ARResT/Interrogate: an interactive immunoprofiler for IG/TR NGS data.

Abstract Motivation The study of immunoglobulins and T cell receptors using next-generation sequencing has finally allowed exploring immune repertoires and responses in their immense variability and complexity. Unsurprisingly, their analysis and interpretation is a highly convoluted task. Results We thus implemented ARResT/Interrogate, a web-based, interactive application. It can organize and filter large amounts of immunogenetic data by numerous criteria, calculate several relevant statistics, and present results in the form of multiple interconnected visualizations. Availability and Implementation ARResT/Interrogate is implemented primarily in R, and is freely available at http://bat.infspire.org/arrest/interrogate/ Supplementary information Supplementary data are available at Bioinformatics online

Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.This work was supported by Ministry of Health of the Czech Republic, grant no. 16-34272A; computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme “Projects of Large Research, Development, and Innovations Infrastructures”. Analyses in Prague (JT, EF and MS) were supported by Ministry of Health, Czech Republic, grant no. 00064203, and by PRIMUS/17/MED/11. Analyses in the Monza (Centro Ricerca Tettamanti, SS, AG and GC) laboratory were supported by the Italian Association for Cancer Research (AIRC) and Comitato Maria Letizia Verga

Pitfalls in TCR gene clonality testing: teaching cases

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis

Improved discrimination of melanotic schwannoma from melanocytic lesions by combined morphological and GNAQ mutational analysis

The histological differential diagnosis between melanotic schwannoma, primary leptomeningeal melanocytic lesions and cellular blue nevus can be challenging. Correct diagnosis of melanotic schwannoma is important to select patients who need clinical evaluation for possible association with Carney complex. Recently, we described the presence of activating codon 209 mutations in the GNAQ gene in primary leptomeningeal melanocytic lesions. Identical codon 209 mutations have been described in blue nevi. The aims of the present study were to (1) perform a histological review of a series of lesions (initially) diagnosed as melanotic schwannoma and analyze them for GNAQ mutations, and (2) test the diagnostic value of GNAQ mutational analysis in the differential diagnosis with leptomeningeal melanocytic lesions. We retrieved 25 cases that were initially diagnosed as melanotic schwannoma. All cases were reviewed using established criteria and analyzed for GNAQ codon 209 mutations. After review, nine cases were classified as melanotic schwannoma. GNAQ mutations were absent in these nine cases. The remaining cases were reclassified as conventional schwannoma (n = 9), melanocytoma (n = 4), blue nevus (n = 1) and lesions that could not be classified with certainty as melanotic schwannoma or melanocytoma (n = 2). GNAQ codon 209 mutations were present in 3/4 melanocytomas and the blue nevus. Including results from our previous study in leptomeningeal melanocytic lesions, GNAQ mutations were highly specific (100%) for leptomeningeal melanocytic lesions compared to melanotic schwannoma (sensitivity 43%). We conclude that a detailed analysis of morphology combined with GNAQ mutational analysis can aid in the differential diagnosis of melanotic schwannoma with leptomeningeal melanocytic lesions

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is under-investigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (IG) and T-cell receptor (TR) rearrangements was performed in paired cHL diagnosis and recurrences of 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal IG rearrangements were detected by next-generation sequencing (NGS) in 69/120 (58%) diagnosis and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24/34 patients (71%). Clonally unrelated cHL was observed in 10/34 patients (29%) as determined by IG-NGS clonality assessment, and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of &gt;2 years, ~60% of cHL patients for which the clonal relationship could be established showed a second primary cHL. Clonal TR gene rearrangements were identified in 14/125 samples (11%), and TCL-associated gene mutations were detected in 7/14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged &gt;50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based IG/TR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.</p

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is under-investigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (IG) and T-cell receptor (TR) rearrangements was performed in paired cHL diagnosis and recurrences of 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal IG rearrangements were detected by next-generation sequencing (NGS) in 69/120 (58%) diagnosis and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24/34 patients (71%). Clonally unrelated cHL was observed in 10/34 patients (29%) as determined by IG-NGS clonality assessment, and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of &gt;2 years, ~60% of cHL patients for which the clonal relationship could be established showed a second primary cHL. Clonal TR gene rearrangements were identified in 14/125 samples (11%), and TCL-associated gene mutations were detected in 7/14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged &gt;50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based IG/TR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.</p

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is under-investigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (IG) and T-cell receptor (TR) rearrangements was performed in paired cHL diagnosis and recurrences of 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal IG rearrangements were detected by next-generation sequencing (NGS) in 69/120 (58%) diagnosis and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24/34 patients (71%). Clonally unrelated cHL was observed in 10/34 patients (29%) as determined by IG-NGS clonality assessment, and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of &gt;2 years, ~60% of cHL patients for which the clonal relationship could be established showed a second primary cHL. Clonal TR gene rearrangements were identified in 14/125 samples (11%), and TCL-associated gene mutations were detected in 7/14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged &gt;50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based IG/TR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.</p

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is under-investigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (IG) and T-cell receptor (TR) rearrangements was performed in paired cHL diagnosis and recurrences of 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal IG rearrangements were detected by next-generation sequencing (NGS) in 69/120 (58%) diagnosis and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24/34 patients (71%). Clonally unrelated cHL was observed in 10/34 patients (29%) as determined by IG-NGS clonality assessment, and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of &gt;2 years, ~60% of cHL patients for which the clonal relationship could be established showed a second primary cHL. Clonal TR gene rearrangements were identified in 14/125 samples (11%), and TCL-associated gene mutations were detected in 7/14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged &gt;50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based IG/TR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.</p

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is under-investigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (IG) and T-cell receptor (TR) rearrangements was performed in paired cHL diagnosis and recurrences of 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal IG rearrangements were detected by next-generation sequencing (NGS) in 69/120 (58%) diagnosis and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24/34 patients (71%). Clonally unrelated cHL was observed in 10/34 patients (29%) as determined by IG-NGS clonality assessment, and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of &gt;2 years, ~60% of cHL patients for which the clonal relationship could be established showed a second primary cHL. Clonal TR gene rearrangements were identified in 14/125 samples (11%), and TCL-associated gene mutations were detected in 7/14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged &gt;50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based IG/TR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.</p

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is under-investigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (IG) and T-cell receptor (TR) rearrangements was performed in paired cHL diagnosis and recurrences of 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal IG rearrangements were detected by next-generation sequencing (NGS) in 69/120 (58%) diagnosis and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24/34 patients (71%). Clonally unrelated cHL was observed in 10/34 patients (29%) as determined by IG-NGS clonality assessment, and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of &gt;2 years, ~60% of cHL patients for which the clonal relationship could be established showed a second primary cHL. Clonal TR gene rearrangements were identified in 14/125 samples (11%), and TCL-associated gene mutations were detected in 7/14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged &gt;50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based IG/TR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.</p