Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important ... more Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.
AimsLarge B cell lymphoma with IRF4 rearrangement (LBCL‐IRF4) is a new entity in the 2017 revised... more AimsLarge B cell lymphoma with IRF4 rearrangement (LBCL‐IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79‐year‐old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL‐IRF4 based on fluorescence in‐situ hybridisation (FISH) for IRF4.Methods and resultsWith FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008.ConclusionsIn addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL‐IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de‐novo LBCL‐IRF4. BCL6 rearrangements were...
Current diagnostic standards for lymphoproliferative disorders include multiple tests for detecti... more Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 ...
Clonality assessment of the immunoglobulin heavy (IGH) and light chain (IGK) using GeneScan analy... more Clonality assessment of the immunoglobulin heavy (IGH) and light chain (IGK) using GeneScan analysis is an important supplementary assay in lymphoma diagnostics. Occasionally cases are encountered with an IGK rearrangement pattern that cannot readily be assigned to a monoclonal lymphoma, whereas the occurrence of bi-clonal lymphomas is rare and the result of the IGH locus of these cases is in line with monoclonality. We subjected three such ambiguous cases to next generation sequencing (NGS)-based clonality assessment. Sequence information of the rearrangements combined with knowledge of the complex organization of the IGK locus resulted in two explanations that can attribute seemingly bi-clonal IGK rearrangements to a single clone. For two cases, this involved inversion rearrangements on the IGK locus, whereas for the third case, cross-reactivity of primers generated an additional clonal product. In conclusion, NGS-based clonality assessment allows the detection of both inversion rearrangements and cross-reactivity of primers, and can therefore facilitate the interpretation of lymphoma cases with complex IGK rearrangement patterns.
3470 Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into line... more 3470 Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into lineage committed cells. Recent data are suggestive for some degree of lineage flexibility in both normal as malignant cells. We here present a boy that presented with T-cell acute lymphoblastic leukemia (T-ALL). During T-ALL treatment, the patient developed in short time a non-Langerhans-cell histiocytose in the ileum, and subsequently a disseminated form of histiocytic sarcoma (a.o. also in the liver). Interestingly, the same clonal T cell receptor (TCR) gene rearrangements were found in all three malignancies (TCRB-VJ, TCRG-VJ and TCRB-DJ) indicating they were related. There were no immunoglobulin rearrangements. To understand the evolution from T-ALL to non-Langerhans-cell histiocytose and to histiocytic sarcoma, an extensive genetic analysis was performed. Using a SNP6.0 array platform we analyzed DNA isolated from T-ALL cells, a bone marrow sample obtained during complete remission fro...
Background Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 ... more Background Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, diagnosis can be difficult. Approximately one third of sarcoma patients however are characterised by recurrent genetic alterations mainly chromosomal translocations, which result in fusion genes, and gene amplifications. Fusion genes are highly useful diagnostic markers and are currently identified in the clinical setting using FISH/RT-PCR. Conversely, conclusive results cannot be achieved in up to 25% of fusion positive patients using these methods. Next generation sequencing (NGS) is a promising tool that can improve soft-tissue sarcoma diagnosis. Methods A novel sarcoma-specific NGS gene capture panel containing probes for 90 fusion genes and 7 genes with frequent copy number changes was designed, optimised and validated in order to improve fusion-positive sarcoma diagnosis. Genomic DNA was extracted from 92 well-characterised FFPE samples and libraries prepared u...
Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-bas... more Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.
SummaryMantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and sev... more SummaryMantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and several other cytogenetic aberrations, including heterozygous loss of chromosomal arms 1p, 6q, 11q and 13q and/or gains of 3q and 8q. The common intervals of chromosomal imbalance have been narrowed down using array‐comparative genomic hybridization (CGH). However, the chromosomal intervals still contain many genes potentially involved in MCL pathogeny. Combined analysis of tiling‐resolution array‐CGH with gene expression profiling on 11 MCL tumours enabled the identification of genomic alterations and their corresponding gene expression profiles. Only subsets of genes located within given cytogenetic anomaly‐intervals showed a concomitant change in mRNA expression level. The genes that showed consistent correlation between DNA copy number and RNA expression levels are likely to be important in MCL pathology. Besides several ‘anonymous genes’, we also identified various fully annotated gene...
Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefor... more Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats (MNR). The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7% when using the MNR panel and 95.0% with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7% when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7% if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype.
ObjectiveTo comparatively analyse the aberrant affinity maturation of the antinuclear and rheumat... more ObjectiveTo comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren’s syndrome (SjS) using an integrated omics workflow.MethodsPeptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS.ResultsAffected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity...
B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphom... more B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymph...
Malignant melanomas are known for their remarkable morphological variation and aberrant immunophe... more Malignant melanomas are known for their remarkable morphological variation and aberrant immunophenotype with loss of lineage-specific markers, especially in recurrences and metastases. Hot spot mutations in BRAF, NRAS, GNAQ, and GNA11 and mutations in KIT are oncogenic events in melanomas. Therefore, genotyping can be a useful ancillary diagnostic tool. We present one case each of recurrent and metastatic melanoma, both showing histological and immunohistochemical features of solitary fibrous tumor (SFT). Mutational analysis detected BRAF and NRAS mutations in the primary and secondary lesions, respectively. This result confirmed the diagnosis of recurrent/metastastic melanoma.
Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important ... more Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.
AimsLarge B cell lymphoma with IRF4 rearrangement (LBCL‐IRF4) is a new entity in the 2017 revised... more AimsLarge B cell lymphoma with IRF4 rearrangement (LBCL‐IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79‐year‐old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL‐IRF4 based on fluorescence in‐situ hybridisation (FISH) for IRF4.Methods and resultsWith FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008.ConclusionsIn addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL‐IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de‐novo LBCL‐IRF4. BCL6 rearrangements were...
Current diagnostic standards for lymphoproliferative disorders include multiple tests for detecti... more Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 ...
Clonality assessment of the immunoglobulin heavy (IGH) and light chain (IGK) using GeneScan analy... more Clonality assessment of the immunoglobulin heavy (IGH) and light chain (IGK) using GeneScan analysis is an important supplementary assay in lymphoma diagnostics. Occasionally cases are encountered with an IGK rearrangement pattern that cannot readily be assigned to a monoclonal lymphoma, whereas the occurrence of bi-clonal lymphomas is rare and the result of the IGH locus of these cases is in line with monoclonality. We subjected three such ambiguous cases to next generation sequencing (NGS)-based clonality assessment. Sequence information of the rearrangements combined with knowledge of the complex organization of the IGK locus resulted in two explanations that can attribute seemingly bi-clonal IGK rearrangements to a single clone. For two cases, this involved inversion rearrangements on the IGK locus, whereas for the third case, cross-reactivity of primers generated an additional clonal product. In conclusion, NGS-based clonality assessment allows the detection of both inversion rearrangements and cross-reactivity of primers, and can therefore facilitate the interpretation of lymphoma cases with complex IGK rearrangement patterns.
3470 Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into line... more 3470 Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into lineage committed cells. Recent data are suggestive for some degree of lineage flexibility in both normal as malignant cells. We here present a boy that presented with T-cell acute lymphoblastic leukemia (T-ALL). During T-ALL treatment, the patient developed in short time a non-Langerhans-cell histiocytose in the ileum, and subsequently a disseminated form of histiocytic sarcoma (a.o. also in the liver). Interestingly, the same clonal T cell receptor (TCR) gene rearrangements were found in all three malignancies (TCRB-VJ, TCRG-VJ and TCRB-DJ) indicating they were related. There were no immunoglobulin rearrangements. To understand the evolution from T-ALL to non-Langerhans-cell histiocytose and to histiocytic sarcoma, an extensive genetic analysis was performed. Using a SNP6.0 array platform we analyzed DNA isolated from T-ALL cells, a bone marrow sample obtained during complete remission fro...
Background Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 ... more Background Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, diagnosis can be difficult. Approximately one third of sarcoma patients however are characterised by recurrent genetic alterations mainly chromosomal translocations, which result in fusion genes, and gene amplifications. Fusion genes are highly useful diagnostic markers and are currently identified in the clinical setting using FISH/RT-PCR. Conversely, conclusive results cannot be achieved in up to 25% of fusion positive patients using these methods. Next generation sequencing (NGS) is a promising tool that can improve soft-tissue sarcoma diagnosis. Methods A novel sarcoma-specific NGS gene capture panel containing probes for 90 fusion genes and 7 genes with frequent copy number changes was designed, optimised and validated in order to improve fusion-positive sarcoma diagnosis. Genomic DNA was extracted from 92 well-characterised FFPE samples and libraries prepared u...
Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-bas... more Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.
SummaryMantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and sev... more SummaryMantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and several other cytogenetic aberrations, including heterozygous loss of chromosomal arms 1p, 6q, 11q and 13q and/or gains of 3q and 8q. The common intervals of chromosomal imbalance have been narrowed down using array‐comparative genomic hybridization (CGH). However, the chromosomal intervals still contain many genes potentially involved in MCL pathogeny. Combined analysis of tiling‐resolution array‐CGH with gene expression profiling on 11 MCL tumours enabled the identification of genomic alterations and their corresponding gene expression profiles. Only subsets of genes located within given cytogenetic anomaly‐intervals showed a concomitant change in mRNA expression level. The genes that showed consistent correlation between DNA copy number and RNA expression levels are likely to be important in MCL pathology. Besides several ‘anonymous genes’, we also identified various fully annotated gene...
Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefor... more Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats (MNR). The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7% when using the MNR panel and 95.0% with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7% when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7% if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype.
ObjectiveTo comparatively analyse the aberrant affinity maturation of the antinuclear and rheumat... more ObjectiveTo comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren’s syndrome (SjS) using an integrated omics workflow.MethodsPeptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS.ResultsAffected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity...
B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphom... more B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymph...
Malignant melanomas are known for their remarkable morphological variation and aberrant immunophe... more Malignant melanomas are known for their remarkable morphological variation and aberrant immunophenotype with loss of lineage-specific markers, especially in recurrences and metastases. Hot spot mutations in BRAF, NRAS, GNAQ, and GNA11 and mutations in KIT are oncogenic events in melanomas. Therefore, genotyping can be a useful ancillary diagnostic tool. We present one case each of recurrent and metastatic melanoma, both showing histological and immunohistochemical features of solitary fibrous tumor (SFT). Mutational analysis detected BRAF and NRAS mutations in the primary and secondary lesions, respectively. This result confirmed the diagnosis of recurrent/metastastic melanoma.
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Papers by Patricia Groenen