Genomic Taxonomy of Aggressive B-Cell Lymphoid Neoplasms.

Aggressive B-cell lymphomas collectively make up half of all lymphoma diagnoses. The current classification system, the 2017 revision of the WHO 4th edition, assigns these tumors into groups based on morphology, immunophenotype, site of disease, and the presence of recurrent chromosomal rearrangements. Accurate and reproducible diagnosis is required for selection of optimal treatment, prognostication, and ongoing basic research and clinical trials aimed at improving outcomes. Ideally, the taxonomy would continue to evolve towards further defining homogeneous groups of tumors sharing targetable biology. Gene expression (GE) profiling of tumors supports the biologic validity of the entities in the current classification and, along with genomic sequencing, is driving the identification of new lymphoma subtypes. In the mid-2000s GE profiling studies identified specific signatures that distinguish aggressive B-cell entities from each other—namely, primary mediastinal large B-cell lymphoma (PMBL) from diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) from DLBCL. The increasingly divergent treatment of these entities makes reliable diagnosis important. Prior to those studies, GE profiling identified 2 distinct subtypes of DLBCL. This binary division of DLBCL into the cell-of-origin groups of germinal center B-cell-like (GCB) and activated B-cell-like (ABC) has been foundational to our understanding of the pathology of DLBCL. However, recent failure of clinical trials to improve outcomes by adding targeted agents to R-CHOP in upfront treatment of ABC-DLBCL highlights that this binary division may not be sufficiently granular to support precision medicine. These signatures have been translated onto tractable technology platforms, including nuclease protection assay, RT-MLPA, and NanoString, allowing potential integration into diagnostic workflows. More recently, GE signatures have been described that identify distinct molecular subtypes within GCB-DLBCL. Working from the standpoint of tumors that have GE profiles sitting between BL and DLBCL (the “molecular high grade” signature [MHG]) or the GE signature of tumors with rearrangement of MYC and BCL2 (the “double hit” signature [DHITsig]), a sizeable group of GCB-DLBCL can be identified with poor prognosis. Finally, a group of DLBCL without mediastinal involvement have been shown to display the PMBL GE signature. These tumors share perturbation of the hallmark pathways of PMBL but arrive at this biology through different genetic mechanisms. These subtypes map onto, and complement, the newly minted genetics-based classifications of DLBCL. In order to arrive at a tractable unified molecular classification for aggressive B-cell lymphoma, ongoing efforts are needed to integrate GE and genetic aberrations across the disease spectrum. Such a classification framework holds the promise of improved diagnostic accuracy and reliability while providing the foundation for improving patient outcomes through precision medicine. Citation Format: David W. Scott. The role of gene expression in the classification of aggressive B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA08.

e19563 Background: Tisagenlecleucel is an anti-CD19 chimeric antigen receptor T-cell therapy approved for refractory diffuse large B-cell lymphoma(DLBCL) in May 2018. However, a recently concluded phase 3 international trial showed that Tisagenlecleucel was not superior to standard salvage therapy with aggressive lymphoma that was refractory to or progressing within 12 months after first-line treatment. Also, recently the availability of long-term outcomes from earlier trials makes it an excellent time to evaluate the Sustained response(SR) and Adverse effects associated with it. Methods: A comprehensive literature search was done of Pubmed, Embase, and Cochrane. Results: We included three trials which included two multicenter trials and one case series. Schuster et al. in their single-center case series demonstrated that Tisagenlecleucel infusion in patients with refractory diffuse large B-cell lymphoma on median follow-up at 28.6 months showed Complete remission(CR): 43%; 95% CI(18-71) Sustained remission(SR): 86%; 95% CI(13 to 98), Severe cytokine-release syndrome(CRS): 18%. On further median follow-up at 60.7 months showed a CR: 46%, overall response rate(ORR): 58%, Progression-free survival(PFS): 31% (95% CI:14-51). Good recovery in humoral and cellular response. The limitation of the trial was a relatively small number of enrolled patients. JULIET trial, a more extensive multicenter phase 2 trial involving 93 patients, demonstrated that Tisagenlecleucel infusion in patients with refractory diffuse large B-cell lymphoma on 14 months median follow-up showed an ORR: 52% (95% CI, 41 to 62), CR: 40%, Partial response(PR): 12%. On further median follow-up at 40.3 months showed CR: 39%, ORR: 53·0% (95% CI 43·5–62·4). Grade 3-4 CRS: 23%. BELINDA Trial also an extensive multicenter phase 3 trial involving 322 patients which involved patients with aggressive B cell lymphoma that was refractory to or progressing within 12 months after first-line therapy showed infusion of Tisagenlecleucel with optional bridging treatment or salvage chemotherapy and autologous HSCT Showed similar Medial event-free survival(MEFS) at three months for both groups. Progression at six weeks was 25.9% in Tisagenlecleucel vs. 13.8% in the control group. Conclusions: Tisagenlecleucel infusion is shown to have a high rate of durable response in relapsed or refractory diffuse large B-cell lymphoma in adults. Long-term follow-up showing satisfactory ORR with good recovery in cellular and humoral function. However, infusion of Tisagenlecleucel with optional bridging therapy or salvage chemotherapy and autologous HSCT inpatient with aggressive B cell lymphoma that was refractory to or progressing within 12 months after first-line therapy showed Tisagenlecleucel is not superior to standard salvage therapy needing additional studies to evaluate the benefits when used as a second-line agent.

Pathology and classification of aggressive mature B-cell lymphomas.

The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model.

Diffuse large B-cell lymphoma (DLBCL): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Select patients with relapsed/refractory aggressive B cell lymphoma may benefit from bridging radiation (bRT) prior to anti-CD19-directed chimeric antigen receptor T cell therapy (CAR-T). Here, we examined patient and treatment factors associated with outcomes and patterns of failure after bRT and CAR-T. We retrospectively reviewed adults with diffuse large B-cell lymphoma (DLBCL) who received bRT prior to axicabtagene ciloleucel, tisagenlecleucel, or lisocabtagene maraleucel between 11/2017-4/2023. Clinical/treatment characteristics, response, and toxicity were extracted. Survival was modeled using Kaplan-Meier or Cox regression models for events distributed over time, or binary logistic regression for disease response. Fisher's Exact Test or Mann-Whitney U methods were used. Of 51 patients, 25.5% had bulky disease and 64.7% had Stage III/IV disease at the time of RT. Comprehensive bRT alone to all disease sites was delivered to 51% of patients, and 29.4% were additionally bridged with systemic therapy. Median follow-up was 10.3 months (95% CI: 7.7-16.4). Overall response rate (ORR) was 82.4% at 30 days post-CAR-T infusion. Median overall survival (OS) was 22.1 months (6.6-not reached) and the median progression-free survival (PFS) was 7.4 months (5.5-30). OS/PFS were 80% (66-99)/78% (64-87) at 1-year, and 59% (44-71)/54% (40-67) at 2-years, respectively. Comprehensive RT to all sites of disease correlated with improved PFS and OS, p ≤ 0.04. Additionally, ECOG ≥2 and Stage III/IV disease predicted poor OS (p ≤ 0.02). Disease bulk, IPI ≥3, and non-GCB histology were poor predictors for disease-specific survival (DSS), p<0.05. The latter two, as well as bRT dose of ≤30 Gy predicted worse PFS (p<0.05). Among patients with advanced stage disease, comprehensive bRT to all sites of disease (n=10) was not associated with improved OS and PFS compared to focal bRT (n=23), p>0.17. No difference was seen in bridging RT vs. chemoRT. Twenty-six patients developed relapse (50.9%), of which 46% was in-field. Risk of in-field relapse correlated with bulky disease (OR=7, 95% CI: 1.2-41, p=0.03) and lack of response at 30 day post-CAR-T evaluation (OR=16.8, 95% CI: 1.6-176, p=0.02), but not with bRT dose (p=0.27). bRT and CART is a good treatment strategy for select patients with aggressive B cell lymphoma. Comprehensive bRT including all sites of disease is associated with improved outcomes.

Positron Emission Tomography (PET) Guided Therapy of Aggressive Lymphomas - Interim PET-Based Outcome Prediction and Treatment Changes in Patients with B Cell Lymphomas Participating in the PETAL Trial

Differential expression of enhancer of zeste homolog 2 (EZH2) protein in small cell and aggressive B-cell non-Hodgkin lymphomas and differential regulation of EZH2 expression by p-ERK1/2 and MYC in aggressive B-cell lymphomas

Aggressive B-cell lymphomas: frequency, immunophenotype, and genetics in a reference laboratory population.

Clinical, Immunophenotypic and Genetic Characteristics of Aggressive (non-Burkitt) B-Cell Lymphoma in a Real Life Cohort

Clinicopathological features of aggressive B-cell lymphomas including B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell and Burkitt lymphomas: a study of 44 patients from Argentina

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), representing around 30% to 40% of all newly diagnosed lymphomas 1. DLBCL is clinically, morphologically and biologically a heterogeneous disease reflected in the highly variable clinical course. The 2008 World Health Organization (WHO) classification of lymphoid malignancies recognizes within the group of DLBCL, not otherwise specified (NOS) several subtypes characterized by unique clinical and pathological features including primary DLBCL of the central nervous system (CNS), primary cutaneous DLBCL, leg type, T-cell histiocyte-rich large cell lymphoma and EBV positive DLBCL of the elderly (Table 1). Nevertheless, most cases of DLBCL fall into the ‘NOS’ category. In the last 15 years our understanding of the genetic changes and biology of DLBCL has increased tremendously 2. Gene expression profiling (GEP) studies have revealed that DLBCL comprises several molecular subgroups that reflect either the stage in B cell development from which the disease originates or the activity of different biological programs 3, 4. The standard initial treatment for DLBCL is combined immunochemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) 5. While durable remissions can be achieved in the majority of cases with this combined regimen, over 30% of patients will not respond or will relapse with resistant disease. A possible explanation for such differences in therapeutic success is the considerable biological heterogeneity of DLBCL. Therefore, there is an ongoing effort to tailor therapy based on specific subtypes of DLBCL, and to identify prognostic markers like BCL2 and MYC. The diagnosis of DLBCL needs to integrate, in addition to classic morphology and immunophenotype, all the new genetic and molecular diagnostic tools. This report attempts to review distinctive pathological characteristics of DLBCL and their clinical significance. DLBCL is characterized by monoclonal rearrangement of immunoglobulin heavy (IGH) and light chain (IGκ-IGλ) genes. Analogous to most B-NHL, DLBCL derives from a mature B cell that has experienced the germinal center (GC) reaction. Based on GEP studies DLBCLs have been divided into two main subgroups based on the putative cells of origin 3, 4. GC B cell-like (GCB)-DLBCL (50% cases) exhibits a transcriptional profile that resembles that of GC B-cell with expression of genes normally detected in GC B-cells such as CD10 and the transcriptional repressor BCL6 and harboring highly mutated immunoglobulin genes with ongoing somatic hypermutations (SHM). Activated B cell-like (ABC)-DLBCL shows several features of B cell receptor (BCR) activated B-cells at a plasmablastic stage, just prior to exit the GC with up-regulation of genes required for plasma cell differentiation (IRF4/MUM1) (Figure 1). These tumors downregulate the GC-specific program, activating at the same time, the NF-κB and BCR signaling pathways 2. Consistent with their late GC origin, these tumors do not show evidence of ongoing SHM. Because not all cases of DLBCL can be characterized as GCB or ABC, a less well-characterized group comprising about 15% of the cases remain unclassifiable. Within the group of mature B cell NHL, DLBCL shows the highest degree of genomic complexity including point mutations and copy number aberrations, and less frequently chromosomal translocations and gene amplification 2. Although some of these lesions might be observed in both GCB and ABC subtypes of DLBCL, most of them are preferentially associated with one or the other subtype of DLBCL, suggesting their potential role for diagnostic, prognostic and therapeutic stratification (Figure 2). The frequent mutations found in DLBCL are thought to be the result of an aberrant function of the physiologic SHM mechanism. Accordingly, mutations in CREBBP/EP300 and MLL2 are preferentially seen in GCB-subtype. EZH2 is required for GC formation and mutations in this gene are restricted to GCB-DLBCL. Translocations resulting in deregulated BCL2 and MYC are almost restricted to GCB-DLBCL. Around 30–40% of GCB-DLBCL carry the t(14;18) translocation, whereas MYC translocations have been identified in 5–15% of DLBCL 6. In contrast, translocations involving the BCL6 locus are present in 35% of DLBCL cases, being more frequent in ABC-DLBCL (25%). Another major transformation mechanism that impairs plasma cell differentiation are mutations and deletions in PRDM1/BLIMP1 identified in 25% of ABC-DLBCL. Constitutive activation of the NF-κB transcription factor complex represents the hallmark of ABC-DLBCL 2. The underlying causes of NF-κB signaling pathway activation are diverse and include gain-of-function mutations in signal transduction components of the BCR (CD79a and CARD11) and toll-like receptor (MYD88) signaling pathways. Loss-of-function mutation and/or deletions in the NF-κB negative regulator TNFAIP3/A20 have been reported in up to 30% of ABC-DLBCL. The diagnosis of DLBCL is usually not difficult. Lymph nodes demonstrate a diffuse proliferation of large lymphoid cells that have totally or partially effaced architecture. Cytological, DLBCL can be centroblastic, immunoblastic or anaplastic in appearance 1. Centroblastic morphology is the most common variant composed of medium to large cells with round to oval vesicular nuclei and fine chromatin with two to four nuclear membrane-bound nucleoli. There is moderate amphophilic to basophilic cytoplasm. In the immunoblastic variant greater than 90% of the cells have an immunoblastic appearance with a large centrally located nucleolus and basophilic or amphophilic cytoplasm. This variant has been associated with the non-germinal center type derivation, an adverse prognosis 4 and recently as a major reservoir for MYC-IGH translocations. In most cases, however; the tumors show an admixture of centroblasts and immunoblasts. The distinction of the immunoblastic variant from the centroblastic variant is not always straightforward and has generally met poor intraobserver and interobserver reproducibility. The anaplastic variant is rather rare and is characterized by large cells with bizarre pleomorphic nuclei. These cells may resemble Hodgkin or RS cells and may show sinusoidal and/or cohesive growth pattern and even mimic undifferentiated carcinoma. The anaplastic morphology is independent of ALK expression in B-cell lymphomas. Other rare morphologic variants exist including the signet ring cell, microvillous and spindle cell variants. The neoplastic cells characteristically express pan B-cell markers including CD19, CD20, CD22, PAX5 and CD79a. Surface and/or cytoplasmic immunoglobulin (IgM > IgG > IgA) can be demonstrated in 50–75% of the cases. Other markers commonly used in the characterization of DLBCL include CD10, BCL6, BCL2 and IRF4/MUM1 1. Aberrant phenotypes are not uncommon in DLBCL and may be responsible for confusion in the diagnosis. Lack of one or more B-cell markers may occur. In 10% of the cases the tumor cells express CD5. This CD5 positive DLBCL usually represent de novo cases, and only rarely are transformed CLL cases. Expression of CD5 is associated with worse prognosis even in the rituximab era. Aberrant expression of cytoplasmic CD3 has been also documented mainly in extranodal DLBCL without the expression of other T-cell markers. DLBCL with cyclin D1 expression lack the characteristic t(11;14) translocation of MCL, have a centroblastic morphology and a post-germinal center phenotype with positivity for IRF4/MUM1. These cases should be distinguished from MCL with blastic or pleomorphic morphology. In situ hybridization for EBERs should be performed in cases with geographic necrosis and/or Reed–Stenberg-like cells to exclude the possibility of an EBV+DLBCL of the elderly. Because of the prognostic value of cell of origin and the increasing effort to tailor therapy based on molecular characteristics of DLBCL, a reliable method to identify GCB and non-GCB subtypes is needed. GEP, which is considered the gold standard to assign the molecular subtypes, is not routinely available and is not cost effective in routine diagnosis. Several studies have attempted to recapitulate the molecular subgroups (GCB vs. non-GCB) using a limited panel of antibodies available in most pathology laboratories (Figure 1). The Hans algorithm has been the most widely used in clinical trials. In this classifier three antibodies are used CD10, BCL6 and IRF4/MUM1. According to this algorithm DLBCL with CD10 expression in more than 30% cells belong to the GCB group. Cases that are CD10 negative, BCL6 positive but IRF4/MUM1 negative are also GCB subtype. Cases that are IRF4/MUM1 positive with or without expression of BCL6 are assigned to the non-GCB subtype. There have been other algorithms attempting to improve the Hans classifier. These include the use of FOXP1 and GCET1 by Choi et al., and the use of LMO2 by Natkunam et al. One study comparing different algorithms showed an 87% concordance with GEP for the Choi method and 86% for Hans scheme 7. In this same study, the Tally algorithm based on FOXP1, GCET1, CD10, IRF4/MUM1 and LMO2 antibodies was demonstrated to be the most robust. The use of BCL6 was excluded from the analysis because it was considered a problematic, poorly reproducible stain. Nevertheless, in our experience BCL6 is reliable and helpful in the routine diagnosis. Accordingly, in the report from the international DLBCL rituximab-CHOP consortium program study, an algorithm based on expression of CD10, FOXP1 and BCL6 was used, which had a 92.6% concordance with GEP 8. Although most studies find that immunohistochemical algorithms correlate with prognosis in DLBCL, everybody agrees that these algorithms are an imperfect substitution for GEP. The improvement of molecular techniques is making possible to use paraffin-embedded material with results comparable to fresh-frozen material. The recent development of a 20-gene assay using NanoString technology in paraffin-embedded tissue is a promising methodology with potential to be used as a routine method for determining cell of origin in DLBCL 9. Translocations involving the MYC oncogene are the molecular hallmark of Burkitt lymphoma (BL). Most cases involve the IGH@ gene on chromosome 14. Less commonly, the light chain genes on chromosome 2 and 22 are involved in the translocation. These translocations in BL are characteristically the sole chromosomal aberration identified 10, 11. Approximately 5 to 15% of DLBCL cases have been reported to carry MYC translocations, whereas 19–38% of cases show MYC low copy number gains 6. Most MYC rearranged DLBCL cases fall into the GCB subtype 10, 12. In contrast to BL, MYC rearrangements in DLBCL are often seen to non-IGH@ partners and with complex karyotypes 10. The prognostic significance of MYC rearrangement alone in DLBCL is controversial. Several studies have demonstrated the association of MYC rearrangement with poorer outcome in DLBCL patients treated with R-CHOP 6. However, recent studies have suggested that the impact of MYC is strongly influenced by BCL2 and that MYC alone does not have a worse prognosis 13, 14. MYC rearranged DLBCL may either arise de novo or may represent a high-grade transformation of a low-grade lymphoma, usually follicular lymphoma. In the latter case the MYC translocation is accompanied by a BCL2 rearrangement, so-called ‘double-hit’ (DH) lymphoma. Approximately 40% of all DH lymphomas represent transformed FL cases 15. However, 60–80% of MYC rearranged bona-fide de novo DLBCL are accompanied either by a BCL2 and/or a BCL6 rearrangement, representing DH or triple hit (TH) lymphomas 12, 14. Altogether DLBCL with DH/TH represent around 3–6% of all DLBCL and are generally refractory to standard chemotherapy regimens and have a poor prognosis 13, 14, 16. Importantly, not all patients with DLBCL morphology and DH/TH may have a dismal prognosis. Recently, attention has focused on the apparent importance of the MYC partner because it has been suggested that only cases with an IG/MYC translocation have an adverse prognosis. The development of FISH assays readily performed in paraffin sections and more recently a monoclonal antibody that specifically recognizes MYC protein have facilitated the recognition of MYC alterations in routine diagnosis. Break-apart FISH probes are considered to be the most sensitive assay for identifying chromosomal rearrangements. In addition, the use of MYC/IGH dual fusion probe helps to confirm the MYC partner 6. However, dual fusion probes for MYC/IGκ and MYC/IGλ are not commercially available. MYC protein expression has been proposed as a rapid and cost-effective screening tool to identify MYC rearrangements. Although some studies agree that finding >70% MYC+ cells correlates with MYC rearrangement 6, 13, a considerable number of cases with MYC rearrangement fall into the low MYC expression group (<40%) 12. A combined approach MYC-FISH and IHC seems advisable. More common than MYC translocations is MYC protein overexpression reported to occur in 25–30% of DLBCL cases 13, 14, 16. Most studies used a cut-off of 40% for MYC positivity. Of note, the negative prognostic impact of MYC protein expression is observed only in patients who simultaneously overexpressed BCL2 protein, so-called ‘double-expresser’ (DE). The cut-off for BCL2 protein overexpression varies in different studies, but most studies required at least 50% of tumor cells. MYC and BCL2 DE have been reported to occur in 19–34% of DLBCL patients, and to have a worse prognosis than patients who do not express any or only one protein 13, 14, 16, but better prognosis than DH/TH DLBCL, which have a dismal outcome. Interestingly, the DE cases appear more common in the ABC subtype, and it has been suggested that this may largely contribute to the known inferior survival of the ABC subtype 16. The morphological distinction between BL and DLBCL has been problematic for pathologists. GEP studies have shown that BL has a characteristic signature but that there are cases within the spectrum of DLBCL and aggressive B-cell lymphomas, which have a similar molecular BL signature or fell into an intermediate category 10, 11. The 2008 WHO classification recognized this problematic an added a provisional category of B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCLU) (Figure 3) 15. BCLU is a disease of older patients presenting with nodal or extranodal disease usually in an advanced clinical stage, high lactate dehydrogenase and frequent bone marrow and CNS infiltration with a dismal prognosis. Interestingly, MYC rearrangements can be detected in 30–50% of the cases usually associated with many more chromosomal aberrations 15. The incidence of DH/TH in BCLU has been reported to be high (32%–78%).The precise morphological boundaries of this category are still not well defined. There is an ongoing discussion about how to classify aggressive lymphomas with DH/TH. One proposal is to continue classifying them based on morphology as DLBCL or BCLU or putting them together as a group of ‘DH/TH lymphomas’ regardless of the morphology of the tumor cells. The understanding of the biology of DLBCL has increased tremendously in the last 15 years. The diagnosis of DLBCL needs to integrate, in addition to standard morphology and immunohistochemistry, all available ancillary techniques. The routine use of FISH and IHC to detect MYC and BCL2 alterations/overexpression is recommended. Patients with DH and double expression of MYC and BCL2 represent poor-risk subsets in which alternative strategies should be explored 5. The distinction of GCB versus ABC-DLBCL has not yet led to differences in primary treatment. The current standard of care for most patients is R-CHOP, which has improved dramatically the outcome of DLBCL. However, for patients who fail R-CHOP, the choice of therapy is very likely to be influenced by the cell of origin 5. For example, bortezomib or BTK inhibitors have been shown to be effective in relapsed ABC-DLBCL but not in GCB subtype. On the contrary treatment with EZH2 inhibitors and BCL6 inhibitors have been shown to be effective in relapsed GCB-DLBCL. Emerging new targeted therapy will certainly influence the diagnosis and treatment of DLBCL in the near future.

Correction of Vitamin D Deficiency in Patients with Aggressive B-Cell Lymphomas during Rituximab-Containing Chemotherapy: Impact on Outcome

Background: High MYC and BCL2 co-expression as detected by immunohistochemical staining (IHC) of fixed biopsy samples identifies a sub-group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcome among patients treated with standard chemotherapy, and the differential expression of MYC and BCL2 among DLBCL subtypes provides a biological basis for the prognostic value of the ‘Cell of Origin’ classification system. Thus quantifying MYC activity and BCL2 expression in formalin-fixed paraffin embedded (FFPE) biopsy specimens could help to identify patients who might benefit from more aggressive chemotherapy. Unfortunately, IHC is not a reproducibly quantitative test due to a number of pre- and post- analytical factors. In contrast, gene expression profiling (GEP) allows for the possibility of better standardization and quantitation of biomarkers in biopsy samples, but traditional GEP has required RNA isolated from frozen tissue. Design: We sought to develop a molecular classifier of MYC activity and BCL2 expression that is applicable to FFPE biopsy samples using the ‘NanoString nCounter’ platform in a 2-stage approach: 1. Discriminate between Burkitt Lymphoma (BL) and DLBCL using a selection of genes specific for each diagnostic category. 2. Quantify MYC and BCL2 expression using statistically justified ‘MYC target’ genes as well as other genes selected with an unbiased approach, those with significant differential expression between MYC IHC High and IHC Low cases. 3. Normalize data to selected housekeeping genes and assess the tissue microenvironment. The initial gene set was developed in silico based on the whole genome gene expression of 56 carefully selected de novo DLBCL that had companion MYC immunostaining. Results: An initial gene set comprising 200 genes was tested on a discovery cohort of FFPE biopsy samples of 42 aggressive B-cell lymphomas (12 Burkitt Lymphoma [BL] and 30 DLBCL). Differential analysis and prediction models were used to construct a classifier comprising 87 genes that resulted in the successful classification of these tumors. We next validated the approach using an independent cohort of FFPE tissue biopsies (12 BL, 7 genetic “double hit” lymphomas, and 38 DLBCL lacking MYC and BCL2 rearrangements). Targeted profiling and molecular classification correctly diagnosed 100% of tumors as either BL or DLBCL. For DLBCLs, the molecular classifier correctly predicted the MYC expression in 87% of cases when compared to a well-validated IHC assay. We conclude that a targeted gene expression profile, using the Nanostring nCounter platform, coupled with a validated molecular classifier can effectively distinguish DLBCL from BL and quantify MYC activity and BCL2 expression in DLBCL. This protocol will be useful for the routine diagnostic and prognostic stratification of aggressive B-cell lymphomas in clinical practice. Citation Format: Christopher D. Carey, Daniel Gusenleitner, Bjoern Chapuy, Heather Sun, Azra Ligon, Alexandra E. Kovach, Long P. Le, Aliyah R. Sohani, Margaret Shipp, Stefano Monti, Scott J. Rodig. A targeted molecular classifier of MYC activity and BCL-2 expression in aggressive B-cell lymphomas, designed for clinical practice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5582. doi:10.1158/1538-7445.AM2014-5582