Cellular immunotherapy in multiple myeloma: lessons from preclinical models.

The majority of multiple myeloma patients relapse with the current treatment strategies, raising the need for alternative therapeutic approaches. Cellular immunotherapy is a rapidly evolving field and currently being translated into clinical trials with encouraging results in several cancer types, including multiple myeloma. Murine multiple myeloma models are of critical importance for the development and refinement of cellular immunotherapy. In this review, we summarize the immune cell changes that occur in multiple myeloma patients and we discuss the cell-based immunotherapies that have been tested in multiple myeloma, with a focus on murine models.

Recent advances in myeloma treatment.

The novel agents including thalidomide, bortezomib and lenalidomide have been incorporated into combination regimens which are moving from the advanced/refractory setting to first-line treatment. For the majority of elderly patients, the following regimens are considered standard: melphalan+prednisone in combination with bortezomib or thalidomide and the combination of lenalidomide+low-dose dex. For transplant-eligible patients novel agents are included in the induction phase before and in the consolidation/maintenance phase after transplant. In the relapsed/refractory setting, combinations of novel agents generate the best results but cumulative toxicity is limiting. Several newer agents such as carfilzomib, pomalidomide and deacetylase inhibitors are entering phase II and III clinical trials. The place of allogeneic stem cell transplantation in the treatment of myeloma remains controversial.

A defined donor activating natural killer cell receptor genotype protects against leukemic relapse after related HLA-identical hematopoietic stem cell transplantation.

Killer cell immunoglobulin-like receptors (KIRs) recognize different groups of Human Leukocyte Antigen (HLA) class I alleles and are expressed by natural killer (NK) cells and some T lymphocytes. NK cell cytotoxicity is triggered by failure to recognize the appropriate HLA class I ligand on target cells. Recently, it has been shown that HLA class I ligand incompatibility in the graft-versus-host (GvH) direction is associated with a better outcome in haploidentical hematopoietic stem cell transplantation (HSCT). Since KIR genotypes are very diverse in the population, we explored whether or not the donor KIR genotype could affect the graft-versus-leukemia (GvL) effect in the related HLA-identical HSCT setting. We determined the KIR and HLA genotypes of 65 HLA-identical patient-donor siblings. We found that the presence of two activating KIRs, 2DS1 and 2DS2, in the donor was significantly associated with a decreased leukemic relapse rate (P=0.03; OR=0.18; 95% CI: 0.037-0.88). Moreover, the probability of relapse at 5 years was significantly lower for patients who received a graft from a donor with the 2DS1(+)2DS2(+) genotype than for those who received a transplant from other donors (17 vs 63%, respectively; P=0.018). In conclusion, this study suggests that a joint effect of these two selected activating KIRs in the donor might confer some protection against leukemic relapse.

Prognostic impact of progression to induction chemotherapy and prior paclitaxel therapy in patients with germ cell tumors receiving salvage high-dose chemotherapy in the last 10 years: a study of the European Society for Blood and Marrow Transplantation Solid Tumors Working Party.

Little is known about the prognostic impact of prior paclitaxel therapy and response to induction chemotherapy defined as the regimen preceding high-dose chemotherapy (HDCT) for the salvage therapy of advanced germ cell tumors. Twenty European Society for Blood and Marrow Transplantation centers contributed data on patients treated between 2002 and 2012. Paclitaxel used in either prior lines of therapy or in induction-mobilization regimens was considered. Multivariable Cox analyses of prespecified factors were undertaken on PFS and overall survival (OS). As of October 2013, data for 324 patients had been contributed to this study. One hundred and ninety-two patients (59.3%) had received paclitaxel. Sixty-one patients (19%) had a progression to induction chemotherapy, 234 (72%) a response (29 (9%) missing or granulocyte colony-stimulating factor without chemotherapy). Both progression to induction chemotherapy and prior paclitaxel were significantly associated with shorter OS univariably (P<0.001 and P=0.032). On multivariable analysis from the model with fully available data (N=216) progression to induction was significantly prognostic for PFS and OS (P=0.003), but prior paclitaxel was not (P=0.674 and P=0.739). These results were confirmed after multiple imputation of missing data. Progression to induction chemotherapy could be demonstrated as an independent prognostic factor, in contrast to prior paclitaxel.

Establishment and characterization of a human stroma-dependent myeloma cell line (MM5.1) and its stroma-independent variant (MM5.2).

Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors. We describe a new human myeloma cell line (MM5.1) that can be maintained in the presence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immunoglobulin kappa light chain RNA sequence as the patient's original tumor cells, has a plasma cell morphology and expresses plasma cell antigens (cytoplasmic kappa light chain, CD38, BB4). Without the presence of stromal factors, MM5.1 cells become apoptotic. A low proliferative effect was observed in the presence of oncostatin M (OSM) but other cytokines (IL-10, IL-11, stem cell factor (SCF) and leukemia inhibitory factor (LIF)) had no effect at all. We observed that MM5.1 cells also grow when physically separated from stromal cell layers by a 0.45 microm microporous membrane or when cultured in conditioned medium from stromal marrow cells. Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Coculturing MM5.1 cells with IL-6 and cytokines that were described to increase the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) had no effect on the growth or survival. A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal supernatants using a sensitive immunoassay. This indicates that sustained proliferation of the MM5.1 cell line depends on a combination of IL6 and at least one, thus far unidentified, stroma-derived factor. After more than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma independently. Molecular analysis revealed clonal identity with the early passage form and Epstein-Barr virus antigen expression was negative. The two variants of this cell line offer a useful model to identify molecular mechanisms involved in clonal evolution towards stroma-independent growth of myeloma cells.

Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation.

Serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-alpha were frequently measured during the first 30 days after allogeneic bone marrow transplantation (BMT) in 84 consecutive adult patients. Major transplant-related complications (MTCs) occurred in 33% of cases and included veno-occlusive liver disease, idiopathic pneumonia syndrome, severe endothelial leakage syndrome and >grade II acute graft-versus-host disease. Compared with patients having minor complications, those with MTCs developed higher levels at times of maximal clinical signs (all cytokines, P<0.001), between days 0-5 post-BMT (IL-6 and IL-8, P<0.05) and days 6-10 (L-6, P<0.001; IL-8 and TNF, P<0.01) post-BMT. We could not discriminate patterns of cytokine release that were specific for any subtype of MTC. Higher levels of IL-8 during days 0-5 were associated (P=0.044) with early (<40 days) death. Multivariate analysis including patient and transplant characteristics as well as post-BMT levels of C-reactive protein showed that high average levels of one or more of the cytokines within the first 10 days post-BMT were independently associated with MTC (Odd's ratio: 2.3 [1.2-4.5], P=0.011). This study shows that systemic release of proinflammatory cytokines contributes to the development of MTC and provides a rationale for pre-emptive anti-inflammatory treatment in selected patients.

The clinical impact of early gram-positive bacteremia and the use of vancomycin after allogeneic bone marrow transplantation.

BACKGROUND: Gram-positive bacteremia (GPB) is an increasing infection after allogeneic bone marrow transplantation (BMT). Our purpose was to identify risk factors for GPB, to evaluate its impact on early mortality and morbidity, and to compare prophylactic with empirical intravenous vancomycin. METHODS AND RESULTS: We studied 89 consecutive BMTs in adult patients. Early GPB occurred in 29% of posttransplantation episodes. T-cell depletion (odds ratio [OR]: 0.18) and vancomycin-prophylaxis (OR: 0.28) reduced the risk of GPB. Mortality at 6 weeks was not significantly different in patients with GPB (15% vs. 9.5%, P = 0.669). GPB was associated with the development of major complications, the use of amphotericin B, and prolonged neutropenia. Vancomycin prophylaxis led to an increased risk of early renal dysfunction (OR: 18.7). CONCLUSION: GPB contributes to overall morbidity during the early post-BMT episode but has no impact on mortality. Vancomycin prophylaxis is effective to reduce GPB but has a negative effect on renal function.

Late and localized extramedullary relapse of a light chain kappa myeloma after syngeneic bone marrow transplantation.

We report the case of a 54-year-old female patient with stage IIIA kappa light chain myeloma (MM) who relapsed 7 years after syngeneic bone marrow transplantation (BMT). The relapse occurred as a voluminous soft tissue plasmacytoma in the leg, developing after local trauma. The patient was successfully treated with local radiotherapy and has remained progression-free for more than 2 years. This case represents one of the longest survivors, in complete remission, after syngeneic transplantation for MM. The presentation of recurrent disease as localized plasmacytoma with extramedullary growth is unusual in the post-transplant setting. Bone Marrow Transplantation (2000) 25, 115-117.

The impact of partial T cell depletion on overall transplant-related toxicity, graft function and survival after HLA-identical allogeneic bone marrow transplantation in standard risk adult patients with leukemia.

In this single-center study, a consecutive cohort of 59 adult patients transplanted with HLA-identical bone marrow and receiving graft-versus-host disease (GVHD) prophylaxis with either standard cyclosporine/methotrexate (n = 33) or partial T cell depletion (E-rosetting) (TCD, n = 26 were analyzed). Only patients with chronic myeloid leukemia in first chronic phase or acute leukemia/myelodysplasia in first or second remission were included. Except for age (median 28 vs 42 years), both groups were comparable in terms of diagnosis, conditioning regimen and growth factor support. TCD significantly reduced >grade II acute GVHD (0 vs 24%, P = 0.02), chronic GVHD (8.5 vs 45%, P = 0.007) and other major bone marrow transplant (BMT)-related complications (4 vs 36%, P = 0.005). TCD decreased overall transplant-related mortality (11.5 vs 36%, P = 0.04). In the TCD group faster neutrophil (13 vs 22 days, P = 0.02) and platelet recoveries (18 vs 26 days, P < 0.001) were noted. The relapse risk was higher after TCD (57.5 vs 21.5%, P = 0.04). Overall survival probability at 10 years was identical in both groups (54 vs 53.5%, P = 0.33). We found a relationship between the number of T cells in the graft and the occurrence of major complications (P < 0.001) and relapse (P = 0.03). This comparative analysis shows that graft-derived T cells have a major role in overall BMT-related toxicity and that partial TCD is an acceptable approach in terms of survival for patients between 40 and 50 years of age.

Chemotherapy-induced acral erythema and acute graft-versus-host disease after allogeneic bone marrow transplantation.

Chemotherapy-induced acral erythema is a rare disorder characterized by a painful and intense erythema of the palms and the soles. In allogeneic bone marrow transplant patients, the differential diagnosis of acute graft-versus-host disease (AGVHD) may be difficult. We describe a case of concurrent acral erythema and AGVHD. The clinical features of both conditions as well as the histological findings on serial skin biopsy specimens are discussed.

The absolute number of circulating CD34+ cells predicts the number of hematopoietic stem cells that can be collected by apheresis.

We report our single institution's effort to standardize the method of collecting peripheral blood progenitor cells (PBPC) used for autologous transplantation. PBPC were mobilized by different types of chemotherapy followed by G-CSF (24 patients) or G-CSF alone (six patients) in 30 patients with various underlying neoplastic diseases. A median of three aphereses (range: 2-7), using the CS3000 cell separator was performed and a blood volume of 101 was processed. We studied the relationship between the absolute numbers of circulating leukocytes, mononuclear cells and CD34+ cells and the amount of PBPC collected during a single apheresis procedure. CD34+ cells were quantified by leukocyte subset analysis based on flow cytometry. Both CFU-GM and CD34+ cell contents of the apheresis products (69 procedures analyzed) correlated strongly: rho = 0.936, P = 0.0001). Regression analysis showed that the total number of CD34+ cells or CFU-GM content of the apheresis products could be predicted (r = 0.915, P = 0.0001) from the absolute number of CD34+ cells in the blood. A number of 10 circulating CD34+ cells per mm3 blood ensured a minimum of 0.5 x 10(6) CD34+ cells per kg, collected on the same day. Of the 30 patients, 17 received an autologous graft that contained only PBPC in 13. Long-term and complete hematological reconstitution was observed in all patients who had a minimum of 2 x 10(6) CD34+ cells per kg reinfused.

An early increase in serum levels of C-reactive protein is an independent risk factor for the occurrence of major complications and 100-day transplant-related mortality after allogeneic bone marrow transplantation.

We monitored levels of C-reactive protein (CRP) in 96 consecutive adult allogeneic BMT patients (age 15-50 years) transplanted in our unit. Major transplant-related complications (MTC) occurred in 32% of cases and included: hepatic veno-occlusive disease, pneumonitis, severe endothelial leakage syndrome and >II acute GVHD. Transplant-related mortality (TRM) before day 100 post-BMT was 13.5%. Variables included in a stepwise logistic regression model were: gender, age, disease category, donor type, T cell depletion, TBI, use of growth factors, bacteremia, mean CRP-levels >50 mg/l between days 0 and 5 (CRP day 0-5) and >100 mg/l between days 6 and 10 (CRP day 6-10) post-BMT. Only high CRP-levels (for MTC and TRM) (P < 0.001) and donor-type (for TRM) (P= 0.02) were independent risk factors. The estimated probability for MTC was 73% (CRP day 6-10 >100 mg/l) vs 17% (CRP day 6-10 <100 mg/l). Using the same cut-off levels, the probabilities for TRM were 36.5% vs 1% in the identical sibling donor situation and 88% vs 12.5% in other donor-type transplants. We conclude that the degree of systemic inflammation, as reflected by CRP-levels, during the first 5-10 days after BMT identifies patients at risk of MTC and TRM. Our data may be useful in selecting patients for clinical trials involving pre-emptive anti-inflammatory treatment.

Monitoring of C-reactive protein after allogeneic bone marrow transplantation identifies patients at risk of severe transplant-related complications and mortality.

Patterns of C-reactive protein (CRP) release were derived from frequent CRP measurements in a cohort of 66 consecutive patients receiving allogeneic bone marrow transplants (BMT) in our unit. Based on a retrospective study of clinical events occurring within the first 40 days after BMT, patients with major transplant-related complications (MTC+ group, n = 22) could be separated from those with fever or mild complications only (MTC- group, n = 44). Treatment-related mortality in the MTC+ group was significantly higher: 32 vs 0% (P < 0.001). Major complications included veno-occlusive liver disease (VOD), severe endothelial leakage syndrome (ELS), pneumonitis and acute GVHD >II. The severity of complications was reflected by the patterns of CRP release with continuously high levels preceding the maximal signs and symptoms of MTC. Univariate analysis showed that, among other variables (sex, age, disease status at transplant, +/- TBI in the conditioning regimen, +/- use of myeloid growth factors after BMT, time to reach PN >200/mm3), three factors were significantly associated with MTC: maximal levels of CRP during the post-transplant episode (CRPmax) (296.6 +/- 91.8 vs 88.9 +/- 55.8 mg/100 ml, P < 0.001), the use of unmanipulated graft (no T depletion) (46.9 vs 12.5%, P < 0.009) and the CRP level on the day of BMT (CRPo) (42.7 +/- 55.4 vs 18.2 +/- 19.5, P = 0.045). In multivariate analysis, using a stepwise logistic regression model including the same variables, CRPmax appeared to be the strongest independent variable (P < 0.001) and a reliable (94% accuracy) parameter to assess the risk of MTC. Based on this model, CRP levels of 200 and 300 mg/100 ml are associated with a risk of 48 and 94% of developing MTC, respectively. We conclude that CRP monitoring after BMT identifies patients at risk of severe transplant-related complications and mortality.

Diagnostic and prognostic value of the MB2 monoclonal antibody in paraffin-embedded bone marrow sections of patients with multiple myeloma and monoclonal gammopathy of undetermined significance.

Routinely processed bone marrow biopsies of 59 patients with untreated multiple myeloma (MM) and of 41 patients with monoclonal gammopathies of undetermined significance (MGUS) were immunocytochemically studied with the MB2 monoclonal antibody. In 54 of 59 biopsies of patients with MM, most neoplastic plasma cells showed strong cytoplasmic positivity to MB2. In contrast, only three biopsies of patients with MGUS contained highly MB2-positive plasma cells, whereas the plasma cells in the remaining biopsies were either negative (18 of 41) or revealed a weak dot-like staining of the cytoplasm (20 of 41). Plasma cells in tonsillar tissue, gastric and duodenal mucosae, and bone marrow with reactive plasmacytosis were not stained with MB2. These findings suggest that MB2 helps to distinguish between MM and MGUS. Because the five MB2-negative patients with MM were all in stage III and had very short survival time, neoplastic plasma cell staining with MB2 could also have a prognostic significance.

Southern blot analysis in a case of Richter's syndrome. Evidence for a postrearrangement heavy chain gene deletion associated with the altered phenotype.

Richter's syndrome (RS) can be defined as the emergence of an aggressive lymphoma in patients suffering from chronic lymphocytic leukemia (CLL). The authors performed immunophenotypic and Southern blot analysis of the peripheral blood and tissue specimen of a patient with RS. Using immunoperoxidase and immunogold-silver staining techniques and a panel of monoclonal antibodies, the authors found that the large cells characteristic of RS showed an altered immunophenotype as compared with the CLL cells and did not express mu heavy chain. Southern blot analysis revealed identical kappa light chain rearrangements in both tumoral cell populations consistent with a common clonal origin. Using the JH probe and several restriction enzymes, the authors also found evidence for a postrearrangement deletion of the heavy chain mu gene. These findings suggest that in this case of RS, a deletion of the heavy chain mu gene resulted in loss of mu expression by the larger cells that were characteristic of RS and was associated with their altered phenotype.

Extramedullary plasmacytoma of the cavernous sinus.

We present the case of an 80-year-old male with an history of multiple myeloma (MM) stage I with extramedullary plasmacytoma of the neck, diagnosed 18 months before and in complete remission after radiation therapy and melphalan-prednisone therapy. He was admitted with signs and symptoms characteristic for cavernous sinus syndrome, including diplopia, exophthalmia, ptosis and orbital pain. Magnetic resonance imaging showed a mass lesion in the cavernous sinus, consistent with relapsing extramedullary plasmacytoma. The patient received palliative radiation therapy and high dose dexamethasone, but treatment failed and the patient died. This case represents one of the few reports of extramedullary plasmacytoma of the cavernous sinus. The development of a clinical presentation of cavernous sinus syndrome in a patient with a history of MM or extramedullary plasmacytoma should raise the suspicion of a plasmacytic involvement of the cavernous sinus.

Progressive paraparesis due to thoracic extramedullary hematopoiesis in myelofibrosis. Case report.

A patient with myelofibrosis who developed a progressive paraparesis caused by spinal cord compression due to thoracic extramedullary hematopoietic tissue is reported. He recovered well after local radiotherapy alone.

Neuroblastoma today.

Notwithstanding the progress made in radiology, nuclear medicine, immunohematology and genetics for more accurate diagnosis and staging in neuroblastoma and the availability of general new efficacious cytostatic drugs, the prognosis of children over 1 yr with advanced disease has remained poor. New refinements in therapeutics with multiagent regimens, massive chemotherapy followed by autologous bone marrow transplantation treatment, with or without immunomagnetic purging, and/or total body irradiation have improved response rate and disease-free survival in metastatic patients, but their effect on long-term survival needs further evaluation.

Experience with high dose chemotherapy and autologous bone marrow transplantation in testicular cancer.

Carboplatin based HDCT followed by ABMT produces reasonable response rates (40-60%) and some durable remissions in patients with advanced and cisplatin resistant testicular cancer. Patients eligible for such an approach are those with primary refractory or recurrent disease and low probability for salvage by conventional dose regimens. The use of recombinant human growth factors and peripheral stem cell transplantation will probably reduce the toxicity and mortality of HDCT.

New drugs and novel mechanisms of action in multiple myeloma in 2013: a report from the International Myeloma Working Group (IMWG).

Treatment in medical oncology is gradually shifting from the use of nonspecific chemotherapeutic agents toward an era of novel targeted therapy in which drugs and their combinations target specific aspects of the biology of tumor cells. Multiple myeloma (MM) has become one of the best examples in this regard, reflected in the identification of new pathogenic mechanisms, together with the development of novel drugs that are being explored from the preclinical setting to the early phases of clinical development. We review the biological rationale for the use of the most important new agents for treating MM and summarize their clinical activity in an increasingly busy field. First, we discuss data from already approved and active agents (including second- and third-generation proteasome inhibitors (PIs), immunomodulatory agents and alkylators). Next, we focus on agents with novel mechanisms of action, such as monoclonal antibodies (MoAbs), cell cycle-specific drugs, deacetylase inhibitors, agents acting on the unfolded protein response, signaling transduction pathway inhibitors and kinase inhibitors. Among this plethora of new agents or mechanisms, some are specially promising: anti-CD38 MoAb, such as daratumumab, are the first antibodies with clinical activity as single agents in MM. Moreover, the kinesin spindle protein inhibitor Arry-520 is effective in monotherapy as well as in combination with dexamethasone in heavily pretreated patients. Immunotherapy against MM is also being explored, and probably the most attractive example of this approach is the combination of the anti-CS1 MoAb elotuzumab with lenalidomide and dexamethasone, which has produced exciting results in the relapsed/refractory setting.