Results from a phase I trial of pembrolizumab plus vorinostat in relapsed/refractory B-cell non-Hodgkin lymphoma.

Outcomes after programmed death-1 (PD-1) blockade in B-cell lymphomas are disappointing with few durable responses. Histone deacetylase inhibitors exhibit favorable immunomodulatory effects and demonstrate synergistic anti-tumor immune responses with anti-PD-1 therapy in preclinical models. We, therefore, developed a phase I study to evaluate the safety and preliminary efficacy of pembrolizumab with vorinostat in relapsed/refractory B-cell lymphomas. Patients were treated in a dose-escalation cohort using a Rolling 6 design followed by an expansion cohort at the recommended phase II dose (R2PD). Fifty-two patients were enrolled (32 Hodgkin and 20 non-Hodgkin lymphoma [NHL]). Here, we report safety data from the dose escalation cohort, and the toxicity and efficacy within NHL patients. Vorinostat was administered twice daily on days 1-5 and 8-12 (dose-level [DL]1: 100 mg; DL2: 200 mg) and pembrolizumab (200 mg) was administered on day 1 of each 3-week cycle. Of six patients treated at DL1, one had a dose-limiting toxicity (DLT) (Stevens-Johnson syndrome [SJS]), and one of six had a DLT at DL2 (thromboembolism); therefore, DL2 was the RP2D. The patient developing SJS was treated with corticosteroids, infliximab, and cyclosporine but ultimately died of invasive fungal infection from the extensive immunosuppression used to treat the SJS. The most common adverse events were hypertension, diarrhea, and cytopenias. Of 20 NHL patients, nine had follicular lymphoma (FL) and 11 had diffuse large B-cell lymphoma (DLBCL). Five DLBCL patients had primary mediastinal B-cell lymphoma (PMBL). The complete and overall response rates (CR and ORR) were 11% and 22% for FL and 45% and 55% for all DLBCL. Amongst DLBCL, the CR and ORR was 80% and 80% for PMBL and 17% and 33% for non-PMBL. In conclusion, pembrolizumab with vorinostat was tolerable and produced responses in relapsed/refractory B-cell NHL, with particularly notable efficacy in PMBL (clinicaltrials gov. Identifier: NCT03150329).

High-grade B-cell lymphoma with concurrent MYC rearrangement and 11q aberrations: clinicopathologic, cytogenetic, and molecular characterization of 4 cases.

High-grade B-cell lymphoma with 11q aberrations (LBL-11q) resembles Burkitt lymphoma (BL), is negative for MYC rearrangement, and harbors chromosome 11q aberrations. Rare cases of high-grade B-cell lymphoma with concurrent MYC rearrangement and 11q aberrations (HGBCL-MYC-11q) have been described. In this study, we report the clinicopathologic, cytogenetic, and molecular findings in 4 such cases. Diagnoses were made on tissue or bone marrow biopsies. Karyotype, fluorescence in situ hybridization, genomic microarray analyses, and next-generation sequencing were performed. All patients were male (median age, 39 years). Three cases were diagnosed as BL, while one was diagnosed as diffuse large B-cell lymphoma. Karyotypes (available in 2 patients) were complex. In 1 patient, copy number analysis showed gains at 1q21.1-q44 and 13q31.3 and loss of 13q34, abnormalities typically seen in BL. All of our cases showed 2 or more mutations that are recurrent in BL, including ID3, TP53, DDX3X, CCND3, FBXO1, and MYC. Two cases showed a GNA13 mutation, commonly seen in LBL-11q. Cases of HGBCL-MYC-11q display overlapping morphologic and immunophenotypic, as well as cytogenetic and molecular features between BL and LBL-11q, with a mutational landscape enriched for mutations recurrent in BL. Concurrent MYC rearrangement with 11q abnormalities is important to recognize, especially as it has implications for their classification.

Low T-cell proportion in the tumor microenvironment is associated with immune escape and poor survival in diffuse large B-cell lymphoma.

The tumor microenvironment (TME) is important in the pathogenesis and prognosis of lymphoma. Previous studies have demonstrated that features of the diffuse large B-cell lymphoma (DLBCL) TME can be associated with prognosis, but questions remain about the mechanisms underlying these TME features, and the interplay between tumor cells and the local TME. Therefore, we performed multispectral immunofluorescence (mIF) using two 6-color panels to interrogate the cellular proportions of T-cell subsets, macrophages, and natural killer cells in 57 cases of de novo DLBCL treated with R-CHOP chemotherapy. We found that very low CD3+ T-cell proportion and low CD4+PD1+ and CD8+PD1+ T cells have poor survival compared to those with a high T-cell proportion. Also, cases with concurrently low TIM3 and PD1 have a poor prognosis. This poor prognosis with low T-cell proportion was validated using immune deconvolution of gene expression profiling data from 351 cases of DLBCL and an additional cohort of 53 cases of DLBCL using routine immunohistochemistry. In addition, cases with loss of B2M, HLA I and/or HLA II protein expression on the tumor cells also had a low T-cell proportion, providing evidence that lack of these proteins allows for immune evasion. Overall, our results show that patients with DLBCL with a low T-cell proportion in the TME have a poor survival when treated with R-CHOP and exhibit mechanisms of immune escape.

Vascular Endothelial Growth Factor as an Immediate-Early Activator of Ultraviolet-Induced Skin Injury.

The negative health consequences of acute ultraviolet (UV) exposure are evident, with reports of 30,000 emergency room visits annually to treat the effects of sunburn in the United States alone. The acute effects of sunburn include erythema, edema, severe pain, and chronic overexposure to UV radiation, leading to skin cancer. Whereas the pain associated with the acute effects of sunburn may be relieved by current interventions, existing post-sunburn treatments are not capable of reversing the cumulative and long-term pathological effects of UV exposure, an unmet clinical need. Here we show that activation of the vascular endothelial growth factor (VEGF) pathway is a direct and immediate consequence of acute UV exposure, and activation of VEGF signaling is necessary for initiating the acute pathological effects of sunburn. In UV-exposed human subjects, VEGF signaling is activated within hours. Topical delivery of VEGF pathway inhibitors, targeted against the ligand VEGF-A (gold nanoparticles conjugated with anti-VEGF antibodies) and small-molecule antagonists of VEGF receptor signaling, prevent the development of erythema and edema in UV-exposed mice. These findings collectively suggest targeting VEGF signaling may reduce the subsequent inflammation and pathology associated with UV-induced skin damage, revealing a new postexposure therapeutic window to potentially inhibit the known detrimental effects of UV on human skin. It is essential to emphasize that these preclinical studies must not be construed as suggesting in any way the use of VEGF inhibitors as a sunburn treatment in humans because warranted future clinical studies and appropriate agency approval are essential in that regard.

Relative frequency and clinicopathologic characteristics of MYC-rearranged follicular lymphoma.

MYC rearrangement is a relatively rare genetic abnormality in follicular lymphoma (FL). In this study, we evaluated the relative frequency of MYC rearrangement in 522 cases of FL and studied their clinicopathologic, cytogenetic, and molecular characteristics. Fluorescence in situ hybridization studies for MYC (break-apart probe), MYC/IGH, IGH/BCL2, and BCL6 rearrangements were performed on tissue microarrays. Immunohistochemical stains for CD10, BCL2, BCL6, and MYC were performed and scored on MYC-rearranged cases. On 4 FL cases, a custom targeted panel of 356 genes was used for mutation analysis. Ten cases (1.9%) were positive for MYC rearrangement. Histologically, 6 of 10 cases were grade 1-2, and 4 cases were grade 3A. By immunohistochemistry, 9 of 9 tested cases were CD10+, all cases were BCL6+, and 9/10 cases were BCL2+. MYC protein staining was low in all cases tested. IGH/BCL2 rearrangement was detected in 5 of 9 cases, whereas BCL6 rearrangement was detected in 3 of 7 tested cases and 4 of 10 cases showed MYC/IGH rearrangement. The most commonly detected mutations in the MYC-positive cases included HLA-B, TNFRSF14, and KMT2D. MYC and/or B2M abnormalities were detected in 2 cases. In conclusion, MYC rearrangement is uncommon in FL and these cases do not appear to have specific histologic characteristics. Molecular analysis showed abnormalities in genes associated with transformation, namely MYC and B2M. Larger studies are needed to evaluate if MYC-rearrangement in FL has prognostic significance.

Validation of the Double-Hit Gene Expression Signature (DLBCL90) in an Independent Cohort of Patients with Diffuse Large B-Cell Lymphoma of Germinal Center Origin.

The prognosis of diffuse large B-cell lymphoma (DLBCL) has been associated with clinical parameters, cell of origin, and various genetic aberrations. Recently, a NanoString gene expression assay (DLBCL90) was developed, which identifies DLBCL cases with an outcome similar to those with double- or triple-hit DLBCL with both MYC and BCL2 rearrangements. This study validates the predictive ability of the DLBCL90 assay in an independent cohort of patients with the germinal center B-cell subtype DLBCL. A customized targeted sequencing panel was used to analyze the mutational profile in these patients. Cases with a double or triple hit by conventional fluorescence in situ hybridization cytogenetic analysis are known to have a poor prognosis, and the DLBCL90 gene expression signature identified these cases, as well as additional cases that would have otherwise been missed by fluorescence in situ hybridization analysis. Our findings validate use of the DLBCL90 assay for identifying high-risk patients for new and innovative therapies.

Double-hit Signature with TP53 Abnormalities Predicts Poor Survival in Patients with Germinal Center Type Diffuse Large B-cell Lymphoma Treated with R-CHOP.

PURPOSE: We performed detailed genomic analysis on 87 cases of de novo diffuse large B-cell lymphoma of germinal center type (GCB DLBCL) to identify characteristics that are associated with survival in those treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). EXPERIMENTAL DESIGN: The cases were extensively characterized by combining the results of IHC, cell-of-origin gene expression profiling (GEP; NanoString), double-hit GEP (DLBCL90), FISH cytogenetic analysis for double/triple-hit lymphoma, copy-number analysis, and targeted deep sequencing using a custom mutation panel of 334 genes. RESULTS: We identified four distinct biologic subgroups with different survivals, and with similarities to the genomic classifications from two large retrospective studies of DLBCL. Patients with the double-hit signature, but no abnormalities of TP53, and those lacking EZH2 mutation and/or BCL2 translocation, had an excellent prognosis. However, patients with an EZB-like profile had an intermediate prognosis, whereas those with TP53 inactivation combined with the double-hit signature had an extremely poor prognosis. This latter finding was validated using two independent cohorts. CONCLUSIONS: We propose a practical schema to use genomic variables to risk-stratify patients with GCB DLBCL. This schema provides a promising new approach to identify high-risk patients for new and innovative therapies.

Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol.

The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.

A vertebrate model to reveal neural substrates underlying the transitions between conscious and unconscious states.

The field of neuropharmacology has not yet achieved a full understanding of how the brain transitions between states of consciousness and drug-induced unconsciousness, or anesthesia. Many small molecules are used to alter human consciousness, but the repertoire of underlying molecular targets, and thereby the genes, are incompletely understood. Here we describe a robust larval zebrafish model of anesthetic action, from sedation to general anesthesia. We use loss of movement under three different conditions, spontaneous movement, electrical stimulation or a tap, as a surrogate for sedation and general anesthesia, respectively. Using these behavioral patterns, we find that larval zebrafish respond to inhalational and IV anesthetics at concentrations similar to mammals. Additionally, known sedative drugs cause loss of spontaneous larval movement but not to the tap response. This robust, highly tractable vertebrate model can be used in the detection of genes and neural substrates involved in the transition from consciousness to unconsciousness.

Association Between Spatial Heterogeneity Within Nonmetastatic Gastroesophageal Adenocarcinomas and Survival.

Importance: Intratumoral heterogeneity has been recognized as a significant barrier in successfully developing targetable biomarkers for gastroesophageal adenocarcinoma (GEA) and may affect neoadjuvant precision medicine approaches. Objective: To describe intratumoral spatial heterogeneity of tumor cell populations in nonmetastatic GEA and its association with survival. Design, Setting, and Participants: This case series retrospectively identified 41 patients with GEA who underwent up-front surgical resection at a tertiary referral cancer center from January 1, 1989, through December 31, 2013. Survival was calculated from date of surgery to date of death through June 1, 2017. Data were analyzed from June 2, 2017, to March 1, 2019. Main Outcomes and Measures: Overall survival, intratumoral clonal composition determined by genomic single-nucleotide variation array and bioinformatic analysis, and intercellular tumoral distances determined by multiprobe fluorescence in situ hybridization. Results: Among the 41 patients included in the analysis (22 men [54%]; mean [SD] age, 63 [12] years), a high proportion (19 [46%]) presented with tumors possessing high intratumoral heterogeneity. Kaplan-Meier analysis demonstrated that cases with an intratumoral clonal composition count of at least 2 exhibited worse survival compared with cases with a clonal composition count of 0 to 1 (univariate hazard ratio, 3.92; 95% CI, 1.27-12.08; P = .02). This finding remained significant on multivariate analysis controlling for stage, Lauren histologic subtype, receipt of adjuvant therapy, and age (multivariate hazard ratio, 4.55; 95% CI, 1.09-19.04; P = .04). Multiprobe fluorescence in situ hybridization demonstrated intratumoral clonal populations coexisting at submillimeter distances with differing relevant oncogenic copy number alterations, such as EGFR, JAK2, FGFR2, MET, CCND1, KRAS, MYC, PIK3CA, CD274, and PDCD1LG2. Conclusions and Relevance: This study found that spatial intratumoral heterogeneity of oncogenic copy number alterations exists before metastatic dissemination, and increased heterogeneity was associated with worse outcomes in resected GEA. Baseline heterogeneity illustrates the challenges in GEA targeted therapy. Further study may offer insight into strategies on combinatorial and/or sequential targeted and immunotherapeutic approaches.

Duplex DNA from Sites of Helicase-Polymerase Uncoupling Links Non-B DNA Structure Formation to Replicative Stress.

BACKGROUND: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. MATERIALS AND METHODS: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. RESULTS: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. CONCLUSION: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation.

Analysis of stochastic fluctuations in responsiveness is a critical step toward personalized anesthesia.

Traditionally, drug dosing is based on a concentration-response relationship estimated in a population. Yet, in specific individuals, decisions based on the population-level effects frequently result in over or under-dosing. Here, we interrogate the relationship between population-based and individual-based responses to anesthetics in mice and zebrafish. The anesthetic state was assessed by quantifying responses to simple stimuli. Individual responses dynamically fluctuated at a fixed drug concentration. These fluctuations exhibited resistance to state transitions. Drug sensitivity varied dramatically across individuals in both species. The amount of noise driving transitions between states, in contrast, was highly conserved in vertebrates separated by 400 million years of evolution. Individual differences in anesthetic sensitivity and stochastic fluctuations in responsiveness complicate the ability to appropriately dose anesthetics to each individual. Identifying the biological substrate of noise, however, may spur novel therapies, assure consistent drug responses, and encourage the shift from population-based to personalized medicine.

Zebrafish: A Pharmacogenetic Model for Anesthesia.

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery.

Outcomes after Allogeneic Stem Cell Transplantation in Patients with Double-Hit and Double-Expressor Lymphoma.

Double-hit lymphomas (DHLs) and double-expressor lymphomas (DELs) are associated with resistance to frontline and salvage immunochemotherapy, as well as autologous stem cell transplantation (SCT). We hypothesized that allogeneic SCT (alloSCT) could overcome the chemoresistance associated with DEL/DHL. We retrospectively studied the impact of DEL/DHL status in a multicenter cohort of patients who underwent alloSCT for relapsed/refractory (rel/ref) aggressive B cell non-Hodgkin lymphoma (B-NHL). Seventy-eight patients transplanted at 3 centers in whom tumor tissue was available for immunohistochemistry and fluorescence in situ hybridization were enrolled; 47% had DEL and 13% had DHL. There were no significant differences in 4-year progression-free (PFS) or overall survival (OS) between patients with DEL compared with patients without DEL (PFS 30% versus 39%, P = .24; OS 31% versus 49%, P = .17) or between patients with DHL compared with patients without DHL (PFS 40% versus 34%, P = .62; OS 50% versus 38%, P = .46). The lack of association between DEL or DHL and outcome was confirmed in multivariable models, although inadequate sample size may have limited our ability to detect significant differences. In our cohort alloSCT produced durable remissions in patients with rel/ref aggressive B-NHL irrespective of DEL and DHL status, justifying its consideration in the treatment of patients with rel/ref DEL/DHL.

Favorable impact of allogeneic stem cell transplantation in patients with therapy-related myelodysplasia regardless of TP53 mutational status.

Therapy-related myelodysplastic syndrome is a long-term complication of cancer treatment in patients receiving cytotoxic therapy, characterized by high-risk genetics and poor outcomes. Allogeneic hematopoietic cell transplantation is the only potential cure for this disease, but the prognostic impact of pre-transplant genetics and clinical features has not yet been fully characterized. We report here the genetic and clinical characteristics and outcomes of a relatively large cohort of patients with therapy-related myelodysplastic syndrome (n=67) who underwent allogeneic transplantation, comparing these patients to similarly treated patients with de novo disease (n=199). The 5-year overall survival was not different between patients with therapy-related and de novo disease (49.9% versus 53.9%; P=0.61) despite a higher proportion of individuals with an Intermediate-2/High International Prognostic Scoring System classification (59.7% versus 43.7%; P=0.003) and high-risk karyotypes (61.2% versus 30.7%; P<0.01) among the patients with therapy-related disease. In mutational analysis, TP53 alteration was the most common abnormality in patients with therapy-related disease (n=18: 30%). Interestingly, the presence of mutations in TP53 or in any other of the high-risk genes (EZH2, ETV6, RUNX1, ASXL1: n=29: 48%) did not significantly affect either overall survival or relapse-free survival. Allogeneic stem-cell transplantation is, therefore, a curative treatment for patients with therapy-related myelodysplastic syndrome, conferring a similar long-term survival to that of patients with de novo disease despite higher-risk features. While TP53 alteration was the most common mutation in therapy-related myelodysplastic syndrome, the finding was not detrimental in our case-series.

Cyclin D1 depletion induces DNA damage in mantle cell lymphoma lines.

Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.

Neural Stem Cell-Based Anticancer Gene Therapy: A First-in-Human Study in Recurrent High-Grade Glioma Patients.

Purpose: Human neural stem cells (NSC) are inherently tumor tropic, making them attractive drug delivery vehicles. Toward this goal, we retrovirally transduced an immortalized, clonal NSC line to stably express cytosine deaminase (HB1.F3.CD.C21; CD-NSCs), which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU).Experimental Design: Recurrent high-grade glioma patients underwent intracranial administration of CD-NSCs during tumor resection or biopsy. Four days later, patients began taking oral 5-FC every 6 hours for 7 days. Study treatment was given only once. A standard 3 + 3 dose escalation schema was used to increase doses of CD-NSCs from 1 × 107 to 5 × 107 and 5-FC from 75 to 150 mg/kg/day. Intracerebral microdialysis was performed to measure brain levels of 5-FC and 5-FU. Serial blood samples were obtained to assess systemic drug concentrations as well as to perform immunologic correlative studies.Results: Fifteen patients underwent study treatment. We saw no dose-limiting toxicity (DLT) due to the CD-NSCs. There was 1 DLT (grade 3 transaminitis) possibly related to 5-FC. We did not see development of anti-CD-NSC antibodies and did not detect CD-NSCs or replication-competent retrovirus in the systemic circulation. Intracerebral microdialysis revealed that CD-NSCs produced 5-FU locally in the brain in a 5-FC dose-dependent manner. Autopsy data indicate that CD-NSCs migrated to distant tumor sites and were nontumorigenic.Conclusions: Collectively, our results from this first-in-human study demonstrate initial safety and proof of concept regarding the ability of NSCs to target brain tumors and locally produce chemotherapy. Clin Cancer Res; 23(12); 2951-60. ©2016 AACR.