Rosaria De Filippi

The classical Hodgkin's lymphoma microenvironment and its role in promoting tumour growth and immune escape.

It has become clear that cancer is not merely a growth of autonomously proliferating cells, but that other non‐malignant cell types are a functional part of the disease. Immune cells, fibroblasts, specialized mesenchymal cells and microvasculature together make up the tumour microenvironment and have functional interactions with tumour cells. Classical Hodgkins lymphoma (cHL) is characterized by only a few malignant cells and an abundance of inflammatory cells. Hodgkin and Reed–Sternberg (HRS) cells are surrounded by T and B cells admixed with plasma cells, macrophages, eosinophils and mast cells. A constitutive activity of NF‐κB and an altered JAK–STAT signalling pathway are part of the biological background associated with the increased expression of cytokines and cytokine receptors seen in HRS cells. Over‐expression of the members of the TNF receptor family, especially CD30 and CD40, is a hallmark of HRS cells. cHL is a tumour where aberrant cytokine production contributes not only to the proliferation of HRS cells but also to the maintenance of an appropriate environment for the tumour cells. In addition, several chemokines contribute to the composition of the inflammatory background in cHL. This review summarizes updated information on the complex interactions between the HRS cells and their tissue microenvironment and highlights the development of newer therapeutic strategies aimed at targeting the non‐malignant inflammatory/immune cellular components of HL that are involved in cancer cell growth and/or immune escape. Copyright

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells.

Increasing evidence suggests that bone marrow‐derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor‐associated neo‐angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF‐α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF‐α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF‐α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF‐α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin‐2, granulocyte‐colony stimulating factor, hepatocyte growth factor, interleukin (IL)‐6, IL‐8, and platelet‐derived growth factor‐BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF‐α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF‐α increased the ability of MSCs to induce migration of MCF‐7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo‐angiogenesis and tumor cell migration. J. Cell. Physiol. 226: 2131–2138, 2011.

Functional coexpression of Interleukin (IL)-7 and its receptor (IL-7R) on Hodgkin and Reed-Sternberg cells: Involvement of IL-7 in tumor cell growth and microenvironmental interactions of Hodgkin's lymphoma.

The clinical and pathological features of classical Hodgkin lymphoma (cHL) mirror an abnormal tissue and systemic immune response due to the production of a variety of cytokines and chemokines by the malignant Hodgkin‐Reed‐Sternberg (H‐RS) cells and/or surrounding reactive cells. Here, we demonstrate that HL‐derived cell lines (L‐428, KM‐H2, HDLM‐2, L‐1236 and L‐540) and primary H‐RS cells from lymph node tissues of HL patients express the IL‐7(R) receptor. IL‐7 appears to be involved in autocrine circuitries of HL because L‐1236, HDLM‐2 and KM‐H2 cells display the constitutive production of IL‐7 and neutralizing anti‐IL‐7 antibodies induces a statistically significant inhibition of their basal proliferation. In addition, IL‐7, either exogenous or fibroblasts‐derived, promotes the clonogenic growth and reduces apoptosis of cultured H‐RS cells, being also able to partially protect these cells from the cytotoxic effects of doxorubicin. We also provide evidence that IL‐7 stimulates IL‐6 secretion from IL‐7R‐expressing fibroblasts from HL‐involved lymph nodes (HLFs), and that a striking increase in IL‐6 secretion can be observed in cocultures of HLFs with L1236 cells. Finally, we show that L‐1236 cells‐derived IL‐7 represents a costimulator for proliferation of purified CD4+CD25+CD127dim/− regulatory T cells (Tregs). Taken together, our data indicates that the IL‐7/IL‐7R axis constitutes an additional signaling pathway between H‐RS cells and their reactive cellular background, thereby affecting proliferation and survival of tumor cells, acting as a cofactor for Tregs expansion and enhancing the microenviromental production of IL‐6, a cytokine associated with the presence of “B” symptoms and a poor outcome in HL patients.

Efficacy and safety of bendamustine for the treatment of patients with recurring Hodgkin lymphoma

The management of patients with Hodgkin lymphoma (HL) recurring after stem cell transplantation (SCT) and multiply relapsed disease remains challenging. We report on 41 such patients who received bendamustine hydrochloride, a bifunctional mechlorethamine derivative mechanistically unrelated to traditional alkylators, after a median of four prior chemotherapy lines, including SCT in 85% of cases. Bendamustine was given at doses of 90–120 mg/m2 every 21 or 28 d. At first assessment (2–4 cycles), the overall response rate (ORR) was 78% with 12 (29%) complete (CR) and 20 (49%) partial responses (PR). Upon treatment prolongation to 6–8 courses, 40% of PRs progressed, yielding a final ORR of 58% with 31% of CRs. Eight patients (two CRs, six PRs) were subsequently allotransplanted. Median progression‐free and overall survival exceeded 11 and 21 months respectively; complete responders displayed a median disease‐free survival above 9 months with a relapse rate of only 30%. Outcomes were independent of disease chemosensitivity, previous transplant and bendamustine dose‐intensity. No life‐threatening or unexpected adverse events occurred. Within the limits of a retrospective analysis and schedule heterogeneity, these results appear very encouraging and prompt prospective trials to confirm bendamustine as a valuable option in the palliative setting and in cytoreductive strategies before allotransplantation.

RD-CODOX-M/IVAC with rituximab and intrathecal liposomal cytarabine in adult Burkitt lymphoma and 'unclassifiable' highly aggressive B-cell lymphoma

Specific trials on adult Burkitt lymphoma (BL) and ‘unclassifiable’ lymphomas with features intermediate between BL and diffuse large B‐cell lymphoma (BL/DLBCL) are advocated which include substantial numbers of older patients, to improve treatment feasibility, while countering risks of systemic and central nervous system (CNS) recurrences. We prospectively evaluated a modified CODOX‐M/IVAC (CODOX‐M: cyclophosphamide, vincristine, doxorubicin, high‐dose methotrexate; IVAC: ifosfamide, etoposide and high‐dose cytarabine) regimen by the addition of rituximab (R) and liposome‐encapsulated cytarabine (D) to increase antitumour activity and halve the number of intrathecal treatments. Thirty adults (40% >60 years) with BL (n = 15) and BL/DLBCL (n = 15) were accrued. Primary endpoints were progression‐free survival (PFS), CNS recurrence, and liposomal cytarabine‐associated toxicity. Eighty percent of patients received the whole treatment programme, the remaining cases received at least three full courses. Application of the RD‐CODOX‐M/IVAC regimen resulted in remarkable 4‐year PFS (78%) and complete remission (CR) rates (93%). However, PFS was significantly lower in patients older than 60 years as compared to younger ones (49%vs 93%, P = 0·03; median, 36 months), despite high actual dose‐intensity, CR rate and tolerability. Reduced‐intensity intratechal prophylaxis through liposomal cytarabine was effective because the CNS failure rate was low (3·4%) and without severe neurological toxicities. The RD‐CODOX‐M/IVAC strategy is feasible and highly effective, but improving outcomes in elderly patients remains a priority.

Biweekly rituximab, cyclophosphamide, vincristine, non-pegylated liposome-encapsulated doxorubicin and prednisone (R-COMP-14) in elderly patients with poor-risk diffuse large B-cell lymphoma and moderate to high 'life threat' impact cardiopathy.

This Phase II study assessed feasibility and efficacy of a biweekly R‐COMP‐14 regimen (rituximab, cyclophosphamide, non‐pegylated liposome‐encapsulated doxorubicin, vincristine and prednisone) in untreated elderly patients with poor‐risk diffuse large B‐cell lymphoma (DLBCL) and moderate to high ‘life threat’ impact NIA/NCI cardiac comorbidity. A total of 208 courses were delivered, with close cardiac monitoring, to 41 patients (median age: 73 years, range: 62–82; 37% >75 years) at a median interval of 15·6 (range, 13–29) days; 67% completed all six scheduled courses. Response rate was 73%, with 68% complete responses (CR); 4‐year disease‐free survival (DFS) and time to treatment failure (TTF) were 72% and 49%, respectively. Failures were due to early death (n = 3), therapy discontinuations (no‐response n = 2; toxicity n = 6), relapse (n = 6) and death in CR (n = 3). Incidence of cardiac grade 3–5 adverse events was 7/41 (17%; 95% confidence interval: 8–31%). Time to progression and overall survival at 4‐years were 77% and 67%, respectively. The Age‐adjusted Charlson Comorbidity Index (aaCCI) correlated with failures (P = 0·007) with patients scoring ≤7 having a longer TTF (66% vs. 29%; P = 0·009). R‐COMP‐14 is feasible and ensures a substantial DFS to poor‐risk DLBCL patients who would have been denied anthracycline‐based treatment due to cardiac morbidity. The aaCCI predicted both treatment discontinuation rate and TTF.

The cumulative amount of serum free light chain is a strong prognosticator in chronic lymphocytic leukemia

Identification of patients at risk of early disease progression is the mainstay of tailored management in chronic lymphocytic leukemia (CLL). Although application of established biomarkers is limited by intrinsic detection/readout complexities, abnormality of κ and λ serum-free light chain ratio [sFLC (κ/λ)] was proposed as a straightforward prognosticator in CLL. By analyzing 449 therapy-naive patients, we show that an abnormal sFLC(κ/λ), along with CD38, ZAP-70, IGHV mutations, cytogenetics and stage, independently predicts treatment-free survival (TFS) but becomes prognostically irrelevant if the cumulative amount of clonal and nonclonal FLCs [sFLC(κ + λ)], a variable associated with cytogenetic risk, exceeds the threshold of 60.6 mg/mL. Patients with sFLC(κ + λ) above cut-off displayed a poorer TFS outcome, irrespective of sFLC(κ/λ). Only ZAP-70, cytogenetics, stage, and TFS remained associated with sFLC(κ + λ) in a multivariate model. By assigning 1 point each for these variables, the 3-year probability of TFS was 94.8%, 84.5%, 61.6%, and 21.1% for patients scoring 0, 1, 2, and 3 + 4, respectively (P < .0001). These data, and the demonstration that monoclonal and polyclonal B cells concur to FLC synthesis in tumor tissues, suggest that sFLC(κ/λ) and sFLC(κ + λ) mirror distinct biologic processes in CLL. sFLC(κ + λ) assessment represents a sensitive and cost-effective tool for identifying CLL patients requiring early treatment.

Tumor flare reactions and response to lenalidomide in patients with refractory classic Hodgkin lymphoma

Patients with Hodgkin lymphoma (HL) failing salvage stem cell transplantation are candidates to investigational strategies. Lenalidomide represents an attractive option as it targets several signaling pathways, which regulate survival of HL cells and their microenvironmental interactions. We report the occurrence of Grades 2 and 3 tumor flare reactions in the first three patients entered a lenalidomide-based compassionate program for treatment-refractory HL. Flares occurred in concomitance of the scheduled week-off lenalidomide and upon withdrawal of symptomatic steroid treatment, and were associated with changes in B-cell regulatory cytokines and the concurrent expansion of polyclonal B-cells. Flares mimicked tumor progression but were effectively managed with anti-inflammatory treatment and followed by a clinical response, suggesting that they may mirror the pleiotropic actions of lenalidomide on HL microenvironment.

TNF receptor-associated factor 1 is a positive regulator of the NF-κB alternative pathway

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is unique among the members of the TRAF family, as it lacks the N-terminal RING/zinc-finger domain. Also the function of TRAF1 is not clearly established, with many papers reporting contradictory results. Here we show that TRAF1 interacts with BAFF receptor, a member of the TNF receptor family, and positively regulates activation of the alternative NF-kappaB pathway. Ectopic expression of TRAF1 causes degradation of TRAF3, stabilization of NIK, and processing of p100 to produce the mature form p52. In addition, we show that knocking-down expression of TRAF1 in the Hodgkins disease derived cell line L1236, interfere with p100 processing and with p52 mediate gene transcription. Collectively these results support a role for TRAF1 as a positive regulator of the NF-kappaB alternative pathway.

Elevation of clonal serum free light chains in patients with HIV-negative primary effusion lymphoma (PEL) and PEL-like lymphoma.

Primary effusion lymphoma (PEL) is a human herpesvirus 8 (HHV-8)-associated malignancy that presents in the serous body cavities as lymphomatous effusions, usually without extracavitary tumour masses (Carbone & Gloghini, 2008). Most cases occur in human immunodeficiency virus type-1 (HIV)-infected patients, but PEL can also develop in HIV-seronegative individuals following prolonged immunosuppressive therapy and/or in association to age-related immunodepression (Carbone & Gloghini, 2008). Other agents, including hepatitis C virus (HCV), may be associated to PEL and HHV-8 negative cases have been also described as HHV8-unrelated PEL-like lymphoma (Paner et al, 2003; Kobayashi et al, 2007; Carbone & Gloghini, 2008). This lymphoma arises from mature post-germinal centre B-cells which, despite having a pre-plasma cell genotype and gene expression signature (Jenner et al, 2003; Carbone & Gloghini, 2008), rarely express surface immunoglobulins (Igs) due to the defective expression of some B-cell-specific transcription factors (Arguello et al, 2003; Di Bartolo et al, 2009). By applying highly sensitive techniques, however, light chain mRNA was frequently demonstrated and a minority (10%) of tumour cells was found to display low levels of cytoplasmic monoclonal light chains (Wakely et al, 2002; Di Bartolo et al, 2009). The prognosis of PEL is poor with a median survival of 6 months also due to the lack of specific prognostic factors and tumour markers to optimize therapy (Nador et al, 1996; Boulanger et al, 2005). This report shows that elevated levels of clonally-restricted serum free light chain (sFLC) can be found in some PEL cases and that variations in such levels may reflect therapyrelated changes in the tumour cell mass, so representing a possible marker for treatment monitoring/modulation. The sFLC assay specifically quantifies circulating light chains unbound to heavy chains into intact Ig molecules and allows disease monitoring in non-secretory/oligo-secretory (NS-OS) multiple myeloma and other situations where, at the tumour plasma cells level, the compromised heavy chains production leads to unbalanced, excess production of clonal light chains or to expression of light chains only (Pratt, 2008). Patients’ characteristics are shown in Table I. Patient 1 had HCV-related cirrhosis and a previous left nephrectomy due to kidney carcinoma. He presented with bulky ascites and computed tomography disclosed a left pleural effusion and a diffuse thickening of the peritoneal membrane with mesenteric nodulations. Peritoneal fluid was removed and a laparoscopic peritoneal biopsy performed. Morphology (Fig 1A, B), immunophenotypic (Fig 1C, D) and molecular studies (Fig 1E) were consistent with HHV-8-unrelated PEL-like lymphoma. The patient was given bortezomib, cyclophosphamide and steroids, which resulted, in complete effusions control after 3 weeks. Abdominal ultrasounds confirmed the clinical response but, 2 weeks after the second course of treatment, effusions recurred leading to death with progressive lymphoma. Patient 2 had been receiving continuous immunosuppression for 9 years after kidney transplantation. He presented with fever, right pleural effusion and bulky ascites. Tumour cell features were consistent with HHV-8 PEL. The patient refused chemotherapy and was given oral thalidomide (200 mg/d), which resulted in ascites and pleural effusion control lasting 3 months. Due to neurotoxicity, thalidomide was reduced to 100 mg/d for 1 month and then withdrawn. Bulky effusions recurred, leading to death despite further treatment. Patient 3 had HCV-related cirrhosis, a severe cardiomyopathy and presented with pleural, pericardial and abdominal effusions containing HHV-8 PEL cells (Table I, Fig 1E). The patient received two courses of cyclophosphamide, vincristine and prednisone leading to substantial control of effusions but bulky disease recurred shortly after the third course of treatment, leading to death 1 month later. The free j and k concentrations (normal ranges, j: 3Æ3–19Æ4 mg/l; k: 5Æ71–26Æ3 mg/l) were assayed by immunonephelometry (Freelite; The Binding Site, Ltd., Birmingham, UK) on cryopreserved ()80 C) serum samples and the FLC j/k ratio was calculated (free j concentration divided by free k concentration, reference range 0Æ26–1Æ65). All of the patients displayed an abnormal baseline sFLC j/k ratio (ranging from 0Æ04 to 0Æ15) due to a k chain clonal involvement, with absolute free k levels ranging from 143Æ5 to 790 mg/l (Table I). Cytoplasmic k chains were documented in tumour cells in Patients 1 and 2, while imunohistochemistry was negative in the remaining case (Table I, Fig 1D). Most interestingly, serial measurements during the course of disease showed changes in sFLC levels that correlated with clinical progress. At the time of maximal response to treatment, levels of free k chains displayed a higher than 75% drop from baseline levels in all patients followed by a rise at tumour progression (Fig 1F). This was paralleled by corresponding modifications of the sFLC j/k Correspondence