International Working Group consensus response evaluation criteria in lymphoma (RECIL 2017).

In recent years, the number of approved and investigational agents that can be safely administered for the treatment of lymphoma patients for a prolonged period of time has substantially increased. Many of these novel agents are evaluated in early-phase clinical trials in patients with a wide range of malignancies, including solid tumors and lymphoma. Furthermore, with the advances in genome sequencing, new "basket" clinical trial designs have emerged that select patients based on the presence of specific genetic alterations across different types of solid tumors and lymphoma. The standard response criteria currently in use for lymphoma are the Lugano Criteria which are based on [18F]2-fluoro-2-deoxy-D-glucose positron emission tomography or bidimensional tumor measurements on computerized tomography scans. These differ from the RECIST criteria used in solid tumors, which use unidimensional measurements. The RECIL group hypothesized that single-dimension measurement could be used to assess response to therapy in lymphoma patients, producing results similar to the standard criteria. We tested this hypothesis by analyzing 47 828 imaging measurements from 2983 individual adult and pediatric lymphoma patients enrolled on 10 multicenter clinical trials and developed new lymphoma response criteria (RECIL 2017). We demonstrate that assessment of tumor burden in lymphoma clinical trials can use the sum of longest diameters of a maximum of three target lesions. Furthermore, we introduced a new provisional category of a minor response. We also clarified response assessment in patients receiving novel immune therapy and targeted agents that generate unique imaging situations.

Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.

The TGF-ß/SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia.

Natural killer (NK) cells are key components of the innate immune system, providing potent antitumor immunity. Here, we show that the tumor growth factor-ß (TGF-ß)/SMAD signaling pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia (ALL). We characterized NK cells in 50 consecutive children with B-ALL at diagnosis, end induction and during maintenance therapy compared with age-matched controls. ALL-NK cells at diagnosis had an inhibitory phenotype associated with impaired function, most notably interferon-γ production and cytotoxicity. By maintenance therapy, these phenotypic and functional abnormalities partially normalized; however, cytotoxicity against autologous blasts remained impaired. We identified ALL-derived TGF-ß1 to be an important mediator of leukemia-induced NK cell dysfunction. The TGF-ß/SMAD signaling pathway was constitutively activated in ALL-NK cells at diagnosis and end induction when compared with healthy controls and patients during maintenance therapy. Culture of ALL blasts with healthy NK cells induced NK dysfunction and an inhibitory phenotype, mediated by activation of the TGF-ß/SMAD signaling pathway, and abrogated by blocking TGF-ß. These data indicate that by regulating the TGF-ß/SMAD pathway, ALL blasts induce changes in NK cells to evade innate immune surveillance, thus highlighting the importance of developing novel therapies to target this inhibitory pathway and restore antileukemic cytotoxicity.

Umbilical cord blood graft engineering: challenges and opportunities.

We are entering a very exciting era in umbilical cord blood transplantation (UCBT), where many of the associated formidable challenges may become treatable by ex vivo graft manipulation and/or adoptive immunotherapy utilizing specific cellular products. We envisage the use of double UCBT rather than single UCBT for most patients; this allows for greater ability to treat larger patients as well as to manipulate the graft. Ex vivo expansion and/or fucosylation of one cord will achieve more rapid engraftment, minimize the period of neutropenia and also give certainty that the other cord will provide long-term engraftment/immune reconstitution. The non-expanded (and future dominant) cord could be chosen for characteristics such as better HLA matching to minimize GvHD, or larger cell counts to enable part of the unit to be utilized for the development of specific cellular therapies such as the production of virus-specific T-cells or chimeric-antigen receptor T-cells which are reviewed in this study.

Robust T cell responses to aspergillosis in chronic granulomatous disease: implications for immunotherapy.

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4(+) T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4-depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4(+) T cell-depleted mice, similar to wild-type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4(+) T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non-depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4(+) T cell response suggest that CD4(+) T cell-based immunotherapy for this disease is unlikely to be beneficial.

Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant.

Adoptive immunotherapy with ex vivo expanded T cells is a promising approach to prevent or treat leukemia. Myeloid leukemias express tumor-associated antigens (TAA) that induce antigen-specific cytotoxic T lymphocyte (CTL) responses in healthy individuals. We explored the feasibility of generating TAA-specific CTLs from stem cell donors of patients with myeloid leukemia to enhance the graft-versus-leukemia effect after stem cell transplantation. CTL lines were manufactured from peripheral blood of 10 healthy donors by stimulation with 15mer peptide libraries of five TAA (proteinase 3 (Pr3), preferentially expressed antigen in melanoma, Wilms tumor gene 1 (WT1), human neutrophil elastase (NE) and melanoma-associated antigen A3) known to be expressed in myeloid leukemias. All CTL lines responded to the mix of five TAA and were multi-specific as assessed by interferon-γ enzyme-linked immunospot. Although donors showed individual patterns of antigen recognition, all responded comparably to the TAAmix. Immunogenic peptides of WT1, Pr3 or NE could be identified by epitope mapping in all donor CTL lines. In vitro experiments showed recognition of partially human leukocyte antigen (HLA)-matched myeloid leukemia blasts. These findings support the development of a single clinical grade multi-tumor antigen-specific T-cell product from the stem cell source, capable of broad reactivity against myeloid malignancies for use in donor-recipient pairs without limitation to a certain HLA-type.

Pediatric Hodgkin lymphoma: are we over-scanning our patients?

Despite the favorable outcome of most pediatric patients with Hodgkin lymphoma (HL), there is rising concern about risks of carcinogenesis from both diagnostic and therapeutic radiation exposure for patients treated on study protocols. Although previous studies have investigated radiation exposure during treatment, radiation from post-treatment surveillance imaging may also increase the likelihood of secondary malignancies. All diagnostic imaging examinations involving ionizing radiation exposure performed for surveillance following completion of therapy were recorded for 99 consecutive pediatric patients diagnosed with HL from 2000 to 2010. Cumulative radiation dosage from these examinations and the frequency of relapse detection by these examinations were recorded. In the first 2 years following completion of therapy, patients in remission received a median of 11 examinations (range 0-26). Only 13 of 99 patients relapsed, 11 within 5 months of treatment completion. No relapse was detected by 1- or 2-view chest radiographs (n = 38 and 296, respectively), abdomen/pelvis computed tomography (CT) scans (n = 211), or positron emission tomography (PET) scans alone (n = 11). However, 10/391 (2.6%) of chest CT scans, 4/364 (1.1%) of neck CT scans, and 3/47 (6.4%) of PET/CT scans detected relapsed disease. Thus, only 17 scans (1.3%) detected relapse in a total of 1358 scans. Mean radiation dosages were 31.97 mSv for Stage 1, 37.76 mSv for Stage 2, 48.08 mSv for Stage 3, and 51.35 mSv for Stage 4 HL. Approximately 1% of surveillance imaging examinations identified relapsed disease. Given the very low rate of relapse detection by surveillance imaging stipulated by current protocols for pediatric HL patients, the financial burden of the tests themselves, the high cure rate, and risks of second malignancy from ionizing radiation exposure, modification of the surveillance strategy is recommended.

Outcome of hematopoietic stem cell transplant as salvage therapy for Hodgkin's lymphoma in adolescents and young adults at a single institution.

For patients with relapsed Hodgkin's lymphoma (HL), high dose chemotherapy with stem cell rescue (HDCT-SCT) may improve survival over chemotherapy alone. We assessed the outcomes of HDCT-SCT in 37 consecutive adolescent and young adult patients with relapsed HL whose malignancy was categorized based on sensitivity to chemotherapy. We determined whether current outcomes supported the use of HDCT-SCT in all of our patients or just those patients with lower-risk characteristics such as chemosensitivity. With a median follow-up of 6.5 years, the 2-year overall survival (OS) was 89% (95% CI: 62-97%) for the chemosensitive patients (n = 21), whereas for patients with resistant disease (n = 16), OS was 53% (95% CI: 25-74%). Both autologous and allogeneic transplants were well tolerated, with 100-day treatment-related mortality under 10%. Our data show encouraging outcomes for patients with chemosensitive relapsed HL who receive hematopoietic stem cell transplant (HSCT) and support the value of the procedure even when the disease is chemoresistant.

CD3(+) and/or CD14(+) depletion from cord blood mononuclear cells before ex vivo expansion culture improves total nucleated cell and CD34(+) cell yields.

Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle in the use of CB is its low cell dose, which is largely responsible for an elevated risk of graft failure and a significantly delayed neutrophil and platelet engraftment. As a positive correlation has been shown between the total nucleated cell (TNC) and CD34(+) cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with MSC in a cytokine cocktail. We show that this strategy can be further improved if CD3(+) and/or CD14(+) cells are first depleted from the CB MNC before ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible as clinical-grade devices and reagents are available. Ultimately, the aim of improving TNC and CD34(+) transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.

T cell therapies following hematopoietic stem cell transplantation: surely there must be a better way than DLI?

Advances in the past few years have significantly improved adoptive immunotherapy strategies available following autologous and allogeneic hematopoietic stem cell transplantation (HSCT). Minimal residual disease, relapsed disease and viral infections remain a significant cause of mortality in patients undergoing HSCT. Novel therapies are critically needed to overcome these management dilemmas, while sparing the graft-versus-tumor effect and avoiding graft-versus-host disease. This review focuses on the T-cell strategies currently available to allay disease while minimizing toxicities in patients who have undergone HSCT.

Reconstitution of adenovirus-specific cell-mediated immunity in pediatric patients after hematopoietic stem cell transplantation.

Adenovirus (adv) is a significant cause of morbidity and mortality in pediatric hematopoietic stem cell transplant recipients, and control of infection seems to require antigen-specific T cells. We evaluated the recovery of adv-specific cellular immunity in this patient population related to degree of T-cell immunosuppressive therapy and compared this to adv cellular immunity of normal donors. Over 12 months, we monitored for adv DNA in stool and blood of patients and in the blood of a normal donor group. Twenty-two pediatric hematopoietic stem cell transplant (HSCT) patients (14 months-20 years) who received matched-related (MRD n=6), mismatched related (Haplo n=6) or matched unrelated donor (MUD n=10) grafts, were followed and results compared to healthy controls (n=8). Adv was detected by polymerase chain reaction in blood and/or stool from 81.8% of patients on at least one occasion post-HSCT, but only 68% of patients developed symptomatic adv infections. Recovery of adv-specific T cells was significantly delayed in the MUD and Haplo recipients, whereas recovery in the MRD group was similar to levels detected in healthy donors within 30 days post-transplant. In conclusion, recipients of alternative donor transplants at our institution have significantly delayed adv-specific cellular immune recovery, which correlates to an increased risk of adv-associated morbidity and mortality.

T cell therapies.

T cell therapies are increasingly used for the treatment of malignancies and viral-associated diseases. Initial studies focused on the use of unmanipulated T cell populations after allogeneic stem cell transplantation. More recently, the use of antigen-specific T cells has been explored. This chapter reviews the clinical experience with polyclonal Epstein-Barr virus (EBV)-specific cytotoxic T cells (CTL) for the treatment of EBV-associated malignancies. Strategies on how to improve the antitumor activity of EBV-specific CTL are being discussed. If effective, these strategies will have broad implications for T cell therapies for a range of human tumors with defined antigens.

Adenovirus infection rates in pediatric recipients of alternate donor allogeneic bone marrow transplants receiving either antithymocyte globulin (ATG) or alemtuzumab (Campath).

Infectious complications due to adenovirus are of increasing concern after allogeneic stem cell transplantation. Over the past 4 years, we have modified our conditioning regimens to use alemtuzumab in preference to anti-thymocyte globulin (ATG) for pediatric patients receiving stem cell transplants from alternate donors. Recent reports in adult studies implicate alemtuzumab as a risk factor for adenovirus infection. We therefore evaluated the incidence of adenovirus infection in pediatric patients receiving either ATG or alemtuzumab in their conditioning regimens. Of the 111 patients evaluated, a total of 54 patients received ATG and 57 patients received alemtuzumab. In total, 35/111 (32%) patients were infected by adenovirus, and 9/111 (8%) had adenovirus disease (AD). Adenovirus infection was greater in the alemtuzumab group than the ATG group (23/57 vs 12/54) (P=0.039) and disseminated AD was more frequent in the alemtuzumab group vs the ATG group (8/57 and 1/54 respectively) (P=0.032). The presence of Grade 3-4 graft-versus-host disease was a risk factor for adenovirus infection. Our findings highlight the fact that adenovirus infection is a frequent complication after stem cell transplantation from alternate donors in the pediatric population and that alemtuzumab increases the risk of infection compared to ATG. This work will help in identifying at-risk populations for our upcoming immunotherapy trial using adoptively transferred donor-derived adenovirus-specific cytotoxic T lymphocytes.

Cytomegalovirus (CMV) infections and CMV-specific cellular immune reconstitution following reduced intensity conditioning allogeneic stem cell transplantation with Alemtuzumab.

We studied the incidence and recurrence of Cytomegalovirus (CMV) infection and reactivation in 38 recipients of Alemtuzumab reduced intensity conditioning-stem cell transplantation, and used CMV-HLA tetramer studies to discover if these events correlated with recovery of circulating CMV-specific CD8+ T cells (cytotoxic T lymphocyte (CTLs)). The cumulative incidence of CMV infection was 60% at 1 year (95% CI, 45-78%) with a median reactivation time of 24 days (range 5-95 days). All patients with CMV reactivation received Ganciclovir or Foscarnet, and only one developed CMV disease. More strikingly, only 8/21 patients had relapse of CMV antigenemia. Tetramer analysis in 13 patients showed that 11 reconstituted CMV CTLs (7/11 by day 30 and 10/11 by day 90). The development of CMV infection was accompanied by a >5-fold rise of CMV CTLs. Recurrence of CMV infection occurred only in the patients who failed to generate a CTL response to the virus. Hence, recipients of SCT using Alemtuzumab-RIC are initially profoundly immunosuppressed and have a high incidence of early CMV reactivation. However, in the majority of patients, infection is transient, and antiviral T cell reconstitution is rapid. Monitoring with CMV-specific CTLs may help identify the subset of patients at risk from recurrent infection or disease.

T-cell immunotherapy for adenoviral infections of stem-cell transplant recipients.

Human adenoviruses are ubiquitous lytic DNA viruses that can be divided into 51 different serotypes, grouped from A to F on the basis of genome size, composition, homology, and organization. Adenovirus infections, although frequent, are rarely fatal in immunocompetent individuals, due to potent innate and adaptive immune responses. By contrast, adenoviruses are a significant cause of morbidity and mortality in immunosuppressed individuals, for whom there are limited treatment options. Since antiviral drugs have variable efficacy in the treatment of severe adenovirus disease, iatrogenic reconstitution with in vitro expanded virus-specific cytotoxic T lymphocytes (CTLs) is an attractive option for prophylaxis and treatment, particularly because the endogenous recovery of adenovirus-specific T cells has proved important in controlling infection in vivo. Thus, we have characterized human T-cell responses to adenovirus in vitro and explored the potential of adoptive T-cell immunotherapy as a prophylactic or therapeutic strategy for adenovirus infections posttransplant.