Genomic profiling for clinical decision making in lymphoid neoplasms.

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

LT and SOX9 expression are associated with gene sets that distinguish Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet radiation (UVR). MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, as the former harbour few DNA mutations and are not related to UVR, and the latter usually arise in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT; coded by MCPyV_gp3) and small T antigen (sT; coded by MCPyV_gp4), promote different carcinogenic pathways. OBJECTIVES: To determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC; to describe the mutational burden and the most frequently mutated genes in both MCC subtypes; and to identify the clinical and molecular factors that may be related to patient survival. METHODS: Ninety-two patients with a diagnosis of MCC were identified from the medical databases of participating centres. To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter technology. For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the GATK Best Practices workflow for somatic mutations. RESULTS: The expression of LT enabled the series to be divided into two groups (LT positive, n = 55; LT negative, n = 37). Genes differentially expressed in LT-negative patients were related to epithelial differentiation, especially SOX9, or proliferation and the cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive patients, and differentially expressed genes in SOX9-positive patients were related to epithelial/squamous differentiation. In LT-positive patients, the mean SNV frequency was 4.3; in LT-negative patients it was 10 (P = 0.03). On multivariate survival analysis, the expression of SNAI1 [hazard ratio (HR) 1.046, 95% confidence interval (CI) 1.007-1.086; P = 0.02] and CDK6 (HR 1.049, 95% CI 1.020-1.080; P = 0.001) were identified as risk factors. CONCLUSIONS: Tumours with weak LT expression tend to co-express genes related to squamous differentiation and the cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.

Merkel cell carcinoma (MCC) is an aggressive form of skin tumour. There are two subtypes of MCC: one of them is related to a virus called Merkel cell polyomavirus (MCPyV); the other one is related to persistent exposure to sunlight. The aim of this research was to find differences between these subtypes in their molecular behaviour (the genes that are expressed and the mutations that may be found). To do this, we carried out two studies, one to investigate gene expression (the process cells use to convert the instructions in our DNA into a functional product such as a protein) and one to look at gene mutations (changes in the DNA sequence). We found that the tumours that were not related to MCPyV expressed genes related to epithelial differentiation (the process by which unspecialized cells gain features characteristics of epithelial cells, which, among other things, make up the outer surface of the body), which means that the origin of both MCC subtypes may be different. We also found that MCPyV-related tumours had fewer mutations. Our findings are important because they help us to understand the biology of the MCC subtypes and could help with the development of new treatments for people diagnosed with skin tumours.

Extranodal natural killer/T-cell lymphoma nasal type in a western population: Molecular profiling identifies new therapeutic targets.

(A) Correlation matrix of unsupervised co-regulated genes, based on the 208 genes included in the NanoString platform. Some of the clusters of co-regulated genes corresponded to the following: Inflammatory cells; Epstein-Barr virus; B-cells; Cytotoxic T-cells; T-cells; and Proliferation. (B) Analysis of genomic alterations by targeted sequencing. Distribution of mutations in the 62 analyzed genes. Rows correspond to sequenced genes, columns represent individual patients. Color coding: green, missense; blue, synonymous; pink, frameshift; violet, Indel; red, stop gained; yellow, UTR.

Primary Effusion Lymphoma, Multicentric Castleman's Disease, and Kaposi's Sarcoma in an HHV-8 and HIV-Positive Patient: A Case Report.

Primary effusion lymphoma (PEL), Kaposi's sarcoma (KS), and multicentric Castleman's disease (MCD) is an uncommon group of diseases included in the same spectrum with related characteristics. The coexistence of all of them in the same individual is a rare occurrence. We present the case of a 25-year-old patient diagnosed with human immunodeficiency virus (HIV) and the development of all these related pathologies. Despite the use of intensive treatment according to the latest recommendations, the evolution was unfavorable. This case reflects the need for new therapies and research in this field.

Clinical and pathological characteristics of peripheral T-cell lymphomas in a Spanish population: a retrospective study.

We investigated the clinicopathological features and prognostic factors of patients with peripheral T-cell lymphoma (PTCL) in 13 sites across Spain. Relevant clinical antecedents, CD30 expression and staining pattern, prognostic indices using the International Prognostic Index and the Intergruppo Italiano Linfomi system, treatments, and clinical outcomes were examined. A sizeable proportion of 175 patients had a history of immune-related disorders (autoimmune 16%, viral infections 17%, chemo/radiotherapy-treated carcinomas 19%). The median progression-free survival (PFS) and overall survival (OS) were 7·9 and 15·8 months, respectively. Prognostic indices influenced PFS and OS, with a higher number of adverse factors resulting in shorter survival (P < 0·001). Complete response (CR) to treatment was associated with better PFS (62·6 vs. 4 months; P < 0·001) and longer OS (67·0 vs. 7·3 months; P < 0·001) compared to no CR. CD30 was expressed across all subtypes; >15% of cells were positive in anaplastic lymphoma kinase-positive and -negative anaplastic large-cell lymphoma and extranodal natural killer PTCL groups. We observed PTCL distribution across subtypes based on haematopathological re-evaluation. Poor prognosis, effect of specific prognostic indices, relevance of histopathological sub-classification, and response level to first-line treatment on outcomes were confirmed. Immune disorders amongst patients require further examination involving genetic studies and identification of associated immunosuppressive factors.

Genetic evolution of in situ follicular neoplasia to aggressive B-cell lymphoma of germinal center subtype.

In situ follicular neoplasia (ISFN) is the earliest morphologically identifiable precursor of follicular lymphoma (FL). Although it is genetically less complex than FL and has low risk for progression, ISFN already harbors secondary genetic alterations, in addition to the defining t(14;18)(q32;q21) translocation. FL, in turn, frequently progresses to diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBL). By BCL2 staining of available reactive lymphoid tissue obtained at any time point in patients with aggressive B-cell lymphoma (BCL), we identified ten paired cases of ISFN and DLBCL/HGBL, including six de novo tumors and four tumors transformed from FL as an intermediate step, and investigated their clonal evolution using microdissection and next-generation sequencing. A clonal relationship between ISFN and aggressive BCL was established by immunoglobulin and/or BCL2 rearrangements and/or the demonstration of shared somatic mutations for all ten cases. Targeted sequencing revealed CREBBP, KMT2D, EZH2, TNFRSF14 and BCL2 as the genes most frequently mutated already in ISFN. Based on the distribution of private and shared mutations, two patterns of clonal evolution were evident. In most cases, the aggressive lymphoma, ISFN and, when present, FL revealed divergent evolution from a common progenitor, whereas linear evolution with sequential accumulation of mutations was less frequent. In conclusion, we demonstrate for the first time that t(14;18)+ aggressive BCL can arise from ISFN without clinically evident FL as an intermediate step and that during this progression, branched evolution is common.

Atypical BCL6/GATA3+ Primary Cutaneous Acral CD8-Positive T-Cell Lymphoma: A Diagnostic Challenge.

ABSTRACT: Primary cutaneous acral CD8-positive T-cell lymphoma consists of slow-growing nodules in acral sites with a histopathology, suggesting high-grade lymphoma despite the indolent clinical course. It has been recently included in WHO-EORTC classification for primary cutaneous lymphomas as a provisional entity. A correct diagnosis of this entity is important because its differential diagnosis include more aggressive cutaneous lymphomas. We present a 53-year-old woman with an indolent solitary nodule on her right leg, which histopathologically showed features of CD8-positive T-cell lymphoma, although with some peculiarities, including epidermotropism, absence of CD68 expression, and positivity for GATA3 and Bcl6 in neoplastic cells. This case could contribute to better define the spectrum of this rare cutaneous lymphoma.

Histiocytic hyperplasia with hemophagocytosis and acute alveolar damage in COVID-19 infection.

The spectrum of COVID-19 infection includes acute respiratory distress syndrome (ARDS) and macrophage activation syndrome (MAS), although the histological basis for these disorders has not been thoroughly explored. Post-mortem pulmonary and bone marrow biopsies were performed in 33 patients. Samples were studied with a combination of morphological and immunohistochemical techniques. Bone marrow studies were also performed in three living patients. Bone marrow post-mortem studies showed striking lesions of histiocytic hyperplasia with hemophagocytosis (HHH) in most (16/17) cases. This was also observed in three alive patients, where it mimicked the changes observed in hemophagocytic histiocytosis. Pulmonary changes included a combination of diffuse alveolar damage with fibrinous microthrombi predominantly involving small vessels, in particular the alveolar capillary. These findings were associated with the analytical and clinical symptoms, which helps us understand the respiratory insufficiency and reveal the histological substrate for the macrophage activation syndrome-like exhibited by these patients. Our results confirm that COVID-19 infection triggers a systemic immune-inflammatory disease and allow specific therapies to be proposed.

Epstein-Barr virus-associated large B-cell lymphoma transformation in marginal zone B-cell lymphoma: a series of four cases.

AIMS: To present four examples of clonally related Epstein-Barr virus (EBV)-associated large-cell transformation of marginal zone lymphoma (MZL) (of nodal, extranodal and splenic types), occurring 120, 11 and 5 months after the initial diagnosis in three instances, and concurrently in one case; and to discuss several interesting features of EBV infection. METHODS AND RESULTS: Somatic mutations were detected by use of a customised panel for next-generation sequencing and polymerase chain reaction studies of IgH in both low-grade and high-grade components of each case. In case 1, the initial biopsy of nodal MZL showed scattered EBV-positive cells, which might constitute an indication of EBV-induced progression. Case 2 showed heterogeneous EBV expression, a phenomenon attributable to loss of the EBV episomes during cell division, or to a secondary superinfection or reactivation of the virus. In case 3, p53 overexpression related to gene mutation and EBV-encoded small RNAs were identified in the same neoplastic component. In case 4, the mucosa-associated lymphoid tissue-type MZL and the high-grade component were identified concurrently in a patient previously treated with methotrexate for an autoimmune disorder. CONCLUSION: These data suggest that the presence of EBV should be added to the list of potential markers to be analysed for MZL prognosis.

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Update on peripheral T-cell lymphomas with T-helper phenotype: Are there too many subtypes?

Follicular helper T (TFH) cells are the providers of T-cell help to B-cells in the development of germinal centers and for the generation of most class-switched antibodies. The markers most commonly associated with TFH activity are IL21, IL4, CD40L, BCL6, SAP, CXCR5/CXCL13, and ICOS. T-cell lymphoma genomic studies have shown that different T-cell lymphoma types express signatures typical for TFH cells, this including angioimmunoblastic T-cell lymphoma (AITL), a related condition termed peripheral T-cell lymphoma with TFH phenotype and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder. Angioimmunoblastic T-cell lymphoma is a well-established entity, a clinically aggressive disease with a survival of 30% OS after 5 years. Molecular and clinical studies have confirmed this as a well-established clinicopathological entity with relatively specific gene mutations, including mutations found in hematopoietic precursor cells and others. Peripheral T-cell lymphoma with TFH phenotype is an associated disorder with histology of PTCL but a TFH phenotype, as defined by the expression of 2-3 immunohistochemical markers. Molecular studies on this entity are showing a partial overlap with AITL. Primary cutaneous CD4+ small/medium lymphoproliferative disorder is an entirely different process that takes place in the skin, showing frank cytologic atypia, monoclonal TCR rearrangement and TFH phenotype in the context of a clinically benign lesion. Here we review the main clinical, molecular and diagnostic features of these three lymphoproliferative processes.

Inflammatory Cells in Atypical Eruption of Lymphocyte Recovery Carry the Same Mutations as Neoplastic Myeloid Cells.

Cutaneous eruption of lymphocyte recovery (ELR) during bone marrow (BM) aplasia recovery after intensive chemotherapy has been reported in very few patients. The presence of skin rashes in patients with acute leukemia who are undergoing intensive chemotherapy and BM transplantation is a diagnostic challenge because of the clinical similarity between drug eruptions, infiltrates related to the relapse of the underlying disease, cutaneous graft-versus-host disease, and ELR. IDH1 mutations have been identified as a recurrent genetic anomaly in acute myeloid leukemia and myelodysplastic syndromes. However, until now, this IDH1 mutation has not been reported as being shared by myeloid cells and non-neoplastic inflammatory cells in this clinical setting. Here, we present the rare case of a woman diagnosed with myelodysplastic syndrome that evolved into an acute myelogenous leukemia with leukemic cutaneous infiltrate. The patient developed ELR after the intensive chemotherapy administered before BM transplantation. The IDH1 mutation was identified in BM cells and in myeloid and inflammatory cells in skin biopsies before allogeneic BM transplantation. We discuss the main aspects of the differential diagnosis of these cutaneous reactions in leukemic patients and the biological significance of the IDH1 mutation.

Activating mutations and translocations in the guanine exchange factor VAV1 in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas frequently associated with poor prognosis and for which genetic mechanisms of transformation remain incompletely understood. Using RNA sequencing and targeted sequencing, here we identify a recurrent in-frame deletion (VAV1 Δ778-786) generated by a focal deletion-driven alternative splicing mechanism as well as novel VAV1 gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) in PTCL. Mechanistically these genetic lesions result in increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (nuclear factor of activated T cells, NFAT) VAV1 effector pathways. These results support a driver oncogenic role for VAV1 signaling in the pathogenesis of PTCL.

PD-1/PD-L1 expression and interaction by automated quantitative immunofluorescent analysis show adverse prognostic impact in patients with diffuse large B-cell lymphoma having T-cell infiltration: a study from the International DLBCL Consortium Program.

Programmed cell death protein 1/programmed cell death protein ligand1 (PD-1/PD-L1) interaction is an important immune checkpoint targeted by anti-PD-1/PD-L1 immunotherapies. However, the observed prognostic significance of PD-1/PD-L1 expression in diffuse large B-cell lymphoma treated with the standard of care has been inconsistent and even contradictory. To clarify the prognostic role of PD-1/PD-L1 expression and interaction in diffuse large B-cell lymphoma, in this study we used 3-marker fluorescent multiplex immunohistochemistry and Automated Quantitative Analysis Technology to assess the CD3+, PD-L1+, and PD-1+CD3+ expression in diagnostic samples and PD-1/PD-L1 interaction as indicated by presence of PD-1+CD3+ cells in the vicinity of PD-L1+ cells, analyzed their prognostic effects in 414 patients with de novo diffuse large B-cell lymphoma, and examined whether PD-1/PD-L1 interaction is required for the prognostic role of PD-1+/PD-L1+ expression. We found that low T-cell tissue cellularity, tissue PD-L1+ expression (irrespective of cell types), PD-1+CD3+ expression, and PD-1/PD-L1 interaction showed hierarchical adverse prognostic effects in the study cohort. PD-1/PD-L1 interaction showed higher sensitivity and specificity than PD-1+ and PD-L1+ expression in predicting inferior prognosis in patients with high CD3+ tissue cellularity ("hot"/inflammatory tumors). However, both PD-1+ and PD-L1+ expression showed adverse prognostic effects independent of PD-1/PD-L1 interaction, and PD-1/PD-L1 interaction showed favorable prognostic effect in PD-L1+ patients without high CD3+ tissue cellularity. Macrophage function and tumor-cell MYC expression may contribute to the PD-1-independent adverse prognostic effect of PD-L1+ expression. In summary, low T-cell tissue cellularity has unfavorable prognostic impact in diffuse large B-cell lymphoma, and tissue PD-L1+ expression and T-cell-derived PD-1+ expression have significant adverse impact only in patients with high T-cell infiltration. PD-1/PD-L1 interaction in tissue is essential but not always responsible for the inhibitory effect of PD-L1+/PD-1+ expression. These results suggest the benefit of PD-1/PD-L1 blockade therapies only in patients with sufficient T-cell infiltration, and the potential of immunofluorescent assays and Automated Quantitative Analysis in the clinical assessment of PD-1/PD-L1 expression and interaction.

CD30-positive primary cutaneous lymphoproliferative disorders: molecular alterations and targeted therapies.

Primary cutaneous CD30-positive T-cell lymphoproliferative disorders are the second most common subgroup of cutaneous T-cell lymphomas. They include two clinically different entities with some overlapping features and borderline cases: lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Molecular studies of primary cutaneous anaplastic large cell lymphoma reveal an increasing level of heterogeneity that is associated with histological and immunophenotypic features of the cases and their response to specific therapies. Here, we review the most significant genetic, epigenetic and molecular alterations described to date in primary cutaneous CD30-positive T-cell lymphoproliferative disorders, and their potential as therapeutic targets.

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Atypical Histiocytic Lesion Preceding a Peripheral T-Cell Lymphoma Involving the Skin Exhibiting the Same Molecular Alterations.

Peripheral T-cell lymphoma (PTCL), not otherwise specified (NOS) is a diagnosis of exclusion, showing extreme cytological and phenotypic heterogeneity. Skin involvement of PTCL may be primary or secondary. Diagnosis of histiocytosis may be difficult, requiring clinical-pathological correlation. We describe a laryngeal atypical histiocytic lesion (AHL) and a nasal PTCL, NOS with cutaneous involvement in the same patient presenting with peculiar histopathologic and immunophenotypic features. The laryngeal neoplasm showed morphological and immunophenotypic evidence of histiocytic differentiation and does not fit any other category of the WHO classification nor the revised classification of histiocytosis. The nasal and cutaneous lesions presented features close to natural killer/T-cell lymphoma and gamma-delta T-cell lymphoma but did not meet accurately the WHO criteria. A somatic activating Q61K mutation was found on exon 3 of the NRAS gene in both AHL and PTCL, NOS. The mutation on NRAS gene in both AHL and PTCL, NOS may suggest a common origin from a precursor cell.

AKT Hyperactivation and the Potential of AKT-Targeted Therapy in Diffuse Large B-Cell Lymphoma.

AKT signaling is important for proliferation and survival of tumor cells. The clinical significance of AKT activation in diffuse large B-cell lymphoma (DLBCL) is not well analyzed. Here, we assessed expression of phosphorylated AKT (p-AKT) in 522 DLBCL patients. We found that high levels of p-AKT nuclear expression, observed in 24.3% of the study cohort, were associated with significantly worse progression-free survival and Myc and Bcl-2 overexpression. However, multivariate analysis indicated that AKT hyperactivation was not an independent factor. miRNA profiling analysis demonstrated that 63 miRNAs directly or indirectly related to the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway were differentially expressed between DLBCLs with high and low p-AKT nuclear expression. We further targeted AKT signaling using a highly selective AKT inhibitor MK-2206 in 26 representative DLBCL cell lines and delineated signaling alterations using a reverse-phase protein array. MK-2206 treatment inhibited lymphoma cell viability, and MK-2206 sensitivity correlated with AKT activation status in DLBCL cells. On MK-2206 treatment, p-AKT levels and downstream targets of AKT signaling were significantly decreased, likely because of the decreased feedback repression; Rictor and phosphatidylinositol 3-kinase expression and other compensatory pathways were also induced. This study demonstrates the clinical and therapeutic implications of AKT hyperactivation in DLBCL and suggests that AKT inhibitors need to be combined with other targeted agents for DLBCL to achieve optimal clinical efficacy.