Genomic landscape of virus-associated cancers.

It has been estimated that 15%-20% of human cancers are attributable to infections, mostly by carcinogenic viruses. The incidence varies worldwide, with a majority affecting developing countries. Here, we present a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures and driver gene mutations and possess a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. For example, compared to the respective virus-negative counterparts, virus-positive cases across different cancer histologies had less often mutations of TP53 and deletions of 9p21.3/ CDKN2 A- CDKN1A ; Epstein-Barr virus-positive (EBV+) gastric cancer had more frequent mutations of EIF4A1 and ARID1A and less marked mismatch repair deficiency signatures; and EBV-positive cHL had fewer somatic genetic lesions of JAK-STAT, NF-κB, PI3K-AKT and HLA-I genes and a less pronounced activity of the aberrant somatic hypermutation signature. In cHL, we also identify germline homozygosity in HLA class I as a potential risk factor for the development of EBV-positive Hodgkin lymphoma. Finally, an analysis of clinical trials of PD-(L)1 inhibitors in four virus-associated cancers suggested an association of viral infection with higher response rate in patients receiving such treatments, which was particularly evident in gastric cancer and head and neck squamous cell carcinoma. These results illustrate the epidemiological, genetic, prognostic, and therapeutic trends across virus-associated malignancies.

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation.

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition.

Identification of novel STAT5B mutations and characterization of TCRß signatures in CD4+ T-cell large granular lymphocyte leukemia.

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRß sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.

Dissecting Clonal Hematopoiesis in Tissues of Classical Hodgkin Lymphoma Patients.

Clonal hematopoiesis predisposes to hematological malignancies. However, clonal hematopoiesis is understudied in classical Hodgkin lymphoma (cHL), a mature B-cell neoplasm exhibiting the most abundant microenvironment. We analyzed clonal hematopoiesis in 40 cHL cases by sequencing microdissected tumor cells and matched normal cells from blood and/or lymph nodes. Five patients had blood and/or tissue clonal hematopoiesis. In three of five patients (all failing first-line therapy), clonal hematopoiesis spread through the tissue microenvironment extensively, and featured mutant DNMT3AR882H , KRASG60D and DNMT3AR882H +TET2Q1274 * in 33%, 92% and 60% of non-neoplastic cells, respectively. In the latter case, DNMT3A/TET2-mutant clonal hematopoiesis seeded the neoplastic clone, which was infected by the Epstein-Barr virus and showed almost no other somatic mutations exome-wide. In the former case, DNMT3A-mutant clonal hematopoiesis did not originate the neoplastic clone despite dominating the blood and B-cell lineage (~94% leukocytes; ~96% mature blood B cells), yet led to NPM1-mutated acute myeloid leukemia 6 years after therapy for cHL. Our results expand to cHL the spectrum of hematologic malignancies associated with clonal hematopoiesis.

Correction: Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphomas.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphomas.

The Epstein-Barr virus (EBV) is linked to various B-cell lymphomas, including Burkitt lymphoma (BL), classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) at frequencies ranging, by routine techniques, from 5 to 10% of cases in DLBCL to >95% in endemic BL. Using higher-sensitivity methods, we recently detected EBV traces in a few EBV-negative BL cases, possibly suggesting a "hit-and-run" mechanism. Here, we used routine and higher-sensitivity methods (qPCR and ddPCR for conserved EBV genomic regions and miRNAs on microdissected tumor cells; EBNA1 mRNA In situ detection by RNAscope) to assess EBV infection in a larger lymphoma cohort [19 BL, 34 DLBCL, 44 cHL, 50 follicular lymphomas (FL), 10 T-lymphoblastic lymphomas (T-LL), 20 hairy cell leukemias (HCL), 10 mantle cell lymphomas (MCL)], as well as in several lymphoma cell lines (9 cHL and 6 BL). qPCR, ddPCR, and RNAscope consistently documented the presence of multiple EBV nucleic acids in rare tumor cells of several cases EBV-negative by conventional methods that all belonged to lymphoma entities clearly related to EBV (BL, 6/9 cases; cHL, 16/32 cases; DLBCL, 11/30 cases), in contrast to fewer cases (3/47 cases) of FL (where the role of EBV is more elusive) and no cases (0/40) of control lymphomas unrelated to EBV (HCL, T-LL, MCL). Similarly, we revealed traces of EBV infection in 4/5 BL and 6/7 HL cell lines otherwise conventionally classified as EBV negative. Interestingly, additional EBV-positive cases (1 DLBCL, 2 cHL) relapsed as EBV-negative by routine methods while showing EBNA1 expression in rare tumor cells by RNAscope. The relapse specimens were clonally identical to their onset biopsies, indicating that the lymphoma clone can largely loose the EBV genome over time but traces of EBV infection are still detectable by high-sensitivity methods. We suggest EBV may contribute to lymphoma pathogenesis more widely than currently acknowledged.

Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma.

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

Genomics of Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is a chronic mature B-cell neoplasm with unique clinicopathologic features and an initial exquisite sensitivity to chemotherapy with purine analogs; however, the disease relapses, often repeatedly. The enigmatic pathogenesis of HCL was recently clarified by the discovery of its underlying genetic cause, the BRAF-V600E kinase-activating mutation, which is somatically and clonally present in almost all patients through the entire disease spectrum and clinical course. By aberrantly activating the RAF-MEK-ERK signaling pathway, BRAF-V600E shapes key biologic features of HCL, including its specific expression signature, hairy morphology, and antiapoptotic behavior. Accompanying mutations of the KLF2 transcription factor or the CDKN1B/p27 cell cycle inhibitor are recurrent in 16% of patients with HCL and likely cooperate with BRAF-V600E in HCL pathogenesis. Conversely, BRAF-V600E is absent in other B-cell neoplasms, including mimickers of HCL that require different treatments (eg, HCL-variant and splenic marginal zone lymphoma). Thus, testing for BRAF-V600E allows for a genetics-based differential diagnosis between HCL and HCL-like tumors, even noninvasively in routine blood samples. BRAF-V600E also represents a new therapeutic target. Patients' leukemic cells exposed ex vivo to BRAF inhibitors are spoiled of their HCL identity and then undergo apoptosis. In clinical trials of patients with HCL who have experienced multiple relapses after purine analogs or who are refractory to purine analogs, a short course of the oral BRAF inhibitor vemurafenib produced an almost 100% response rate, including complete remission rates of 35% to 42%, without myelotoxicity. To further improve on these results, it will be important to clarify the mechanisms of incomplete leukemic cell eradication by vemurafenib and to explore chemotherapy-free combinations of a BRAF inhibitor with other targeted agents (eg, a MEK inhibitor and/or an anti-CD20 monoclonal antibody).

BRAF inhibitors reverse the unique molecular signature and phenotype of hairy cell leukemia and exert potent antileukemic activity.

Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL.

Constant activation of the RAF-MEK-ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia.

The BRAF-V600E mutation defines genetically hairy cell leukemia among B-cell leukemias and lymphomas. In solid tumors, BRAF-V600E is known to aberrantly activate the oncogenic MEK-ERK pathway, and targeted BRAF and/or MEK inhibitors have shown remarkable efficacy in clinical trials in melanoma patients. However, the MEK-ERK pathway status in hairy cell leukemia has not been thoroughly investigated. We assessed phospho-ERK expression in 37 patients with hairy cell leukemia and 44 patients with neoplasms mimicking hairy cell leukemia (40 splenic marginal zone lymphoma, 2 hairy cell leukemia-variant and 2 splenic lymphoma/leukemia unclassifiable) using immunohistochemistry on routine biopsies and/or Western blotting on purified leukemic cells, and correlated the phospho-ERK status with the BRAF-V600E mutation status. Besides confirming the constant presence of BRAF-V600E in all patients with hairy cell leukemia, we observed ubiquitous phospho-ERK expression in this malignancy. Conversely, all 44 cases with neoplasms mimicking hairy cell leukemia were devoid of BRAF-V600E and none expressed phospho-ERK. Furthermore, the two exceptionally rare cases of non-hairy cell leukemia unclassifiable chronic B-cell neoplasms previously reported to be BRAF-V600E(+) on allele-specific polymerase chain reaction lacked phospho-ERK expression as well, suggesting the presence of the mutation in only a small part of the leukemic clone in these cases. In conclusion, our findings support the use of phospho-ERK immunohistochemistry in the differential diagnosis between hairy cell leukemia and its mimics, and establish the MEK-ERK pathway as a rational therapeutic target in this malignancy.

Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation.

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.

BRAF mutations in hairy-cell leukemia.

BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box.

TM7SF2 gene encodes 3beta-hydroxysterol Delta(14)-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter-luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Disruption of the gene encoding 3beta-hydroxysterol Delta-reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis.

Tm7sf2 gene encodes 3beta-hydroxysterol Delta(14)-reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Delta(14)-unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C-terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Delta(14)-reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3beta-hydroxysterol Delta(14)-reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3beta-hydroxysterol Delta(14)-reductase activity were detectable in liver microsomes of Tm7sf2((-/-)) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2((-/-)) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3beta-hydroxysterol Delta(14)-reductase activity determined in liver nuclei showed comparable values in wild-type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2((-/-)) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell-cycle progression are downregulated in the liver of Tm7sf2((-/-)) mice, whereas genes involved in xenobiotic metabolism are upregulated.

GL15 and U251 glioblastoma-derived human cell lines are peculiarly susceptible to induction of mitotic death by very low concentrations of okadaic acid.

The challenging possibility of selectively inducing mitotic death in tumor cells by combining genotoxic agents with the inhibition of G2 checkpoints of the cell cycle is the subject of intensive investigation. We show that very low concentrations (3.5 and 5 nM) of okadaic acid induce mitotic death in two glioblastoma cell lines, in the absence of genotoxic agents. At the concentrations used, the main target of okadaic acid action is protein phosphatase 2A (PP2A), an enzyme deeply involved in the negative control of cell-cycle progression. The peculiar susceptibility of glioblastoma cells to induction of mitotic death by very low concentrations of okadaic acid must be related to an impairment of PP2A activity and to a specific deficiency in some cell-cycle checkpoints. In addition to its ability to induce abnormal mitoses in actively proliferating glioblastoma cells, okadaic acid possesses the ability to force semi-confluent glioblastoma cells to the M phase of the cell cycle, where they show the same abnormalities observed in actively proliferating glioblastoma cells. In semi-confluent cells the induction of mitotic death involves the activity of both the extracellular signal regulated kinases (ERKs) and the M-phase promoting factor: okadaic acid overstimulates ERK activity, and PD98059 (inhibitor of ERK activation) as well as roscovitine (S)-isomer (specific inhibitor of M-phase promoting factor activity) counteract the induction of mitotic death. Our results show that, without the use of genotoxic agents, it is possible to induce mitotic death in glioblastoma cells by activating the same uncontrolled pathways responsible for the uncontrolled proliferation.