Genetic Characterization of Primary Mediastinal B-Cell Lymphoma: Pathogenesis and Patient Outcomes.

PURPOSE: Primary mediastinal large B-cell lymphoma (PMBCL) is a rare aggressive lymphoma predominantly affecting young female patients. Large-scale genomic investigations and genetic markers for risk stratification are lacking. PATIENTS AND METHODS: To elucidate the full spectrum of genomic alterations, samples from 340 patients with previously untreated PMBCL were investigated by whole-genome (n = 20), whole-exome (n = 78), and targeted (n = 308) sequencing. Statistically significant prognostic variables were identified using a multivariable Cox regression model and confirmed by L1/L2 regularized regressions. RESULTS: Whole-genome sequencing revealed a commonly disrupted p53 pathway with nonredundant somatic structural variations (SVs) in TP53-related genes (TP63, TP73, and WWOX) and identified novel SVs facilitating immune evasion (DOCK8 and CD83). Integration of mutation and copy-number data expanded the repertoire of known PMBCL alterations (eg, ARID1A, P2RY8, and PLXNC1) with a previously unrecognized role for epigenetic/chromatin modifiers. Multivariable analysis identified six genetic lesions with significant prognostic impact. CD58 mutations (31%) showed the strongest association with worse PFS (hazard ratio [HR], 2.52 [95% CI, 1.50 to 4.21]; P < .001) and overall survival (HR, 2.33 [95% CI, 1.14 to 4.76]; P = .02). IPI high-risk patients with mutated CD58 demonstrated a particularly poor prognosis, with 5-year PFS and OS rates of 41% and 58%, respectively. The adverse prognostic significance of the CD58 mutation status was predominantly observed in patients treated with nonintensified regimens, indicating that dose intensification may, to some extent, mitigate the impact of this high-risk marker. By contrast, DUSP2-mutated patients (24%) displayed durable responses (PFS: HR, 0.2 [95% CI, 0.07 to 0.55]; P = .002) and prolonged OS (HR, 0.11 [95% CI, 0.01 to 0.78]; P = .028). Upon CHOP-like treatment, these patients had very favorable outcome, with 5-year PFS and OS rates of 93% and 98%, respectively. CONCLUSION: This large-scale genomic characterization of PMBCL identified novel treatment targets and genetic lesions for refined risk stratification. DUSP2 and CD58 mutation analyses may guide treatment decisions between rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone and dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab.

A variant of KSHV-associated inflammatory cytokine syndrome in elderly men of Mediterranean descent.

The spectrum of HHV-8-associated disorders includes Kaposi's sarcoma, primary effusion lymphoma, multicentric Castleman's disease, and the recently described KSHV inflammatory cytokine syndrome (KICS), a life-threatening disorder complicating HIV infection. There have been no reports in the literature concerning non-immunosuppressed individuals affected with KICS. We report here a KICS-like illness occurring in two elderly Greek men without HIV infection or other recognizable cause of immunosuppression.

Cost-Utility Analysis of Pegaspargase for the Treatment of Acute Lymphoblastic Leukemia in Greece.

BACKGROUND AND OBJECTIVE: Acute lymphoblastic leukemia (ALL) is an acute, rapidly progressing and life-threatening form of cancer involving immature lymphocytes called lymphoblasts. ALL is the most common subtype of leukemia in children and adolescents. The aim of the present study was to assess the cost-utility of pegaspargase versus L-asparaginase, both followed by Erwinase in the therapy sequence, as a treatment option for pediatric, adolescent, and adult patients with ALL in Greece. METHODS: A published cost-utility model comprising a decision tree and a state-transition Markov model was adapted from a public payer perspective to compare a pegaspargase treatment sequence with an L-asparaginase sequence. Efficacy and safety data, as well as utility values, were extracted from the published literature. Direct costs pertaining to drug acquisition, administration, and management of hypersensitivity were considered in the analysis (€2020). Model-extrapolated outcomes included quality-adjusted life-years (QALYs), costs, and incremental cost-effectiveness ratios (ICER). All future outcomes were discounted at 3.5% per annum. Sensitivity analyses were used to explore the impact of changing input data. RESULTS: The analysis showed that the pegaspargase sequence was estimated to produce 0.05 additional QALYs (18.12 vs. 18.07) and lower cost of - €1698 compared with L-asparaginase, indicating that the pegaspargase sequence was a dominant treatment strategy (improved outcomes with reduced costs) compared with L-asparaginase. Deterministic sensitivity analysis confirmed the cost-effective profile of pegaspargase. At the defined willingness-to-pay threshold of €54,000/QALY gained, probabilistic sensitivity analysis showed that pegaspargase had a 100% probability of being cost effective relative to the L-asparaginase sequence. CONCLUSION: The pegaspargase sequence was found to be less costly and more effective (in terms of QALYs) in relation to the L-asparaginase sequence, representing a dominant strategic option for Greek public payers in ALL.

Real-life Experience With Rituximab-CHOP Every 21 or 14 Days in Primary Mediastinal Large B-cell Lymphoma.

BACKGROUND/AIM: Primary mediastinal large B-cell lymphoma (PMLBCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL), whose prognosis has greatly improved since the incorporation of the anti-CD20 monoclonal antibody rituximab into current therapeutic regimens. Evidence, however, on the optimal time interval between consecutive chemoimmunotherapy (CIT) cycles is still scarce. This study aimed to evaluate the efficacy outcomes of the more commonly administered 3-weekly regimens to the biweekly ones in a PMLBCL patients' population, who were mostly treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone every 21 days (R-CHOP-21) or R-CHOP-14. PATIENTS AND METHODS: We retrospectively studied our cohort of consecutively treated PMLBCL patients, focusing on their treatment density, in order to determine possible differences in treatment outcomes. RESULTS: CIT, in the form of both R-CHOP-21 as well as R-CHOP-14 (or similar regimens), is highly active in PMLBCL, with low rates of early treatment failure. In our cohort of patients, R-CHOP-14 did not result in a meaningful improvement of freedom from progression (FFP) or overall survival (OS). CONCLUSION: Both R-CHOP-14 and R-CHOP-21 are probably equally effective in PMLBCL, yet further, prospective, randomized studies are warranted to clarify whether dose-dense regimens can be associated with better disease control and long-term results.

Real-life experience with the combination of polatuzumab vedotin, rituximab, and bendamustine in aggressive B-cell lymphomas.

Transplant-ineligible relapsed/refractory (rr) diffuse large B-cell lymphoma (DLBCL) patients represent an unmet medical need. Polatuzumab vedotin (Pola), an anti-CD79b antibody-drug-conjugate (ADG), with bendamustine- rituximab(BR) has recently gained approval for these patients, both in the USA and Europe, based on the GO29365 phase IIb trial. Real-life data with Pola are extremely limited. We report the outcomes of 61 Greek patients, who received Pola-(B)R mainly within a compassionate use program. Treatment was given for up to six 21-day cycles. Bendamustine was omitted in three cases due to previous short-lived responses. Fourty-nine rrDLBCL(efficacy cohort-EC) and 58 rr aggressive B-NHL (safety cohort-SC) patients received at least 1 Pola-BR cycle. Twenty-one (43%) patients of the EC responded with 12/49 (25%) CR and 9/49 (18%) PR as best response. Median progression-free survival, overall survival and duration of response were 4.0, 8.5, and 8.5 months respectively, while 55% of patients experienced a grade ≥3 adverse event, mainly hematologic. Treatment discontinuations and death during treatment were mainly due to disease progression. Twenty-two (41%) patients received further treatment; 11/22 are still alive, including one after CAR-T cells, and two after stem cell transplantation. Our data confirm that Pola-BR is a promising treatment for rrDLBCL patients, inducing an adequate response rate with acceptable toxicity. Pola-BR could be used as bridging therapy before further consolidative treatments.

Candida albicans Induces Metabolic Reprogramming in Human NK Cells and Responds to Perforin with a Zinc Depletion Response.

As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin.

Human Invariant Natural Killer T cells possess immune-modulating functions during Aspergillus infection.

Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d(+) antigen presenting cells using flow cytometry and multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d(+) subpopulations, CD1d(+)CD1c(+) mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d(+)CD1c(+) mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection.

Polymorphonuclear neutrophils and granulocytic myeloid-derived suppressor cells inhibit natural killer cell activity toward Aspergillus fumigatus.

Invasive aspergillosis is a devastating infectious disease in immunocompromised patients. Besides neutrophils and macrophages, natural killer (NK) cells have recently emerged as important players in immunity to this infection. It was shown that NK cells comprise an essential role in the clearance of Aspergillus fumigatus (A. fumigatus) in neutropenic but not in nonneutropenic mice. However, the antifungal activity of NK cells and their regulation have not been fully characterized. In this study, we investigated the interplay between polymorphonuclear neutrophils (PMNs) or granulocyte myeloid-derived suppressor cells (Gr-MDSCs) with NK cells. Both cell types exhibited an equal inhibitory effect on NK cell activation through downregulation of NKp30 expression on the cell surface and cytotoxicity towards the cell line K562. Furthermore, we showed that NK cell activation and antifungal cytotoxicity were impaired when NK cells had been cultured in the presence of PMNs or Gr-MDSCs before fungal stimulation. Besides the reduced cytotoxicity a decreased release of interferon gamma (IFNγ), a key player in the clearance of an A. fumigatus infection, was observed. Thus, inhibition of NK cell activity by PMNs or Gr-MDSCs might impair an effective anti-fungal immune response during recovery from conditions such as hematopoietic stem cell transplantation.

Pathogenic fungi regulate immunity by inducing neutrophilic myeloid-derived suppressor cells.

Despite continuous contact with fungi, immunocompetent individuals rarely develop pro-inflammatory antifungal immune responses. The underlying tolerogenic mechanisms are incompletely understood. Using both mouse models and human patients, we show that infection with the human pathogenic fungi Aspergillus fumigatus and Candida albicans induces a distinct subset of neutrophilic myeloid-derived suppressor cells (MDSCs), which functionally suppress T and NK cell responses. Mechanistically, pathogenic fungi induce neutrophilic MDSCs through the pattern recognition receptor Dectin-1 and its downstream adaptor protein CARD9. Fungal MDSC induction is further dependent on pathways downstream of Dectin-1 signaling, notably reactive oxygen species (ROS) generation as well as caspase-8 activity and interleukin-1 (IL-1) production. Additionally, exogenous IL-1ß induces MDSCs to comparable levels observed during C. albicans infection. Adoptive transfer and survival experiments show that MDSCs are protective during invasive C. albicans infection, but not A. fumigatus infection. These studies define an innate immune mechanism by which pathogenic fungi regulate host defense.

Human dendritic cell subsets display distinct interactions with the pathogenic mould Aspergillus fumigatus.

The mould Aspergillus fumigatus is primarily an opportunistic pathogen of immunocompromised patients. Once fungal spores have been inhaled they encounter cells of the innate immune system, which include dendritic cells (DCs). DCs are the key antigen-presenting cells of the immune system and distinct subtypes, which differ in terms of origin, morphology and function. This study has systematically compared the interactions between A. fumigatus and myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and monocyte-derived DCs (moDCs). Analyses were performed by time-lapse video microscopy, scanning electron microscopy, plating assays, flow cytometry, 25-plex ELISA and transwell assays. The three subsets of DCs displayed distinct responses to the fungus with mDCs and moDCs showing the greatest similarities. mDCs and moDCs both produced rough convolutions and occasionally phagocytic cups upon exposure to A. fumigatus whereas pDCs maintained a smooth appearance. Both mDCs and moDCs phagocytosed conidia and germ tubes, while pDCs did not phagocytose any fungi. Analysis of cytokine release and maturation markers revealed specific differences in pro- and anti-inflammatory patterns between the different DC subsets. These distinct characteristics between the DC subsets highlight their differences and suggest specific roles of moDCs, mDCs and pDCs during their interaction with A. fumigatus in vivo.

Human natural killer cells acting as phagocytes against Candida albicans and mounting an inflammatory response that modulates neutrophil antifungal activity.

BACKGROUND: Natural killer (NK) cells are innate lymphocytes with potent cytotoxic activity. Whereas activity of NK cells has been demonstrated against the fungal pathogens Aspergillus fumigatus and Cryptococcus neoformans, little was known about their interaction with Candida albicans. METHODS: Primary human NK cells were isolated from buffy coats, primed with a cytokine cocktail and used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry, and enzyme-linked immunosorbent assay. RESULTS: Human NK cells actively recognized C. albicans, resulting in degranulation and secretion of granulocyte-macrophage colony-stimulating factor, interferon γ, and tumor necrosis factor α . Uniquely, activation of NK cells was triggered by actin-dependent phagocytosis. Antifungal activity of NK cells against C. albicans could be detected and mainly attributed to secreted perforin. However, NK cells were unable to inhibit filamentation of C. albicans. Human polymorphonuclear neutrophils (PMNs) counteracted the proinflammatory reaction of NK cells by preventing direct contact between NK cells and the fungal pathogen. Activation of PMNs was enhanced in the presence of NK cells, resulting in increased fungicidal activity. CONCLUSIONS: Our results show a unique pattern of NK cell interaction with C. albicans, which involves direct proinflammatory activation and modulation of PMN activity. For the first time, phagocytosis of a pathogen is shown to contribute to NK cell activation.

Direct interaction studies between Aspergillus fumigatus and human immune cells; what have we learned about pathogenicity and host immunity?

Invasive aspergillosis is a significant threat to health and is a major cause of mortality in immunocompromised individuals. Understanding the interaction between the fungus and the immune system is important in determining how the immunocompetent host remains disease free. Several studies examining the direct interaction between Aspergillus fumigatus and purified innate immune cells have been conducted to measure the responses of both the host cells and the pathogen. It has been revealed that innate immune cells have different modes of action ranging from effective fungal killing by neutrophils to the less aggressive response of dendritic cells. Natural killer cells do not phagocytose the fungus unlike the other innate immune cells mentioned but appear to mediate their antifungal effect through the release of gamma interferon. Transcriptional analysis of A. fumigatus interacting with these cells has indicated that it can adapt to the harsh microenvironment of the phagosome and produces toxins, ribotoxin and gliotoxin, that can induce cell death in the majority of innate immune cells. These data point toward potential novel antifungal treatments including the use of innate immune cells as antifungal vaccines.

Human NK cells display important antifungal activity against Aspergillus fumigatus, which is directly mediated by IFN-γ release.

Despite the strong interest in the NK cell-mediated immunity toward malignant cells and viruses, there is a relative lack of data on the interplay between NK cells and filamentous fungi, especially Aspergillus fumigatus, which is the major cause of invasive aspergillosis. By studying the in vitro interaction between human NK cells and A. fumigatus, we found only germinated morphologies to be highly immunogenic, able to induce a Th1-like response, and capable of upregulating cytokines such as IFN-γ and TNF-α. Moreover, priming NK cells with human rIL-2 and stimulating NK cells by direct NK cell-pathogen contact were essential to induce damage against A. fumigatus. However, the most interesting finding was that NK cells did not mediate anti-Aspergillus cytotoxicity through degranulation of their cytotoxic proteins (perforin, granzymes, granulysine), but via an alternative mechanism involving soluble factor(s). To our knowledge, our study is the first to demonstrate that IFN-γ, released by NK cells, directly damages A. fumigatus, attributing new properties to both human NK cells and IFN-γ and suggesting them as possible therapeutic tools against IA.

Disseminated intravascular B-cell lymphoma: clinicopathological features and outcome of three cases treated with anthracycline-based immunochemotherapy.

The purpose of this study was to evaluate the use of combination anthracycline-based immunochemotherapy in intravascular lymphoma (IVL). This is an extremely rare, disseminated, and aggressive extranodal CD20(+) non-Hodgkin's lymphoma (NHL) with poor outcome following anthracycline-based chemotherapy. From a population of 700 newly diagnosed patients with NHL who were registered and followed up at our unit between 1990 and 2005, three cases (0.4%) have been classified as IVL. Among the patients, there were two men and one woman, with a median age of 52 years. We have assessed the clinicopathological characteristics, response to therapy, and outcome. All patients presented with systemic symptoms and disseminated disease. All patients received anthracycline-based chemotherapy in combination with the anti-CD20 monoclonal antibody rituximab (immunochemotherapy). Complete remission was achieved in all three patients, and currently all remain progression free with a follow-up of 24-45 months. In conclusion, anthracycline-based immunochemotherapy induces durable remissions in patients with IVL, an ultimately fatal disease, suggesting that the clinical course of this disease may be altered with immunochemotherapy.