Michele Magni

Stereotyped B-cell receptor is an independent risk factor of chronic lymphocytic leukemia transformation to Richter syndrome.

Purpose: Few biological prognosticators are useful for prediction of Richter syndrome (RS), representing the transformation of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. Stereotyped B-cell receptors (BCR) may have prognostic effect in CLL progression. We tested the prognostic effect of stereotyped BCR for predicting RS transformation. Experimental Design: The prevalence of stereotyped BCR was compared in RS (n = 69) versus nontransformed CLL (n = 714) by a case-control analysis. Subsequently, the effect of stereotyped BCR at CLL diagnosis on risk of RS transformation was actuarially assessed in a consecutive CLL series (n = 753). Results: RS (n = 69) displayed a higher prevalence of stereotyped BCR (P < 0.001) compared with nontransformed CLL. The actuarial risk of RS transformation was significantly higher in CLL carrying stereotyped BCR (P < 0.001). Among BCR subsets most represented in CLL, subset 8 using IGHV4-39/IGHD6-13/IGHJ5 carried the highest risk of RS transformation [hazard ratio (HR), 24.50; P < 0.001]. Multivariate analysis selected stereotyped BCR (HR, 3.33; P = 0.001) and IGHV4-39 usage (HR, 4.03; P = 0.004) as independent predictors of RS transformation. The combination of IGHV4-39 usage and stereotyped BCR in the same patient identified CLL with a very high risk of RS transformation (5-year risk, 68.7%). The risk carried by stereotyped BCR and IGHV4-39 usage was specific for RS transformation and had no effect on CLL progression without transformation. Conclusions: Analysis of BCR features may help identify CLL patients at risk of RS. A close monitoring and a careful biopsy policy may help early recognition of RS in CLL patients using stereotyped BCR, particularly if combined with IGHV4-39.

Risk Factors for the Development of Secondary Malignancy After High-Dose Chemotherapy and Autograft, With or Without Rituximab: A 20-Year Retrospective Follow-Up Study in Patients With Lymphoma

PURPOSE High-dose chemotherapy with peripheral blood progenitor cell (PBPC) autograft is effective in high-risk lymphoma, particularly with the addition of rituximab; however, it is associated with risk of secondary malignancy. These issues have been addressed in a series of 1,347 patients with lymphoma treated with a high-dose sequential (HDS) program. PATIENTS AND METHODS A total of 1,024 patients with B-cell lymphoma, 234 patients with Hodgkins lymphoma, and 89 patients with T-cell lymphoma were treated with HDS between 1985 and 2005 at 11 Gruppo Italiano Terapie Innovative Linfomi centers. HDS was given as salvage treatment to 707 patients (52%); 655 patients (49%) received a modified HDS, with high-dose cytarabine and two consecutive PBPC harvests. Rituximab-supplemented HDS was given to 523 patients (39%). RESULTS At a median follow-up of 7 years, the median overall survival (OS) was 16.2 years; in B-cell lymphoma the OS was significantly superior with rituximab HDS compared to HDS alone. The cumulative incidence at 5 and 10 years of secondary myelodysplasia/acute leukemia (sMDS/AL) were 3.09% and 4.52%, respectively, that of solid tumors were 2.54% and 6.79%, respectively. Factors associated with sMDS/AL were male sex and use of the second harvest PBPC for the graft; factors found to be associated with solid tumor were advanced age, post-HDS radiotherapy, and rituximab addition to HDS. Despite the increased risk of solid tumors, rituximab addition to HDS was still associated with survival advantages. CONCLUSION This analysis has relevant implications for the design and use of intensive chemoimmunotherapy with autograft. In addition, it offers useful insights toward the understanding and prevention of tumor development.

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.

Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Increasing evidence argues that the success of an anticancer treatment may rely on immunoadjuvant side effects including the induction of immunogenic tumor cell death. Based on the assumption that this death mechanism is a similar prerequisite for the efficacy of an active immunotherapy using killed tumor cells, we examined a vaccination strategy using dendritic cells (DC) loaded with apoptotic and necrotic cell bodies derived from autologous tumors. Using this approach, clinical and immunologic responses were achieved in 6 of 18 patients with relapsed indolent non-Hodgkins lymphoma (NHL). The present report illustrates an impaired ability of the neoplastic cells used to vaccinate nonresponders to undergo immunogenic death on exposure to a cell death protocol based on heat shock, γ-ray, and UVC ray. Interestingly, when compared with doxorubicin, this treatment increased surface translocation of calreticulin and cellular release of high-mobility group box 1 and ATP in histologically distinct NHL cell lines. In contrast, treated lymphoma cells from responders displayed higher amounts of calreticulin and heat shock protein 90 (HSP90) compared with those from nonresponders and boosted the production of specific antibodies when loaded into DCs for vaccination. Accordingly, the extent of calreticulin and HSP90 surface expression in the DC antigenic cargo was significantly associated with the clinical and immunologic responses achieved. Our results indicate that a positive clinical effect is obtained when immunogenically killed autologous neoplastic cells are used for the generation of a DC-based vaccine. Therapeutic improvements may thus be accomplished by circumventing the tumor-impaired ability to undergo immunogenic death and prime the antitumor immune response.

Boosting T Cell-Mediated Immunity to Tyrosinase by Vaccinia Virus-Transduced, CD34+-Derived Dendritic Cell Vaccination: A Phase I Trial in Metastatic Melanoma

Purpose: Six American Joint Committee on Cancer stage IV melanoma patients were enrolled into a Phase I study of vaccination with autologous CD34+-derived dendritic cells transduced with a modified vaccinia Ankara virus encoding human tyrosinase gene (MVA-hTyr). Experimental Design: Patients received a first intravenous injection of 1 × 108 MVA-hTyr–transduced dendritic cells, followed by three s.c. injections at a 14-day interval. Results: Treatment was well tolerated, except for low-grade fever (three of six patients), mild erythema at injection site (five of six), and vitiligo (two of six). A partial response, involving shrinkage of an s.c. nodule, later surgically removed, was observed in 1 patient, who then remained disease-free (>850 days). By human lymphocyte antigen tetramer analysis, significant and often long-lasting increases in frequency of T cells directed to tyrosinase368–376 but not to gp100209–217 were documented in periphery of 4 of 5 HLA-A*0201+ patients, a few days after vaccine administration. In addition, maturation phenotype of tyrosinase-specific T cell shifted toward the T effector memory/T terminally differentiate stages (CCR7−CD45RA−/+) in synchrony with the T-cell frequency peaks. By enzyme-linked immunospot in peripheral blood of five HLA-A*0201+ patients, we found that the vaccine could induce interferon γ-releasing effector cells directed to HLA-A*0201/tyrosinase368–376 and to vaccinia virus HLA-A*0201/H3L184–192 epitopes. Moreover, an interferon γ response after vaccination was elicited even against the HLA-DRB1–1501/tyrosinase386–406 epitope in one out of two HLA-A* DRB1–01501+ patients. Conclusions: These results indicate that vaccination with MVA-hTyr–transduced dendritic cells is well tolerated, can possibly produce clinical responses, and activates tyrosinase- and vaccinia virus-specific T cells in vivo. These data suggest a broad utility of the MVA vector for targeting tumor-associated antigens to dendritic cells for tumor immunotherapy.

Prolonged survival in poor-risk diffuse large B-cell lymphoma following front-line treatment with rituximab-supplemented, early-intensified chemotherapy with multiple autologous hematopoietic stem cell support: a multicenter study by GITIL (Gruppo Italiano Terapie Innovative nei Linfomi)

A prospective multicenter program was performed to evaluate the combination of rituximab and high-dose (hd) sequential chemotherapy delivered with multiple autologous peripheral blood progenitor cell (PBPC) support (R-HDS-maps regimen) in previously untreated patients with diffuse large B-cell lymphoma (DLB-CL) and age-adjusted International Prognostic Score (aaIPI) score 2-3. R-HDS-maps includes: (i) three APO courses; (ii) sequential administration of hd-cyclophosphamide (CY), hd-Ara-C, both supplemented with rituximab, hd-etoposide/cisplatin, PBPC harvests, following hd-CY and hd-Ara-C; (iii) hd-mitoxantrone (hd-Mito)/L-Pam + 2 further rituximab doses; (iv) involved-field radiotherapy. PBPC rescue was scheduled following Ara-C, etoposide/cisplatin and Mito/L-Pam. Between 1999 and 2004, 112 consecutive patients aged <65 years (74 score 2, 38 score 3) entered the study protocol. There were five early and two late toxic deaths. Overall 90 patients (80%) reached clinical remission (CR); at a median 48 months follow-up, 87 (78%) patients are alive, 82 (73%) in continuous CR, with 4 year overall survival (OS) and event-free survival (EFS) projections of 76% (CI 68–85%) and 73% (CI 64–81%), respectively. There were no significant differences in OS and EFS between subgroups with Germinal-Center and Activated B-cell phenotype. Thus, life expectancy of younger patients with aaIPI 2-3 DLB-CL is improved with the early administration of rituximab-supplemented intensive chemotherapy compared with the poor outcome following conventional chemotherapy.

Rituximab Improves the Efficacy of High-Dose Chemotherapy With Autograft for High-Risk Follicular and Diffuse Large B-Cell Lymphoma: A Multicenter Gruppo Italiano Terapie Innnovative nei Linfomi Survey

PURPOSE To investigate the impact of adding rituximab to intensive chemotherapy with peripheral-blood progenitor cell (PBPC) autograft for high-risk diffuse large B-cell lymphoma (DLB-CL) and follicular lymphoma (FL). PATIENTS AND METHODS Data were collected from 10 centers associated with Gruppo Italiano Terapie Innnovative nei Linfomi for 522 patients with DLB-CL and 223 patients with FL (median age, 47 years) who received the original or a modified high-dose sequential (HDS) chemotherapy regimen. HDS was delivered to 396 patients without (R-) and to 349 patients with (R+) rituximab; 154 (39%) and 178 patients (51%) in the R- and R+ subsets, respectively, underwent HDS for relapsed/refractory disease. RESULTS A total of 355 R- (90%) and 309 R+ patients (88%) completed the final PBPC autograft. Early treatment-related mortality was 3.3% for R- and 2.8% for R+ (P = not significant). Two parameters significantly influenced the outcome: disease status at HDS, with 5-year overall survival (OS) projections of 69% versus 57% for diagnosis versus refractory/relapsed status, respectively, and rituximab addition, with 5-year OS of 69% versus 60% in the R+ versus R- groups, respectively. In the multivariate analysis, these two variables maintained an independent prognostic value. The marked benefit of rituximab was evident in patients receiving HDS as salvage treatment: the 5-year OS projections for R+ versus R- were, respectively, 64% versus 38%, for patients with refractory disease or early relapse and 71% versus 57%, for patients with late relapse, partial response, or second/third relapse. CONCLUSION The results of this large series indicate that rituximab should be included in the current practice of PBPC autograft for DLB-CL and FL.

High-Dose Yttrium-90–Ibritumomab Tiuxetan With Tandem Stem-Cell Reinfusion: An Outpatient Preparative Regimen for Autologous Hematopoietic Cell Transplantation

PURPOSE To develop high-dose myeloablative therapy for CD20(+) non-Hodgkins lymphoma (NHL) as a safe and widely applicable regimen. PATIENTS AND METHODS Patients with relapsed/refractory (n = 25) or de novo high-risk (n = 5) NHL received one myeloablative dose of yttrium-90 ((90)Y)-ibritumomab tiuxetan after five chemotherapy courses, including three cycles of anthracycline- or platinum-containing regimens, one cycle of cyclophosphamide (4 to 7 g/m(2)), and one cycle of cytarabine (12 to 24 g/m(2)). The only exclusion criteria were CNS lymphoma and Eastern Cooperative Oncology Group performance status of more than 3. Primary end points were overall survival (OS) and event-free survival (EFS). Secondary end points included safety and applicability of high-dose (90)Y-ibritumomab tiuxetan. To minimize hematologic toxicity, stem cells were reinfused at days 7 and 14 after (90)Y-ibritumomab tiuxetan. RESULTS Thirteen patients received (90)Y-ibritumomab tiuxetan 0.8 mCi/kg, and 17 patients received 1.2 mCi/kg. At 1.2 mCi/kg, the radiation absorbed by critical nonhematologic organs approached the protocol-defined upper safety limit, defining this as the recommended dose for subsequent studies. Hematologic toxicity was mild to moderate and of short duration. Infections occurred in 27% of patients (none had a severity grade greater than 3). After a median observation time of 30 months (range, 22 to 48 months), no myeloid secondary malignancy or chromosomal abnormality was observed, the OS rate was 87%, and the EFS rate was 69%. CONCLUSION High-dose (90)Y-ibritumomab tiuxetan seems to be an innovative myeloablative regimen with unprecedented short-term toxicity and wide applicability. Further studies are required to assess its long-term safety and role in the management of CD20(+) NHL.

High-dose ara-C with autologous peripheral blood progenitor cell support induces a marked progenitor cell mobilization: an indication for patients at risk for low mobilization

A high-dose (HD) chemotherapy scheme was designed for the collection of large numbers of peripheral blood progenitor cells (PBPC) in lymphoma patients who were candidates for myeloablative therapy with autograft. The scheme included the sequential administration of HD cyclophosphamide (CY) (7 g/m2) and HD ara-C (2 g/m2 twice a day for 6 consecutive days), followed by final consolidation with PBPC autograft. PBPC harvests were scheduled following both HD CY and HD ara-C. To minimize hematologic toxicity, small aliquots of PBPC (⩽3 × 106 CD34+ cells/kg) collected following HD CY were reinfused following HD ara-C. The treatment was delivered to 112 patients (median age: 43 years) with lymphoid malignancies (107 non-Hodgkins lymphoma, four Hodgkins lymphoma, one amyloidosis); 75 patients were at disease onset, whereas 37 had relapsed or were refractory after first-line conventional therapy. PBPC mobilization was assessed in terms of peak values of circulating CD34+ cells/μl, as well as total CD34+ cells/kg collected. In a few patients CFU-GM/kg were also evaluated. At the time of maximal mobilization following HD CY, 93 ‘high-mobilizer’ patients had >20 circulating CD34+ cells/μl, whereas the remaining 19 ‘low-mobilizer’ patients did not reach this cut-off value. In spite of poor mobilization after HD CY, 16 out of 19 low mobilizers provided good harvests following HD ara-C; overall, median collected CD34+ cells × 106/kg were 1.4 (0–3.1) and 10.2 (0–37) after HD CY and HD ara-C, respectively (P = 0.00007). Similar patterns were observed when PBPC were evaluated by CFU-GM/kg. Complete and durable hemopoietic reconstitution followed autograft with post HD ara-C PBPC. Within the high-mobilizer group, 88 patients received HD ara-C and 79 (90%) still showed high mobilization; overall, median collected CD34+cells × 106/kg were 17.8 (range 3–94) and 19 (range 0–107) after HD CY and HD ara-C respectively (P = NS). Thus, the scheme allowed sufficient PBPC collections for autografting in low mobilizer patients; in addition, the scheme could be considered whenever extensive chemotherapy debulking is needed prior to PBPC collection.

Granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor infusion makes high-dose etoposide a safe outpatient regimen that is effective in lymphoma and myeloma patients.

PURPOSE This study assessed the efficacy of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human granulocyte colony-stimulating factor (rhG-CSF) in ameliorating the extent and duration of hematologic toxicity after high-dose etoposide cancer therapy. PATIENTS AND METHODS Thirty-two non-Hodgkins lymphoma and myeloma patients were treated with 2 to 2.4 g/m2 etoposide infused intravenously (IV) during a 10- to 12-hour period, followed 72 hours later by subcutaneous administration of rhGM-CSF or rhG-CSF. Hematologic toxicity was compared with that observed in 29 patients who were treated with high-dose etoposide without growth factors. RESULTS The median duration of grade 4 neutropenia in growth factor-treated patients was 3 days, and granulocyte counts never decreased to less than 100/microL in approximately half of the patients. The corresponding figures in the control patients were 8 and 3 days, respectively (P < .0001). No effect was observed in platelet and RBC recovery. Growth factor-treated patients became eligible to receive additional myelotoxic chemotherapy a median of 5 days earlier than controls. Nonhematologic toxicity was minimal. Grade 1 mucositis was observed in two of 61 patients (3%). Antitumor activity assessed within 1 month after etoposide administration was documented in 58% of 38 assessable patients. Finally, high-dose etoposide expanded and mobilized the pool of peripheral-blood hematopoietic progenitors. CONCLUSION The use of rhGM-CSF or rhG-CSF makes high-dose etoposide a safe outpatient regimen and should encourage the inclusion of this highly effective and well-tolerated drug in novel treatment strategies that use high-dose therapy early in the clinical course of chemosensitive tumors.