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Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer–magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Abstract Background The Bland-Altman plot with the limits of agreement has been widely used as an absolute index for assessing test-retest reliability or reproducibility between two measurements. We have observed that in the settings where the relative index such as concordance correlation coefficient (CCC) or intraclass correlation coefficient is employed, the limits of agreement approach may be inconsistent with the scaled index. Particularly, the broad width of the limits of agreement may indicate a lack of agreement when the two measurements are highly concordant but an acceptable difference is not known and the common variance of the data is large. This research aims to create a novel, CCC-based guidance for graphical evaluation of reproducibility or reliability. Methods The concordance correlation coefficient is used to create a 100(1-α)% reference band from two measurements. Simulation studies and real examples, including the peak expiratory flow rate data in Bland and Altman’s paper and the test-retest reproducibility data of the Radiomics study, are implemented to assess the use of the reference band. Results In the absence of an acceptable difference between measurements, we found that the limits of agreement may not be consistent with the concordance correlation coefficient. Our simulation study results and real data application show that the proposed method can provide practitioners with a novel graphical evaluation that is consistent with results from the concordance correlation coefficient. Conclusions Our proposed novel scaled index-based guidance can be used for the graphical evaluation of reproducibility or reliability and may have advantages over the limits of agreement in settings where the concordance correlation coefficient is employed.

The objective of this research focuses on the development of a statistical methodology able to answer the question of whether variation in the intake of sulfur amino acids (SAA) affects the metabolic process. Traditional approaches, which evaluate specific biomarkers after a series of preprocessing procedures, have been criticized as not being fully informative, as well as inappropriate for translation of methodology. Rather than focusing on particular biomarkers, our proposed methodology involves the multifractal analysis that measures the inhomogeneity of regularity of the proton nuclear magnetic resonance (1H-NMR) spectrum by wavelet-based multifractal spectrum. With two different statistical models (Model-I and Model-II), three different geometric features of the multifractal spectrum of each 1H-NMR spectrum (spectral mode, left slope, and broadness) are employed to evaluate the effect of SAA and discriminate 1H-NMR spectra associated with different treatments. The investigated effects of SAA include group effect (high and low doses of SAA), depletion/repletion effect, and time over data effect. The 1H-NMR spectra analysis outcomes show that group effect is significant for both models. The hourly variation in time and depletion/repletion effects does not show noticeable differences for the three features in Model-I. However, these two effects are significant for the spectral mode feature in Model-II. The 1H-NMR spectra of the SAA low groups exhibit highly regular patterns with more variability than that of the SAA high groups for both models. Moreover, the discriminatory analysis conducted using the support vector machine and the principal components analysis shows that the 1H-NMR spectra of SAA high and low groups can be easily discriminatory for both models, while the spectra of depletion and repletion within these groups are discriminatory for Model-I and Model-II. Therefore, the study outcomes imply that the amount of SAA is important and that SAA intake affects mostly the hourly variation of the metabolic process and the difference between depletion and repletion each day. In conclusion, the proposed multifractal analysis of 1H-NMR spectra provides a novel tool to investigate metabolic processes.

CD19 CAR T-cell therapy with axicabtagene ciloleucel (axi-cel) for relapsed or refractory (R/R) large B cell lymphoma (LBCL) may lead to durable remissions, however, prolonged cytopenias and infections may occur. In this single center retrospective study of 85 patients, we characterized immune reconstitution and infections for patients remaining in remission after axi-cel for LBCL. Prolonged cytopenias (those occurring at or after day 30 following infusion) were common with >= grade 3 neutropenia seen in 21/70 (30-0%) patients at day 30 and persisting in 3/31 (9-7%) patients at 1 year. B cells were undetectable in 30/34 (88-2%) patients at day 30, but were detected in 11/19 (57-9%) at 1 year. Median IgG levels reached a nadir at day 180. By contrast, CD4 T cells decreased from baseline and were persistently low with a median CD4 count of 155 cells/μl at 1 year after axi-cel (n=19, range 33 – 269). In total, 23/85 (27-1%) patients received IVIG after axi-cel, and 34/85 (40-0%) received G-CSF. Infections in the first 30 days occurred in 31/85 (36-5%) patients, of which 11/85 (12-9%) required intravenous antibiotics or hospitalization (“severe”) and were associated with cytokine release syndrome (CRS), neurotoxicity, tocilizumab use, corticosteroid use, and bridging therapy on univariate analyses. After day 30, 7 severe infections occurred, with no late deaths due to infection. Prolonged cytopenias are common following axi-cel therapy for LBCL and typically recover with time. Most patients experience profound and prolonged CD4 T cell immunosuppression without severe infection.

In older patients with acute myeloid leukemia, the more frequent presence of biologically inherent therapy-resistant disease and increased comorbidities translate to poor overall survival and therapeutic challenges. Optimal front-line therapies for older patients with acute myeloid leukemia remain controversial. We retrospectively evaluated survival outcomes in 980 elderly (≥70 years) acute myeloid leukemia patients from a single institution between 1995 and 2016. Four treatment categories were compared: high-intensity (daunorubicin/cytarabine or equivalent), hypomethylating agent, low-intensity (low-dose cytarabine or similar without hypomethylating agents), and supportive care therapy (including hydroxyurea). At a median follow up of 20.5 months, the median overall survival for the entire cohort was 7.1 months. Multivariate analysis identified secondary acute myeloid leukemia, poor-risk cytogenetics, performance status, front-line therapy, age, white blood cell count, platelet count, and hemoglobin level at diagnosis as having an impact on survival. High-intensity therapy was used in 360 patients (36.7%), hypomethylating agent in 255 (26.0%), low-intensity therapy in 91 (9.3%), and supportive care in 274 (28.0%). Pairwise comparisons between hypomethylating agent therapy and the three other treatment groups demonstrated statistically significant superior median overall survival with hypomethylating agent [14.4 months) vs. high-intensity therapy 10.8 months, hazard ratio 1.35, 95% confidence interval (CI): 1.10-1.65; P =0.004], low-intensity therapy (5.9 months, hazard ratio 2.01, 95%CI: 1.53-2.62; P

Abstract Background Predicting recurrence after stereotactic body radiotherapy (SBRT) in non-small cell lung cancer (NSCLC) patients is problematic, but critical for the decision of following treatment. This study aims to investigate the association of imaging features derived from the first follow-up computed tomography (CT) on lung cancer patient outcomes following SBRT, and identify patients at high risk of recurrence. Methods Fifty nine biopsy-proven non-small cell lung cancer patients were qualified for this study. The first follow-up CTs were performed about 3 months after SBRT (median time: 91 days). Imaging features included 34 manually scored radiological features (semantics) describing the lesion, lung and thorax and 219 quantitative imaging features (radiomics) extracted automatically after delineation of the lesion. Cox proportional hazard models and Harrel’s C-index were used to explore predictors of overall survival (OS), recurrence-free survival (RFS), and loco-regional recurrence-free survival (LR-RFS). Five-fold cross validation was performed on the final prognostic model. Results The median follow-up time was 42 months. The model for OS contained Eastern Cooperative Oncology Group (ECOG) performance status (HR = 3.13, 95% CI: 1.17–8.41), vascular involvement (HR = 3.21, 95% CI: 1.29–8.03), lymphadenopathy (HR = 3.59, 95% CI: 1.58–8.16) and the 1st principle component of radiomic features (HR = 1.24, 95% CI: 1.02–1.51). The model for RFS contained vascular involvement (HR = 3.06, 95% CI: 1.40–6.70), vessel attachment (HR = 3.46, 95% CI: 1.65–7.25), pleural retraction (HR = 3.24, 95% CI: 1.41–7.42), lymphadenopathy (HR = 6.41, 95% CI: 2.58–15.90) and relative enhancement (HR = 1.40, 95% CI: 1.00–1.96). The model for LR-RFS contained vascular involvement (HR = 4.96, 95% CI: 2.23–11.03), lymphadenopathy (HR = 2.64, 95% CI: 1.19–5.82), circularity (F13, HR = 1.60, 95% CI: 1.10–2.32) and 3D Laws feature (F92, HR = 1.96, 95% CI: 1.35–2.83). Five-fold cross-validated the areas under the receiver operating characteristic curves (AUC) of these three models were all above 0.8. Conclusions Our analysis reveals disease progression could be prognosticated as early as 3 months after SBRT using CT imaging features, and these features would be helpful in clinical decision-making.

Acute graft- vs. -host disease (GVHD) is an important cause of morbidity and death after allogeneic hematopoietic cell transplantation (HCT). We identify a new approach to prevent GVHD that impairs monocyte-derived dendritic cell (moDC) alloactivation of T cells, yet preserves graft- vs.-leukemia (GVL). Exceeding endoplasmic reticulum (ER) capacity results in a spliced form of X-box binding protein-1 (XBP-1s). XBP-1s mediates ER stress and inflammatory responses. We demonstrate that siRNA targeting XBP-1 in moDCs abrogates their stimulation of allogeneic T cells. B-I09, an inositol-requiring enzyme-1α (IRE1α) inhibitor that prevents XBP-1 splicing, reduces human moDC migration, allo-stimulatory potency, and curtails moDC IL-1β, TGFβ, and p40 cytokines, suppressing Th1 and Th17 cell priming. B-I09-treated moDCs reduce responder T cell activation via calcium flux without interfering with regulatory T cell (Treg) function or GVL effects by cytotoxic T lymphocytes (CTL) and NK cells. In a human T cell mediated xenogeneic GVHD model, B-I09 inhibition of XBP-1s reduced target-organ damage and pathogenic Th1 and Th17 cells without impacting donor Tregs or anti-tumor CTL. DC XBP-1s inhibition provides an innovative strategy to prevent GVHD and retain GVL.

T-helper 1 and T-helper 17 lymphocytes mediate acute graft-versus-host disease (GvHD). Interleukin 12 is critical for T-helper 1 differentiation and interleukin 23 for T-helper 17 maintenance. Interleukin 12 and 23 are heterodimeric cytokines that share the p40 subunit (IL-12/IL-23p40). In a randomized, blinded, placebo-controlled trial, we examined the biological impact and clinical outcomes following IL-12/IL-23p40 neutralization using ustekinumab. Thirty patients received peripheral blood mobilized hematopoietic cell transplantation (HCT) from HLA-matched sibling or unrelated donors, received sirolimus plus tacrolimus as GvHD prophylaxis, and were randomized to ustekinumab versus placebo with 1:1 allocation after stratification by donor type. The primary end point of the trial was the mean percentage (%) T-regulatory (Treg) cells on day 30 post HCT. Ustekinumab was delivered by subcutaneous injection on day −1 and day +20 after transplantation. On day 30 post transplant, no significant difference in % Treg was observed. Ustekinumab suppressed serum IL-12/IL-23p40 levels. Host-reactive donor alloresponse at days 30 and 90 after transplantation was polarized with significant reduction in IL-17 and IFN-α production and increase in IL-4. No toxicity attributed to ustekinumab was observed. Overall survival and National Institute of Health moderate/severe chronic GvHD-free, relapse-free survival were significantly improved among ustekinumab-treated patients. No significant improvements were observed in acute or chronic GvHD, relapse, or non-relapse mortality. These data provide first evidence that IL-12/IL-23p40 neutralization can polarize donor anti-host alloresponse in vivo and provide initial clinical efficacy evidence to be tested in subsequent trials. (Trial registered at clinicaltrials.gov identifier: 01713400.)

Graft-versus-host disease (GvHD) remains a major cause of transplant-related mortality. Interleukin-2 (IL-2) plus sirolimus (SIR) synergistically reduces acute GvHD in rodents and promotes regulatory T cells. This phase II trial tested the hypothesis that IL-2 would facilitate STAT5 phosphorylation in donor T cells, expand regulatory T cells, and ameliorate GvHD. Between 16th April 2014 and 19th December 2015, 20 patients received IL-2 (200,000 IU/m2 thrice weekly, days 0 to +90) with SIR (5–14 ng/mL) and tacrolimus (TAC) (3–7 ng/mL) after HLA-matched related or unrelated allogeneic hematopoietic cell transplantation (HCT). The study was designed to capture an increase in regulatory T cells from 16.0% to more than 23.2% at day +30. IL-2/SIR/TAC significantly increased regulatory T cells at day +30 compared to our published data with SIR/TAC (23.8% vs. 16.0%, P=0.0016; 0.052 k/uL vs. 0.037 k/uL, P=0.0163), achieving the primary study end point. However, adding IL-2 to SIR/TAC led to a fall in regulatory T cells by day +90 and did not reduce acute or chronic GvHD. Patients who discontinued IL-2 before day +100 showed a suggested trend toward less grade II-IV acute GvHD (16.7% vs. 50%, P=0.1475). We surmise that the reported accumulation of IL-2 receptors in circulation over time may neutralize IL-2, lead to progressive loss of regulatory T cells, and offset its clinical efficacy. The amount of phospho-STAT3+ CD4+ T cells correlated with donor T-cell activation and acute GvHD incidence despite early T-cell STAT5 phosphorylation by IL-2. Optimizing IL-2 dosing and overcoming cytokine sequestration by soluble IL-2 receptor may sustain lasting regulatory T cells after transplantation. However, an approach to target STAT3 is needed to enhance GvHD prevention. (clinicaltrials.gov identifier: 01927120).

Effective pharmacological strategies employed in allogeneic hematopoietic cell transplantation should prevent serious chronic graft-versus-host disease and facilitate donor-recipient immune tolerance. Based on demonstrated pro-tolerogenic activity, sirolimus (rapamycin) is an agent with promise to achieve these goals. In a long-term follow-up analysis of a randomized phase II trial comparing sirolimus/tacrolimus versus methotrexate/tacrolimus for graft-versus-host disease prevention in matched sibling or unrelated donor transplant, we examined the impact of prolonged sirolimus administration (≥ 1 year post-transplant). Median follow-up time for surviving patients at time of this analysis was 41 months (range 27–60) for sirolimus/tacrolimus and 49 months (range 29–63) for methotrexate/tacrolimus. Sirolimus/tacrolimus patients had significantly lower National Institutes of Health Consensus moderate-severe chronic graft-versus-host disease (34% vs. 65%; P=0.004) and late acute graft-versus-host disease (20% vs. 43%; P=0.04). While sirolimus/tacrolimus patients had lower prednisone exposure and earlier discontinuation of tacrolimus (median time to tacrolimus discontinuation 368 days vs. 821 days; P=0.002), there was no significant difference in complete immune suppression discontinuation (60-month estimate: 43% vs. 31%; P=0.78). Prolonged sirolimus administration represents a viable approach to mitigate risk for moderate-severe chronic and late acute graft-versus-host disease. Further study of determinants of successful immune suppression discontinuation is needed.

Background There is evidence suggesting that sirolimus, in combination with tacrolimus, is active in the prevention of graft-versus-host disease. Sirolimus-based immune suppression may suppress alloreactive T cells, while sparing the survival and function of regulatory T cells.Design and Methods We conducted a randomized trial to compare the impact of sirolimus/tacrolimus against that of methotrexate/tacrolimus on the prevention of graft-versus-host disease and regulatory T-cell reconstitution.Results Seventy-four patients were randomized 1:1 to sirolimus/tacrolimus or methotrexate/tacrolimus, stratified for type of donor (sibling or unrelated) and the patients' age. The rate of grade II-IV acute graft-versus-host disease at 100 days was 43% (95% CI: 27-59%) in the sirolimus/tacrolimus group and 89% (95% CI: 72-96%) in the methotrexate/tacrolimus group (P

Background The 2005 National Institutes of Health Consensus Development Conference on chronic graft-versus-host disease proposed major changes in the classification and grading of severity of chronic graft-versus-host disease.Design and Methods We aimed to study the association of the proposed chronic graft-versus-host disease classification and global severity with transplantation outcomes among a consecutive series of patients who received pharmacokinetically-targeted doses of intravenous busulfan and fludarabine conditioning followed by transplantation of allogeneic peripheral blood stem cells.Results From a total cohort (n = 242) of patients surviving more than 100 days after hematopoietic stem cell transplantation, 181 (75% of those at risk) had some manifestations of graft-versus-host disease after day 100. Of these, at onset 13 (7%) had late acute graft-versus-host disease, 62 (34%) had classic chronic graft-versus-host disease, and 106 (59%) had the overlap subtype of chronic graft-versus-host disease. The global severity of the chronic graft-versus-host disease was mild in 25% of cases, moderate in 46%, and severe in 29%. Multivariable modeling demonstrated the independent association of global severity of chronic graft-versus-host disease with overall survival (moderate/severe versus mild; HR 2.9, 95% CI 1.8–4.7, P