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Rita Shaknovich

Publications and source records attributed to Rita Shaknovich.

7 recordsLinked to original sources
PubMed2021

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

Circulating Tumor DNA↗
PubMed2006

Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

The transit of T cell-activated B cells through the germinal center (GC) is controlled by sequential activation and repression of key transcription factors, executing the pre- and post-GC B cell program. B cell lymphoma (BCL) 6 and IFN regulatory factor (IRF) 8 are necessary for GC formation and for its molecular activity in Pax5+PU.1+ B cells. IRF4, which is highly expressed in BCL6- GC B cells, is necessary for class switch recombination and the plasma cell differentiation at exit from the GC. In this study, we show at the single-cell level broad coexpression of IRF4 with BCL6, Pax5, IRF8, and PU.1 in pre- and post-GC B cells in human and mouse. IRF4 is down-regulated in BCL6+ human GC founder cells (IgD+CD38+), is absent in GC centroblasts, and is re-expressed in positive regulatory domain 1-positive centrocytes, which are negative for all the B cell transcription factors. Activated (CD30+) and activation-induced cytidine deaminase-positive extrafollicular blasts coexpress Pax5 and IRF4. PU.1-negative plasma cells and CD30+ blasts uniquely display the conformational epitope of IRF4 recognized by the MUM1 Ab, an epitope that is absent from any other IRF4+PU.1+ lymphoid and hemopoietic subsets. Low grade B cell lymphomas, representing the malignant counterpart of pre- and post-GC B cells, accordingly express IRF4. However, a fraction of BCL6+ diffuse large B cell lymphomas express IRF4 bearing the MUM1 epitope, indicative of a posttranscriptional modification of IRF4 not seen in the normal counterpart.

Animals↗
PubMed2006

Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells.

Activation-induced cytidine deaminase (AID) is necessary for immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR) in T-dependent immune response in germinal centers (GCs). The structural similarity of AID with RNA-editing enzymes and its largely cytoplasmic location have fueled controversial views of its mode of interaction with DNA. We show that AID, a mature B-cell-restricted cytoplasmic antigen, is relocated into the nucleus in 2.5% of CDKN1B(-), CCNB1(-) GC cells. The GC dark zone and the outer zone (OZ), but not the light zone, contain nuclear and cytoplasmic AID(+) blasts. AID(+) cells in the OZ are in contact with T cells and CD23(-) follicular dendritic cells. In addition, AID is expressed in extrafollicular large proliferating B cells, 14% of which have nuclear AID. GC and extrafollicular AID(+) cells express E47 but not the inhibiting BHLH protein Id2. Outside the GC, AID(+) B cells are in contact with T cells and show partial evidence of CD40 plus bcr stimulation-dependent signature (CCL22, JunB, cMYC, CD30) but lack early and late plasma cell markers. The distribution of nuclear AID is consistent with the topography of SHM and CSR inside the GC and in extrafollicular activated B cells.

Animals↗
PubMed2006

Identification of rare Epstein-Barr virus infected memory B cells and plasma cells in non-monomorphic post-transplant lymphoproliferative disorders and the signature of viral signaling.

BACKGROUND AND OBJECTIVES: In early and polymorphic post-transplant lymphoproliferative disorders (PTLD) Epstein-Barr virus (EBV), through its latency proteins, drives the proliferation of B lymphocytes, a process which in immunocompetent individuals leads to the establishment of latently infected memory B cells. DESIGN AND METHODS: We analyzed 11 cases, which included early and polymorphic PTLD, and 12 controls for latency of EBV infection and their antigenic profile. RESULTS: We identified a minority of terminally differentiated EBER+ IRTA1+ memory B cells and EBER+ CD138+ PRDM1+ plasma cells in these samples. These elements were identified both in PTLD and in tumor-free tonsils from post-transplant patients but not in EBV- control tonsils. The expression of EBV latency proteins is heterogeneous, and is associated with activation of the NF-kB pathway. EBV signaling (through EBNA2, LMP1 and LMP2A) and NF-kB activation correlated with upregulation of target proteins: cMYC, JunB, CCL22, TRAF1 and IRF4. EBV-infected lymphocytes in early and polymorphic PTLDs represent a mixture of latencies II, III and, in at least 1/3 of infected cells, of latency 0. INTERPRETATION AND CONCLUSIONS: EBV infection correlates with NF-kB activation, with EBV-dependent cell signaling, and lastly, with the presence of EBV-infected plasma cells and memory cells.

Adolescent↗
PubMed2005

PRDM1/Blimp-1 is expressed in human B-lymphocytes committed to the plasma cell lineage.

PRDM1/Blimp-1 (in human and mouse, respectively) has a central role in determining and shaping the secretory arm of mature B-cell differentiation. In this study, a mouse monoclonal antibody that recognizes PRDM1 was used to detail its distribution in normal human lymphoid tissue and in lymphoid neoplasms that correspond to different stages of B-cell differentiation. PRDM1 was expressed in germinal centre blasts that co-express Pax5, CD19, CD20, and CD10, but not BCL6 or MTA-3. Pax5 was downregulated and full plasma cell morphology and phenotype were acquired by PRDM1+, nuclear cREL-, pre-plasma cells upon exit from the germinal centre. Activated extrafollicular B-cells (CD30+, Pax5+) were largely PRDM1-. PRDM1 was also absent in tissue histiocytes and the majority of resting T-cells and S-100+ antigen-presenting cells. PRDM1 and CD138 were expressed simultaneously in human lymphomas with plasma cell differentiation, but not in marginal zone lymphomas or chronic lymphocytic leukaemias. A minority of diffuse large B-cell lymphomas expressed PRDM1 and Hodgkin lymphomas were largely PRDM1-. Infiltrating T-cells in PRDM1- B-cell lymphomas expressed PRDM1. In conclusion, PRDM1 staining is a reliable and informative assay to define plasma cell commitment and differentiation in human normal and neoplastic B-cell lineages.

Animals↗