A diagnostic microdosing approach to investigate platinum sensitivity in non-small cell lung cancer.

The platinum-based drugs cisplatin, carboplatin and oxaliplatin are often used for chemotherapy, but drug resistance is common. The prediction of resistance to these drugs via genomics is a challenging problem since hundreds of genes are involved. A possible alternative is to use mass spectrometry to determine the propensity for cells to form drug-DNA adducts-the pharmacodynamic drug-target complex for this class of drugs. The feasibility of predictive diagnostic microdosing was assessed in non-small cell lung cancer (NSCLC) cell culture and a pilot clinical trial. Accelerator mass spectrometry (AMS) was used to quantify [14 C]carboplatin-DNA monoadduct levels in the cell lines induced by microdoses and therapeutic doses of carboplatin, followed by correlation with carboplatin IC50 values for each cell line. The adduct levels in cell culture experiments were linearly proportional to dose (R2 = 0.95, p < 0.0001) and correlated with IC50 across all cell lines for microdose and therapeutically relevant carboplatin concentrations (p = 0.02 and p = 0.01, respectively). A pilot microdosing clinical trial was conducted to define protocols and gather preliminary data. Plasma pharmacokinetics (PK) and [14 C]carboplatin-DNA adducts in white blood cells and tumor tissues from six NSCLC patients were quantified via AMS. The blood plasma half-life of [14 C]carboplatin administered as a microdose was consistent with the known PK of therapeutic dosing. The optimal [14 C]carboplatin formulation for the microdose was 107 dpm/kg of body weight and 1% of the therapeutic dose for the total mass of carboplatin. No microdose-associated toxicity was observed in the patients. Additional accruals are required to significantly correlate adduct levels with response.

DNA Adducts from Anticancer Drugs as Candidate Predictive Markers for Precision Medicine.

Biomarker-driven drug selection plays a central role in cancer drug discovery and development, and in diagnostic strategies to improve the use of traditional chemotherapeutic drugs. DNA-modifying anticancer drugs are still used as first line medication, but drawbacks such as resistance and side effects remain an issue. Monitoring the formation and level of DNA modifications induced by anticancer drugs is a potential strategy for stratifying patients and predicting drug efficacy. In this perspective, preclinical and clinical data concerning the relationship between drug-induced DNA adducts and biological response for platinum drugs and combination therapies, nitrogen mustards and half-mustards, hypoxia-activated drugs, reductase-activated drugs, and minor groove binding agents are presented and discussed. Aspects including measurement strategies, identification of adducts, and biological factors that influence the predictive relationship between DNA modification and biological response are addressed. A positive correlation between DNA adduct levels and response was observed for the majority of the studies, demonstrating the high potential of using DNA adducts from anticancer drugs as mechanism-based biomarkers of susceptibility, especially as bioanalysis approaches with higher sensitivity and throughput emerge.

Diagnostic Microdosing Approach to Study Gemcitabine Resistance.

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic "microdoses" of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of 14C-labeled gemcitabine for 4-24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity.

I-SPY COVID adaptive platform trial for COVID-19 acute respiratory failure: rationale, design and operations.

INTRODUCTION: The COVID-19 pandemic brought an urgent need to discover novel effective therapeutics for patients hospitalised with severe COVID-19. The Investigation of Serial studies to Predict Your Therapeutic Response with Imaging And moLecular Analysis (ISPY COVID-19 trial) was designed and implemented in early 2020 to evaluate investigational agents rapidly and simultaneously on a phase 2 adaptive platform. This manuscript outlines the design, rationale, implementation and challenges of the ISPY COVID-19 trial during the first phase of trial activity from April 2020 until December 2021. METHODS AND ANALYSIS: The ISPY COVID-19 Trial is a multicentre open-label phase 2 platform trial in the USA designed to evaluate therapeutics that may have a large effect on improving outcomes from severe COVID-19. The ISPY COVID-19 Trial network includes academic and community hospitals with significant geographical diversity across the country. Enrolled patients are randomised to receive one of up to four investigational agents or a control and are evaluated for a family of two primary outcomes-time to recovery and mortality. The statistical design uses a Bayesian model with 'stopping' and 'graduation' criteria designed to efficiently discard ineffective therapies and graduate promising agents for definitive efficacy trials. Each investigational agent arm enrols to a maximum of 125 patients per arm and is compared with concurrent controls. As of December 2021, 11 investigational agent arms had been activated, and 8 arms were complete. Enrolment and adaptation of the trial design are ongoing. ETHICS AND DISSEMINATION: ISPY COVID-19 operates under a central institutional review board via Wake Forest School of Medicine IRB00066805. Data generated from this trial will be reported in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: NCT04488081.

The extent of amotosalen photodegradation during photochemical treatment of platelet components correlates with the level of pathogen inactivation.

BACKGROUND: Amotosalen plus ultraviolet A (UVA) light inactivates a broad range of bacteria, viruses, protozoa, and leukocytes in platelet (PLT) and plasma components. Upon UVA illumination a small fraction of amotosalen reacts with the nucleic acid of contaminating pathogens and residual white blood cells and the remaining fraction undergoes photodegradation into defined photoproducts. The levels of amotosalen and photoproducts can be accurately quantified. STUDY DESIGN AND METHODS: This study evaluated the relationship between the extent of photodegradation of amotosalen and the level of pathogen inactivation in PLT components. PLT components containing of 3.78×10(11) to 7.23×10(11) PLTs in 300 to 450 mL of 35% to 50% plasma and 50% to 65% PLT additive solution and up to 5×10(6) red blood cells (RBCs)/mL were prepared. Each component was contaminated with 10(5) to 10(6) colony-forming units/mL Klebsiella pneumoniae and treated with 115 to 200 µmol/L amotosalen and 0 to 3 J/cm2 UVA light. For each treatment condition, the level of K. pneumoniae inactivation (log-reduction) was measured by microbiologic methods. The initial and postillumination amotosalen concentrations (µmol/L) were determined by high-performance liquid chromatography. RESULTS: For a defined set of treatment conditions, the extent of amotosalen photodegradation was consistent and reproducible. The bacterial log-reduction correlated linearly with the extent of amotosalen photodegradation with a regression correlation coefficient (r2) between 0.845 and 0.890 regardless of the treatment variables such as PLT content, component volume, plasma content, RBC content, initial amotosalen concentration, and UVA dose. CONCLUSION: This study demonstrated that the extent of amotosalen photodegradation can serve as an intrinsic actinometer which directly correlated with the level of pathogen inactivation.

Oxaliplatin-DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy.

FOLFOX is one of the most effective treatments for advanced colorectal cancer. However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by subtherapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in colorectal cancer cell lines and a pilot clinical study. Four colorectal cancer cell lines were cultured with therapeutically relevant (100 µmol/L) or diagnostic microdose (1 µmol/L) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six colorectal cancer patients received by intravenous route a diagnostic microdose containing [14C]oxaliplatin prior to treatment, as well as a second [14C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

Radiocarbon Tracers in Toxicology and Medicine: Recent Advances in Technology and Science.

This review summarizes recent developments in radiocarbon tracer technology and applications. Technologies covered include accelerator mass spectrometry (AMS), including conversion of samples to graphite, and rapid combustion to carbon dioxide to enable direct liquid sample analysis, coupling to HPLC for real-time AMS analysis, and combined molecular mass spectrometry and AMS for analyte identification and quantitation. Laser-based alternatives, such as cavity ring down spectrometry, are emerging to enable lower cost, higher throughput measurements of biological samples. Applications covered include radiocarbon dating, use of environmental atomic bomb pulse radiocarbon content for cell and protein age determination and turnover studies, and carbon source identification. Low dose toxicology applications reviewed include studies of naphthalene-DNA adduct formation, benzo[a]pyrene pharmacokinetics in humans, and triclocarban exposure and risk assessment. Cancer-related studies covered include the use of radiocarbon-labeled cells for better defining mechanisms of metastasis and the use of drug-DNA adducts as predictive biomarkers of response to chemotherapy.

Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [14C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [14C]carboplatin or [14C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer.

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Personalized medicine for targeted and platinum-based chemotherapy of lung and bladder cancer.

The personalized medicine revolution is occurring for cancer chemotherapy. Biomarkers are increasingly capable of distinguishing genotypic or phenotypic traits of individual tumors, and are being linked to the selection of treatment protocols. This review covers the molecular basis for biomarkers of response to targeted and cytotoxic lung and bladder cancer treatment with an emphasis on platinum-based chemotherapy. Platinum derivatives are a class of drugs commonly employed against solid tumors that kill cells by covalent attachment to DNA. Platinum-DNA adduct levels in patient tissues have been correlated to response and survival. The sensitivity and precision of adduct detection has increased to the point of enabling subtherapeutic dosing for diagnostics applications, termed diagnostic microdosing, prior to the initiation of full-dose therapy. The clinical status of this unique phenotypic marker for lung and bladder cancer applications is detailed along with discussion of future applications.

Amotosalen interactions with platelet and plasma components: absence of neoantigen formation after photochemical treatment.

BACKGROUND: The INTERCEPT Blood System (Baxter Healthcare Corp., and Cerus Corp.) is a photochemical treatment (PCT) process that uses amotosalen (S-59) and ultraviolet A (UVA) illumination to inactivate a broad spectrum of pathogens. STUDY DESIGN AND METHODS: To evaluate the potential of the process to create neoantigens, the amounts of residual amotosalen and photoproducts present in PCT platelets (PLTs) and PCT plasma were quantified. The initial amount of amotosalen was 150 micromol per L. After illumination with 3 J per cm2 UVA and before transfusion, a compound adsorption device was used to substantially reduce the amounts of free amotosalen and unreactive photodegradation products. Patient serum samples from Phase III clinical trials were assayed by enzyme-linked immunosorbent assay (ELISA) for antibodies to potential amotosalen neoantigens. RESULTS: After PCT, 15 percent of the starting amount of amotosalen remains bound to PLTs, and 15 to 22 percent remains bound to plasma components. The majority of bound amotosalen is associated with lipid. Less than 1 percent of PLT-bound amotosalen and approximately 2 percent of plasma-bound amotosalen can be extracted into the water-soluble protein fraction. In seven Phase III clinical trials, 523 patients received more than 8000 units of PCT PLTs or PCT plasma. None of the patients exhibited clinical or laboratory manifestations of neoantigenicity. Furthermore, no other alteration of PLT membrane proteins was identified based on testing for lymphocytotoxic antibodies and PLT-specific alloantibodies. CONCLUSION: These results indicate that no neoantigens were detected by ELISA after PCT, suggesting that transfusion of PCT PLTs or PCT plasma does not induce adverse immunologic responses.

Recovery and life span of 111indium-radiolabeled platelets treated with pathogen inactivation with amotosalen HCl (S-59) and ultraviolet A light.

BACKGROUND: A photochemical treatment (PCT) method to inactivate pathogens in platelet concentrates has been developed. The system uses a psoralen, amotosalen HCl, coupled with ultraviolet A (UVA) illumination. STUDY DESIGN AND METHODS: Three sequential clinical trials evaluated viability of PCT platelets prepared with a prototype device. Posttransfusion recovery and lifespan of (111)Indium-labeled autologous 5 day-old platelets in healthy subjects was assessed. In the first study, 23 subjects received transfusions of autologous PCT and/or control platelets. In a second study, 16 of these subjects received PCT platelets processed with a Compound Adsorption Device (CAD) (PCT-CAD) to reduce patient exposure to residual amotosalen. In the third study, the effect of gamma-irradiation on PCT platelets was studied. Data from control transfusions from Study A were used for paired comparisons in the latter 2 studies. RESULTS: Mean PCT-CAD platelet recovery for the 16 subjects with paired data was 42.5 +/- 8.7% versus 50.3 +/- 7.7% for control platelets, mean difference of 7.8% (p < 0.01). Mean lifespan for PCT-CAD platelets was 4.8 days (+/-1.3) versus 6.0 days (+/-1.2) for control platelets, mean difference of 1.3 days (p < 0.01). Platelet recovery and lifespan were similar to PCT-CAD for PCT without CAD treatment and PCT-CAD with gamma-irradiation. CONCLUSION: Viability of 5 day-old PCT platelets was less than for control platelets. However, both were within ranges reported for 5 day-old platelets.