta families. However, no differences in TCR V beta expression were found between the naive and HIV-1 infected chimpanzees, unlike the TCR V beta repertoire perturbation found in HIV-1 infected human subjects. This finding suggests that a complete TCR repertoire in HIV-1 infected chimpanzees is associated with the maintenance CD4+ T-cell numbers and lack of progression to AIDS.