Carolina Terragna

Prognostic relevance of 18-F FDG PET/CT in newly diagnosed multiple myeloma patients treated with up-front autologous transplantation

We prospectively analyzed the prognostic relevance of positron emission tomography-computed tomography (PET/CT) at diagnosis, after thalidomide-dexamethasone (TD) induction therapy and double autotransplantation (ASCT) in 192 newly diagnosed multiple myeloma (MM) patients. Presence at baseline of at least 3 focal lesions (FLs; 44% of cases), a standardized uptake value (SUV) > 4.2 (46%), and extramedullary disease (EMD; 6%) adversely affected 4-year estimates of progression-free survival (PFS; ≥ 3 FLs: 50%; SUV > 4.2: 43%; presence of EMD: 28%). SUV > 4.2 and EMD were also correlated with shorter overall survival (OS; 4-year rates: 77% and 66%, respectively). Persistence of SUV > 4.2 after TD induction was an early predictor for shorter PFS. Three months after ASCT, PET/CT was negative in 65% of patients whose 4-year rates of PFS and OS were superior to those of PET-positive patients (PFS: 66% and OS: 89%). In a multivariate analysis, both EMD and SUV > 4.2 at baseline and persistence of fluorodeoxyglucose (FDG) uptake after ASCT were independent variables adversely affecting PFS. PET/CT involvement at diagnosis, after novel agent-based induction and subsequent ASCT is a reliable predictor of prognosis in MM patients. This study is registered at www.clinicaltrials.gov as NTC01341262.

Molecular Remission After Allogeneic or Autologous Transplantation of Hematopoietic Stem Cells for Multiple Myeloma

PURPOSE To assess the clinical relevance of minimal residual disease (MRD) in patients with multiple myeloma (MM), 50 patients were monitored while they were in complete clinical remission (CCR) after autologous or allogeneic stem-cell transplantation. PATIENTS AND METHODS Stringent molecular monitoring using clonal markers based on rearranged immunoglobulin heavy-chain genes was performed in 44 of 50 MM patients in CCR. Molecular clinical remission (MCR) was defined as more than one consecutive negative polymerase chain reaction (PCR) test result. RESULTS Twelve (27%) of 44 molecularly monitored patients achieved MCR; four of the 12 became PCR-positive, and one of these four relapsed. In comparison with patients who did not achieve MCR, patients who achieved MCR had a significantly lower relapse rate (41% v 16%; P <.05) and longer relapse-free survival (35 v 110 months; P <.005). Fourteen of 26 patients in CCR who had received allografts were evaluated on a molecular basis: seven (50%) of the 14 achieved MCR and did not relapse; one of the seven remaining patients relapsed. Thirty of 47 patients in CCR who received autografts were evaluated on a molecular basis: five (16%) of the 30 achieved MCR; two of these five became PCR-negative, and one of these two relapsed. Ten of the 25 remaining patients later relapsed. For these nonrandomized groups, the higher MCR rate after allograft procedures was statistically significant (P <.01; Fishers exact test). CONCLUSION MCR can be obtained in a relatively high proportion of MM patients who have achieved CCR after undergoing allograft procedures and in a smaller fraction of patients after undergoing autograft procedures. In approximately one fourth of MM patients who achieve CCR after transplantation, it may be possible to keep the disease burden constantly below the PCR threshold. Because MCR was associated with prolonged relapse-free survival, these patients could have a relatively favorable clinical outcome.

Generation and functional characterization of human dendritic cells derived from CD34 ˛ cells mobilized into peripheral blood: comparison with bone marrow CD34 ˛ cells

Dendritic cells (DCs) are the most powerful professional antigen‐presenting cells (APC), specializing in capturing antigens and stimulating T‐cell‐dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady‐state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony‐stimulating factor (G‐CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony‐forming unit‐dendritic cells (CFU‐DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM‐CSF and TNF‐α with or without SCF and FLT‐3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early‐acting growth factors SCF and FLT‐3L with IL‐4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+CD14− cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL‐4‐stimulated DCs was significantly higher than those generated in the absence of IL‐4. Furthermore, autologous serum collected during G‐CSF treatment was more efficient than fetal calf serum (FCS) or two different serum‐free media for large‐scale production of DCs. Thus, our comparative studies indicate that G‐CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early‐acting and intermediate‐late‐acting colony‐stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.

Phase I/II clinical trial of sequential subcutaneous and intravenous delivery of dendritic cell vaccination for refractory multiple myeloma using patient-specific tumour idiotype protein or idiotype (VDJ)-derived class I-restricted peptides

Fifteen multiple myeloma (MM) patients who had failed maintenance therapy after tandem autologous stem cell transplantation underwent anti‐idiotype (Id) vaccination with dendritic cells (DCs). CD14+‐derived DCs were loaded with the autologous Id as whole protein (=6) or Id‐derived class I‐restricted peptides (=9) and keyhole limpet hemocyanin (KLH). Vaccination consisted of three subcutaneous (sc) and two intravenous injections of increasing DC doses at 2 weeks interval. DC therapy was well tolerated. Most patients developed both humoral and T‐cell responses to KLH, suggesting immunocompetence. Eight of 15 patients developed an Id‐specific T‐cell proliferative response, 8/15 increased interferon‐γ‐secreting T cells and 4/15 showed an Id‐positive delayed‐type hypersensitivity test. Anti‐Id cytotoxic T‐lymphocyte precursors increased after DC vaccination in 2/2 evaluable patients. A more robust T‐cell response was observed after sc DC injections and increased Id‐specific T‐cell proliferation was found up to 1 year after vaccination. VDJ‐derived peptides were as effective as the whole protein in stimulating T‐cell responses. Clinically, 7/15 patients have stable disease after a median follow‐up of 26 months, one patient achieved durable partial remission after 40 months, and seven patients progressed. In conclusion, sc injections of cryopreserved Id‐pulsed DCs were safe and, in contrast with intravenous administrations, induced anti‐MM T‐cell responses.

Long-term results of the GIMEMA VEL-03-096 trial in MM patients receiving VTD consolidation after ASCT: MRD kinetics' impact on survival

Polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis is a useful prognostic tool in multiple myeloma (MM), although its long-term impact still needs to be addressed. This report presents the updated results of the GIMEMA-VEL-03-096 trial. Thirty-nine MM patients receiving bortezomib–thalidomide–dexamethasone after autologous transplantation were monitored for MRD by both nested and real-time quantitative-PCR until relapse. Our data confirm the strong impact of MRD on survival: overall survival was 72% at 8 years median follow-up for patients in major MRD response versus 48% for those experiencing MRD persistence (P=0.041). In addition, MRD kinetics resulted predictive for relapse: indeed median remission duration was not reached for patients in major MRD response, 38 months for those experiencing MRD reappearance and 9 months for patients with MRD persistence (P<0.001). Moreover: (1) 26 patients achieving major MRD response (67%) benefit of excellent disease control (median TNT: 42 months); (2) MRD reappearance heralds relapse, with a TNT comparable to that of MRD persistence (9 versus 10 months, P=0.706); (3) the median lag between MRD reappearance and need for salvage treatment is 9 months. These results suggest the usefulness of a long-term MRD monitoring in MM patients and the need for maintenance or pre-emptive treatments ensuring durable responses.

Comparison Between Patients With Philadelphia-Positive Chronic Phase Chronic Myeloid Leukemia Who Obtained a Complete Cytogenetic Response Within 1 Year of Imatinib Therapy and Those Who Achieved Such a Response After 12 Months of Treatment

PURPOSE Imatinib mesylate is a potent inhibitor of BCR-ABL, the constitutively active tyrosine kinase protein critical for the pathogenesis of chronic myeloid leukemia. PATIENTS AND METHODS We reviewed 284 patients with late chronic-phase Philadelphia chromosome (Ph) -positive chronic myeloid leukemia treated with imatinib 400 mg daily after interferon-alpha failure. In a retrospective study, we evaluated the pattern and rapidity of the response to imatinib, comparing the cytogenetic and molecular responses, progression-free and overall survival rates in patients who obtained a complete cytogenetic response within 1 year of treatment (early responders), and in patients where a complete cytogenetic response was detected after 12 months (late responders). RESULTS After 3 or 4 years of treatment, the molecular response of the late cytogenetic responders was similar to that of the early cytogenetic responders. At 36 months of treatment the amount of residual disease measured by standardized quantitative reverse-transcriptase polymerase chain reaction was 0.00047 in late responders versus 0.00022 in early responders, and at 48 months it was 0.00019 versus 0.00026 (median values, P value = nonsignificant). The estimated 4-year progression-free survival rate was 88% for early responders and 100% for late responders, while the estimated 4-year overall survival rates were 92% and 100% for early and late responders, respectively. CONCLUSION The sensitivity and the response (cytogenic and molecular) to imatinib may require 1 year or more. Long-term follow-up results continue to improve in terms of rates and durability of the complete cytogenetic response, major or complete molecular response, and progression-free and overall survival.

PET/CT Improves the Definition of Complete Response and Allows to Detect Otherwise Unidentifiable Skeletal Progression in Multiple Myeloma

Purpose: To evaluate the role of 18F-FDG PET/CT in 282 symptomatic multiple myeloma patients treated up-front between 2002 and 2012. Experimental Design: All patients were studied by PET/CT at baseline, during posttreatment follow-up, and at the time of relapse. Their median duration of follow-up was 67 months. Results: Forty-two percent of the patients at diagnosis had >3 focal lesions, and in 50% SUVmax was >4.2; extramedullary disease was present in 5%. On multivariate analysis, ISS stage 3, SUVmax >4.2, and failure to achieve best complete response (CR) were the leading factors independently associated with shorter progression-free survival (PFS) and overall survival (OS). These 3 variables were used to construct a prognostic scoring system based on the number of risk factors. After treatment, PET/CT negativity (PET-neg) was observed in 70% of patients, whereas conventionally defined CR was achieved in 53%. Attainment of PET-neg favorably influenced PFS and OS. PET-neg was an independent predictor of prolonged PFS and OS for patients with conventionally defined CR. Sixty-three percent of patients experienced relapse or progression; in 12%, skeletal progression was exclusively detected by systematic PET/CT performed during follow-up. A multivariate analysis revealed that persistence of SUVmax >4.2 following first-line treatment was independently associated with exclusive PET/CT progression. Conclusions: PET/CT combined with ISS stage and achievement or not of CR on first-line therapy sorted patients into different prognostic groups. PET/CT led to a more careful evaluation of CR. Finally, in patients with persistent high glucose metabolism after first-line treatment, PET/CT can be recommended during follow-up, to screen for otherwise unidentifiable progression. Clin Cancer Res; 21(19); 4384–90. ©2015 AACR.

Expression of CD86 in acute myelogenous leukemia is a marker of dendritic/monocytic lineage

OBJECTIVE The aim of this study was to determine whether expression of the CD86 costimulatory molecule in acute myeloid leukemia (AML) can identify blast cells committed to the monocytic/dendritic lineage. MATERIAL AND METHODS One hundred ten consecutive AML patients observed at diagnosis were studied by flow cytometry. In selected experiments, in vitro cultures with CD34(+)CD86(+) or CD34(-)CD86(+) blasts were performed in the presence of granulocyte-macrophage colony-stimulating actor (GM-CSF) with or without tumor necrosis factor-alpha (TNF-alpha) or GM-CSF + interleukin-4 (IL-4), respectively, to induce a dendritic differentiation, documented by morphologic and immunophenotypic assays. T-cell alloreactivity to CD86(+) AML cells and leukemic dendritic cells (AML-DC) was tested in mixed leukocyte cultures and anti-leukemic cytotoxic assays. RESULTS CD86 was expressed in 54% AML, whereas CD80 and CD1a were only occasionally positive. CD86(+) AML samples included M5 and M4, but also 47% M0-M1 FAB types, and were more frequently CD14(+) (p < 0.00001) and CD34(-) (p = 0.00005) than CD86(-)AML. Six different patterns of CD86(+) AML were identified, according to CD34 or CD14 total or partial coexpression. Four samples enriched in CD34(+)CD86(+) AML cells differentiated into AML-DC CD86(+), CD80(+), CD40(+), CD11c(+), HLA-DR(++), CD14(+/-) that also were CD1a(+) or CD83(+), after 6 days of in vitro culture with GM-CSF +/- TNF-alpha. CD34(-)CD86(+) AML cells differentiated into AML-DC after 3 to 5 days (n = 5 experiments), and trisomy 8 was found in two AML and AML-DC samples by fluorescence in situ hybridization analysis. Finally, AML-DC induced more potent allo-T-cell proliferation, cytokine release, and anti-leukemic cytotoxicity than CD86(+) blasts. CONCLUSIONS In AML, CD86 is a marker of monocytic/dendritic lineage. Because CD86(+) blasts may differentiate into DC rapidly, CD86(+)AML patients could be optimal candidates for immunotherapy studies, both in autologous and allogeneic settings.

c-MYC oncoprotein dictates transcriptional profiles of ATP-binding cassette transporter genes in Chronic Myelogenous Leukemia CD34+ hematopoietic progenitor cells

Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. ©2011 AACR.

Generation of Dendritic Cells from Positively Selected CD14 + Monocytes for Anti-tumor Immunotherapy

Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 x -, 10 x -, 50 x 10(6) cells and 10 x -, 50 x 10(6) cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-alpha or, more recently, by a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1+/-5.7% to 95.5+/-3.2% (recovery 67.9+/-15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6+/-6.6% of the cells were mature DCs. We obtained 2.89+/-1 x 10(8) DCs/leukapheresis which represented 24.5+/-9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31+/-10.9 of initial CD14+ cells) of DCs than TNF-alpha alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of pre-loaded DCs. The recovery of thawed, viable DCs, was 78+/-10%. Thus, positive selection of CD14+ monocytes allows the generation of a uniform population of mature pre-loaded DCs which can be cryopreserved with no effects on phenotype and function and are suitable for clinical trials. Based on these results, a DCs-based phase II trial of anti-Id vaccination with VDJ-derived HLA class I-restricted peptides and KLH is underway for lymphoma patients.