Successful Treatment of Kaposi Sarcoma-Associated Herpesvirus Inflammatory Cytokine Syndrome After Kidney-Liver Transplant: Correlations With the Human Herpesvirus 8 miRNome and Specific T Cell Response.

After transplant, patient infection with human herpesvirus 8 (HHV-8) and Kaposi sarcoma-associated herpesvirus (KSHV) is known to cause aggressive tumors and severe nonneoplastic complications. These latter syndromes are driven by HHV-8/KSHV lytic reactivations and related hyperinflammatory host responses typically characterized by high viral loads, elevated levels of cytokines and other inflammation biomarkers, cytopenia, organ failure, high fever, and worsening conditions (with no evidence of B cell neoplasias). These disorders are associated with a high mortality rate, often due to lack of prompt diagnosis, effective therapeutic approaches, and adequate follow-up. These features resemble most of those defining the so-called KSHV-associated inflammatory cytokine syndrome (KICS), which was recently recognized in patients positive for human immunodeficiency virus (HIV). In this report, we describe-for the first time-a case of a KICS-like nonneoplastic recurrent complication occurring after transplant in an HIV-negative patient that was successfully treated by a combination of anti-CD20 monoclonal therapy, antivirals, and modification of the immunosuppressive regimen. In addition to clinical and laboratory findings collected during 3-year follow-up, we report novel experimental data on HHV-8-specific T cell dynamics and circulating microRNA profile, showing correlations with clinical course and other laboratory markers (including viral load, C-reactive protein, and cytokine levels), providing useful information about abnormal cellular and cytokine dynamics underlying HHV-8-associated inflammatory disorders in posttransplant patients.

Trends in immune cell function assay and donor-specific HLA antibodies in kidney transplantation: A 3-year prospective study.

The immune cell function assay (ICFA) and de novo anti-donor-specific HLA antibodies (DSA) have been proposed as assays for immune monitoring in renal transplantation, but longitudinal studies examining the modification of both parameters over time and their relation with clinical events are lacking. We prospectively measured longitudinal changes in ICFA and DSA levels in 55 kidney transplant recipients over 3-year follow-up (534 visits) and analyzed their relation with the risk of developing acute rejections or infections. Seven patients (12.7%) developed biopsy-proven acute rejection, and 20 (36.4%) developed viral infections. At 3 years posttransplant, 28% of the patients had developed de novo DSA. ICFA levels peaked at 1-2 months posttransplant (p = 0.005) and leveled off thereafter. They were not associated with the risk of acute rejections, viral infections or development of de novo DSA. Instead, the incidence of de novo DSA was higher in patients who previously had viral infections (adjusted-odds ratio of de novo DSA associated with prior infections: 6.03 [95% CI, 1.64-22.06; p = 0.007]). Our prospective, longitudinal study does not support using ICFA to quantify the immune risk in kidney transplantation. Further studies are needed to confirm the relationship between viral infections and the subsequent development of de novo DSA.

Prevalence of human herpesvirus-6 chromosomal integration (CIHHV-6) in Italian solid organ and allogeneic stem cell transplant patients.

The unique phenomenon of human herpesvirus-6 (HHV-6) chromosomal integration (CIHHV-6) may account for clinical drawbacks in transplant setting, being misinterpreted as active infection and leading to unnecessary and potentially harmful treatments. We have investigated the prevalence of CIHHV-6 in 205 consecutive solid organ (SO) and allogeneic stem cell transplant (alloSCT) Italian patients. Fifty-two (38.5%) of 135 solid organ transplant (SOT) and 16 (22.8%) of 70 alloSCT patients resulted positive for plasma HHV-6 DNA by real-time polymerase chain reaction. Seven SOT and three alloSCT patients presented HHV-6-related diseases, requiring antivirals. Two further patients (0.9%) were identified, presenting high HHV-6 loads. The quantification of HHV-6 on hair follicles disclosed the integrated state, allowing the discontinuation of antivirals. Before starting specific treatments, CIHHV-6 should be excluded in transplant patients with HHV-6 viremia by the comparison of HHV-6 loads on different fluids and tissues. Pretransplantation screening of donors and recipients may further prevent the misdiagnosis of CIHHV-6.

Angiogenesis in multiple myeloma: correlation between in vitro endothelial colonies growth (CFU-En) and clinical-biological features.

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/10(6) BM mononucleated cells in untreated MM samples (2.07 s.d. +/- 1.3) was significantly higher than in normal BM (0.28 +/- 0.48), while no difference was seen between normal BM and MGUS (0.28 +/- 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 +/- 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 +/- 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.

Hepatitis C virus infection in subsets of neoplastic lymphoproliferations not associated with cryoglobulinemia.

Hepatitis C virus (HCV) is both hepatotropic and lymphotropic and a clear-cut association has been proposed between HCV infection and mixed cryoglobulinemia (MC), a benign lymphoproliferative disorder, which sometimes evolves into a frank malignant B cell non-Hodgkin's lymphoma (B-NHL). Moreover, in the presence of antibodies to HCV, as well as of HCV-specific genomes has been reported in the sera of over 37% patients with B-NHL, not associated with MC. Thus, we decided to perform both a serologic and a molecular study to give insights into a possible relationship between HCV infection and neoplastic lymphoproliferations. We used ELISA and RIBA tests to show that anti-HCV antibodies were present in the serum of 29 out of 69 unselected B-NHL patients (42%), while seropositivity in a healthy population was about 1%. The prevalence of anti-HCV antibodies was low in definite subsets of B lymphoid disorders, including multiple myeloma, Waldenström's macroglobulinemia and monoclonal gammopathies of undetermined significance. Then, using reverse transcriptase polymerase chain reaction, we detected HCV sequences directly in the pathologic lymph node biopsies in 13 out of 34 B-NHL cases, and in particular in six out of eight low-grade lymphomas of MALT type and in five out of eight centroblastic-centrocytic follicular lymphomas. In contrast, the peripheral blood samples from 10 B cell chronic lymphocytic leukemia patients resulted negative for the presence of HCV genomes. Similarly, viral sequences were absent in 10 T cell NHL, while only one out of the 14 Hodgkin's disease cases tested resulted positive. Finally, we used a PCR-based assay to characterize the genotypes (I-IV) present in the positive lymphomatous tissues. The presence of both serologic and molecular markers of HCV infection in a high percentage of certain types of B-NHL, not associated with cryoglobulinemia, and its absence from other lymphoproliferative diseases extends the spectrum of HCV-associated lymphoproliferations arguing in favor of some role of this viral infection in the pathogenesis of the malignant proliferation of definite B lymphoid populations.

Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.

In search of a possible involvement of viral agents in angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD) and AILD-like lymphoma (AILD-L), we studied the presence of human herpesvirus-6 (HHV-6) in the lymph node biopsies of 12 cases by polymerase chain reaction (PCR). Given the rarity of this lymphoproliferative disorder, we investigated archival specimens, consisting of formalin-fixed and paraffin-embedded tissues, obtained from patients with a clinical and histologic diagnosis of AILD and AILD-L. HHV-6 sequences were detected in the lymph node biopsies of 7 out of the 12 AILD and AILD-L cases examined. HHV-6 sequences were identified also in the involved liver and spleen tissues of one patient and in the PBMCs of two patients, all carrying viral sequences in the affected lymph nodes. We also used PCR to characterize the HHV-6 genomes, showing that two different viral strains are represented in the pathologic tissues. This study provides evidence of the presence of HHV-6 specific sequences in an unexpectedly high proportion of our series of AILD and AILD-L cases, suggesting a possible involvement of this lymphotropic virus in the pathogenesis of at least some cases of the disease.

Late-appearing PML/RARalpha fusion transcript with coincidental t(12;13)(p13.2;q14) in acute promyelocytic leukemia lacking the t(15;17) cytogenetic anomaly.

The late appearance of a cytogenetic/molecular hallmark in human leukemias is a rare event. We report on a case of acute myeloid leukemia with morphology, immunophenotype and clinical features typical of promyelocytic subtype (APL), in which the specific PML/RARalpha gene rearrangement was molecularly detected only at second relapse of disease, without cytogenetic evidence of the t(15;17). The emergence of the PML/RARalpha gene may be therapy-related or may represent the exceptional result of a clonal evolution during progression of neoplasia. At second relapse, a novel cell clone bearing a t(12;13)(p13.2;q14) was also observed and a molecular deletion and rearrangement of a locus at 13q14, distinct from retinoblastoma (Rb1) locus, was found. In this unusual case, the PML/RARalpha product seems to be not essential for the expression of the promyelocytic phenotype at diagnosis and, when detectable, it is not the sole genetic defect.

Human herpes virus 6 and human herpes virus 8 DNA sequences in brains of multiple sclerosis patients, normal adults and children.

In order to determine whether the newly discovered human herpesviruses (HHVs) are involved in multiple sclerosis (MS), we investigated by polymerase chain reaction the presence of specific deoxyribonucleic acid (DNA) sequences belonging to human herpesvirus 6 (HHV-6) and to human herpesvirus 8 (HHV-8), in the peripheral blood mononuclear cells (PBMCs), and in the brain and spinal cord plaques from MS patients. Normal adult and stillborn children's brains were investigated as controls. PBMCs from 56 MS patients contained HHV-6 DNA in only 3 cases and in none were there HHV-8 sequences. The cerebral DNA from 5 MS patients was positive for HHV-8 and not for HHV-6 sequences, while the nervous tissue of one patient who died with neuromyelitis optica was positive for HHV-6 and negative for HHV-8. The brains of 4/8 adult controls were positive for HHV-6, as were 3/8 for HHV-8; none of the 7 stillborn children's cerebral tissue contained HHV-6 sequences, while 2 contained HHV-8 DNA. Although these data do not support a hypothesis that there is a role for these two HHVs in the pathogenesis of MS, nevertheless it may be suggested that (1) the two viruses possess strong neurotropism and the central nervous system seems to be a reservoir for them (2) HHV-6 infection is probably not transmitted maternally, but is acquired later in infancy.

Double P53 point mutation in extramedullary blast crisis of chronic myelogenous leukemia.

A patient with Philadelphia-positive chronic myelogenous leukemia (CML) evolved in extramedullary blast crisis, was studied for the presence of alterations of the P53 tumor suppressor gene in the different stages of disease progression. No P53 gene aberrations were detected during the chronic and accelerated phases. Two identical missense point mutations, involving codons 249 and 281 and leading to the amino acid substitutions Arg-Ser and Thy-Asp, were identified in cells of an extramedullary mass and then in peripheral blood blast crisis cells. The data indicate that the medullary and extramedullary blast cells belong to the same cellular clone. They also strongly suggest that in this case, the alteration of P53 gene is strictly related to the progression of the disease, although this mechanism is certainly neither the only nor the most frequent molecular event leading to the acute phase.

P53 gene mutations in chronic myelogenous leukemia medullary and extramedullary blast crisis.

Molecular alterations of the P53 gene were investigated in 27 unselected patients with chronic myelogenous leukemia (CML) blast crisis. A rearrangement of the P53 gene was evident by Southern blotting in 3 cases, one of which also showed the same alteration in the chronic phase. Single strand conformation polymorphism and sequencing analysis showed point mutations in 4 blast crisis cases. Of interest, P53 point mutations were evident in all the 3 cases of extramedullary blast crisis examined and the same point mutation was found in the myeloblastoma tissues and in the subsequent peripheral blast cells. These data indicate that: a) P53 gene mutations occur in a significant but not a large number of CML acute phase cases; b) P53 gene point mutations seem to correlate strongly with the infrequent extramedullary presentation of the blast crisis; c) the presence of the same P53 gene point mutation in extramedullary and bone marrow blast cells confirms the common clonal origin of the two cellular populations.

An encephalitic episode in a multiple sclerosis patient with human herpesvirus 6 latent infection.

All the human herpesviruses may cause central nervous system (CNS) diseases, including benign aseptic meningitis or fatal encephalitis. It has recently been stated that human herpesvirus 6 (HHV-6) may also be neuropathogenic in children after primary infection, while in the adult, cases of fatal encephalitis have been reported only in immune-compromised hosts such as AIDS patients, and in one case of an immunosuppressed bone marrow transplant patient. We describe a multiple sclerosis (MS) patient, carrier of HHV-6 latent infection, who experienced an acute inflammation of the CNS diagnosed as encephalitis. HHV-6 specific genomic sequences have been detected by PCR in the patient's PBMCs DNA collected before and during the encephalitis. The PCR performed in the CSF in course of the acute episode was positive, while the CSF collected before the encephalitis was negative. This finding is consistent with an acute encephalopathy caused by the reactivation of a HHV-6 latent infection within the CNS, in a patient with altered immune response due to MS.

Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

ß-HHVs and HHV-8 in Lymphoproliferative Disorders.

Similarly to Epstein-Barr virus (EBV), the human herpesvirus-8 (HHV-8) is a γ-herpesvirus, recently recognized to be associated with the occurrence of rare B cell lymphomas and atypical lymphoproliferations, especially in the human immunodeficiency virus (HIV) infected subjects. Moreover, the human herpesvirus-6 (HHV-6), a ß-herpesvirus, has been shown to be implicated in some non-malignant lymph node proliferations, such as the Rosai Dorfman disease, and in a proportion of Hodgkin's lymphoma cases. HHV-6 has a wide cellular tropism and it might play a role in the pathogenesis of a wide variety of human diseases, but given its ubiquity, disease associations are difficult to prove and its role in hematological malignancies is still controversial. The involvement of another ß-herpesvirus, the human cytomegalovirus (HCMV), has not yet been proven in human cancer, even though recent findings have suggested its potential role in the development of CD4(+) large granular lymphocyte (LGL) lymphocytosis. Here, we review the current knowledge on the pathogenetic role of HHV-8 and human ß-herpesviruses in human lymphoproliferative disorders.