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Clinical Practice Points • Aggressive NK-cell leukemia/lymphoma (ANKL) is a rare natural killer (NK)-cell malignancy, and although it typically presents as rapidly progressive disease, cases may present more indolently, progressing over months rather than weeks. • Unlike the more common extranodal NK-cell leukemia/lymphoma, ANKL may not involve the nasopharynx and can be Epstein-Barr virus (EBV) negative. • Diagnosing ANKL can be challenging. Although not yet standard of care, next-generation sequencing and digital image analysis can be used to clarify this rare diagnosis and confirm EBV negativity. • Treatment of ANKL utilizes asparaginase-based regimens, unlike the anthracycline-based regimens used in peripheral T-cell lymphomas or myeloid disorders. Ensuring the correct diagnosis is crucial in determining the appropriate treatment regimen for this uncommon disease.

PURPOSE Extranodal marginal zone B-cell lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT) that affects the ocular adnexa, also known as ocular adnexal MALT lymphomas (OAML), are low-grade lymphomas that mostly affect elderly individuals. This study was conducted to explore the genetic and microbial drivers of OMAL, and unique morphometric phenotypes associated with these mutations and infections. MATERIALS AND METHODS In this study, we performed targeted deep sequencing of 8 OAML cases to identify its potential genetic and microbial drivers. We additionally performed computational digital image analysis of cases to determine if morphologic features corresponded to genetic mutations and disease biology. RESULTS We identified TBL1XR1 as recurrently mutated in OAML (4/8), and mutations in several other oncogenes, tumor suppressors, transcription regulators, and chromatin remodeling genes. Morphologically, OAML cases with mutations in TBL1XR1 showed lymphoma cells with significantly lower circularity and solidity by computational digital image analysis (p-value

Post-transplant lymphoproliferative disorders (PTLD) are diseases occurring in immunocompromised patients after hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT). Although PTLD occurs rarely, it may be associated with poor outcomes. In most cases, PTLD is driven by Epstein-Barr virus (EBV) infection. Few studies have investigated the mutational landscape and gene expression profile of PTLD. In our study, we performed targeted deep sequencing and RNA-sequencing (RNA-Seq) on 16 cases of florid follicular hyperplasia (FFH) type PTLD and 15 cases of other PTLD types that include: ten monomorphic (M-PTLD), three polymorphic (P-PTLD), and two classic Hodgkin lymphoma type PTLDs (CHL-PTLD). Our study identified recurrent mutations in JAK3 in five of 15 PTLD cases and one of 16 FFH-PTLD cases, as well as 16 other genes that were mutated in M-PTLD, P-PTLD, CHL-PTLD and FFH-PTLD. Digital image analysis demonstrated significant differences in single cell area, major axis, and diameter when comparing cases of M-PTLD and P-PTLD to FFH-PTLD. No morphometric relationship was identified with regards to a specific genetic mutation. Our findings suggest that immune regulatory pathways play an essential role in PTLD, with the JAK/STAT pathway affected in many PTLDs.

Introduction Rapid technological advancements in clinical molecular genetics have increased our diagnostic and prognostic capabilities in health care. Understanding these assays, as well as how they may change over time, is critical for pathologists, clinicians, and translational researchers alike. Methods This review provides a practical summary and basic reference for current molecular genetic technologies, as well as new testing methodologies that are in use, gaining momentum, or anticipated to contribute more broadly in the future. Results Here, we discuss DNA and RNA based methodologies including classic assays such as the polymerase chain reaction (PCR), Sanger sequencing, and microarrays, to more cutting-edge next-generation sequencing (NGS) based assays and emerging molecular technologies such as cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), and NGS-based detection of infectious disease organisms. Conclusion This review serves as a basic foundation for knowledge in current and emerging clinical molecular genetic technologies.

Transposable elements (TEs) are selfish genetic units that typically encode proteins that enable their proliferation in the genome and spread across individual hosts. Here we review a growing number of studies that suggest that TE proteins have often been co-opted or 'domesticated' by their host as adaptations to a variety of evolutionary conflicts. In particular, TE-derived proteins have been recurrently repurposed as part of defense systems that protect prokaryotes and eukaryotes against the proliferation of infectious or invasive agents, including viruses and TEs themselves. We argue that the domestication of TE proteins may often be the only evolutionary path toward the mitigation of the cost incurred by their own selfish activities.

Angioimmunoblastic T-cell lymphoma is a uniquely aggressive mature T-cell neoplasm. In recent years, recurrent genetic mutations in RHOA, TET2, DNMT3A and IDH2 have been identified which characterize this disease. However, a deep molecular study assessing both DNA mutations and RNA expression profile, combined with digital image analysis is lacking. We designed a study to assess the significance of molecular and morphologic features by high resolution digital image analysis in cases of AITL. Eighteen separate tissues from ten patients with AITL were identified and analyzed in this study. Our results confirm recurrent mutations in RHOA, TET2, DNMT3A, and IDH2, and demonstrate increased DNA mutations in coding, promoter and CTCF binding sites in RHOA mutated AITLs versus RHOA non-mutated cases as well as increased overall survival in RHOA mutated patients. In addition, single cell computational digital image analysis morphologically characterized RHOA mutated AITL cells as distinct from cells from RHOA mutation negative patients. Computational analysis of single cell morphological parameters revealed RHOA mutated cells have reduced eccentricity (more circular) compared to RHOA non-mutated AITL cells. Overall, this study expands our understanding of AITL and demonstrates that there are specific cell biologic and morphologic manifestations of RHOA mutations in cases of AITL. KeyPoints: 1) RHOA mutated AITL show distinctly increased and more deleterious mutations than RHOA unmutated cases 2) RHOA mutated T-cells have distinct morphologic features from RHOA unmutated T-cells Disclosures Ohgami: Agilent technologies: Other: received support/funding.

Post-transplant lymphoproliferative disorders (PTLD) are diverse in morphology, immunophenotype and clinical outcomes, occurring in immune compromised patients after hematopoietic stem cell transplantation (HCT) or single organ transplantation (SOT). Predicting the outcome of PTLDs is complex. In most of cases PTLD is driven by Epstein-Barr virus (EBV) infection, though the etiology of EBV negative cases is unknown. There have been only few studies investigating the mutational landscape and gene expression of PTLD. In our study we performed targeted DNA deep sequencing and RNA expression profiling (RNA-Seq) on the hematopoietic lymphoid tissues of 15 post-transplant patients with PTLD and 16 post-transplant patients with follicular hyperplasia (FH) after HCT or SOT. Recurrent mutations in JAK3 were identified in five of 16 PTLD cases and one of 16 follicular hyperplasia cases. We additionally identified mutations in the PIK3CD gene in four PTLD cases and NOTCH1 mutations in 2 PTLD and four FH cases. 17 other genes were commonly mutated in both groups. Gene expression profiles differed between both groups. Genes involved in regulating the immune system were unregulated in PTLD. We also saw a stronger expression of T-cells and a weaker expression of B-cells within the PTLD group when compared with FH. Our findings suggest an important role for immune regulatory pathways in the pathogenesis of PTLD, with the JAK/STAT pathway being a central recurrent key metabolic pathway. Figure Disclosures Ohgami: Agilent technologies: Other: received support/funding.