In patients with suspected lymphoma, the tissue biopsy provides lymphoma confirmation, classification, and prognostic factors, including genetic changes. We developed a deep learning algorithm to ...detect MYC rearrangement in scanned histological slides of diffuse large B-cell lymphoma. The H&E-stained slides of 287 cases from 11 hospitals were used for training and evaluation. The overall sensitivity to detect MYC rearrangement was 0.93 and the specificity 0.52, showing that prediction of MYC translocation based on morphology alone was possible in 93% of MYC-rearranged cases. This would allow a simple and fast prescreening, saving approximately 34% of genetic tests with the current algorithm.
Aims
Large 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 results
With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008.
Conclusions
In 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 found in two cases of LBCL‐IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL‐IRF4 should also be considered in older patients and at localisations other than the head and neck region.
Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the ...clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing–based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein–Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing–based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.
One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for ...clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.
Digital pathology with whole-slide imaging (WSI) has a large potential to make the process of expert consultation and expert panel diagnosis more rapid and more efficient. However, comparison with ...the current methods is necessary for validation of the technique. In this study, we determined if digital assessment of whole-slide images of hematopathology specimens with a focus on the assessment of lymphoma can be used for consultation and panel diagnostics. Ninety-three histological specimens with a suspicion for lymphoma were assessed both with conventional microscopy and digital microscopy with a wash out period between assessments. A consensus diagnosis was based on full concordance between the pathologists or, in case of discordances, was reached at a joint session at a multi-headed microscope. In 81% of the cases, there was a full concordance between digital and light microscopical assessment for all three pathologists. Discordances between conventional microscopy and digital pathology were present in 3% of assessments. In comparison with the consensus diagnosis, discordant diagnoses were made in 5 cases with digital microscopy and in 3 cases with light microscopy. The reported level of confidence and need for additional investigations were similar between assessment by conventional and by digital microscopy. In conclusion, the performance of assessment by digital pathology is in general comparable with that of conventional light microscopy and pathologists feel confident using digital pathology for this subspecialty.
Patients with newly diagnosed high-risk Burkitt lymphoma are treated with high-intensity immune-chemotherapy regimens such as R-CODOX-M/R-IVAC or with lower-intensity regimens such as DA-EPOCH-R. The ...aim of this study was to make a formal comparison between these regimens.
This multicentre, phase 3, open-label, randomised study was done in 26 clinical centres in the Netherlands, Belgium, and Switzerland. Eligible patients were aged 18-75 years with newly diagnosed high-risk Burkitt lymphoma without CNS involvement. Patients were randomly assigned to two cycles of R-CODOX-M/R-IVAC (R-CODOX-M: rituximab 375 mg/m
on day 1 and 9, cyclophosphamide 800 mg/m
on day 1, cyclophosphamide 200 mg/m
on days 2-5, vincristine 1·5 mg/m
on days 1 and 8, doxorubicin 40 mg/m
on day 1, and methotrexate 3000 mg/m
on day 10; R-IVAC: rituximab 375 mg/m
on days 3 and 7, iphosphamide 1500 mg/m
on days 1-5, etoposide 60 mg/m
on days 1-5, and cytarabin 2000 mg/m
on day 1 and 2) or six cycles of DA-EPOCH-R (dose-adjusted etoposide 50-124 mg/m
on days 1-4, prednisolone 120 mg/m
on days 1-5, vincristine 0·4 mg/m
on days 1-4, dose-adjusted cyclophosphamide 480-1866 mg/m
on day 5, dose-adjusted doxorubicin 10-24·8 mg/m
on days 1-4, rituximab 375 mg/m
on days 1 and 5). Patients older than 65 years received a dose modified R-CODOX-M/R-IVAC. All drugs were intravenous except for prednisolone, which was oral. Patients also received four intrathecal CNS administrations with cytarabin (70 mg) and four with methotrexate (15 mg). Patients were stratified by centre, leukemic disease, and HIV-positivity. The primary endpoint was progression-fee survival. All analyses were done by modified intention-to-treat, excluding randomly assigned patients who were subsequently found to have CNS involvement or diagnosis other than Burkitt lymphoma at study entry. This study is registered with the European Clinical Trial Register, EudraCT2013-004394-27.
Due to a slow accrual, the study was closed prematurely on Nov 15, 2021. Between Aug 4, 2014, and Sept 17, 2021, 89 patients were enrolled and randomly assigned to receive R-CODOX-M/R-IVAC (n=46) or DA-EPOCH-R (n=43). Five patients were excluded after random assignment (three in the R-CODOX-M/R-IVAC group one diagnosis other than Burkitt lymphoma at study entry according to local pathology and two CNS involvement and two in the DA-EPOCH-R group one diagnosis other than Burkitt lymphoma at study entry according to local pathology and one CNS involvement. 84 remaining patients were included in the modified intention-to-treat analysis. 73 (87%) of 84 patients were male, 76 (90%) presented with stage III or IV disease, and nine (11%) had HIV-positive Burkitt lymphoma. Median patient age was 52 years (IQR 37-64). With a median follow-up of 28·5 months (IQR 13·2-43·7), 2-year progression-free survival was 76% (95% CI 60-86%) in the R-CODOX-M/R-IVAC group and 70% (54-82%) in the DA-EPOCH-R group (hazard ratio 1·42, 95% CI 0·63-3·18; p=0·40). There were two deaths in the R-CODOX-M/R-IVAC group (one infection treatment related and one due to disease progression not treatment related) and one death in the DA-EPOCH-R group (COVID-19 infection treatment related). In the R-CODOX-M/R-IVAC group, four patients went off-protocol because of toxic effects, versus none in the DA-EPOCH-R group. Patients treated with R-CODOX-M/R-IVAC had more infectious adverse events (24 56% of 43 patients had at least one grade 3-5 infection vs 14 34% of 41 patients in the DA-EPOCH-R group).
The trial stopped early, but the available data suggest that while DA-EPOCH-R did not result in superior progression-free survival compared with R-CODOX-M/R-IVAC, it was associated with fewer toxic effects and need for supportive care. DA-EPOCH-R appears to be an additional valid therapeutic option for patients with high-risk Burkitt lymphoma without CNS involvement.
The Dutch Cancer Society and the Schumacher-Kramer Foundation.
Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B‐cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although ...clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high‐throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B‐cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B‐cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B‐cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one‐quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.
Aims
Current immunohistochemical methods to study the expression of multiple proteins in a single tissue section suffer from several limitations. In this article, we report on sequential ...immunohistochemistry (S‐IHC), a novel, easy method that allows the study of numerous proteins in a single tissue section, while requiring very limited optimization.
Methods and results
In S‐IHC, a tissue section is stained for multiple antibodies, with intermediate scanning of the section and elution of chromogen and antibodies. Overlays are made of the digital images, allowing assessment of multiple proteins in the same tissue section. We used S‐IHC to study nine nodular lymphocyte‐predominant Hodgkin lymphomas (NLPHLs) and 10 T‐cell‐rich and histiocyte‐rich diffuse large B‐cell lymphomas (T/HRBCLs) for expression of cyclin D1, CD20, and CD68. We observed cyclin D1 expression in single tumour cells in 44% of NLPHLs and 60% of T/HRBCLs. Comparison of S‐IHC with classic single immunohistochemical staining revealed discrepancies in eight cases (42%), demonstrating the difficulty of differentiating tumour cells from histiocytes on morphological grounds, and stressing the additional value of S‐IHC.
Conclusions
For research and diagnostic purposes, S‐IHC is a promising technique that assesses the expression of numerous proteins in single tissue sections with complete architectural information, allowing phenotypic characterization of single cells.
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