Francesca Fiore

Early chemotherapy intensification with BEACOPP in advanced-stage Hodgkin lymphoma patients with a interim-PET positive after two ABVD courses

Interim 2‐[18F]Fluoro‐2‐deoxy‐D‐glucose Positron Emission Tomography performed after two chemotherapy cycles (PET‐2) is the most reliable predictor of treatment outcome in ABVD‐treated Hodgkin Lymphoma (HL) patients. We retrospectively analysed the treatment outcome of a therapeutic strategy based on PET‐2 results: positive patients switched to BEACOPP, while negative patients continued with ABVD. Between January 2006 and December 2007, 219 newly diagnosed HL patients admitted to nine centres were enrolled; 54 patients, unfit to receive this treatment were excluded from the analysis. PET‐2 scans were reviewed by a central panel of nuclear medicine experts, according to the Deauville score ( Meignan, 2009 ). After a median follow up of 34 months (12–52) the 2‐year failure free survival (FFS) and overall survival for the entire cohort of 165 patients were 88% and 98%; the FFS was 65% for PET‐2 positive and 92% for PET‐2 negative patients. For 154 patients in which treatment was correctly given according to PET‐2 review, the 2‐year FFS was 91%: 62% for PET‐2 positive and 95% for PET‐2 negative patients. Conclusions: this strategy, with BEACOPP intensification only in PET‐2 positive patients, showed better results than ABVD‐treated historic controls, sparing BEACOPP toxicity to the majority of patients (Clinical Trials.gov Identifier NCT00877747).

Effector γδ T cells and tumor cells as immune targets of zoledronic acid in multiple myeloma

The aim of this study was to investigate the in vitro immunomodulatory effects of zoledronic acid (Zol) on peripheral blood Vγ9/Vδ2 (γδ) T cells of normal donors and multiple myeloma (MM) patients. γδ T cells were stimulated with Zol and low doses of interleukin-2 (IL-2), and then analyzed for proliferation, cytokine production, and generation of effector activity against myeloma cell lines and primary myeloma cells. Proliferation of γδ T cells was observed in 100% of normal donors and 50% of MM patients. γδ T cells produced IFN-γ, surface mobilized the CD107a and CD107b antigens, and exerted direct cell-to-cell antimyeloma activity irrespective of the ability to proliferate to Zol and IL-2. The memory phenotype was predominant in the MM γδ T cells that proliferated in response to Zol (responders), whereas effector cells were predominant in those that did not (nonresponders). Zol induced antimyeloma activity through the monocyte-dependent activation of γδ T cells and by enhancing the immunosensitivity of myeloma cells to γδ T cells. Mevastatin, a specific inhibitor of hydroxy-methylglutaryl-CoA reductase, completely abrogated this antimyeloma activity.

Acute myeloid leukemia cells constitutively express the immunoregulatory enzyme indoleamine 2,3-dioxygenase

Acute myeloid leukemia cells constitutively express the immunoregulatory enzyme indoleamine 2,3-dioxygenase

Exposure to myeloma cell lysates affects the immune competence of dendritic cells and favors the induction of Tr1-like regulatory T cells

The aim of this work was to investigate the interactions of tumor cells with dendritic cells (DC) in normal donors and patients with multiple myeloma (MM). Normal and MM DC internalized necrotic lysates derived from myeloma cell lines (MCL) with high efficiency, whereas necrotic lysates from primary myeloma cells (PMC) were internalized with significantly lower efficiency. A positive correlation was found between susceptibility to internalization and the ability to induce DC maturation. After PMC exposure, DC produced large amounts of IL‐10 and measurable amounts of IL‐4 but no detectable IL‐12. Two rounds of exposure to PMC‐treated DC generated autologous T cells with low proliferative capacity, decreased IFN‐γ production and increased IL‐10 production in the absence of IL‐4 production. These data indicate that myeloma cells can affect host immunity by priming DC towards a maturation state favoring the generation of T cells with a regulatory rather than an effector phenotype.

Long-term follow-up of idiotype vaccination in human myeloma as a maintenance therapy after high-dose chemotherapy

The aim of this work was to evaluate the long-term immunological and clinical impact of idiotype (Id) vaccination in multiple myeloma (MM) patients in first remission after high-dose chemotherapy. A total of 15 patients received a series of subcutaneous (s.c.) injections of autologous Id, conjugated to keyhole limpet hemocyanin (KLH) and in association with low doses of GM-CSF. The median duration of follow-up was 110 months from diagnosis. The vaccine induced immune responses that lasted almost 2 years after the end of treatment. Antibody responses included anti-KLH IgM and IgG (90% of patients), anti-KLH IgE (30%), anti-GM-CSF IgG (20%), anti-Id IgG (20%), and anti-Id IgE (30%). Id-specific delayed type hypersensitivity skin tests were positive in 85% of tested patients. Following vaccination, a progressive recovery of T-cell receptor (TCR) diversity was observed and the loss of oligoclonality was significantly correlated with the remission duration. Although Id/KLH conjugates did not eliminate the residual tumor burden, the median progression-free survival, and overall survival were 40 and 82 months, respectively. A retrospective case-matched analysis showed similar results in patients treated with IFN-α alone or in association with steroids. This vaccine formulation can overcome Id-specific immune tolerance by inducing clinical responses that are worthy of further investigation.

Interim positron emission tomography scan in Hodgkin lymphoma: definitions, interpretation rules, and clinical validation.

[18F]-Fluoro-2-deoxy-d-glucose positron emission tomography (18F-FDG PET) scan performed early during the therapy for Hodgkin lymphoma (HL) has a high prognostic value and correlates with survival. Several ongoing trials are under way to investigate the value of treatment adaptation based on early 18F-FDG PET results both in early and advanced-stage HL. In the former, in order to reduce the number of patients in whom involved-field radiotherapy is required for optimal treatment, and in the latter to identify the small subset (about 20%) of patients with a dismal prognosis who should be treated very aggressively while sparing the toxic effects of the therapy in most of them. However some issues still remain unsettled, including the standardization of interpretation rules for the interim-positron emission tomography (PET) scan, the optimal time to perform this examination during therapy, and the impact of a risk-adapted therapeutic strategy on the overall outcome of therapy in HL. International efforts are now underway to reach a consensus among experts on a set of simple, reproducible rules for PET interpretation, and attempts are being made to launch retrospective clinical studies so as to validate these rules.

Immune Modulation by Zoledronic Acid in Human Myeloma: An Advantageous Cross-Talk between Vγ9Vδ2 T Cells, αβ CD8+ T Cells, Regulatory T Cells, and Dendritic Cells

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8+ αβ T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8+ αβ antitumor effector T cells.

Hepatitis B Virus Reactivation and Efficacy of Prophylaxis with Lamivudine in Patients Undergoing Allogeneic Stem Cell Transplantation

Patients previously infected with hepatitis B virus (HBV) undergoing an allograft and recipients from HBV carrier donors are at risk of posttransplant viral reactivation. The role of prophylaxis with lamivudine remains unclear. One hundred seventeen patients, with a median age of 52 years (20-67 years), with various hematologic malignancies transplanted between 1999 and 2007 entered the study. Eighty-seven recipients negative for HBV surface antigen (HBsAg), antihepatitis B core antigen antibodies (anti-HBc), and HBV-DNA with HBsAg and HBV-DNA negative donors were defined as at low risk of HBV reactivation, whereas all the remaining 30 patients were defined as at high risk. Patients at high risk transplanted in 2005 or after received lamivudine to prevent HBV reactivation as per the Italian guidelines by the Associazione Italiana per lo Studio del Fegato (AISF). Patients at low risk did not experience HBV reactivation/hepatitis. Among the recipients at high risk, 11 of 25 anti-HBc positive, those HBsAg positive (2 of 2) or negative but transplanted from HBsAg positive donors (3 of 3) were treated with lamivudine. None of these developed HBV reactivation/hepatitis after a median follow-up of 40 months (17-55 months). Hepatitis developed in 3 anti-HBc positive untreated patients conditioned with a reduced-intensity regimen. Hepatitis B was not observed in recipients at low risk, transplanted from HBsAg negative/anti-HBc positive or negative donors. Lamivudine was effective in controlling reactivation in: HBsAg positive recipients, in patients transplanted from HBsAg positive donors and in HBsAg negative/antiHBc positive recipients, who showed a significant risk of reactivation if not given prophylaxis (NCT 00876148).

Comprehensive assessment of the TCRBV repertoire in small T-cell samples by means of an improved and convenient multiplex PCR method

OBJECTIVE Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a hosts immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional alphabeta T cells in small T-cell samples. MATERIALS AND METHODS The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of beta variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility. RESULTS Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 x 10(5) T cells. CONCLUSION We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.

Peripheral Blood CD34+ Percentage at Hematological Recovery after Chemotherapy Is a Good Early Predictor of Harvest: A Single-Center Experience

Several algorithms for early prediction of poor-mobilizing patients after chemotherapy and granulocyte colony-stimulating factor administration have been proposed. They generally define peripheral blood cut-off levels of CD34+ cells at a fixed day after starting chemotherapy, mostly with cyclophosphamide. To define an algorithm for early addition of plerixafor regardless of the chemotherapy regimen used, we retrospectively analyzed 280 chemomobilization attempts in 236 patients treated at our institution between 2002 and 2012. In multivariate analysis, CD34+ absolute count and CD34+ percentage upon total leukocyte count at day 1 (defined as the first day in which leukocytes reached a value > 1 × 10(9)/L) were the only factors able to predict a total harvest ≥ 2 × 10(6) CD34+/kg. In patients with day 1 CD34+ lower than 20/μL, the CD34+ percentage was a more reliable predictor of stem cell harvest in the following days than CD34+ absolute count. Upon definition of the best CD34+ cut-off value for identification of poor-mobilizing patients, an algorithm was set up to guide plerixafor administration. It was prospectively validated in 20 patients in 2013 with encouraging results in terms of low incidences of both mobilization failure and plerixafor use. Large prospective trials that define the most cost-effective strategy for just-in-time rescue plerixafor are warranted.