Variability In Clinical Response Research Articles

P76 Management of aciclovir-resistant mucocutaneous herpes simplex virus infection: a systematic review

Abstract Aciclovir resistance results in immunocompromised hosts due to long-term antiviral therapy and ongoing viral replication. Aciclovir resistance in herpes simplex virus (HSV) poses a major concern, as this can lead to chronic and recalcitrant infection that significantly impacts on the quality of life. While different treatment options exist, there are currently no standardized clinical treatment guidelines for mucocutaneous aciclovir-resistant (ACV-R) HSV infection. This systematic review seeks to identify safe and effective treatments for mucocutaneous ACV-R HSV infection. For this systematic review, PubMed/MEDLINE and Embase were searched in September 2022 for all articles published between 1 January 1980 and 1 September 2022. Studies were included if they described the use of a medication for the treatment of mucocutaneous ACV-R HSV infection. From the initial search that yielded 1066 studies, 86 met the inclusion criteria and were selected. Of the 86 studies, 6 were clinical trials and 80 were case reports/series. A total of 318 patients were included in our review. Foscarnet [intravenous (IV) and topical] was studied in 5 clinical trials and 25 case reports/series, leading to complete response in most patients, with adverse effects including renal toxicity and electrolyte abnormalities. Vidarabine (IV) was found to be inferior to foscarnet in a clinical trial and reported in two case reports. Common side-effects included nausea, anaemia and renal impairment. Trifluridine (topical) was studied in a clinical trial and a case report, showing complete response in some patients. Cidofovir (IV, topical, intralesional) was studied in 21 case reports/series, showing complete response in most patients, with renal impairment as the most common adverse effect. Imiquimod (topical) was studied in eight case reports/series, showing complete response and no side-effects. Pritelivir (oral) was reported in two case reports/series, showing complete response and no side-effects. Combination treatment was reported in 10 case reports/series with variable clinical response. Our review also identified reports of less widely used treatments. These include artesunate, zidovudine, varicella-zoster virus vaccine, leflunomide and surgical excision, which achieved variable response rates but have not been widely studied. While foscarnet and cidofovir have the most robust evidence, IV administration of these agents is associated with significant side-effects and may be limiting. Intralesional and topical formulations of cidofovir, trifluridine and imiquimod are alternative options appropriate for outpatient management. Pritelivir is a novel and promising treatment of mucocutaneous ACV-R HSV infection.

BI03 (P76) Management of aciclovir-resistant mucocutaneous herpes simplex virus infection: a systematic review

Abstract Aciclovir resistance results in immunocompromised hosts due to long-term antiviral therapy and ongoing viral replication. Aciclovir resistance in herpes simplex virus (HSV) poses a major concern, as this can lead to chronic and recalcitrant infection that significantly impacts on quality of life. While different treatment options exist, there are currently no standardized clinical treatment guidelines for mucocutaneous aciclovir-resistant (ACV-R) HSV infection. This systematic review seeks to identify safe and effective treatments for mucocutaneous ACV-R HSV infection. For this systematic review, PubMed/MEDLINE and Embase were searched in September 2022 for all articles published between 1 January 1980 and 1 September 2022. Studies were included if they described the use of medications for the treatment of mucocutaneous ACV-R HSV infection. From the initial search that yielded 1066 studies, 86 met the inclusion criteria and were selected. Of the 86 studies, six were clinical trials and 80 were case reports/series. A total of 318 patients were included in our review. Foscarnet [intravenous (IV) and topical] was studied in five clinical trials and 25 case reports/series, leading to complete response in most patients, with adverse effects including renal toxicity and electrolyte abnormalities. Vidarabine (IV) was found to be inferior to foscarnet in a clinical trial and reported in two case reports. Common side-effects included nausea, anaemia and renal impairment. Trifluridine (topical) was studied in a clinical trial and a case report, showing complete response in some patients. Cidofovir (IV, topical, intralesional) was studied in 21 case reports/series, showing complete response in most patients, with renal impairment as the most common adverse effect. Imiquimod (topical) was studied in eight case reports/series, showing complete response and no side-effects. Pritelivir (oral) was reported in two case reports/series, showing complete response and no side-effects. Combination treatment was reported in 10 case reports/series with variable clinical response. Our review also identified reports of less widely used treatments. These include artesunate, zidovudine, varicella-zoster virus vaccine, leflunomide and surgical excision, which achieved variable response rates but have not been widely studied. While foscarnet and cidofovir have the most robust evidence, IV administration of these agents is associated with significant side-effects and may be limiting. Intralesional and topical formulations of cidofovir, trifluridine and imiquimod are alternative options appropriate for outpatient management. Pritelivir is a novel and promising treatment of mucocutaneous ACV-R HSV infection.

Improving the Accuracy of Biologic Drug Survival Data: The Increasing Prevalence of Off-Label Dosing Must Be Considered when Reporting Drug Effectiveness and Safety

Improving the Accuracy of Biologic Drug Survival Data: The Increasing Prevalence of Off-Label Dosing Must Be Considered when Reporting Drug Effectiveness and Safety

EPS3.01 Analysis of synthetic derivatives with reduced lipophilicity as candidate potentiators towards better treatment alternatives for cystic fibrosis

Objectives: KALYDECO (ivacaftor/VX-770), a potentiator that enhances channel gating of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) protein and certain CFTR gating mutants was first approved to treat Cystic Fibrosis (CF). Thereafter, Orkambi, a combination of ivacaftor and the trafficking corrector lumacaftor was approved for patients with homozygous F508del mutation. Unfortunately, ORKAMBI mediates modest and variable clinical responses which relates to a negative impact of VX-770 on the metabolic stability of F508del-CFTR. VX-770 was also shown to exert a negative effect on the stability of other membrane solute carriers. The destabilizing effect of VX-770 also correlated with its predicted lipophilicity, suggesting the need for discovery and optimization of efficacious potentiators to maximize the clinical benefit in CF.

(018) Immunohistochemical Characterization of the Innervation of the Vulvar Vestibule in Patients with Suspected Neuroproliferative Vestibulodynia

Abstract Introduction Provoked vestibulodynia (PVD) is a chronic pain condition characterized by allodynia localized to the vestibule, the endodermal tissue surrounding the vaginal introitus. The finding of increased densities of nerve fibers in the vestibular mucosa of patients with PVD has led to the identification of a neuroproliferative subtype. The etiology of PVD, including neuroproliferative vestibulodynia (NPV), is not fully known which has led to challenges in adequately managing this condition. Significantly, the microscopic innervation of the vestibule remains incompletely described despite the preliminary data which support the role of peripheral innervation in vestibular pain pathogenesis. Objective To characterize the types and distribution of nerves present in vestibule samples from patients with suspected NPV using a suite of markers of general (protein gene product 9.5, PGP9.5), sensory (calcitonin gene-related peptide, CGRP) and autonomic innervation (vasoactive intestinal polypeptide, VIP and tyrosine hydroxylase, TH) and compare this to cadaveric vestibule tissues; and to investigate whether there is an association between this innervation and NPV etiology using markers of neuroproliferation (nerve growth factor, NGF) and immune activation (c-kit). Methods Archival formalin-fixed, paraffin embedded vestibulectomy samples were obtained from six patients who had previous immunohistochemical staining to confirm NPV diagnosis. All samples were stained with nerve markers PGP9.5 and CGRP, in addition to NGF, and mast cell marker c-kit. A subset of samples was stained with nerve markers TH and VIP to further elucidate specific types of innervation present. Vestibule samples were obtained from female cadaveric donors and were stained with the same suite of antibodies for comparison. All tissues were serially sectioned at 5 μm. Results Nerve fibers immunoreactive for all nerve markers were identified in both patient and cadaveric vestibule. Patient samples were characterized by the proliferation of PGP9.5-positive nerve fibers and c-kit positive mast cells which were identified throughout the tissue stroma, including in proximity to nerve bundles. NGF expression was localized to a subset of nerves including those that demonstrated co-expression of sensory and autonomic nerve markers. Cells immunoreactive for NGF were also observed, including those that showed co-expression with putative mast cells that stained positively for c-kit. Relative to the cadaveric tissues, the patient samples were characterized by increased densities of CGRP, TH, and VIP-immunoreactive fibers which were observed throughout the vestibular mucosa. Some heterogeneity in patterns of innervation was observed between the patient samples, including increased densities of autonomic fibers in one patient as indicated by increased VIP and TH-immunoreactive nerve fibers. Conclusions Our results support the existence of a neuroproliferative subtype of PVD that is characterized by the hyperinnervation of the vestibular mucosa and neuro-immune interactions. This investigation highlights that the proliferation of other nerve fiber types in addition to nociceptive, CGRP-positive fibers, including adrenergic and cholinergic nerve fibers, may be important in PVD etiology in some patients. Heterogeneity in patterns of innervation across individuals with NPV could explain, in part, variability in clinical response to treatment and should be further elucidated in ongoing research efforts. Disclosure No

Therapeutic drug monitoring of disease-modifying antirheumatic drugs in circulating leukocytes in immune-mediated inflammatory diseases.

The treatment of immune-mediated inflammatory diseases (IMIDs) is one of the main challenges of modern medicine. Although a number of disease-modifying antirheumatic drugs (DMARDs) are available, there is wide variability in clinical response to treatment among individuals. Therapeutic drug monitoring (TDM) has been proposed to optimize treatment; however, some patients still experience unsatisfactory outcomes, although the blood concentrations of drugs in these patients remain in the therapeutic range. One possible reason for this is that the conventional samples (e.g., whole blood or plasma) used in TDM may not accurately reflect drug concentrations or concentrations of their metabolites at the target site. Hence, more refined TDM approaches to guide clinical decisions related to dose optimization are necessary. Circulating leukocytes or white blood cells have a critical role in driving the inflammatory process. They are recruited to the site of injury, infection and inflammation, and the main target of small molecule DMARDs is within immune cells. Given this, assaying drug concentrations in leukocytes has been proposed to be of possible relevance to the interpretation of outcomes. This review focuses on the clinical implications and challenges of drug monitoring of DMARDs in peripheral blood leukocytes from therapeutic or toxicological perspectives in IMIDs.

Associations of plasma aprepitant and its N-dealkylated metabolite with cachexia status and clinical responses in head and neck cancer patients.

Oral aprepitant has a large interindividual variation in clinical responses in advanced cancer. This study aimed to characterize plasma aprepitant and its N-dealkylated metabolite (ND-AP) based on the cachexia status and clinical responses in head and neck cancer patients. Fifty-three head and neck cancer patients receiving cisplatin-based chemotherapy with oral aprepitant were enrolled. Plasma concentrations of total and free aprepitant and ND-AP were determined at 24h after a 3-day aprepitant treatment. The clinical responses to aprepitant and degrees of cachexia status were assessed using a questionnaire and Glasgow Prognostic Score (GPS). Serum albumin level was negatively correlated with the plasma concentrations of total and free aprepitant but not ND-AP. The serum albumin level had a negative correlation with the metabolic ratio of aprepitant. The patients with GPS 1 or 2 had higher plasma concentrations of total and free aprepitant than those with GPS 0. No difference was observed in the plasma concentration of ND-AP between the GPS classifications. The plasma interleukin-6 level was higher in patients with GPS 1 or 2 than 0. The absolute plasma concentration of free ND-AP was higher in patients without the delayed nausea, and its concentration to determine the occurrence was 18.9ng/mL. The occurrence of delayed nausea had no relation with absolute plasma aprepitant. Cancer patients with a lower serum albumin and progressive cachectic condition had a higher plasma aprepitant level. In contrast, plasma free ND-AP but not aprepitant was related to the antiemetic efficacy of oral aprepitant.

Characterizing the innervation of the vulvar vestibule and the immunohistochemical features of neuroproliferative vestibulodynia.

Provoked vestibulodynia (PVD) is a chronic pain condition characterized by allodynia localized to the vulvar vestibule. The finding of increased densities of nerve fibers in the vestibular mucosa of patients with PVD has led to the identification of a neuroproliferative subtype. The etiology of PVD, including neuroproliferative vestibulodynia (NPV), is not fully understood. The gross and microscopic innervation of the vulvar vestibule remains incompletely described, despite the preliminary data supporting the role of peripheral innervation in PVD. To characterize the gross anatomic and microscopic innervation of the vulvar vestibule through cadaveric dissection and immunohistochemistry. The pudendal nerve and inferior hypogastric plexus (IHP) were dissected using 6 cadaveric donors. Histology and immunohistochemistry were used to confirm patterns of innervation identified gross anatomically. Immunohistochemistry was performed on vestibulectomy specimens obtained from 6 patients diagnosed with NPV and compared with cadaveric vestibular tissues. Outcomes included (1) dissection of pelvic innervation and (2) immunohistochemical localization of markers for the following: general innervation protein gene product 9.5 (PGP9.5), sensory innervation (calcitonin gene-related peptide), autonomic innervation (vasoactive intestinal polypeptide, tyrosine hydroxylase), neuroproliferation (nerve growth factor [NGF]), and immune activation (C-kit). Perineal (pudendal) nerve branches were traced to the external wall of the vulvar vestibule. Some anatomic heterogeneity was observed in perineal nerve-branching patterns. Fibers from the IHP were identified in close proximity to the vulvar vestibule. Autonomic and sensory nerve fibers were identified in both patient and cadaveric vulvar vestibule samples. Patient samples were characterized by the proliferation of PGP9.5-positive nerve fibers and C-kit-positive mast cells, which were in proximity to neve bundles and showed coexpression with putative NGF-positive cells. NGF expression was localized to a subset of nerves, including those that demonstrated co-expression of sensory and autonomic nerve markers. Increased densities of autonomic fibers positive for vasoactive intestinal polypeptide and tyrosine hydroxylase were observed in 1 patient sample. Heterogeneity in gross and microscopic patterns of innervation could explain variability in clinical response to treatment and should be used to inform future therapeutic interventions. This study used a combination of approaches to elucidate the innervation of the vulvar vestibule, including in NPV. The small sample size is a limitation. The vulvar vestibule contains both sensory and autonomic innervation, which may originate from the pudendal nerve and IHP. Our results support the existence of a neuroproliferative subtype that is characterized by the proliferation of sensory and autonomic nerve fibers and neuroimmune interactions.

Dynamics and specificities of T cells in cancer immunotherapy.

Recent advances in cancer immunotherapy - ranging from immune-checkpoint blockade therapy to adoptive cellular therapy and vaccines - have revolutionized cancer treatment paradigms, yet the variability in clinical responses to these agents has motivated intense interest in understanding how the T cell landscape evolves with respect to response to immune intervention. Over the past decade, the advent of multidimensional single-cell technologies has provided the unprecedented ability to dissect the constellation of cell states of lymphocytes within a tumour microenvironment. In particular, the rapidly expanding capacity to definitively link intratumoural phenotypes with the antigen specificity of T cells provided by T cell receptors (TCRs) has now made it possible to focus on investigating the properties of T cells with tumour-specific reactivity. Moreover, the assessment of TCR clonality has enabled a molecular approach to track the trajectories, clonal dynamics and phenotypic changes of antitumour T cells over the course of immunotherapeutic intervention. Here, we review the current knowledge on the cellular states and antigen specificities of antitumour T cells and examine how fine characterization of T cell dynamics in patients has provided meaningful insights into the mechanisms underlying effective cancer immunotherapy. We highlight those T cell subsets associated with productive T cell responses and discuss how diverse immunotherapies might leverage the pre-existing tumour-reactive T cell pool or instruct de novo generation of antitumour specificities. Future studies aimed at elucidating the factors associated with the elicitation of productive antitumour T cell immunity are anticipated to instruct the design of more efficacious treatment strategies.

GUT MICROBIAL METABOLISM OF 5-ASA IS PROSPECTIVELY ASSOCIATED WITH TREATMENT FAILURE IN PATIENTS WITH IBD

Variation in clinical response to 5-aminosalicylic acid (5-ASA) has been attributed in part to its inactivation by gut microbes. Recently, in the Inflammatory Bowel Disease (IBD) Multi’omics Database (IBDMDB), a multicenter year-long cohort of 100+ participants with IBD, we identified 12 gut microbial enzymes from two protein families that convert 5-ASA to N-acetyl 5-ASA, a compound that lacks anti-inflammatory effects. Within the IBDMDB, we then found that a subset of these enzymes was cross-sectionally linked with greater risk of treatment failure, defined by corticosteroid use. The aim of this study was to prospectively associate these drug-degrading gut microbial enzymes with treatment failure and to confirm in vitro conversion of 5-ASA by these enzymes.

Humanized mouse models for immuno-oncology research.

Immunotherapy has emerged as a promising treatment paradigm for many malignancies and is transforming the drug development landscape. Although immunotherapeutic agents have demonstrated clinical efficacy, they are associated with variable clinical responses, and substantial gaps remain in our understanding of their mechanisms of action and specific biomarkers of response. Currently, the number of preclinical models that faithfully recapitulate interactions between the human immune system and tumours and enable evaluation of human-specific immunotherapies in vivo is limited. Humanized mice, a term that refers to immunodeficient mice co-engrafted with human tumours and immune components, provide several advantages for immuno-oncology research. In this Review, we discuss the benefits and challenges of the currently available humanized mice, including specific interactions between engrafted human tumours and immune components, the development and survival of human innate immune populations in these mice, and approaches to study mice engrafted with matched patient tumours and immune cells. We highlight the latest advances in the generation of humanized mouse models, with the aim of providing a guide for their application to immuno-oncology studies with potential for clinical translation.

Integrative Analysis of Genetic and Tumor Microenvironment Topology to Delineate Prognostic Subtypes in Mantle Cell Lymphoma

BACKGROUND Mantle Cell Lymphoma (MCL) is a B-cell lymphoma characterized by t(11;14) leading to CyclinD1 overexpression with highly variable clinical response. Although the tumor intrinsic mechanisms have been well studied and the role of the tumor microenvironment (TME) in survival and therapeutic responses are marginally examined, understanding how distinct tumors regulate their respective immune landscapes has been constrained, by a lack of appropriate experimental techniques that recapitulate the heterogeneity of the TME in MCL. Herein, we integrated high-throughput genomics and characterized TME using a highly multiplexed, spatially resolved, single-cell digital pathology approach termed imaging mass cytometry (IMC) to delineate the tumor-immune landscape. Thus, comprehensive whole exome sequencing and TME imaging analysis demonstrated prognostic subtypes elucidating the interplay between tumor intrinsic and extrinsic factors in MCL. METHODS MCL cohort with complete clinicopathological information and treatment protocol (n=580) was utilized from the North America Mantle Cell Lymphoma Consortium. These cases have been reviewed by at least two hematopathologists to confirm the MCL diagnosis. Whole exome sequencing (WES) was performed on 179 formalin-fixed tissue embedded (FFPE) samples. 44 samples have matched microarray data. Genetic driver lesions were identified using VarScan 2.4, MutSigCV 1.3.01, and GISTIC 2.0. Formalin-fixed paraffin-embedded (FFPE) tissue tumor microarrays (TMA) consisting of 55 MCL cases with duplicate or triplicate cores were generated. To perform the TME analysis, we designed a 42 marker IMC panel which includes canonical markers for immune cell subsets and markers for two additional highly mutated/aberrated genes in MCL (ATM and TP53). TMA was stained with antibodies tagged with heavy metal isotopes, laser ablated to generate spatial digital maps of the tissue and unsupervised clustering was performed to identify immune cell types using the Phenograph algorithm. Survival analysis was performed using the Kaplan-Meier method (logrank test). RESULTS Initial genomic analysis (n=179) suggested at least two prognostic subtypes, dependent on ATM or TP53 mutation status, and the latter associated with inferior-clinical outcome. Transcriptional profiling in a subset identified enrichment of gene signatures associated with B-cell receptor (BCR), MYC Targets, apoptosis, and CCND1 upregulation in the TP53-mutant subgroup. TME analysis showed that the TP53 positive tumor cell populations exhibited a receded T cell population (CD4+ and CD8+). Interestingly, the TP53-positive tumor cells expressed high protein levels of p-STAT-3, p-NFKB, and HLA expression compared to ATM-positive tumor cells. Additionally, unsupervised analysis using NMF consensus clustering showed four patient subgroups wherein the worst survival subgroup (OS: 45 months vs 62 months, p < 0.05) was independent of TP53 mutations and highly enriched in ATM mutations, 17p11.1-13.3 deletions, and depleted in CD8 naïve T cell and Macrophages M1. Whereas the good survival group (OS: 75 months) was highly enriched in ATM and TP53 mutations but deficient in 17p deletions. CONCLUSION While most MCL studies looked at the MCL heterogeneity at the genomic level, this study shows the integration of genomic and TME factors to elucidate prognostic MCL subtypes from paraffin samples at a single cell resolution. The current analysis suggests that the TP53 mutated MCL subgroup has reduced T-cell enrichment in the TME and high protein expression of oncogenes. A patient subgroup highly enriched in ATM mutations, independent of the TP53 mutations and depleted immune inflitration showed the worst prognosis. Thus, this study suggests a new angle of ATM mutations in MCL pathogenesis which could improve risk stratification. Overall, this study may unravel distinct mechanisms of tumorigenesis with relevant clinical and biological implications.

Phase 1 Trial of AUC-Targeted Melphalan in Myeloma Patients Undergoing Autologous Transplant (The MyMel Study)

Standard 200 mg/m2 high dose melphalan (HDM) yields a 500% variation in plasma melphalan exposure, i.e. area under the curve (AUC), leading to unpredictable DNA damage and widely variable clinical responses. Strategies to predict melphalan AUC have been unsuccessful given population PK models (Nath, Br J Clin Pharmacol 2010 & Cho Clin Pharmacol Ther 2017) are ineffective on an individual patient level and only explain ~20% of the variability observed, and test dose predictions have failed to accurately represent melphalan AUC for doses >100 mg/m2. PK directed dose adjustment of HDM would require a 24-hour turnaround for sample collection, shipping, processing and data analysis. Previously we described methods for measuring melphalan PK after the Day -2 HDM dose, allowing for real-time dose adjustments (Sweiss, Br J Clin Pharmacol 2020). Here, we set out to perform the first phase 1 study of AUC-targeted HDM in ASCT (NCT04483206). This multicenter phase 1 trial (Fig 1) is designed to test 7 AUC ranges (13-21 mg*h/L) to determine the maximum tolerated systemic exposure (MTSE). The feasibility portion focuses on the first 2 AUCs (phase A, n=20) in patients with ≥2L therapy, where we measure accuracy of achieving target AUCs of 13.5 and 14.5 mg∗h/L, respectively. Patients receive HDM 100 mg/m2 on day -3 with PK measurement and subsequent dose-adjustment on day -1 to achieve the total target AUC. 7 blood samples are drawn, processed and shipped frozen for LC-MS/MS analysis. PK parameter estimations are determined from noncompartmental analysis (NCA) and per FDA guidance used to adjust doses. Nonlinear mixed effects modeling (NONMEM) is performed in parallel to compare PK estimations. Using observed first-dose HDM clearance (Clobs) from NCA and assuming that Cl is consistent between doses, linear dose-proportional adjustments are performed to achieve the target AUC0-inf. After a planned FDA review of these cohorts, we will begin to dose escalate to determine the MTSE with up to 5 AUC levels (15.5-19.5 mg∗h/L). Bone marrow (BM) MRD status will be assessed at Day 90 as part of the Phase B primary objective. Correlatives include measuring DNA damage in circulating PBMCs post-HDM to correlate with AUC, piloting a DNA fiber combing assay on primary MM cells from patient BM samples to quantify DNA replicative stress, and assessing PROs using NCI PRO-CTCAE v.1.0. We have enrolled 8 patients into the first AUC cohort with 110 blood samples collected. Median age was 69 (56-75) years, with 7 (87.5%) males, and 4 (50%) Blacks. The median day -3 HDM Clobs and AUC0-inf were 22.05 (16.3-38.2) L/hr and 9.53 (6.97-11.82) mg∗h/L, respectively. All shipped blood samples were received in suitable frozen condition, and in 7 (87.5%) of 8 patients, LC-MS/MS run with NCA estimation and dose reporting was done within 12 hours of receiving samples. One patient whose samples were delayed from shipping had the dose reported on day-1 resulting in 5 of 7 day-1 samples collected. Covariates of HDM PK (i.e., FFM, hematocrit) were entered into our developed PK model to further enrich our data prediction. All 8 day -1 doses were adjusted, with a resulting median total BSA-based HDM dose of 139.6 (110.93-192.6) mg/m2. After adjustment, median day -1 Clobs and AUC0-inf were 24.48 (19.3-35.8) L/hr and 3.84 (1.7-7) mg∗h/L, respectively. For accuracy of dose adjustment, 107 (97.3%) samples were evaluable for PK parameter estimation, with 3 concentrations excluded (n=3 patients). One subject whose last 2 day -1 samples were not collected was removed from analysis as it resulted in 19% AUC extrapolation and 33% variance between day -3 and -1 Cl values. Of 7 patients, 6 (85.7%) achieved the target AUC, with less than 10% variance in Cl between doses. (Fig 2). In the one patient, it was assessed that the day -1 Cl deviated by 24% as a result of prolonged time from drug admixture to completion of infusion leading to drug degradation. Estimated melphalan Cl generated from NCA and NONMEM correlated well (r=0.99). This is the first trial in MM implementing a novel personalized dosing approach using drug exposure as the target to define each cohort. We demonstrate feasibility of real-time PK sampling, shipping, processing, NCA PK parameter estimation, and dose adjustment of HDM. So far we report a high rate of accuracy in achieving the target AUC which we hypothesize will deliver reliable DNA damage to MM cells. Enrollment is active and mature data will be presented at the conference. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Diagnostic Gene Expression Signature for Bladder Cancer Can Stratify Cases into Prescribed Molecular Subtypes and Predict Outcome.

Bladder cancer is a biologically heterogeneous disease with variable clinical presentations, outcomes and responses to therapy. Thus, the clinical utility of single biomarkers for the detection and prediction of biological behavior of bladder cancer is limited. We have previously identified and validated a bladder cancer diagnostic signature composed of 10 biomarkers, which has been incorporated into a multiplex immunoassay bladder cancer test, Oncuria™. In this study, we evaluate whether these 10 biomarkers can assist in the prediction of bladder cancer clinical outcomes. Tumor gene expression and patient survival data from bladder cancer cases from The Cancer Genome Atlas (TCGA) were analyzed. Alignment between the mRNA expression of 10 biomarkers and the TCGA 2017 subtype classification was assessed. Kaplan–Meier analysis of multiple gene expression datasets indicated that high expression of the combined 10 biomarkers correlated with a significant reduction in overall survival. The analysis of three independent, publicly available gene expression datasets confirmed that multiplex prognostic models outperformed single biomarkers. In total, 8 of the 10 biomarkers from the Oncuria™ test were significantly associated with either luminal or basal molecular subtypes, and thus, the test has the potential to assist in the prediction of clinical outcome.

IL-17A Increases Doxorubicin Efficacy in Triple Negative Breast Cancer

Due to lack of targetable receptors and intertumoral heterogeneity, triple negative breast cancer (TNBC) remains particularly difficult to treat. Doxorubicin (DOX) is typically used as nonselective neoadjuvant chemotherapy, but the diversity of treatment efficacy remains unclear. Comparable to variability in clinical response, an experimental model of TNBC using a 4T1 syngeneic mouse model was found to elicit a differential response to a seven-day treatment regimen of DOX. Single-cell RNA sequencing identified an increase in T cells in tumors that responded to DOX treatment compared to tumors that continued to grow uninhibited. Additionally, compared to resistant tumors, DOX sensitive tumors contained significantly more CD4 T helper cells (339%), γδ T cells (727%), Naïve T cells (278%), and activated CD8 T cells (130%). Furthermore, transcriptional profiles of tumor infiltrated T cells in DOX responsive tumors revealed decreased exhaustion, increased chemokine/cytokine expression, and increased activation and cytotoxic activity. γδ T cell derived IL-17A was identified to be highly abundant in the sensitive tumor microenvironment. IL-17A was also found to directly increase sensitivity of TNBC cells in combination with DOX treatment. In TNBC tumors sensitive to DOX, increased IL-17A levels lead to a direct effect on cancer cell responsiveness and chronic stimulation of tumor infiltrated T cells leading to improved chemotherapeutic efficacy. IL-17A’s role as a chemosensitive cytokine in TNBC may offer new opportunities for treating chemoresistant breast tumors and other cancer types.

Predicting Normal Tissue Radiotoxicity in Radiotherapy Patients Based on the Individual Radiosensitivity Using Three-Color Fluorescence In Situ Hybridization: A Literature Review

Background: The variability of clinical response in radiotherapy (RT) patients revealed that individual radiosensitivity exists in humans. Although several techniques for radiosensitivity assessment are available, fluorescence in situ hybridization (FISH) has proven to be the most reliable cytogenetic technique. This study will review the use of three-color FISH to evaluate the individual radiosensitivity in cancer patients, particularly breast and prostate cancer patients. Also, it will explain factors that should be considered when using this assay in RT patients.Methods: We used the “radiosensitivity”, “fluorescence in situ hybridization”, “FISH”, and “cancer” as medical subject headings. Non-English articles were excluded. Only articles written in English with a full-text PDF format could be found and using three-color FISH for individual radiosensitivity prediction in radiotherapy patients as subjects were included in this review.Results: : In total, 1,905 articles were retrieved from PubMed and PubMed Central databases from 1990 to 2020. The articles were screened, and those that met the inclusion criteria and did not meet the exclusion criteria were reviewed. Finally, we evaluated eight articles in this reviewConclusions: Appropriate assays such as three-color FISH for individual radiosensitivity assessment could optimize the effectiveness of RT treatment and predict the severity of normal tissue toxicity reactions in RT patients.

P604: CATALASE EXPRESSION IN LEUKEMIA CELLS IS CONTROLLED BY GENETIC AND EPIGENETIC MECHANISMS

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in Western countries. It is an incurable disease characterized by an extremely variable clinical course and response to treatment. We have recently shown that high catalase (CAT) expression identifies a more aggressive clinical behavior in CLL. However, molecular mechanisms controlling catalase expression in leukemia cells are still poorly characterized. The rs1001179 single nucleotide polymorphism (SNP) as well as DNA methylation in the CAT promoter have been shown to regulate CAT expression in various cell types. Aims: With the aim to characterize regulatory mechanisms underlying differential expression of catalase in CLL, in this study we investigated the role of rs1001179 SNP and CpG Island II methylation encompassing this SNP in the regulation of CAT expression. Methods: Peripheral blood mononuclear cells (PBMC) from 55 CLL patients and 50 healthy donors (HD) were used for the study. CAT mRNA levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). DNA samples were genotyped for the rs1001179 SNP using Restriction Fragment Length Polymorphism (RFLP)-PCR. Bioinformatic analysis was used to predict transcription factor (TF) binding to SNP sequences. Chromatin Immunoprecipitation (ChIP)-qPCR was performed to validate predicted TF binding sites. Methylation levels of CpG sites within the CAT promoter was determined by pyrosequencing of bisulfite-converted DNA. Results: The rs1001179 SNP genotyping showed that CLL cells harboring the T allele exhibited a significantly higher catalase expression compared with cells bearing the CC genotype. Interestingly, bioinformatic analysis predicted that the SNP provide distinct binding sites for various TF, including ETS-1 and GR-β. ChIP assay confirmed that CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β, but not to the other analyzed TFs. In addition, CLL cells exhibited lower methylation levels within the CAT promoter compared with HD B cells, in line with the higher catalase mRNA and protein levels expressed by CLL versus HD B cells. Moreover, methylation levels negatively correlated with CAT expression in CLL cells. Remarkably, inhibition of methyltransferase activity in leukemic cells induced a significant increase of CAT mRNA and protein levels, thus showing that DNA methylation could control catalase expression in CLL. Moreover, expression of DNA methyl transferase 1 (DNMT1) resulted significantly reduced in CLL cells compared with HD B cells and inversely correlated with CAT expression in CLL, thus suggesting that differences in methylation levels underlying catalase expression could be driven by the DNMT1 enzyme. Finally, modeling analysis showed that the CT/TT genotypes exhibited a lower methylation and a higher CAT expression level, suggesting that the rs1001179 T allele and methylation cooperate in inducing CAT gene expression. Summary/Conclusion: Our data show that SNP as well as methylation of the catalase promoter are involved in controlling catalase expression in CLL. The key advance of this study is to provide new insights into the regulatory mechanisms underlying differential expression of catalase in leukemic cells, which is of clinical relevance in CLL. Moreover, our study may form the basis for future challenges aimed at developing combinatorial therapies targeting catalase regulatory pathways.

Impact of the novel CYP2C:TG haplotype and CYP2B6 variants on sertraline exposure in a large patient population.

Sertraline is a commonly used SSRI antidepressant drug, metabolized by CYP2C19 and CYP2B6, that exhibits a substantial interindividual variation in clinical response, of which only a part can be attributed to known genetic variants. In the current study we have examined the role of a newly discovered ultrarapid CYP2C:TG haplotype and CYP2B6 variants in order to identify the possible missing heritability for such variation in sertraline response in a large patient population (n = 840). Compared to the reference group (CYP2C19*1/*1, n = 160), sertraline exposure was increased by 128% in CYP2C19 PMs (n = 29, p < 0.001) but decreased by about 20% in CYP2C19 ultrarapid metabolizers (Ums) (homozygous carriers of CYP2C19*17 and/or CYP2C:TG haplotype) with the diplotypes CYP2C19*17/*17, CYP2C:TG/TG, or CYP2C19*17/CYP2C:TG (n = 135, p < 0.003, p = 0.022, p < 0.003, respectively). Interestingly, in patients carrying the increased function CYP2B6*4 allele, and also carrying the CYP2C19*17 and CYP2C:TG alleles (n = 10), sertraline exposure was 35.4% lower compared to the reference group, whereas in subjects being poor metabolizers (PM) in both the CYP2C19 and CYP2B6 gene, the sertraline concentrations were raised by 189%. In summary, the CYP2C19 variants including the CYP2C:TG haplotype had a significant impact on sertraline metabolism, as well as the CYP2B6*4, *6, and *9 alleles. Knowing the CYP2B6 and CYP2C19 genotype, including the CYP2C:TG haplotype status, can prospectively be useful to clinicians in making more appropriate sertraline dosing decisions

A Validated HPLC-MS/MS Method for Quantification of Fingolimod and Fingolimod-Phosphate in Human Plasma: Application to Patients with Relapsing–Remitting Multiple Sclerosis

Fingolimod is a sphingosine 1-phosphate-receptor modulator approved for the oral treatment of relapsing–remitting multiple sclerosis (RRMS), a form of MS characterized by a pattern of exacerbation of neurological symptoms followed by recovery. Here, we validated a simple and rapid liquid chromatography–tandem mass spectrometry method for the measurement of the concentrations of Fingolimod and its active metabolite Fingolimod-Phosphate (Fingolimod-P) in human plasma. The lower limits of quantification were set at 0.3 and 1.5 ng/mL for Fingolimod and Fingolimod-P, respectively, and the linearity was in the range 0.3–150 ng Fingolimod/mL and 1.5–150 ng Fingolimod-P/mL. After protein precipitation, the extraction recoveries of both analytes were always above 60% with minimal matrix effect. The method was accurate and precise, satisfying the criteria set in the European Medicine Agency guidelines for bioanalytical method validation. The method was then applied to measure Fingolimod and Fingolimod-P concentrations in the plasma of 15 RRMS patients under chronic treatment with Fingolimod, administered daily at the dose of 0.5 mg for up to 24 months. No significant differences were observed between samples collected at 6, 12 and 24 months for both analytes, indicating that the drug’s bioavailability was unaffected by multiple daily doses up to 24 months. The levels of Fingolimod-P were about two-fold higher than the levels of the parent compound. The availability of this analytical method can allow the monitoring of the impact of plasma levels of the drug and its metabolite on inter-individual variability in clinical responses.

Abstract 1879: A novel implantable device to in vivo assess anti-cancer agents

Abstract Background: The high variability in clinical responses observed in cancer patients highlights the need of a tailored therapeutic approach. A possible modality is to assess drugs sensitivity directly in the patients, by introducing small drug delivering devices in tumor sites for a very short period and then looking at the local anti-tumor effect. Here, we present the design of an innovative drug eluting device and its test with the BTK inhibitor ibrutinib as an example of small molecules. Methods: Mathematical models considered factors related to drug (MW), physical properties, desired concentrations in surrounding tissue, polymers and tissue physical features to identify the optimal polymers and the drug loading for the desired release profile over 24h. In vitro proliferation was measured with the MTT assay, in vivo experiments done in NOD-SCID mice (license TI05/2021), and immunohistochemistry on FFPE xenograft sections stained for Ki67 and cleaved caspase 3 (CASP-3). Results: Device was designed as an arrow-shaped cylinder, with flat end and flatter sections to be filled with the drug-eluting polymers. Prototypes were built in nylon6,6, a biocompatible but stable polymer. Ibrutinib was incorporated in low MW polyester poly-ε-caprolactone (PCL) as biopolymer by solvent casting. Polymeric coating onto devices was done with a dedicated automatic device.Devices loaded with biopolymer and different concentrations of ibrutinib or “empty” biopolymers were first in vitro tested using diffuse large B cell lymphoma (DLBCL) cell lines. Over 72h, devices with drug inhibited proliferation of the ibrutinib-sensitive TMD8 and OCI-Ly10 cell lines, but not of the ibrutinib-resistant SU-DHL-2 and U2932. No effect was seen with devices with ibrutinib-free biopolymers. Devices, empty or loaded with ibrutinib (5μg), were then inserted in xenografts of ibrutinib-sensitive cell line OCI-Ly10 and ibrutinib resistant U2932. After 24h, mice were sacrificed and xenografts analyzed. By Ki67 and CASP-3 a reduced cell proliferation and an increased apoptosis in the region surrounding the device was observed in the ibrutinib-sensitive xenografts, conversely nor reduced cell proliferation nor apoptosis induction were identified in the ibrutinib-resistant xenografts. Conclusions: We have created a prototype of a device that can locally release drugs allowing the evaluation of anti-tumor activity, optimizing cures tailored to single patient. The system can be further developed to include multiple drugs, including e.g. antibodies. Citation Format: Giuseppe Perale, Eugenio Gaudio, Tommaso Casalini, Luca Aresu, Anna Rita De Corso, Filippo Spriano, Chiara Tarantelli, Anastasios Stathis, Andrea Castrovinci, Francesco Bertoni. A novel implantable device to in vivo assess anti-cancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1879.