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Childhood cancer survivors (CCS) experience significant morbidity due to treatment- related late effects and benefit from late-effects surveillance. Adherence to screening recommendations is suboptimal. Survivorship care programs often struggle with resource limitations and may benefit from understanding institution-level financial outcomes associated with patient adherence to justify programmatic development and growth. The purpose of this study is to examine how CCS adherence to screening recommendations relates to the cost of care, insurance status, and institution-level financial outcomes. A retrospective chart review of 286 patients, followed in a structured survivorship program, assessed adherence to the Children’s Oncology Group follow-up guidelines by comparing recommended versus performed screening procedures for each patient. Procedure cost estimates were based on insurance status. Institutional profit margins and profit opportunity loss were calculated. Bivariate statistics tested adherent versus nonadherent subgroup differences on cost variables. A generalized linear model predicted the likelihood of adherence based on cost of recommended procedures, controlling for age, gender, race, and insurance. Adherence to recommended surveillance procedures was 50.2%. Nonadherence was associated with higher costs of recommended screening procedures compared to the adherent group estimates ($2,469.84 vs. $1,211.44). Failure to perform the recommended tests resulted in no difference in reimbursement to the health system between groups ($1,249.63 vs. $1,211.08). For the nonadherent group, this represented $1,055.13 in “lost profit opportunity” per visit for patients, which totaled $311,850 in lost profit opportunity due to nonadherence in this subgroup. In the final model, nonadherence was related to higher cost of recommended procedures (p < .0001), older age at visit (p = .04), Black race (p = .02), and government-sponsored insurance (p = .03). Understanding institutional financial outcomes related to patient adherence may help inform survivorship care programs and resource allocation. Potential financial burden to patients associated with complex care recommendations is also warranted.

Peer support has begun to gain traction as a mechanism for improving well-being in medicine. In this paper, we share our experience building, training, and piloting a peer support team based on a "critical incident stress management" model. The HOPES team (Helping Our Peers Endure Stress) is dedicated to, and composed entirely of, members of our division of pediatric hematology/oncology. Peer support will not solve all the well-being problems afflicting medicine. It is, however, a very good place to start.

Over 80% of children diagnosed with cancer are now cured. The burgeoning population of survivors of childhood cancer experiences high rates of morbidity and mortality due to 'late-effects' of treatment. These can be defined as any consequence of treatment that persists beyond or develops after the completion of cancer therapy. Awareness of late-effects is critically important for pediatricians and adult providers alike, as late-effects impact children in proximity to cancer treatment, as well as adults many decades removed. This review presents the importance of lifelong follow-up care for survivors, highlights existing screening guidelines, and reviews various models of survivor care.National and international guidelines have been developed to standardize screening for survivors, and multiple models of survivorship care exist. The optimal model likely depends on individual factors, including the survivor's needs and preferences, as well as local resources. Key strategies for the successful care of survivors include accurate risk-stratification for specific late-effects, individualized screening plans, education of survivors and professionals, clear communication between providers, and well coordinated transition of care across services.Early identification and management of late-effects are important for survivors of childhood cancer. Providers should be familiar with the risks for specific late-effects and have access to screening guidelines. The strengths and weaknesses of care models, along with individual circumstances, should be considered in designing the optimal approach to care for each survivor.

Following the discovery of a fetal hepatic tumor, labor was induced at 38 weeks, and a phenotypically normal female was delivered vaginally. A serum alpha-fetoprotein level at birth was 373,170 ng/mL. Postnatal magnetic resonance imaging confirmed a mass in the right lobe of the liver, and a percutaneous core biopsy revealed an epithelial type hepatoblastoma with predominantly embryonal histology. Methylation testing revealed hypomethylation at imprinting center 2, consistent with a diagnosis of Beckwith-Wiedemann syndrome. This case suggests that Beckwith-Wiedemann syndrome testing should be considered in all patients with hepatoblastoma, even in the absence of other phenotypic stigmata.

Multicystic dysplastic kidney is a rare urinary anomaly characterized by multiple non-communicating cysts resulting in a non-functional kidney. In addition to association with hypertension and contralateral renal anomalies, children with multicystic dysplastic kidney have an increased risk of Wilms tumor. Cohort studies and systematic reviews are hampered in estimating the true risk of this association due to the rarity and infrequent reporting of the condition. We present a case of a 2-year-old male child with an antenatal diagnosis of multicystic dysplastic kidney undergoing surveillance ultrasonography who presented with a symptomatic Wilms tumor. Level of evidence: Not applicable for this multicentre audit.

Background Childhood cancer survivors (CCSs) are at high risk of morbidity and mortality from long-term complications of their cancer treatment. The Children's Oncology Group developed screening guidelines to enable the early identification of and intervention for late effects of cancer treatment. There is a paucity of data on the adherence of CCSs to screening recommendations. Procedure A retrospective analysis of medical records to evaluate the rate of adherence of CCSs to the personalized, risk-based recommendations provided to them in the context of a structured long-term follow-up program over a 3-year period. Results Two hundred eighty-six CCSs visited the survivorship clinic 542 times during the 3-year study period. The overall rate of adherence to recommended screening was 74.2%. Using a univariate model and greater age at diagnosis and at screening recommendation were associated with decreased screening adherence. Gender, cancer diagnosis, radiation therapy, anthracycline exposure, and hematopoietic stem cell transplant were not significantly associated with adherence. In a multivariate model, age over 18 years at the time of the visit was significantly associated with decreased adherence (P < 0.0329) (odds ratio: 1.53, 95% confidence interval: 1.04–2.25). Conclusions Adherence to recommended screening tests is suboptimal among CCSs, with lower rates of adherence in CCSs older than 18 years of age compared with those younger than 18 years of age. Given the morbidity and mortality from the late effects of therapy among young adult CCSs, it is critically important to identify and remove barriers to late-effects screening among CCSs.

An 18-year-old girl is diagnosed with Ewing sarcoma. The treatment of her cancer will include 8.4 g/m2 of cyclophosphamide and 63 g/m2 of ifosfamide. She has questions about the impact the treatment may have on her future fertility and what options are available to preserve her fertility. Available fertility preservation techniques are discussed, including embryo cryopreservation, oocyte cryopreservation, and ovarian tissue cryopreservation.

Background Cardiovascular disease is prevalent among childhood cancer survivors (CCS). Arterial stiffness measured by pulse wave velocity (PWV) may be predictive of cardiovascular morbidity. Increased PWV has been seen in adults following chemotherapy. Purpose To evaluate PWV in a cohort of CCS and healthy controls. Patients and Methods All participants were >6 years old. CCS were >12 months off-therapy and free of cardiac disease, diabetes, and kidney dysfunction. Height, weight, blood pressure (BP), medications, cancer diagnosis, age at diagnosis, time off therapy, chemotherapy, and radiation exposures were recorded. PWV was measured on all participants. Results Sixty-eight CCS (mean 17.3 ± 6 years, 52.9% male), and 51 controls (mean 18.4 ± 5.5 years, 37.3% male) were evaluated. Among CCS, 34% had lymphoma, 44% leukemia, and 22% solid tumors, and 49% were exposed to radiation. CCS were off therapy 7 ± 4.2 years. Both groups were statistically similar in age, BMI, and BP. CCS ≥18 years old had significantly higher PWV compared to controls ≥18 years old (6.37 ± 0.89 vs. 5.76 ± 0.88 m/sec, P = 0.012). The relationship persisted in a regression model adjusted for age, sex, and BMI z-score (β = 0.52, 95%CI 0.051–0.979, P = 0.03). Seventy percent of CCS ≥18 had elevated PWV compared to established norms. Radiation therapy, anthracycline dose, and chemotherapy exposures were not predictive of increased PWV in CCS. Conclusions CCS ≥18 demonstrated prematurely elevated PVW. Further studies are needed to determine the predictive value of PWV in this population and its utility as a screening modality. Pediatr Blood Cancer 2015;62:1832–1837. © 2015 Wiley Periodicals, Inc.

Background Childhood sarcomas are rare and require complex interdisciplinary care including surgery, chemotherapy, and radiation. The goal of this study was to determine if racial or ethnic disparities exist for pediatric sarcoma patients in the United States. Methods The United States' National Cancer Institute's Surveillance, Epidemiology, and End Results database was used to identify patients aged 0-21 diagnosed with primary sarcomas from 1973 to 2012. Patients were considered by race and ethnicity. Survival curves were computed using the Kaplan-Meier method and the log-rank test. Results A total of 11,502 patients were included in this study. When stratified by race, non-Hispanic black and Hispanic patients were significantly more likely to present with advanced stage disease than white patients. White patients were more likely to receive radiation therapy than black and Hispanic patients (P = 0.01). There was no significant difference between patients who underwent surgery (P = 0.21). Overall survival was better for white patients than black or Hispanic ones. Despite the overall 5-year survival improvement during the study period (56.2%-70.3%), survival disparities between race and ethnicity have grown. Conclusions Racial and ethnic disparities do exist with respect to stage, treatment, and survival of these rare tumors. Black and Hispanic patients are presenting at more advanced stage and have overall worse survival. This survival disparity has widened over the past 4 decades.

e18125 Background: The 2010 Dependent Coverage Provision (DCP) of the Affordable Care Act (ACA) allowed young adults to remain on their parents' health insurance until age 26 years (y), whereas pre-DCP the cut-off varied, but was most often 18 y. Our objective was to compare rates of insurance disenrollment among patients with cancer who were DCP-eligible compared to those who were not. Methods: A retrospective cohort from the OptumLabs Data Warehouse, which includes claims data for privately insured enrollees in a large US health plan, was used. Patients born between 1982-93 diagnosed with cancer between 2000-15 were included. In the Recent Cohort, patients who turned 19 in 2010-12 (who were always eligible to stay on parents’ insurance) were matched to patients who turned 19 in 2007-09 (who were not protected by the DCP when they turned 19). Sensitivity analyses paired patients in an Earlier Cohort, who turned 19 (in 2001-03 and 2004-06), none of whom were eligible for the DCP when they turned 19. Patients were matched on cancer type, diagnosis date, and additional clinical characteristics. Using a time to loss of coverage analysis (defined as > 90 d interruption in insurance enrollment), hazard ratios (HR) were calculated using Cox proportional hazards models. Difference-in-difference between pairs from the Recent and Earlier cohorts was evaluated. Results: Of the 3,013 patients who turned 19 in 2010-12, 2,829 were matched. Median time to disenrollment was 26 months (m) compared to 22 m among patients who turned 19 in 2007-09 (HR 0.88, 95% CI 0.81-0.95, p = 0.0009). In the 4,489 pairs of patients who turned 19 between 2001-06, median time to disenrollment was 20 m among both the younger and older patients in the pair (p = 0.047). In grouped analyses, the difference-in-difference between the Recent and Earlier sets of pairs displayed a 14% reduction in the hazard for losing coverage (p < 0.0001), favoring those who turned 19 after DCP became available. Conclusions: In pediatric cancer patients and survivors, a vulnerable population that needs continuous insurance coverage, these data suggest that the DCP of the ACA lowers the insurance drop-out rate.

Since the 1970s, outcomes for childhood cancer have shown remarkable and steady improvements. Five-year overall survival from childhood cancer now exceeds 80% and as many as 1 in 500 young American adults are survivors of childhood cancer. Despite these successes, nearly a quarter of survivors will have multiple severe, disabling or life-threatening conditions by age 50, over 90% have measurable end-organ damage and almost 20% will die within 30 years of diagnosis. The complications faced by survivors, and the care they require, are multisystem and complex. The most common complications include endocrine, cardiac, musculoskeletal and pulmonary complications, as well as treatment-related secondary neoplasms. Survivors also often face societal, emotional and psychological barriers, such as learning challenges, school difficulties, and problems obtaining insurance, and almost one in five suffer from stress-related mental disorders such as post-traumatic stress symptoms. Extensive, intricate, long-term medical and psychological follow-up is required to maintain survivors’ health and quality of life.

The efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic leukemia (ALL) cell lines and primary samples has facilitated identification of the antileukemia activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and endpoint analysis at high disease burden. In this study, we report that bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This approach provides a noninvasive, longitudinal measure of leukemia burden and localization that enhances the sensitivity of treatment response detection and provides greater insight into the mechanism of action of antileukemia agents. In addition, this study reveals significant cell line– and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Overall, this study demonstrates that the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects at earlier, previously unquantifiable disease burdens and thus provides the means to standardize and expedite the evaluation of anti-ALL activity in preclinical xenograft studies.

The last decade has brought increasing recognition that the wellness of health care providers has an impact on the quality of care, patient satisfaction, and health care economics. This review will describe models of burnout, discuss the impact of burnout on medicine with a focus on pediatric hematologists/oncologists, and present interventions that may help ameliorate physician burnout.

Since the 1950s, the overall survival (OS) of children with cancer has gone from almost zero to approaching 80%. While there have been notable successes in treating solid tumors such as Wilms tumor, some childhood solid tumors, exemplified by diseases like high-risk neuroblastoma and metastatic sarcomas, have continued to elude effective therapy[1]. With the use of megatherapy techniques such as tandem transplantation, dose-escalation has been pushed to the edge of dose-limiting toxicities, and any further improvements in event-free survival (EFS) will have to be achieved through novel therapeutic approaches. In this chapter, we will review the status of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) for many pediatric solid tumor types. The vast majority of the clinical experience in transplant for pediatric solid tumors is in the autologous setting, so we will review some general principles of autologous HSCT, followed by an examination of HSCT for diseases such as Hodgkin disease, Ewing sarcoma, and neuroblastoma. Finally, we will look to the future of cell-based therapies by considering some experimental approaches to effector cell therapies. (1) Principles of autologous HSCT Prior to the introduction of high-dose chemotherapy (HDC) with autologous stem cell rescue (also called autologous HSCT), marrow tolerance was the limiting factor in the escalation of chemotherapy for the treatment of malignancies. With the ability to safely harvest, store and re-infuse a patient’s own hematopoietic stem cells, doses of cytotoxic therapies for cancer could safely proceed beyond marrow tolerance, thereby allowing more intense treatment of certain malignancies. Two approaches to the use of HDC with stem cell rescue include: (1) myeloablative regimens, meaning that no hematopoietic recovery can occur without the stored HSCs; and (2) sub-myeloablative HDC regimens in which stem cell rescue is used to speed recovery, decrease toxicity and decrease the interval between courses of chemotherapy, although it is not absolutely required for engraftment[2–3]. Although the increased treatment intensity may improve disease-free survival for patients with some malignancies, this must be balanced with the increased treatment-related mortality associated with the higher doses of cytotoxic agents, as well as the potential late effects of more intense cytotoxic treatments and radiotherapeutic regimens in young children. Criteria that may help define circumstances in which HDC with stem cell rescue would be most beneficial include: (1) a tumor with good response to induction chemotherapy, but a poor 3 or 5-year EFS; and (2) a HDC regimen that can utilize multiple agents active against the disease, especially if the agents differ from those used during induction therapy. Although the use of HDC with stem cell rescue is controversial in most diseases, diseases such as Hodgkin disease and high-risk neuroblastoma (discussed below) meet the design criteria listed above and have demonstrated improved outcomes in clinical trials.

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL.

Understanding the pathogenesis of leukemia in the context of lymphopoiesis may reveal novel therapeutic targets. Previously, we have shown that mTOR inhibitors (MTI) show activity in vitro and in preclinical models of both human and murine precursor B acute lymphoblastic leukemia (pre-B ALL), inhibiting cell proliferation and inducing apoptosis. These MTI-mediated effects can be reversed by interleukin-7 (IL-7), an important regulator of early B-cell development. This observation led us to examine the contribution of signaling via the IL-7Rα chain, which is shared by the receptor complexes of IL-7 and thymic stromal-derived lymphopoietin (TSLP). TSLP is closely related to IL-7 and active in lymphopoiesis, but an effect of TSLP on leukemia cells has not been described. We examined the effect of TSLP on pre-B ALL cells and their response to MTIs. Here, we show that TSLP stimulates proliferation of pre-B ALL cell lines. TSLP also partially reverses the effects of MTI on proliferation, apoptosis, and ribosomal protein S6 and 4E-BP1 phosphorylation in cell lines, with similar biological effects seen in some primary human lymphoblast samples. These data show that TSLP can promote survival of pre-B ALL cells and antagonize the effects of MTIs. These findings suggest that IL-7Rα chain is responsible for transducing the survival signal that overcomes MTI-mediated growth inhibition in pre-B ALL. Thus, further exploration of the IL-7Rα pathway may identify potential therapeutic targets in the treatment of ALL. Our data illustrate that growth-factor–mediated signaling may provide one mechanism of MTI resistance. [Cancer Res 2007;67(20):9963–70]

Lateral sinus thrombosis (LST), a rare complication of otitis media, is managed by antibiotics, surgery and anticoagulation. Traditionally, post-operative anticoagulation has been achieved by intravenous unfractionated heparin followed by oral warfarin. Fractionated, or low-molecular weight heparin derivatives (LMWH) have been introduced recently. There has been minimal literature to date regarding their use for the management of LST. We present use of the LMWH enoxaparin (Lovenox) for otogenic LST in two children, both of whom experienced hemorrhagic complications. On this basis and in the context of a literature review, we urge caution when using LMWH for pediatric otogenic LST.

Cardiovascular disease is prevalent among childhood cancer survivors (CCS). Arterial stiffness measured by pulse wave velocity (PWV) may be predictive of cardiovascular morbidity. Increased PWV has been seen in adults following chemotherapy.To evaluate PWV in a cohort of CCS and healthy controls.All participants were6 years old. CCS were12 months off-therapy and free of cardiac disease, diabetes, and kidney dysfunction. Height, weight, blood pressure (BP), medications, cancer diagnosis, age at diagnosis, time off therapy, chemotherapy, and radiation exposures were recorded. PWV was measured on all participants.Sixty-eight CCS (mean 17.3 ± 6 years, 52.9% male), and 51 controls (mean 18.4 ± 5.5 years, 37.3% male) were evaluated. Among CCS, 34% had lymphoma, 44% leukemia, and 22% solid tumors, and 49% were exposed to radiation. CCS were off therapy 7 ± 4.2 years. Both groups were statistically similar in age, BMI, and BP. CCS ≥ 18 years old had significantly higher PWV compared to controls ≥ 18 years old (6.37 ± 0.89 vs. 5.76 ± 0.88 m/sec, P = 0.012). The relationship persisted in a regression model adjusted for age, sex, and BMI z-score (β = 0.52, 95%CI 0.051-0.979, P = 0.03). Seventy percent of CCS ≥ 18 had elevated PWV compared to established norms. Radiation therapy, anthracycline dose, and chemotherapy exposures were not predictive of increased PWV in CCS.CCS ≥ 18 demonstrated prematurely elevated PVW. Further studies are needed to determine the predictive value of PWV in this population and its utility as a screening modality.

Opsoclonus myoclonus ataxia syndrome (OMAS) can be refractory to standard therapies and devastating. Alternative treatments are imperative. A 14-month-old male diagnosed with neuroblastoma and paraneoplastic OMAS achieved complete cancer remission with chemotherapy. The OMAS, however, persisted over the subsequent 4 years despite numerous immune-modulatory and immunosuppressive therapies. The patient ultimately achieved complete remission following therapeutic plasma exchange (TPE) combined with rituximab and intravenous immunoglobulin. After three asymptomatic years, he relapsed. Upon reintroducing TPE and rituximab plus oral prednisolone, the patient rapidly achieved a second complete remission. This case offers proof-of-principle for the potential efficacy of TPE for neuroblastoma-associated OMAS.

As the cure rate for adolescents with cancer has improved, the focus on future reproductive potential has increased. Despite a concerted effort to reduce the impact of cancer treatment on fertility through the alteration of therapy and the implementation of protective measures, many adolescent women with cancer remain at risk for impaired reproductive potential. Although the only standard-of-care approach to fertility preservation in this population remains embryo cryopreservation, there has been intense development of oocyte and ovarian cryopreservation as viable alternatives. This article focuses on the developing modalities of fertility preservation for adolescent women diagnosed with cancer.

Survivors of childhood cancer represent a rapidly growing population of patients, some of whom experience temporary or permanent premature ovarian failure (POF) as a consequence of their disease or treatment. Although the risks and benefits of exogenous hormones have been extensively explored in menopausal women 50 years of age and older, there is scant data on the long-term safety of exogenous hormones in childhood cancer survivors. Although there are certainly benefits that can be achieved through hormone replacement for this unique population, many of these patients also have very long hormone exposure times and a markedly increased baseline risk for second malignancies, including breast cancer. Given the significant potential risks, hormone replacement should not be reflexively instituted in childhood cancer survivors with POF. It should only be considered following a thorough, balanced discussion of the risks and benefits of hormone replacement with each patient.

Background A significant iron burden may occur after only 10 blood transfusions in patients with hematologic disorders. Children with acute lymphoblastic leukemia (ALL) routinely receive blood transfusions during therapy, although few studies to date have quantified transfusion-related iron burden in these patients. This study quantifies the transfused blood volume and resultant iron load in a large cohort of pediatric patients with ALL, and evaluates risk factors that may impact transfusion volume. Methods This single institution retrospective study evaluated 107 patients who completed therapy for ALL between July 1995 and March 2007. Age, weight, and hemoglobin at presentation, ALL risk category, leukemia cell type, and volume of blood transfusions were collected from medical records. Results Patients received an average of 115 ml/kg of blood (77 mg/kg iron) during treatment. There was a significant association between the volume of packed red blood cells and ALL risk category. Patients with standard-risk disease received 90 ml/kg (60 mg/kg iron), patients with high-risk disease 196 ml/kg (131 mg/kg iron) and patients with T-cell disease 114 ml/kg (76 mg/kg iron). There was no correlation between age or hemoglobin at presentation with amount of blood received. Conclusions Patients with ALL often receive a substantial amount of iron during therapy, with patients with high-risk disease receiving the greatest load. As iron overload has an overlapping toxicity profile with chemotherapy and is treatable, screening for increased iron burden and iron-related morbidities should be considered during long-term follow-up of patients with ALL, particularly in those with high-risk ALL. Pediatr Blood Cancer 2011;56:368–371. © 2010 Wiley-Liss, Inc.

Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To address this issue, we created a series of CARs that contain the T cell receptor-ζ (TCR-ζ) signal transduction domain with the CD28 and/or CD137 (4-1BB) intracellular domains in tandem. After short-term expansion, primary human T cells were subjected to lentiviral gene transfer, resulting in large numbers of cells with >85% CAR expression. In an immunodeficient mouse xenograft model of primary human pre-B-cell acute lymphoblastic leukemia, human T cells expressing anti-CD19 CARs containing CD137 exhibited the greatest antileukemic efficacy and prolonged (>6 months) survival in vivo, and were significantly more effective than cells expressing CARs containing TCR-ζ alone or CD28-ζ signaling receptors. We uncovered a previously unrecognized, antigen-independent effect of CARs expressing the CD137 cytoplasmic domain that likely contributes to the enhanced antileukemic efficacy and survival in tumor bearing mice. Furthermore, our studies revealed significant discrepancies between in vitro and in vivo surrogate measures of CAR efficacy. Together these results suggest that incorporation of the CD137 signaling domain in CARs should improve the persistence of CARs in the hematologic malignancies and hence maximize their antitumor activity.

Despite considerable success in treating newly diagnosed childhood acute lymphoblastic leukemia (ALL), relapsed disease remains a significant clinical challenge. Using a NOD/SCID mouse xenograft model, we report that immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODNs) stimulate significant immune activity against primary human ALL cells in vivo. The administration of CpG ODNs induced a significant reduction in systemic leukemia burden, mediated continued disease control, and significantly improved survival of mice with established human ALL. The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODNs and correlated with the production of IL-12p70, IFN-α, and IFN-γ by the host. In addition, depletion of natural killer cells by anti–asialo-GM1 treatment significantly reduced the in vivo antileukemic activity of CpG ODN. This antileukemia effect was not limited to the xenograft model because natural killer cell–dependent killing of ALL by human peripheral blood mononuclear cells (PBMCs) was also increased by CpG ODN stimulation. These results suggest that CpG ODNs have potential as therapeutic agents for the treatment of ALL.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While current therapy is effective for the majority of children, relapsed ALL remains the fifth most common pediatric malignancy and responds very poorly to treatment. In addition, about 50% of ALL cases occur in adults, and while remission is achieved in many patients the majority of these will succumb to relapsed disease. In most cases relapse is treated with more intensive chemotherapy or hematopoietic cell transplantation, therapies that are associated with significant toxicity. In both patient populations, therefore, there is an urgent need for novel approaches to inhibit disease progression. Immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODN) have been shown to induce strong anti-tumor immune activity in a variety of model systems and are currently being evaluated in clinical trials. We have previously reported that CpG ODN stimulation of ALL cells alters their antigen presentation ability and enhances Th1 responses by allogeneic T cells. In this study, using NOD-SCID mice bearing xenografts of primary human ALL or human ALL cell lines, we demonstrate that CpG ODN stimulate significant immune activity against ALL cells in vivo. A single intravenous injection of 300micrograms Class B CpG ODN induced a significant reduction in percentage of human ALL cells in peripheral blood (P

Biologically targeted cancer agents, including signal transduction inhibitors, have shown great promise in treating hematologic malignancies. However, used as single agents, these drugs may not be curative secondary to innate or acquired cellular resistance. Thus, acute lymphoblastic leukemia (ALL) and other cancer cells may become resistant to rapamycin, an mTOR inhibitor (MTI), following extended exposure to the drug. A strategy to overcome such resistance is to combine targeted agents, and thereby inhibit multiple targets simultaneously. Previously, we have shown activity of MTI in models of both human and murine ALL. In mouse models, treatment of ALL with MTI prolongs survival but may not cure disease. IL-7, a lymphoid growth factor important in the regulation of progenitor B cell development and proliferation, can reverse the inhibitory effects of MTI on human and murine pre-B ALL cells. We wished to further explore the mechanisms by which IL-7-mediated signaling protects ALL cells from the inhibitory effects of MTI, through the investigation of modulators of growth factor signaling in ALL. Thus, we have evaluated the impact of LY294002, an inhibitor of phosphatidyl inositol-3 kinase (PI3K). PI3K is a critical signaling molecule in cell survival and proliferation, with one of its central roles being signal transduction from growth factor receptors to the activation of AKT (an upstream regulator of mTOR). PI3K/AKT pathway over-activation has been implicated in many different cancers. Treatment of ALL cell lines with the PI3K inhibitor LY294002 markedly decreased cell proliferation in a dose-dependent manner. More importantly, the inhibitory effects of LY294002 were additive or synergistic with the inhibitory effects of MTI, and prevented the ability of IL-7 to reverse the inhibitory effects of rapamycin. Treatment of pre-B ALL cell lines with 2.5 μM LY294002 resulted in decreased proliferation to 20–45% of baseline as compared to untreated cells, whereas treatment with a higher dose (5 μM) reduced cell proliferation to 10–20%. Combinations of LY294002 and rapamycin, even at low doses, inhibited cell proliferation to a greater degree than each drug individually. Co-treatment with 2.5 μM LY294002 and low dose rapamycin (1 ng/ml) resulted in profound inhibition of proliferation to

T-bodies that combine the antigen recognition capability of single chain antibody molecules (scFv) with the signal transduction domains of activating receptors can be used to generate T cells with novel, engineered specificities independent of their endogenous TCR. We have developed a series of T-bodies with specificity towards CD19, a molecule with expression that appears restricted to normal and malignant B cells. These T-bodies contain the activating signals from the costimulatory T cell receptors, CD28 and/or 4-1BB, in tandem with the TCR-ζ signal transduction domain. Using lentiviral gene transfer and T cells expansion via bead-immobilized anti-CD3 and anti-CD28 antibodies, we can generate T cells 95% of which express the T-bodies. Lentiviral transduction combined with this efficient T cell expansion system has the potential to produce >1011 antigen-specific T cells without the need for a cell selection step. Interestingly, we have noted significant constitutive signaling effects of T-body overexpression on transduced T cells that may been overlooked previously due to lower transduction efficiency. Despite the observed constitutive effects, we show that lentiviral-engineered T cells expressing anti-CD19 T bodies efficiently kill CD19 expressing cell lines and primary B-ALL cells in an antigen-specific manner. T-body engineered T cells also produce cytokines in response to antigen triggering with different patterns of cytokine production by T cells engineered with different T-bodies. In particular, a T-body receptor containing the 4-1BB signal transduction domain combined with the TCR-ζ domain stimulates a >6-fold increase in IL-6 production compared with other receptor combinations in CD4+ T cells. In contrast, a CD28 and TCR-ζ domain containing T body uniquely enhances IL-2 (>3-fold) and TNF-α (> 7-fold) secretion by CD8+ T cells. These results indicate that the functional characteristics of our tested T-body signaling constructs are distinct and depend upon the particular receptor and T cell subset combination. We are also comparing the anti-tumor response generated by adoptively transferred T-body engineered T cells in leukemia bearing immunodeficient mice as a pre-clinical, therapeutic model. In addition to elucidating important functional differences of our lentiviral constructs, these studies indicate that an important variable in clinical trials will be optimizing the cytosolic signaling domain for the desired T cell subset. The present results suggest that distinct combinatorial signaling domains may be required for optimal function in CD4 and CD8 engineered T cells.