Angela M. B. Collie

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation

Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes with CK20-positive Merkel cell carcinoma, including RB1 mutations restricted to Merkel cell polyomavirus-negative tumors. However, some CK20-negative Merkel cell carcinomas harbor mutations not previously described in Merkel cell carcinoma. Hence, CK20-negative Merkel cell carcinomas harbor diverse oncogenic drivers which may represent therapeutic targets in individual tumors.

Molecular Subtype Classification of Formalin-Fixed, Paraffin-Embedded Diffuse Large B-Cell Lymphoma Samples on the ICEPlex® System

Microarray gene expression profiling (GEP) has been used to identify molecular subtypes of diffuse large B-cell lymphoma (DLBCL) based on the similarity of GEP to a putative “cell of origin” (COO) and defines two molecular subtypes: activated B-cell-like (ABC) DLBCL and germinal centre B-cell-like (GCB) DLBCL (Alizadeh, et al 2000). This dichotomization has prognostic and biological significance, with the ABC subtype having a worse outcome and distinct pathobiology that includes activation of the B-cell receptor and nuclear factor (NF)-κB pathways (Lenz, et al 2008). Attempts to reduce this subclassification to practice using immunohistochemistry are fraught with technical and interpretive issues such that a need for practical quantitative molecular assays exists (Coutinho, et al 2013, de Jong, et al 2007, de Jong, et al 2009, Salles, et al 2011). Indeed, studies have refined this COO concept using limited gene sets, including a 14-gene model to assign ABC and GCB DLBCL subtype developed by Wright et al (2003) as well as a recently-described DLBCL subtyping assay based on parsimonious digital gene expression (Nanostring) technology (Scott, et al 2014). In order to support clinical trials for therapies targeting populations enriched in ABC DLBCL and to offer prognostic information for DLBCL patients as part of our clinical service, we developed a novel multiplex, single-tube, gene expression assay on the ICEPlex® system (PrimeraDx, Mansfield, MA), which allows differentiation between GCB and ABC DLBCL subtypes in formalin-fixed paraffin-embedded (FFPE) specimens using a Food and Drug Administration-cleared platform.

Comparison between melanoma gene expression score and fluorescence in situ hybridization for the classification of melanocytic lesions

Melanoma accounts for most skin cancer-related deaths and has an increasing incidence. Accurate diagnosis and distinction from atypical nevi can be at times difficult using light microscopy alone. Fluorescence in situ hybridization (FISH) and melanoma gene expression score (myPath, Myriad Genetics) have emerged as ancillary tools to further aid in this differential diagnosis. Our aim in this study was to correlate FISH results, gene expression score, consensus histopathologic impression and clinical outcome on a series of 117 challenging melanocytic lesions collected from three separate institutions. The lesions were separated into two groups: 39 histopathologically unequivocal lesions (15 malignant, 24 benign) and 78 challenging lesions interpreted by expert consensus (27 favor malignant, 30 favor benign, and 21 ambiguous). Melanoma-FISH was performed using probes for 6p25, 11q13, 8q24, and 9p21/CEP9 and scored according to established criteria. Analysis by myPath gene expression score was performed and interpreted by the manufacturer as ‘benign’, ‘indeterminate,’ or ‘malignant’. In the unequivocal group, melanoma-FISH and myPath score showed 97 and 83% agreement with the histopathologic diagnosis, respectively, with 93 and 62% sensitivity, 100 and 95% specificity, and 80% inter-test agreement. In the challenging group, FISH and the myPath score showed 70 and 64% agreement with the histopathologic interpretation, respectively, with 70% inter-test agreement and similar sensitivities and specificities. The inter-test agreement was 73% overall, excluding indeterminate results. Discordant test results occurred in 27/117 cases from both unequivocal and challenging groups. Melanoma-FISH and gene expression score are valuable ancillary tools, though both have limitations and return discordant results in a subset of cases. Follow-up studies with more extensive clinical outcome data are warranted to establish the accuracy of these tests for the classification of melanocytic lesions.

Dual expression of MYC and BCL2 proteins predicts worse outcomes in diffuse large B-cell lymphoma

Abstract Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma. However, the immunohistochemistry protocols for dual-expression staining and the scoring cut-offs vary by study. Sixty-nine cases of diffuse large B-cell lymphoma were evaluated for MYC and BCL2 protein expression using various cut-offs that have been recommended in prior studies. Independent of the International Prognostic Index risk group, cases with dual protein expression of BCL2 and MYC using ≥50%/40% cut-offs and ≥70%/40% had significantly shorter overall survival than cases without. It was verified in this patient population that the use of BCL2 and MYC immunohistochemistry, performed with available in vitro diagnostic-cleared antibodies, provides rapid prognostic information in patients with de novo diffuse large B-cell lymphoma. This study has practical implications for diagnostic laboratories and serves as a guide for implementation in the setting of future clinical trials.

Flow Cytometric Analysis of Cerebrospinal Fluid Has Low Diagnostic Yield in Samples Without Atypical Morphology or Prior History of Hematologic Malignancy

OBJECTIVES To identify pretest characteristics of cerebrospinal fluid (CSF) specimens that will allow the rational use of flow cytometric analysis (FCA) in the diagnosis of hematologic malignancy. METHODS Retrospective data were collected on 501 consecutive CSF samples submitted for FCA. RESULTS A positive diagnosis of hematologic malignancy was made in 41 specimens (8.2%). Blasts or atypical lymphocytes were noted on Wright-stained slides in 98% of FCA-positive specimens (40/41), and a history of a hematologic malignancy was present in 89% of specimens (34/38). All FCA-positive specimens had atypical morphology or history of hematologic malignancy. Four hundred six specimens (81%) were FCA negative. Of FCA-negative specimens, 7% (30/406) had atypical morphology, and 3% (12/404) had future central nervous system involvement seen within 30 days. CONCLUSIONS These data support a policy in which FCA of CSF is actively discouraged unless atypical lymphocytes or blasts are seen or a history of hematologic malignancy is present.

Cutaneous intravascular extramedullary hematopoiesis in a patient with post-polycythemia vera myelofibrosis

Cutaneous extramedullary hematopoiesis represents a rare manifestation of myeloproliferative disorders with less than 45 reported patients in the literature. Cutaneous extramedullary hematopoiesis is most often seen in the context of primary myelofibrosis but has also been identified in myelofibrosis evolving from polycythemia vera or essential thrombocytosis. Herein, we report a case of intravascular extramedullary hematopoiesis diagnosed in a cutaneous biopsy of a 67-year-old woman with post-polycythemia vera myelofibrosis. To our knowledge, this is the first reported case of cutaneous intravascular extramedullary hematopoiesis diagnosed by skin biopsy. Extramedullary hematopoiesis involves production of blood cells outside the bone marrow. In early fetal development, extramedullary hematopoiesis is physiologic and involves the liver and, to a lesser extent, the spleen.1,2 In the postnatal period, extramedullary hematopoiesis can present with the clinical visual of a ‘blueberry muffin baby’. In such cases, it is associated with congenital viral infections, including cytomegalovirus and Rubella, as well as hematopoietic disorders.3– 5 In adults, extramedullary hematopoiesis can be a reactive response to a loss of marrow elements or to an underlying hematologic abnormality, but is also associated with myeloproliferative neoplasms.6 Myeloproliferative neoplasms are defined as clonal proliferations of myeloid stem cells and include chronic myelogenous leukemia BCR-ABL1 positive (CML), primary myelofibrosis, polycythemia vera, and essential thrombocythemia, among others.7 Primary myelofibrosis is separated into a prefibrotic phase, characterized by proliferation of megakaryocytes and granulocytes in the bone marrow, followed by a fibrotic phase, which is characterized by collagen and reticulin fibrosis in the bone marrow. Intravascular hematopoiesis can also be seen in the bone marrow 7. In the later stages of primary myelofibrosis, extramedullary hematopoiesis most commonly involves the spleen followed by the liver, but it has also been reported in the lymph nodes, kidney, bladder, central nervous system, lungs, gastrointestinal system, and skin.6– 8 Polycythemia vera is characterized by three phases, including a pre-polycythemic phase, a polycythemic phase with increased red cell mass, and a post-polycythemia vera myelofibrosis phase.7,9,10 One study demonstrated that 6% of polycythemia vera patients will progress to myelofibrosis within 15 years.11 Essential thrombocythemia involves proliferation of cells of megakaryocyte lineage with corresponding thrombocytosis. It can also evolve to myelofibrosis and has rarely been associated with extramedullary hematopoiesis.7,9,12

Analysis of Peripheral T-cell Lymphoma Diagnostic Workup in the United States

Micro‐Abstract With increased understanding of the unique entities of peripheral T‐cell lymphoma (PTCL), subtype‐specific approaches are emerging, and more precise diagnoses are becoming increasingly important. Using data from a large prospective cohort study, we examined the diagnostic patterns of PTCL in the United States. We found that the workup for PTCL varies widely and often lacks important phenotypic information to fully characterize the lymphoma. Background: With increased understanding of the unique entities, subtype‐specific approaches for peripheral T‐cell lymphoma (PTCL) are emerging, and more precise diagnoses are becoming increasingly important. Patients and Methods: We analyzed the approach to the histopathologic diagnosis of PTCL using data from the comprehensive oncology measures of peripheral T‐cell lymphoma (COMPLETE) study. The COMPLETE trial is a large prospective cohort study of patients with newly diagnosed PTCL in the United States. Results: A total of 499 patients were enrolled from 40 academic and 15 community‐based centers. Baseline assessment forms were collected for 493 patients, of which 435 (88%) were available for analysis. The most common diagnoses were PTCL, not otherwise specified (PTCL‐NOS), anaplastic large cell lymphoma, and angioimmunoblastic T‐cell lymphoma (AITL). A mean of 10 markers (range, 0‐21) was assessed per patient. CD30 was assessed frequently but not uniformly in cases that were not anaplastic large cell lymphoma. Only 17% of PTCL‐NOS cases were assessed for PD1. CXCL13 was a relatively sensitive marker in AITL, expressed in 84% of tested cases; however, only 3% of PTCL‐NOS cases were tested. T follicular helper cell marker assessment differed between academic and community practices, with PD1 more often evaluated by academic centers in cases of AITL (62% vs. 12%; P = .01). Conclusion: The diagnostic workup for PTCL in the United States varies widely and often lacks important phenotypic information to fully characterize the lymphoma. Gaps in testing of selected markers should be filled, given the impending revision to the World Health Organization classification. The accuracy of diagnosis will become increasingly important as we enter the era of targeted treatment for PTCL.

Molecular characteristics of diffuse large B-cell lymphoma, not otherwise specified

Abstract Diffuse large B-cell lymphoma (DLBCL) is a clinically and pathologically heterogeneous entity. Multiple molecular methods have been used to further understand and define this heterogeneity. Gene profiling has identified “cell-of-origin” molecular subtypes of DLBCL based on expression patterns similar to normal B-cells. These molecular subtypes are germinal center B-cell–like DLBCL and activated B-cell–like DLBCL and have been correlated with prognosis. These subtypes have also been associated with additional molecular alterations and preferential activation of specific oncogenic pathways. Other array-based molecular techniques such as microRNA, comparative genomic hybridization, and single nucleotide polymorphism arrays have also been applied to identify recurrent microRNA and gene copy number alterations. Most recently, next-generation whole-exome and whole-genome sequencing have provided further insight into the spectrum of genetic alterations in DLBCL. The biologic significance of these discoveries continues to be elucidated; however, emerging data are already informing treatment strategies that may target altered B-cell receptor signaling pathways and dysregulated epigenetic pathways. It will be important for pathologists to be familiar with the advances in our understanding of the molecular pathogenesis of DLBCL because they will certainly impact routine diagnosis, classification, and therapeutic decisions.

Testicular Myeloid Sarcoma: A Rare Manifestation of Acute Myeloid Leukemia in an Infant

Myeloid sarcoma manifesting in the testis is rare and may occur concomitantly with bone marrow disease or as a separate entity. We describe our experience with a 6-month-old boy who presented with painless scrotal swelling and was found to have bilateral testicular masses on ultrasonography. The patient underwent unilateral radical inguinal orchiectomy. Surgical pathology revealed myeloid sarcoma of the testicle. He developed peripheral blood involvement 1 week postoperatively. Bone marrow biopsy showed acute myeloid leukemia. He is in remission after 2 cycles of induction chemotherapy, local radiation therapy, and allogeneic bone marrow transplantation.