Giovanna Andreola

Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

Vaccination of Metastatic Melanoma Patients With Autologous Tumor-Derived Heat Shock Protein gp96-Peptide Complexes: Clinical and Immunologic Findings

PURPOSE To determine the immunogenicity and antitumor activity of a vaccine consisting of autologous, tumor-derived heat shock protein gp96-peptide complexes (HSPPC-96, Oncophage; Antigenics, Inc, Woburn, MA) in metastatic (American Joint Committee on Cancer stage IV) melanoma patients. PATIENTS AND METHODS Sixty-four patients had surgical resection of metastatic tissue required for vaccine production, 42 patients were able to receive the vaccine, and 39 were assessable after one cycle of vaccination (four weekly injections). In 21 patients, a second cycle (four biweekly injections) was given because no progression occurred. Antigen-specific antimelanoma T-cell response was assessed by enzyme-linked immunospot (ELISPOT) assay on peripheral blood mononuclear cells (PBMCs) obtained before and after vaccination. Immunohistochemical analyses of tumor tissues were also performed. RESULTS No treatment-related toxicity was observed. Of 28 patients with measurable disease, two had a complete response (CR) and three had stable disease (SD) at the end of follow-up. Duration of CR was 559+ and 703+ days, whereas SD lasted for 153, 191, and 272 days, respectively. ELISPOT assay with PBMCs of 23 subjects showed a significantly increased number of postvaccination melanoma-specific T-cell spots in 11 patients, with clinical responders displaying a high frequency of increased T-cell activity. Immunohistochemical staining of melanoma tissues from which vaccine was produced revealed high expression of both HLA class I and melanoma antigens in seven of eight clinical responders (two with CR, three with SD, and the three with long-term disease-free survival) and in four of 12 nonresponders. CONCLUSION Vaccination of metastatic melanoma patients with autologous HSPPC-96 is feasible and devoid of significant toxicity. This vaccine induced clinical and tumor-specific T-cell responses in a significant minority of patients.

Cytokines in cancer therapy

Cytokines are crucial factors in the activation and development of immune response, including responses against tumor cells. Interleukin (IL)-2, a T-cell growth factor, has been largely used to activate T and NK cells in vivo and to maintain such an activation for therapeutic purposes. When given to patients, IL-2 was shown to cause clinical responses, especially in metastatic melanoma and renal cancer patients, though its mechanism of action could not be completely elucidated. Cytokines (IL-2, IL-12, GM-CSF) are also used as natural adjuvants of vaccines of various formulation to help in activating and maintaining an antitumor immune response. This review summarizes findings deriving from the use of cytokines in cancer therapy and provides insights into future approaches when a more appropriate use of cytokines, together with new vaccines, is likely to improve clinical outcome.

Rituximab and Subcutaneous 2-Chloro-2′-Deoxyadenosine as Therapy in Untreated and Relapsed Waldenström's Macroglobulinemia

With the aim to assess the efficacy of subcutaneous cladribine in combination with rituximab, 29 newly diagnosed/pretreated WM patients were enrolled in a multicenter phase II trial. Intended therapy consisted of rituximab on day 1 followed by s.c. cladribine 0.1 mg/kg for 5 consecutive days, administered monthly for 4 cycles. The expression of genes involved in cladribine metabolism was evaluated. With a median follow-up of 50 months the ORR rate observed was 89.6% without any difference between newly or pretreated patients (P=.522). The therapy was well tolerated; no major infections were observed, no patients developed transformation to high-grade NHL nor myelodysplasia. Low expression levels of hCNT1 correlated with the failure to achieve a CR (P=.024). The combination of rituximab/cladribine is safe and effective in WM patients requiring treatment. The pharmacogenomic analysis suggests that hCNT1 might be beneficial in predicting clinical response to such a combination treatment.

Filgrastim XM02 (Tevagrastim®) after autologous stem cell transplantation compared to lenograstim: favourable cost-efficacy analysis

Purpose Granulocyte colony-stimulating factors (G-CSFs), filgrastim and lenograstim, are recognised to be useful in accelerating engraftment after autologous stem cell transplantation. Several forms of biosimilar non-glycosylated G-CSF have been approved by the European Medicines Agency, with limited published data supporting the clinical equivalence in peripheral blood stem cell mobilisation and recovery after autologous stem cell transplantation. Method With the aim of comparing cost-effective strategies in the use of G-CSF after autologous stem cell transplantation, we retrospectively evaluated 32 patients consecutively treated with biosimilar filgrastim XM02 (Tevagrastim) and 26 with lenograstim. All patients received G-CSF (biosimilar or lenograstim) at a dosage of 5 mcg/kg/day subcutaneously from day 5 to absolute neutrophil count of 1500/mmc for three days. Results The median time to absolute neutrophil count engraftment was 11 days for the filgrastim XM02 group and 12 days for the lenograstim group. As for platelets recovery, the median time was 12 days in both groups. The median number of G-CSF vials used for patients was 9.5 for Tevagrastim and 10.5 for lenograstim, reflecting a mean estimated cost of about 556.1 euros for Tevagrastim versus 932.2 euros for lenograstim (p< 0.001). The median days of febrile neutropenia were 1.5 and 1 for filgrastim XM02 and lenograstim, respectively. No adverse event related to the use of XM02 filgrastim was recorded. Conclusion In our experience, filgrastim XM02 and lenograstim showed comparable efficacy in shortening the period of neutropenia after cytoreduction and autologous stem cell transplantation, with a favourable cost effect for filgrastim XM02.

Peripheral T-lymphocyte subsets in patients treated with Rituximab-Chlorambucil combination therapy for indolent NHL.

The anti-CD20 chimeric monoclonal antibody, rituximab, is a well-established treatment for newly diagnosed or pretreated follicular non-Hodgkin’s lymphoma (NHL) patients and is able to induce molecular response in about 30% of the cases when used as single agent [1]. The addition of rituximab to chemotherapy improves the clinical and molecular remission rate and prolongs the time to treatment failure [2, 3] without any increase in infectious complications in randomized controlled trials. Prolonged rituximab treatment is associated with a depletion of circulating B cells; however, data are not available on T-cells peripheral subset after a rituximab chemotherapy combination treatment. We retrospectively investigated the effects of rituximab– chlorambucil combination therapy on peripheral blood lymphocyte subsets in 25 low-grade or follicular B-cell NHL patients (12 newly diagnosed and 13 relapsed/ refractory). The regimen [4] consisted of chlorambucil 6 mg/m daily for six consecutive weeks, in association with a standard four-weekly rituximab as induction phase. After revaluation, the responding patients received four additional cycles with chlorambucil (6 mg/m daily for 2 weeks monthly) plus rituximab (once monthly). All patients completed the planned treatment and were evaluated for response and toxicity. After induction, we observed absolute G2 and G3 lymphopenia in 13 and 6 patients, respectively. After consolidation, ten patients had G2 and five patients had G3 absolute lymphopenia. After a median of 9 months (3–30 months) from the end of treatment, six patients showed G2 lymphopenia. As expected, after the first administration of rituximab, immunophenotypic analysis showed a reduction of CD19/ 20+ positive cells below 0.1% in all the patients; that reduction was still present at revaluation after induction and consolidation therapy. As shown in Fig. 1, all the patients presented a significant CD4+ reduction (p<0.001), independent of the levels at baseline (median absolute CD4+ count, 246 cell/ μl), and this trend was maintained during the therapy (median absolute CD4+ count, 216 cell/μl). At median follow-up of 9 months in 50% of the patients, absolute CD4+ T cells had recovered within normal range. On the other hand, we observed no significant reduction of CD8+ and CD56+ absolute values. Infections were rare and none was fatal: two patients developed cutaneous HZV, one had perianal abscess, and one patient was hospitalized for HBV reactivation. To our knowledge, this is the first report on the T-cell profile in patients receiving a rituximab chemotherapy combined chemotherapy. Our experience suggests that chlorambucil–rituximab combination therapy induces a selective decrease of absolute CD4+ T cells during treatment. However, no major infection was observed; the lack of a correlation between the drop in CD4+ T cell count and an increase in infection rate could be related to the relative short period of absolute CD4+ low count. On the contrary, as recently observed by Kaplan et al. [5] in AIDSrelated lymphomas, this effect might justify the increased infectious death rates observed with rituximab-CHOP treatment. Ann Hematol (2006) 85:813–814 DOI 10.1007/s00277-006-0170-9

A retrospective international study on primary extranodal marginal zone lymphoma of the lung (BALT lymphoma) on behalf of International Extranodal Lymphoma Study Group (IELSG)

Primary lymphoma of the lung is a rare entity. Clinical features, optimal treatment, role of surgery and outcomes are not well defined, and the follow‐up is variable in published data. Clinical data of 205 patients who were confirmed to have bronchus mucosa‐associated lymphoid tissue lymphoma from December 1986 to December 2011 in 17 different centres worldwide were evaluated. Fifty‐five per cent of the patients were female. The median age at diagnosis was 62 (range 28–88) years. Only 9% had a history of exposure to toxic substances, while about 45% of the patients had a history of smoking. Ten per cent of the patients had autoimmune disease at presentation, and 19% patients had a reported preexisting lung disease. Treatment modalities included surgery alone in 63 patients (30%), radiotherapy in 3 (2%), antibiotics in 1 (1%) and systemic treatment in 128 (62%). Patients receiving a local approach, mainly surgical resection, experienced significantly improved progression‐free survival (p = 0.003) versus those receiving a systemic treatment. There were no other significant differences among treatment modalities. The survival data confirm the indolent nature of the disease. Local therapy (surgery or radiotherapy) results in long‐term disease‐free survival for patients with localized disease. Systemic treatment, including alkylating‐containing regimens, can be reserved to patients in relapse after incomplete surgical excision or for patients with advanced disease. Copyright

Chlorambucil-rituximab as first-line therapy in patients affected by follicular non-Hodgkin's lymphoma: a retrospective single-centre study.

Rituximab, a chimeric monoclonal antibody directed against the CD20 antigen, has been shown to be active in newly diagnosed and relapsed patients with follicular lymphoma (FL), both as monotherapy and in combination with chemotherapy. Many studies suggest that the prognosis of patients with FL may improve when it is used in combination with chemotherapy. Despite these advances, the disease remains essentially incurable with standard therapy, and novel approaches to treatment are needed because optimal therapy is not defined. The combination of chlorambucil–rituximab is one of several standard treatment options for FL. Here, we considered data arising from 75 patients with newly diagnosed FL at the European Institute of Oncology treated with the combination of rituximab plus chlorambucil. The aim of this study was to evaluate the efficacy and safety of chlorambucil and rituximab, delivered 6 mg/m2/day orally for 6 weeks and 375 mg/m2 in a standard 4‐weekly schedule, respectively. Patients responding to the induction therapy received a prolonged therapy with four additional cycles of chlorambucil plus rituximab. Seventy‐one patients (94.6%) completed the treatment; four patients discontinued treatment because of grade 3–4 hematological toxicity. The overall response rate was 97.3% including 74.7% of complete responses. Only two patients had a stable disease at revaluation after treatment. With a median follow‐up of 57 months, 72 patients (96%) are alive. Median event‐free survival (EFS) and median overall survival (OS) were not reached; 5‐year OS rate was 98.4%. The 5‐year EFS was 71.3%. By univariate and multivariate analyses, elevated beta‐2 microglobulin levels and partial responses to therapy were correlated with worse EFS. These results suggest that the combination of chlorambucil and rituximab is an active and safe regimen in patients with newly diagnosed FL, principally in those with low tumour burden and favourable prognostic factors. Copyright

Chlorambucil – rituximab as first line combination therapy in follicular non-Hodgkin's lymphoma: A clinical and biological analysis

The achievement of a molecular response (marrow blood clearance of BCL-2/IgHþ positive neoplastic cells) seems to be associated with a longer event free survival (EFS) and might prolong overall survival (OS) [1] in patients affected by follicular NHLs. The chimeric monoclonal anti-CD20 antibody, rituximab, has become a well established treatment for newly diagnosed or pre-treated indolent NHL patients; the addition of rituximab to chemotherapy improves the clinical and molecular remission rate and prolongs the time to treatment failure [2]. We previously demonstrated the feasibility and efficacy of chlorambucil plus rituximab combination therapy in the treatment of newly diagnosed and relapsed/ refractory indolent B-cell NHL patients [3]. Here we present our experience evaluating the clinical and biological activity of the combination as first line treatment in follicular NHL patients. Twenty-five consecutive newly diagnosed patients affected by grade 1-2 (WHO classification [4]) follicular lymphoma treated at European Institute of Oncology between November 2001 and June 2004 were retrospectively investigated. BCL-2/IgH translocation on marrow blood was evaluated by quantitative and qualitative PCR at diagnosis and by qualitative PCR after the induction and maintenance therapy and every 3 months during follow-up. Treatment consisted of oral chlorambucil (leukeran) 6 mg/sqm administered daily for 6 consecutive weeks in association with standard 4-weekly rituximab (375 mg/smq i.v) administration schedule. Eight weeks later, patients clinically re-evaluated as responding received a consolidation therapy with chlorambucil 6 mg/sqm daily for 2 weeks every month (4 cycles) plus rituximab (375 mg/smq i.v) once every month. In patients BCL-2/IgH positive at diagnosis, molecular CR was defined as the achievement of a clinical CR and qualitative PCR negativity in marrow blood. Patients’ main clinical characteristics are summarized in Table I. The median time from diagnosis to beginning of therapy was 27 days (range 10 – 48). All patients completed the planned treatment without any delay or dose reduction (either in induction or in maintenance therapy) and were fully assessed for clinical and biological response. Overall response rate was 96% (24/25) with a high rate of CR (88%). Only one patient (BCL-2/IgH negative at baseline) had a SD at revaluation after treatment. With a median follow-up of 36 months (range 19 – 50), three patients relapsed at 6.7, 19.7, and 31 months respectively. Ten patients had a positive PCR BCL-2/IgH translocation in marrow blood at diagnosis; nine out of 10 patients had a low level (5100 pg/ml) of BCL-2/IgHþ cells by quantitative PCR. All treated patients obtained a clearance of BCL-2/IgH translocation in marrow blood; eight out of 10 remain in molecular CR. In terms of DSF, we did not observe any correlation between relapse rate and Ann Arbor stage, FLIPI score, marrow involvement at trephine biopsy, RQ-PCR at diagnosis whereas 2/10

R-ESHAP plus pegfilgrastim as an effective peripheral stem cell mobilization regimen for autologous stem-cell transplantation in patients with relapsed/refractory diffuse large B-cell lymphoma.

Stem cell (SC) mobilization is significantly influenced by the mobilization schedule in patients with lymphoma. We evaluated data from 30 patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) undergoing SC mobilization. All received R-ESHAP plus a single dose of pegfilgrastim. All patients collected ⩾ 2 × 10(6) CD34+cells/kg, 80% of them at least 5 × 10(6) CD34+cells/kg. Adverse effects of the regimen included myelosuppression and neutropenic fever. Herein, our results suggest that R-ESHAP plus pegfilgrastim is a highly effective mobilization strategy in patients affected by DLBCL associated with a low incidence of adverse events.