Sung Choi

Integrative Clinical Sequencing in the Management of Refractory or Relapsed Cancer in Youth

IMPORTANCEnCancer is caused by a diverse array of somatic and germline genomic aberrations. Advances in genomic sequencing technologies have improved the ability to detect these molecular aberrations with greater sensitivity. However, integrating them into clinical management in an individualized manner has proven challenging.nnnOBJECTIVEnTo evaluate the use of integrative clinical sequencing and genetic counseling in the assessment and treatment of children and young adults with cancer.nnnDESIGN, SETTING, AND PARTICIPANTSnSingle-site, observational, consecutive case series (May 2012-October 2014) involving 102 children and young adults (mean age, 10.6 years; median age, 11.5 years, range, 0-22 years) with relapsed, refractory, or rare cancer.nnnEXPOSURESnParticipants underwent integrative clinical exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing and genetic counseling. Results were discussed by a precision medicine tumor board, which made recommendations to families and their physicians.nnnMAIN OUTCOMES AND MEASURESnProportion of patients with potentially actionable findings, results of clinical actions based on integrative clinical sequencing, and estimated proportion of patients or their families at risk of future cancer.nnnRESULTSnOf the 104 screened patients, 102 enrolled with 91 (89%) having adequate tumor tissue to complete sequencing. Only the 91 patients were included in all calculations, including 28 (31%) with hematological malignancies and 63 (69%) with solid tumors. Forty-two patients (46%) had actionable findings that changed their cancer management: 15 of 28 (54%) with hematological malignancies and 27 of 63 (43%) with solid tumors. Individualized actions were taken in 23 of the 91 (25%) based on actionable integrative clinical sequencing findings, including change in treatment for 14 patients (15%) and genetic counseling for future risk for 9 patients (10%). Nine of 91 (10%) of the personalized clinical interventions resulted in ongoing partial clinical remission of 8 to 16 months or helped sustain complete clinical remission of 6 to 21 months. All 9 patients and families with actionable incidental genetic findings agreed to genetic counseling and screening.nnnCONCLUSIONS AND RELEVANCEnIn this single-center case series involving young patients with relapsed or refractory cancer, incorporation of integrative clinical sequencing data into clinical management was feasible, revealed potentially actionable findings in 46% of patients, and was associated with change in treatment and family genetic counseling for a small proportion of patients. The lack of a control group limited assessing whether better clinical outcomes resulted from this approach than outcomes that would have occurred with standard care.

Sirolimus is associated with veno-occlusive disease of the liver after myeloablative allogeneic stem cell transplantation

Sirolimus is an effective agent used in graft-versus-host disease (GVHD) prophylaxis after allogeneic transplantation. It also has antiproliferative effects on vascular endothelium when used to coat coronary artery stents. We noted an excess of veno-occlusive disease (VOD) in a clinical trial, and retrospectively reviewed the records of 488 patients to determine the association between sirolimus and VOD. When used with cyclophosphamide/total body irradiation (Cy/TBI) conditioning, sirolimus is associated with an increased incidence of VOD (OR 2.35, P = .005). The concomitant use of methotrexate further increased this rate (OR 3.23, P < .001), while sirolimus without methotrexate was not associated with an increased risk of VOD (OR 1.55, P = .33). Mortality after VOD diagnosis was unaffected, and overall treatment-related mortality was lowest when sirolimus was used without methotrexate. Similar findings were noted in matched, related, and unrelated as well as mismatched donor subgroups. When used with busulfan-based conditioning, sirolimus use was associated with an even higher rate of VOD (OR 8.8, P = .008). Our findings suggest that sirolimus use is associated with VOD after TBI-based transplantation when used with methotrexate after transplantation. Sirolimus-based GVHD prophylaxis without methotrexate is associated with the greatest overall survival. Myeloablative doses of busulfan should not be used with sirolimus-based immunosuppression.

Tacrolimus/sirolimus vs tacrolimus/methotrexate as GVHD prophylaxis after matched, related donor allogeneic HCT

Grades 2-4 acute graft-versus-host disease (GVHD) occurs in approximately 35% of matched, related donor (MRD) allogeneic hematopoietic cell transplantation (HCT) recipients. We sought to determine if the combination of tacrolimus and sirolimus (Tac/Sir) was more effective than tacrolimus and methotrexate (Tac/Mtx) in preventing acute GVHD and early mortality after allogeneic MRD HCT in a phase 3, multicenter trial. The primary end point of the trial was to compare 114-day grades 2-4 acute GVHD-free survival using an intention-to-treat analysis of 304 randomized subjects. There was no difference in the probability of day 114 grades 2-4 acute GVHD-free survival (67% vs 62%, P = .38). Grades 2-4 GVHD was similar in the Tac/Sir and Tac/Mtx arms (26% vs 34%, P = .48). Neutrophil and platelet engraftment were more rapid in the Tac/Sir arm (14 vs 16 days, P < .001; 16 vs 19 days, P = .03). Oropharyngeal mucositis was less severe in the Tac/Sir arm (peak Oral Mucositis Assessment Scale score 0.70 vs 0.96, P < .001), but otherwise toxicity was similar. Chronic GVHD, relapse-free survival, and overall survival at 2 years were no different between study arms (53% vs 45%, P = .06; 53% vs 54%, P = .77; and 59% vs 63%, P = .36). Based on similar long-term outcomes, more rapid engraftment, and less oropharyngeal mucositis, the combination of Tac/Sir is an acceptable alternative to Tac/Mtx after MRD HCT. This study was funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; and the trial was registered at www.clinicaltrials.gov as #NCT00406393.

HDAC inhibition and graft versus host disease

Histone deacetylase (HDAC) inhibitors are currently used clinically as anticancer drugs. Recent data have demonstrated that some of these drugs have potent antiinflammatory or immunomodulatory effects at noncytotoxic doses. The immunomodulatory effects have shown potential for therapeutic benefit after allogeneic bone marrow transplantation in several experimental models of graft versus host disease (GVHD). These effects, at least in part, result from the ability of HDAC inhibitors (HDACi) to suppress the function of host antigen presenting cells such as dendritic cells (DC). HDACi reduce the dendritic cell (DC) responses, in part, by enhancing the expression of indoleamine 2,3-dioxygenase (IDO) in a signal transducer and activator of transcription-3 (STAT-3) dependent manner. They also alter the function of other immune cells such as T regulatory cells and natural killer (NK) cells, which also play important roles in the biology of GVHD. Based on these observations, a clinical trial has been launched to evaluate the impact of HDAC inhibitors on clinical GVHD. The experimental, mechanistic studies along with the brief preliminary observations from the ongoing clinical trial are discussed in this review.

Host-derived CD8+ dendritic cells are required for induction of optimal graft-versus-tumor responses after experimental allogeneic bone marrow transplantation

The graft-versus-tumor (GVT) effect after allogeneic hematopoietic cell transplantation (allo-HCT) represents an effective form of immunotherapy against many malignancies. Meaningful separation of the potentially curative GVT responses from graft-versus-host disease (GVHD), the most serious toxicity following T-cell replete allo-HCT, has been an elusive goal. GVHD is initiated by alloantigens, although both alloantigens and tumor-specific antigens (TSAs) initiate GVT responses. Emerging data have illuminated a role for antigen-presenting cells (APCs) in inducing alloantigen-specific responses. By using multiple clinically relevant murine models, we show that a specific subset of host-derived APCs-CD8(+) dendritic cells (DCs)-enhances TSA responses and is required for optimal induction of GVT. Stimulation of TLR3, which among host hematopoietic APC subsets is predominantly expressed on CD8(+) DCs, enhanced GVT without exacerbating GVHD. Thus, strategies that modulate host APC subsets without direct manipulation of donor T cells could augment GVT responses and enhance the efficacy of allo-HCT.

Infectious Risk after Allogeneic Hematopoietic Cell Transplantation Complicated by Acute Graft-versus-Host Disease

The occurrence of infections after allogeneic hematopoietic stem cell transplantation (HCT) is nearly universal. However, the relationship between infections and graft-versus-host disease (GVHD) is complex and attribution of infectious-related mortality is highly inconsistent, making comparison of infectious complication rates across allogeneic HCT clinical studies difficult. We categorized infectious complications from diagnosis or 1 year before HCT (whichever occurred later) through 2 years after HCT according to timing, frequency, causative organism, severity, and contribution to mortality for 431 consecutive patients who underwent allogeneic HCT from 2008 to 2011. We then assessed the contribution of risk factors, such as the frequency of pre-HCT infections and post-HCT GVHD, on post-HCT infection frequency and severity. We found that each pre-HCT bacterial infection/year leads to an additional 2.15 post-HCT bacterial infection/year (Pu2009=u2009.004). Pre-HCT viral and fungal infections were not predictors for post-HCT infections. Acute GVHD (aGVHD) significantly increased the risk of developing life-threatening (hazard ratio [HR], 1.97; 95% confidence interval [CI], 1.33 to 2.90) and fatal (HR, 2.8; 95% CI, 1.10 to 7.08) infections. Furthermore, patients who develop aGVHD experienced ~60% more infections than patients who never develop aGVHD. Quantification of infection frequency and severity for patients with and without GVHD may facilitate comparison of infectious outcomes across allogeneic HCT trials.

Allogeneic transplantation with myeloablative FluBu4 conditioning improves survival compared to reduced intensity FluBu2 conditioning for acute myeloid leukemia in remission

The optimal intensity of conditioning for allogeneic hematopoietic stem cell transplantation (HCT) in acute myeloid leukemia (AML) remains undefined. Traditionally, myeloablative conditioning regimens improve disease control, but at the risk of greater nonrelapse mortality. Because fludarabine with myeloablative doses of intravenous busulfan using pharmacokinetic monitoring has excellent tolerability, we reasoned that this regimen would limit relapse without substantially elevating toxicity when compared to reduced intensity conditioning. We retrospectively analyzed 148 consecutive AML patients in remission receiving T cell replete HCT conditioned with fludarabine and intravenous busulfan at doses defined as reduced (6.4xa0mg/kg; FluBu2, nu2009=u200963) or myeloablative (12.8xa0mg/kg; FluBu4, nu2009=u200985). Early and late nonrelapse mortality (NRM) was similar among FluBu4 and FluBu2 recipients, respectively (dayu2009+u2009100: 4 vs 0xa0%; 5xa0years: 19 vs 22xa0%; pu2009=u20090.54). NRM did not differ between FluBu4 and FluBu2 in patients >50xa0years of age (24 vs 22xa0%, pu2009=u20090.75). Relapse was lower in recipients of FluBu4 (5xa0years: 30 vs 49xa0%; pu2009=u20090.04), especially in patients with poor risk cytogenetics (22 vs 59xa0%; pu2009=u20090.02) and those >50xa0years of age (28 vs 51xa0%; pu2009=u20090.02). Overall survival favored FluBu4 recipients at 5xa0years (53 vs 34xa0%, pu2009=u20090.02), a finding confirmed in multivariate analysis (HR: 0.57; 95xa0% CI: 0.34–0.95; pu2009=u20090.03). These data suggest that myeloablative FluBu4 may provide equivalent NRM, reduced relapse, and improved survival compared to FluBu2, emphasizing the importance of busulfan dose in conditioning for AML.

Double Umbilical Cord Blood Transplantation after Novel Myeloablative Conditioning Using a Regimen of Fludarabine, Busulfan, and Total Lymphoid Irradiation

We conducted a pilot study evaluating double umbilical cord blood transplantation (dCBT) after myeloablative conditioning with fludarabine and busulfan 3.2xa0mg/kg i.v.xa0× 4, followed by total lymphoid irradiation at 400xa0cGy (FluBu4/TLI) for any indicated hematological disorder for patients without a suitable donor. Twenty patients with predominantly high-risk disease underwent dCBT according to protocol. The regimen was well tolerated, with mucositis as the primary observed toxicity (nxa0=xa019). The cumulative incidence of neutrophil engraftment was 89% (95% confidence interval [CI], 64% to 97%), with a median time to recovery of 16xa0days (range, 12 to 31xa0days). All evaluable patients with neutrophil engraftment achieved complete donor chimerism by day 40. The cumulative incidence of grades III and IV acute graft-versus-host disease (GVHD) at day 100 was 10% (95% CI, 2% to 27%), and the cumulative incidence of chronic GVHD was 35% (95% CI, 16% to 55%) by the end of the study. At 1xa0year, the cumulative incidence of treatment-related mortality (TRM) was 35% (95% CI, 16% to 55%). The leading cause of nonrelapse mortality was acute GVHD (nxa0=xa04), followed by graft failure (nxa0=xa02) and chronic GVHD (nxa0=xa01). TRM was significantly associated with a pretransplantation hematopoietic cell transplantation-specific comorbidity index score ≥ 3 (Pxa0=xa0.005). At 1xa0year, disease relapse occurred in 6 patients and overall survival was 40% (95% CI, 19% to 60%). We conclude that FluBu4/TLI is an adequate preparative regiment before dCBT, providing high engraftment rates and relatively early neutrophil recovery. The best survival outcomes were seen in patients without significant comorbidities before transplantation, and outcomes are comparable to previously published dCBT studies.

Ikaros deficiency in host hematopoietic cells separates GVL from GVHD after experimental allogeneic hematopoietic cell transplantation

The graft-versus-leukemia (GVL) effect following allogeneic hematopoietic stem cell transplantation (allo-HCT) is critical for its curative potential. Hwever, GVL is tightly linked to graft-versus-host disease (GVHD). Among hematological malignancies, acute lymphoblastic leukemia (ALL) is the most resistant to GVL, although the reasons for this remain poorly understood. Clinical studies have identified alterations in Ikaros (Ik) transcription factor as the major marker associated with poor outcomes in ALL. We have shown that the absence of Ik in professional host-derived hematopoietic antigen-presenting cells (APCs) exacerbates GVHD. However, whether Ik expression plays a role in resistance to GVL is not known. In this study we used multiple clinically relevant murine models of allo-HCT to explore whether Ik expression in hematopoietic APCs and/or leukemic cells is critical for increasing resistance to GVL and thus inducing relapse. We found that Ik deficiency in host APCs failed to enhance GVL despite increased GVHD severity. Mechanistic studies with bone marrow (BM) chimeras and tetramer analyses demonstrated reduced tumor-specific immunodominant (gag+) antigen responses in the [B6Ik−/−→B6] group. Loss of GVL was observed when both the leukemia cells and the host APCs were deficient in Ik. We found that calreticulin (CRT) expression in host antigen-presenting dendritic cells (DCs) of Ik−/− animals was significantly lower than in wild-type animals. Rescuing CRT expression in Ik−/− DCs improved leukemic-specific cytotoxic T cell function. Together, our data demonstrate that the absence of Ikaros in host hematopoietic cells promotes resistance to GVL despite increasing GVHD and thus provides a potential mechanism for the poor outcome of Ik−/− ALL patients.

Donor Tregs suppress the good with the bad after allogeneic BMT

The therapeutic potential of allogeneic bone marrow transplanation (BMT) relies on the graft versus tumor (GVT) effect involving lloreactive donor T cells. Donor leukocyte infusions (DLI) following llogeneic BMT provide an unequivocal evidence for the potency VT mediated by donor alloreactive T effector cells (Teffs) [1]. Howver, alloreactive donor Teffs also cause graft versus host disease GVHD), a major complication of allogeneic BMT. As such, GVHD nd GVT activity are tightly linked and mechanisms that separate VHD from GVT are not well understood. In this issue, Choi et al. 13] explore the role of donor T cell subsets, the Teffs and donor egulatory T cells (Tregs), in the tumor microenvironment followng delayed DLI. The data suggest while Tregs may suppress the ad, GVHD, they also have the potential to suppress the good, GVT esponses, from the accumulation of donor Tregs in the tumors ollowing DLI after allogeneic BMT. CD4+CD25+ Tregs constitute approximately 5–7% of periphral CD4+ T cells and play a critical role in negatively regulating mmune responses [2]. The naturally occurring Tregs express the ranscription factor forkhead box protein P3 (FoxP3) [3], and supress immune effector responses by using multiple mechanisms epending on the context [4]. Tregs suppress the activation and roliferation of many cell types, including T cells, B cells, dendritic ells, natural killer cells, and NKT cells in vivo and/or in vitro [5,6]. Experimental murine models have clearly demonstrated the eneficial effects of Tregs in the abrogation of GVHD [7]. Tregs have onetheless been reported not to mitigate GVT responses [8]. Howver, the exact mechanisms of Treg function in GVHD and GVT ctivity are not well-understood. Regulatory T cells in the tumor icroenvironment have been shown to decrease tumor clearance nd promote tumor burden in the context of non-BMT models. They lay an important role in reducing the immune surveillance against umors [9,10]. The role of Tregs, especially in the context of the umor microenvironment following allogeneic BMT, is not known. Choi et al. demonstrate that delayed DLI in tolerant mice brogated GVHD and induced GVT responses. Intriguingly, the antiumor effects were not durable and disappeared over time despite