Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination.

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.

Prophylactic maintenance with venetoclax/azacitidine after reduced-intensity conditioning allogeneic transplant for high-risk MDS and AML.

ABSTRACT: We conducted a phase 1 trial assessing safety and efficacy of prophylactic maintenance therapy with venetoclax and azacitidine (Ven/Aza) for patients with high-risk myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) undergoing reduced intensity allogeneic stem cell transplantation (allo-SCT) after Ven and fludarabine/busulfan conditioning (Ven/FluBu2 allo-SCT) with tacrolimus and methotrexate as graft-versus-host disease (GVHD) prophylaxis. Among 27 patients who underwent Ven/FluBu2 allo-SCT (55.6% with prior Ven exposure, and 96% with positive molecular measurable residual disease), 22 received maintenance therapy with Aza 36 mg/m2 intravenously on days 1 to 5, and Ven 400 mg by mouth on days 1 to 14 per assigned dose schedule/level (42-day cycles × 8, or 28-day cycles × 12). During maintenance, the most common grade 3-4 adverse events were leukopenia, neutropenia, and thrombocytopenia, which were transient and manageable. Infections were uncommon (n = 4, all grade 1-2). The 1-year and 2-year moderate/severe chronic GVHD rates were 4% (95% confidence interval [CI], 0.3%-18%) and 22% (95% CI, 9%-40%), respectively. After a median follow-up of 25 months among survivors, the median overall survival (OS) was not reached. Among the 22 patients who received Ven/Aza maintenance, the 2-year OS, progression-free survival, nonrelapse mortality, and cumulative incidence of relapse rates were 67% (95% CI, 43%-83%), 59% (95% CI, 36%-76%), 0%, and 41% (95% CI, 20%-61%), respectively. Immune monitoring demonstrated no significant impact on T-cell expansion but identified reduced B-cell expansion compared with controls. This study demonstrates prophylactic Ven/Aza maintenance can be safely administered for patients with high-risk MDS/AML, but a randomized study is required to properly assess any potential benefit. This trial was registered at www.clinicaltrials.gov as #NCT03613532.

Clinical trial links oncolytic immunoactivation to survival in glioblastoma.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM)1,2. Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV)3. In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue4. These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

Inaugural Results of the Individualized Screening Trial of Innovative Glioblastoma Therapy: A Phase II Platform Trial for Newly Diagnosed Glioblastoma Using Bayesian Adaptive Randomization.

PURPOSE: The Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) is a phase II platform trial that uses response adaptive randomization and genomic profiling to efficiently identify novel therapies for phase III testing. Three initial experimental arms (abemaciclib [a cyclin-dependent kinase [CDK]4/6 inhibitor], neratinib [an epidermal growth factor receptor [EGFR]/human epidermal growth factor receptor 2 inhibitor], and CC-115 [a deoxyribonucleic acid-dependent protein kinase/mammalian target of rapamycin inhibitor]) were simultaneously evaluated against a common control arm. We report the results for each arm and examine the feasibility and conduct of the adaptive platform design. PATIENTS AND METHODS: Patients with newly diagnosed O6-methylguanine-DNA methyltransferase-unmethylated glioblastoma were eligible if they had tumor genotyping to identify prespecified biomarker subpopulations of dominant glioblastoma signaling pathways (EGFR, phosphatidylinositol 3-kinase, and CDK). Initial random assignment was 1:1:1:1 between control (radiation therapy and temozolomide) and the experimental arms. Subsequent Bayesian adaptive randomization was incorporated on the basis of biomarker-specific progression-free survival (PFS) data. The primary end point was overall survival (OS), and one-sided P values are reported. The trial is registered with ClinicalTrials.gov (identifier: NCT02977780). RESULTS: Two hundred thirty-seven patients were treated (71 control; 73 abemaciclib; 81 neratinib; 12 CC-115) in years 2017-2021. Abemaciclib and neratinib were well tolerated, but CC-115 was associated with ≥ grade 3 treatment-related toxicity in 58% of patients. PFS was significantly longer with abemaciclib (hazard ratio [HR], 0.72; 95% CI, 0.49 to 1.06; one-sided P = .046) and neratinib (HR, 0.72; 95% CI, 0.50 to 1.02; one-sided P = .033) relative to the control arm but there was no PFS benefit with CC-115 (one-sided P = .523). None of the experimental therapies demonstrated a significant OS benefit (P > .05). CONCLUSION: The INSIGhT design enabled efficient simultaneous testing of three experimental agents using a shared control arm and adaptive randomization. Two investigational arms had superior PFS compared with the control arm, but none demonstrated an OS benefit. The INSIGhT design may promote improved and more efficient therapeutic discovery in glioblastoma. New arms have been added to the trial.

Intratumoral drug-releasing microdevices allow in situ high-throughput pharmaco phenotyping in patients with gliomas.

The lack of reliable predictive biomarkers to guide effective therapy is a major obstacle to the advancement of therapy for high-grade gliomas, particularly glioblastoma (GBM), one of the few cancers whose prognosis has not improved over the past several decades. With this pilot clinical trial (number NCT04135807), we provide first-in-human evidence that drug-releasing intratumoral microdevices (IMDs) can be safely and effectively used to obtain patient-specific, high-throughput molecular and histopathological drug response profiling. These data can complement other strategies to inform the selection of drugs based on their observed antitumor effect in situ. IMDs are integrated into surgical practice during tumor resection and remain in situ only for the duration of the otherwise standard operation (2 to 3 hours). None of the six enrolled patients experienced adverse events related to the IMD, and the exposed tissue was usable for downstream analysis for 11 out of 12 retrieved specimens. Analysis of the specimens provided preliminary evidence of the robustness of the readout, compatibility with a wide array of techniques for molecular tissue interrogation, and promising similarities with the available observed clinical-radiological responses to temozolomide. From an investigational aspect, the amount of information obtained with IMDs allows characterization of tissue effects of any drugs of interest, within the physiological context of the intact tumor, and without affecting the standard surgical workflow.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia.

The importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

Autopsy findings from patients diagnosed with COVID-19 demonstrate unique morphological patterns in bone marrow and lymph node.

AIMS: The identification of haemophagocytosis in bone marrow (BM) is recurrently identified in patients with severe COVID-19. These initial COVID-19 autopsy studies have afforded valuable insight into the pathophysiology of this disease; however, only a limited number of case series have focused on lymphoid or haematopoietic tissues. METHODS: BM and lymph node (LN) specimens were obtained from adult autopsies performed between 1 April 2020 and 1 June 2020, for which the decedent had tested positive for SARS-CoV-2. Tissue sections (H&E, CD3, CD20, CD21, CD138, CD163, MUM1, kappa/lambda light chains in situ hybridisation) were examined by two haematopathologists, who recorded morphological features in a blinded fashion. Haemophagocytic lymphohistiocytosis (HLH) was assessed based on HLH 2004 criteria. RESULTS: The BM demonstrated a haemophagocytic pattern in 9 out of 25 patients (36%). The HLH pattern was associated with longer hospitalisation, BM plasmacytosis, LN follicular hyperplasia and lower aspartate aminotransferase (AST), as well as ferritin at demise. LN examination showed increased plasmacytoid cells in 20 of 25 patients (80%). This pattern was associated with a low absolute monocyte count at diagnosis, lower white cell count and lower absolute neutrophil count at demise, and lower ferritin and AST at demise. CONCLUSIONS: Autopsy results demonstrate distinct morphological patterns in BM, with or without haemophagocytic macrophages, and in LN, with or without increased plasmacytoid cells. Since only a minority of patients met diagnostic criteria for HLH, the observed BM haemophagocytic macrophages may be more indicative of an overall inflammatory state.

Clonal Hematopoiesis in Young Women Treated for Breast Cancer.

PURPOSE: Young women treated for breast cancer with cytotoxic therapies are at risk for clonal hematopoiesis of indeterminate potential (CHIP), a condition in which blood cells carrying a somatic mutation associated with hematologic malignancy comprise at least 4% of the total blood system. CHIP has primarily been studied in older patient cohorts with limited clinical phenotyping. EXPERIMENTAL DESIGN: We performed targeted sequencing on longitudinal blood samples to characterize the clonal hematopoietic landscape of 878 women treated for breast cancer enrolled in the prospective Young Women's Breast Cancer Study. RESULTS: We identified somatic driver mutations in 252 study subjects (28.7%), but only 24 (2.7%) had clones large enough to meet criteria for CHIP. The most commonly mutated genes were DNMT3A and TET2, similar to mutations observed in noncancer cohorts. At 9-year median follow-up, we found no association between the presence of a somatic blood mutation (regardless of clone size) and adverse breast cancer (distant relapse-free survival) or non-breast cancer-related outcomes in this cohort. A subset of paired blood samples obtained over 4 years showed no evidence of mutant clonal expansion, regardless of genotype. Finally, we identified a subset of patients with likely germline mutations in genes known to contribute to inherited cancer risk, such as TP53 and ATM. CONCLUSIONS: Our data show that for young women with early-stage breast cancer, CHIP is uncommon after cytotoxic exposure, is unlikely to contribute to adverse outcomes over the decade-long follow-up and may not require additional monitoring if discovered incidentally.

Targeting MET and FGFR in Relapsed or Refractory Acute Myeloid Leukemia: Preclinical and Clinical Findings, and Signal Transduction Correlates.

PURPOSE: Patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) have poor outcomes and require new therapies. In AML, autocrine production of hepatocyte growth factor (HGF) drives MET signaling that promotes myeloblast growth and survival, making MET an attractive therapeutic target. MET inhibition exhibits activity in AML preclinical studies, but HGF upregulation by the FGFR pathway is a common mechanism of resistance. PATIENTS AND METHODS: We performed preclinical studies followed by a Phase I trial to investigate the safety and biological activity of the MET inhibitor merestinib in combination with the FGFR inhibitor LY2874455 for patients with R/R AML. Study Cohort 1 underwent a safety lead-in to determine a tolerable dose of single-agent merestinib. In Cohort 2, dose-escalation of merestinib and LY2874455 was performed following a 3+3 design. Correlative studies were conducted. RESULTS: The primary dose-limiting toxicity (DLT) observed for merestinib alone or with LY2874455 was reversible grade 3 transaminase elevation, occurring in 2 of 16 patients. Eight patients had stable disease and one achieved complete remission (CR) without measurable residual disease. Although the MTD of combination therapy could not be determined due to drug supply discontinuation, single-agent merestinib administered at 80 mg daily was safe and biologically active. Correlative studies showed therapeutic plasma levels of merestinib, on-target attenuation of MET signaling in leukemic blood, and increased HGF expression in bone marrow aspirate samples of refractory disease. CONCLUSIONS: We provide prospective, preliminary evidence that MET and FGFR are biologically active and safely targetable pathways in AML.

Comparison of demographics, disease characteristics, and outcomes between Black and White patients with myelodysplastic syndromes: A population-based study.

Racial disparities in cancer care and outcomes have been well documented in various malignancies, with Black patients having the highest death rate and shortest survival of any racial/ethnic group in the United States (US) for most cancers. However, there have been limited studies on racial/ethnic disparities in myelodysplastic syndromes (MDS). Our study characterized and compared differences in baseline demographics, clinical characteristics, socioeconomic factors, and overall survival (OS) between Black and White patients with MDS in the US. We used the Surveillance, Epidemiology, and End Results (SEER) Program and included 37,562 patients (Black, 8.1 %; White, 91.9 %) diagnosed between 2001 and 2013. We observed significant differences in baseline characteristics between cohorts. In a univariate analysis, Black race was associated with longer survival (hazard ratio [HR]: 0.83; 95 % confidence interval [CI], 0.79-0.86; p < 0.001). The association between race and survival was attenuated but remained significant in various models to adjust for differences in baseline characteristics (HR in multivariable analysis, 0.92; 95 % CI, 0.87-0.96); p < 0.001). Subgroup analysis by histology revealed differences in the association between race and OS. Refractory anemia (RA), RA with ring sideroblasts, and MDS-not otherwise specified, a category in SEER representing a poorly defined MDS subset for 52 % of cases in our study, favored Black patients. RA with excess blasts favored White patients. The overall finding that Black race is associated with better OS outcomes, when compared with White patients, needs to be interpreted with caution and nuanced by histology. Additional research to explore these associations is warranted.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

Patient perspectives on testing for clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP), an emerging biomarker for personalized risk-directed interventions, is increased in cancer survivors. However, little is known about patient preferences for CHIP testing. We surveyed participants in a prospective cohort study of young women with breast cancer (BC). The emailed survey included an introduction to CHIP and a vignette eliciting participants' preferences for CHIP testing, considering sequentially: population-based 10-year risk of BC recurrence, hematologic malignancy, and heart disease; increased CHIP-associated risks; current CHIP management; dedicated CHIP clinic; and hypothetical CHIP treatment. Preference changes were evaluated using the McNemar test. The survey response rate was 82.2% (528/642). Median age at time of survey was 46 years and median time from diagnosis was 108 months. Only 5.9% had prior knowledge of CHIP. After vignette presentation, most survivors (87.1%) recommended CHIP testing for the vignette patient. Presented next with CHIP-independent, population-based risks, 11.1% shifted their preference from testing to not testing. After receiving information about CHIP-associated risks, an additional 10.1% shifted their preference to testing. Preference for testing increased if vignette patient was offered a CHIP clinic or hypothetical CHIP treatment, with 7.2% and 14.1% switching preferences toward testing, respectively. Finally, 75.8% of participants desired CHIP testing for themselves. Among participants, 28.2% reported that learning about CHIP caused at least moderate anxiety. Most young survivors favored CHIP testing, with preferences influenced by risk presentation and potential management strategies. Our findings highlight the importance of risk communication and psychosocial support when considering biomarkers for future risk in cancer survivors. This trial has been registered at www.clinicaltrials.gov as #NCT01468246.

Peripheral blood CD3+ T-cell gene expression biomarkers correlate with clinical frailty in patients with haematological malignancies.

Older patients with cancer often receive treatment regimens based on their age without considering other objective factors that may influence outcomes. Assessment of frailty can identify older patients who are robust and therefore more likely to benefit from intensive treatment, or conversely, frail and might instead be offered alternative approaches. However, such assessment requires specialised training and dedicated clinical resources. Alternative quantitative biomarkers associated with frailty are lacking. Here, we asked if expression signatures of 74 immune cell, ageing, and senescence-related messenger RNAs in purified peripheral blood T cells could identify associations with clinical frailty in patients with haematological malignancies. We studied 69 patients between the ages of 36 and 92 years (median 76 years) with leukaemia, lymphoma, or multiple myeloma, across two institutions. Expression of four genes (aryl hydrocarbon receptor [AHR], CD27, CD28, and interleukin-2 receptor subunit alpha [IL2RA; CD25]) in T cells was associated with frailty, independent of age. An expression-based regression model had 76% sensitivity and 90% specificity to assign a patient as robust. These data identify measurable peripheral blood correlates of clinical frailty and suggest biomarkers for future prospective assessment.

Changes in Surgical Pathology Case Volume and Amendment Rates During the COVID-19 Pandemic.

OBJECTIVES: Surgical pathology volume decreased during the peak of the coronavirus disease 2019 (COVID-19) pandemic. We looked at the 4 months with the greatest reduction in surgical pathology volume during the COVID-19 pandemic and compared them with those same months in 2019 to determine changes in specimen volume. We compared the amendment rates during those periods and types of amendments issued (identification [ID], report defect [RD], diagnostic information [DI]). METHODS: All pathology reports between March to June 2019 and March to June 2020 were extracted from the pathology information system. All amendments issued were extracted over the same period and then subclassified by two pathologists. RESULTS: There was a 52.1% reduction in surgical pathology volume between the 4-month periods in 2019 and 2020 (P = .04). The amendment rate was 0.9% in 2019 compared with 1.4% in 2020, representing a 65.5% increase in amendments overall. There was a 53.3% reduction in amendments issued for ID, a 3.8% reduction in RD, and a 23.2% increase in amendments issued for DI. The change in amendments was not statistically significant. CONCLUSIONS: These findings suggest that a reduction in workload would not improve error rates. The circumstances of the pandemic highlight the many factors contributing to error rates in surgical pathology.

Historical expectations with DNA methyltransferase inhibitor monotherapy in MDS: when is combination therapy truly "promising"?

DNA methyltransferase inhibitors (DNMTIs) for patients with higher risk myelodysplastic syndromes (HR-MDS) have low complete remission rates and are not curative. Early DNMTI combination clinical trials in HR-MDS are often termed "promising," but many randomized trials subsequently failed to show benefit. Clearer understanding of when a combination is likely to improve upon DNMTI monotherapy would inform randomized studies. We reviewed MDS azacitidine or decitabine monotherapy studies. We collected baseline demographics including International Prognostic Scoring System (IPSS) risk, DNMTI, disease characteristics; and response variables including survival and marrow and hematologic responses. Aggregate estimates across studies were calculated using meta-analyses techniques. Using a binomial design, we estimated the necessary operating characteristics to design a phase 2 study showing improved efficacy of a combination over monotherapy. Among 1908 patients, the overall response rate (ORR) was 24% (n = 464; 95% confidence interval [CI], 0.22-0.26): 267 complete response (CR, 14%), 68 partial response (4%), and 129 marrow complete remission (7%). Among 1604 patients for whom a hematologic response was reported, 476 (30%; 95% CI, 0.27-0.32) reported hematologic improvement (HI). More patients treated with azacitidine achieved HI (38%; 95% CI, 0.35-0.41) compared with decitabine (15%; 95% CI, 0.13-0.19), whereas the marrow ORR rate was higher with decitabine (29%; 95% CI, 0.26-0.33) compared with azacitidine (21%; 95% CI, 0.19-0.23). CR rates were similar between DNMTIs: 13% with azacitidine and 16% with decitabine. Variables that influence MDS response include the specific DNMTI backbone and the distribution of IPSS risk of patients enrolled on a trial. Considering these factors can help identify which early combination approaches are worth assessing in larger randomized trials.

Cancer patient survival can be parametrized to improve trial precision and reveal time-dependent therapeutic effects.

Individual participant data (IPD) from oncology clinical trials is invaluable for identifying factors that influence trial success and failure, improving trial design and interpretation, and comparing pre-clinical studies to clinical outcomes. However, the IPD used to generate published survival curves are not generally publicly available. We impute survival IPD from ~500 arms of Phase 3 oncology trials (representing ~220,000 events) and find that they are well fit by a two-parameter Weibull distribution. Use of Weibull functions with overall survival significantly increases the precision of small arms typical of early phase trials: analysis of a 50-patient trial arm using parametric forms is as precise as traditional, non-parametric analysis of a 90-patient arm. We also show that frequent deviations from the Cox proportional hazards assumption, particularly in trials of immune checkpoint inhibitors, arise from time-dependent therapeutic effects. Trial duration therefore has an underappreciated impact on the likelihood of success.