Integration of RNA Sequencing, Whole Exome Sequencing, and Flow Cytometry Into Routine Diagnostic Workup of Pediatric Lymphomas.

The study was conducted to assess the feasibility of integrating state-of-the-art sequencing techniques and flow cytometry into diagnostic workup of pediatric lymphoma. RNA sequencing (RNAseq), whole exome sequencing, and flow cytometry were implemented into routine diagnostic workup of pediatric biopsies with lymphoma in the differential diagnosis. Within 1 year, biopsies from 110 children (122 specimens) were analyzed because of suspected malignant lymphoma. The experience with a standardized workflow combining histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies is reported. Flow cytometry was performed with fresh tissue in 83% (102/122) of specimens and allowed rapid diagnosis of T-cell and B-cell non-Hodgkin lymphomas. RNAseq was performed in all non-Hodgkin lymphoma biopsies and 42% (19/45) of Hodgkin lymphoma samples. RNAseq detected all but one of the translocations found by fluorescence in situ hybridization and PCR. RNAseq and whole exome sequencing identified additional genetic abnormalities not detected by conventional approaches. Finally, 3 cases are highlighted to exemplify how synergy between different diagnostic techniques and specialists can be achieved. This study demonstrates the feasibility and discusses the added value of integrating modern sequencing techniques and flow cytometry into a workflow for routine diagnostic workup of lymphoma. The inclusion of RNA and DNA sequencing not only supports diagnostics but also will lay the ground for the development of novel research-based treatment strategies for pediatric lymphoma patients.

CloneRetriever: An Automated Algorithm to Identify Clonal B and T Cell Gene Rearrangements by Next-Generation Sequencing for the Diagnosis of Lymphoid Malignancies.

BACKGROUND: Clonal immunoglobulin and T-cell receptor rearrangements serve as tumor-specific markers that have become mainstays of the diagnosis and monitoring of lymphoid malignancy. Next-generation sequencing (NGS) techniques targeting these loci have been successfully applied to lymphoblastic leukemia and multiple myeloma for minimal residual disease detection. However, adoption of NGS for primary diagnosis remains limited. METHODS: We addressed the bioinformatics challenges associated with immune cell sequencing and clone detection by designing a novel web tool, CloneRetriever (CR), which uses machine-learning principles to generate clone classification schemes that are customizable, and can be applied to large datasets. CR has 2 applications-a "validation" mode to derive a clonality classifier, and a "live" mode to screen for clones by applying a validated and/or customized classifier. In this study, CR-generated multiple classifiers using 2 datasets comprising 106 annotated patient samples. A custom classifier was then applied to 36 unannotated samples. RESULTS: The optimal classifier for clonality required clonal dominance ≥4.5× above background, read representation ≥8% of all reads, and technical replicate agreement. Depending on the dataset and analysis step, the optimal algorithm yielded sensitivities of 81%-90%, specificities of 97%-100%, areas under the curve of 91%-94%, positive predictive values of 92-100%, and negative predictive values of 88%-98%. Customization of the algorithms yielded 95%-100% concordance with gold-standard clonality determination, including rescue of indeterminate samples. Application to a set of unknowns showed concordance rates of 83%-96%. CONCLUSIONS: CR is an out-of-the-box ready and user-friendly software designed to identify clonal rearrangements in large NGS datasets for the diagnosis of lymphoid malignancies.

Core needle biopsy is an inferior tool for diagnosing cervical lymphoma compared to lymph node excision.

BACKGROUND: In Denmark, fine needle aspiration is the standardized tool for obtaining tissue samples from lymph nodes (LN) of the neck. However, because of a low specificity toward lymphomas, LNs suspicious for this disease are often surgically removed and examined. International studies have implied that a core needle biopsy (CNB) is sufficient for detecting lymphomas, thereby potentially avoiding surgery. However, all studies have been conducted retrospectively and the goal of this prospective study was to find the true sensitivity of CNB. MATERIAL AND METHODS: Fifty-seven patients were enrolled in the study, one was excluded due to lack of CNB material. LNs suspected for lymphoma were surgically removed from the neck, whereafter a CNB was obtained from the removed LN. The CNB and the remaining part of the LN were sent to the Department of Pathology for further processing and the samples were blinded and examined by two pathologists separately. A consensus diagnosis was reached in cases with divergent diagnostic proposals. Sensitivity of the CNB method in comparison to whole tissue sections for lymphoma diagnosis was calculated. RESULTS: The CNB method gave the correct diagnosis in 66% of lymphoma cases, was inconclusive in 14% and gave an incorrect lymphoma subtype in 18%. In 2% the CNB wrongly resulted in a benign diagnosis. CNB was correct in all the non-lymphoma cases; thereby retaining a specificity of 100%. CONCLUSION: This prospective study found a sensitivity of 66% for diagnosing lymphoma with a CNB. As the CNB in this study was obtained under optimal conditions, unlike in clinical practice, we conclude that CNB cannot be recommended as a standard tool for diagnosing lymphomas.

Advances in Lymphoma Molecular Diagnostics.

Lymphomas encompass a diverse group of malignant lymphoid neoplasms. Over recent years much scientific effort has been undertaken to identify and understand molecular changes in lymphomas, resulting in a wide range of genetic alterations that have been reported across all types of lymphomas. As many of these changes are now incorporated into the World Health Organization's defined criteria for the diagnostic evaluation of patients with lymphoid neoplasms, their accurate identification is crucial. Even if many alterations are not routinely evaluated in daily clinical practice, they may still have implications in risk stratification, treatment, prognosis or disease monitoring. Moreover, some alterations can be used for targeted treatment. Therefore, these advances in lymphoma molecular diagnostics in some cases have led to changes in treatment algorithms. Here, we give an overview of and discuss advances in molecular techniques in current clinical practice, as well as highlight some of them in a clinical context.

PreservCyt Is an Optimal Fixative that Permits Cytologic and Molecular Analyses of Vitreoretinal Lymphoma Biopsies.

Purpose: Vitreoretinal lymphoma (VRL) is a potentially fatal intraocular malignancy. Diagnosis is hampered by poor preservation of morphology and DNA/RNA integrity, which precludes adjunctive molecular analysis. We aimed to determine the optimum fixative protocol for VRL biopsies that permits cytology, IHC/flow cytometry and molecular analyses.Methods: Six fixatives were compared on cultured Pfeiffer cells used as a cellular model. Cells were fixed and evaluated on cellular morphology, antibody staining, DNA/RNA amount and integrity. VRL clinical cases were used as validation and proof-of-concept.Results: PreservCyt was the best fixative for preserving cellular morphology and high-quality RNA/DNA from vitreous fluid biopsies. Cells from clinical VRL cases fixed with PreservCyt showed adequate cellular morphology and IHC positivity. Sufficient DNA was obtained for IgH clonality and MYD88 mutation detection using remnant cytological fluid.Conclusions: PreservCyt maintains good morphology and RNA/DNA integrity suggesting that it is a suitable fixative for VRL diagnosis and molecular analysis.

Hodgkin Reed-Sternberg-Like Cells in Non-Hodgkin Lymphoma.

Reed-Sternberg cells (RSCs) are hallmarks of classic Hodgkin lymphoma (cHL). However, cells with a similar morphology and immunophenotype, so-called Reed-Sternberg-like cells (RSLCs), are occasionally seen in both B cell and T cell non-Hodgkin Lymphomas (NHLs). In NHLs, RSLCs are usually present as scattered elements or in small clusters, and the typical background microenviroment of cHL is usually absent. Nevertheless, in NHLs, the phenotype of RSLCs is very similar to typical RSCs, staining positive for CD30 and EBV, and often for B cell lineage markers, and negative for CD45/LCA. Due to different therapeutic approaches and prognostication, it is mandatory to distinguish between cHL and NHLs. Herein, NHL types in which RSLCs can be detected along with clinicopathological correlation are described. Moreover, the main helpful clues in the differential diagnosis with cHL are summarized.