Matteo Carrabba

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

PURPOSEnTo 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.nnnPATIENTS AND METHODSnSixty-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.nnnRESULTSnNo 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.nnnCONCLUSIONnVaccination 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.

Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8+ T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8+ T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-γ and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A27–35 in three of five HLA-A*0201 melanoma patients, and of CEA571–579 and EpCAM263–271 in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer 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.

Recurrent ETNK1 mutations in atypical chronic myeloid leukemia

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.

Low TCR avidity and lack of tumor cell recognition in CD8 + T cells primed with the CEA-analogue CAP1-6D peptide

The use of “altered peptide ligands” (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8+ T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201+ healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- γ release CEA+CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.

Treatment approaches for primary CNS lymphomas

Importance of the field: Primary central nervous system lymphomas (PCNSL) are rare but potentially curable tumours. The overall outcome for PCNSL patients is unsatisfactory and several therapeutic questions remain open. Modest progress in outcome reflects difficulties in conducting randomized trials and scarce molecular and biological knowledge. Areas covered in this review: This review describes conventional and investigational treatments for PCNSL and focuses on the main questions for future clinical trials. PubMed and the authors’ own files were utilized for references search. The terms ‘PCNSL’, ‘primary AND CNS lymphoma’, and ‘CNS AND lymphoma’ were used for PubMed queries. All papers published in English before November 2009 were considered. What the reader will gain: This review illustrates how the paradigm for PCNSL treatment changed during the 1990s from radiotherapy alone to the establishment of high-dose methotrexate–cytarabine combination as standard approach. We present promising data from Phase II studies and discuss questions for randomized trials. Finally, we offer a 5-year scenario for the management of PCNSL. Take-home message: The methotrexate–cytarabine combination should currently be considered as the reference treatment for PCNSL. Well-designed randomized trials and biological studies deriving from the use of novel technologies will be crucial to further improve outcome in these patients.

A New Clinicobiological Scoring System for the Prediction of Infection-Related Mortality and Survival after Allogeneic Hematopoietic Stem Cell Transplantation

Infection-related mortality (IRM) is a substantial component of nonrelapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). No scores have been developed to predict IRM before transplantation. Pretransplantation clinical and biochemical data were collected from a study cohort of 607 adult patients undergoing allo-HSCT between January 2009 and February 2017. In a training set of 273 patients, multivariate analysis revealed that age >60 years (Pu2009=u2009.003), cytomegalovirus host/donor serostatus different from negative/negative (Pu2009<u2009.001), pretransplantation IgA level <1.11u2009g/L (Pu2009=u2009.004), and pretransplantation IgM level <.305u2009g/L (Pu2009=u2009.028) were independent predictors of increased IRM. Based on these results, we developed and subsequently validated a 3-tiered weighted prognostic index for IRM in a retrospective set of patients (nu2009=u2009219) and a prospective set of patients (nu2009=u2009115). Patients were assigned to 3 different IRM risk classes based on this index score. The score significantly predicted IRM in the training set, retrospective validation set, and prospective validation set (Pu2009<u2009.001, .044,u2009and .011, respectively). In the training set, 100-day IRM was 5% for the low-risk group, 11% for the intermediate-riak group, and 16% for the high-risk groups. In the retrospective validation set, the respective 100-day IRM values were 7%, 17%, and 28%, and in the prospective set, they were 0%, 5%, and 7%. This score predicted also overall survival (Pu2009<u2009.001 in the training set, P < 041 in the retrospective validation set, and P < .023 in the prospective validation set). Because pretransplantation levels of IgA/IgM can be modulated by the supplementation of enriched immunoglobulins, these results suggest the possibility of prophylactic interventional studies to improve transplantation outcomes.

Post-remission intensive treatment after induction chemotherapy is feasible in selected elderly patients with acute myeloid leukemia and age ≥75 years: a retrospective analysis of the Rete Ematologica Lombarda.

response was 2 months (range, 1–8 months). The median proportion of bone marrow involvement with lymphomplasmacytic lymphoma (LPL) at the time of initiation of therapy was 80% (range, 40–90%). The median proportion at the end of therapy was 10% (range, 0–90%). The median time to progression (TTP) for the entire cohort was 9.7 months (range, 1–44 months; Fig. 1). The median time to progression for the four patients who were previously untreated was 18.6 months (range, 5–37 months). The median DOR for responders was 7.3 months (range, 1–43 months; Fig. 2). Grade 3–4 toxicities that required dose modifications/delays or interruptions included neuropathy (26%), cytopenias (20%), bacteremia (7%), rituximab reactions (7%), and atrial fibrillation (7%). One patient (7%) discontinued therapy due to toxicity with a G3 E. coli bacteremia after cycle 1 of therapy. This data demonstrate that this regimen is highly effective in WM even in patients who cannot tolerate or cannot receive rituximab. New therapeutic options such as ibrutinib or everolimus maybe used more frequently in patients with WM in the near future. However, these agents do not always induce a significant bone marrow response in comparison to the IgM response observed in the serum [7,8]. However, the significant bone marrow response in many patients with CyBorD can make it an attractive option for achieving complete remissions in patients who do not achieve adequate bone marrow responses with other agents. In addition, in the era of highly expensive combinations of chemotherapeutic agents, the combination of CyBorD may provide a less expensive and highly effective alternative that can be used more broadly in many developing countries with high responses. As with other retrospective studies, ours has many limitations including a small number of patients, selection bias, and different dosing schemas within this cohort. Despite this, it provides preliminary evidence for a highly effective regimen for WM that should be further validated in larger prospective trials. HOURY LEBLEBJIAN, KIMBERLY NOONAN, CLAUDIA PABA-PRADA, STEVEN P. TREON, JORGE J. CASTILLO,* AND IRENE M. GHOBRIAL* Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts Conflict of interest: IMG is on advisory Board for Novartis and Millennium. J.J.C. and I.M.G. are co-senior authors and contributed equally to this work. Contract grant sponsors: Kirsch Lab, Heje fellowship. *Correspondence to: Irene M. Ghobrial, MD, Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Av, Boston, MA 02115. E-mail: irene_ghobrial@dfci.harvard.edu or Jorge Castillo, MD, Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Av, Boston, MA 02115. E-mail: JorgeJ_castillo@dfci.harvard.edu Received for publication: 10 February 2015; Accepted: 17 February 2015 Published online: 19 February 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.23985

Clinical Impact of Pretransplant Multidrug-Resistant Gram-Negative Colonization in Autologous and Allogeneic Hematopoietic Stem Cell Transplantation

Multidrug-resistant Gram-negative bacteria (MDR-GNB) are an emerging cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Three-hundred forty-eight consecutive patients transplanted at our hospital from July 2012 to January 2016 were screened for a pretransplant MDR-GNB colonization and evaluated for clinical outcomes. A pretransplant MDR-GNB colonization was found in 16.9% of allo-HSCT and in 9.6% of auto-HSCT recipients. Both in auto- and in allo-HSCT, carriers of a MDR-GNB showed no significant differences in overall survival (OS), transplant-related mortality (TRM), or infection-related mortality (IRM) compared with noncarriers. OS at 2 years for carriers compared with noncarriers was 85% versus 81% (Pu2009=u2009.262) in auto-HSCT and 50% versus 43% (Pu2009=u2009.091) in allo-HSCT. TRM at 2 years was 14% versus 5% (Pu2009=u2009.405) in auto-HSCT and 31% versus 25% (Pu2009=u2009.301) in allo-HSCT. IRM at 2 years was 14% versus 2% (Pu2009=u2009.142) in auto-HSCT and 23% versus 14% (Pu2009=u2009.304) in allo-HSCT. In multivariate analysis, only grade III to IV acute graft-versus-host disease was an independent factor for reduced OS (Pu2009<u2009.001) and increased TRM (Pu2009<u2009.001) and IRM (Pu2009<u2009.001). During the first year after transplant, we collected 73 GNB bloodstream infectious (BSI) episodes in 54 patients, 42.4% of which sustained by a MDR-GNB. Rectal swabs positivity associated with the pathogen causing subsequent MDR-GNB BSI episodes in 13 of 31 (41.9%). Overall, OS at 4 months from MDR-GNB BSI episode onset was of 67.9%, with a 14-day attributed mortality of 12.9%, not being significantly different between carriers and noncarriers (Pu2009=u2009.207). We conclude that in this extended single-center experience, a pretransplant MDR-GNB colonization did not significantly influence OS, TRM, and IRM both in auto- and allo-HSCT settings and that MDR-GNB attributed mortality can be controlled in carriers when an early pre-emptive antimicrobial therapy is started in case of neutropenic fever.