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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.

Chemical structures, patient data, mouse survival curves, additional biomarker data

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.

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

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., Chemical Research in Toxicology, 30 (1), ISSN:0893-228X, ISSN:1520-5010

Gemcitabine metabolites cause 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 hours. 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.

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.

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 × 1011 to 7.23 × 1011 PLTs in 300 to 450 mL of 35% to 50% plasma and 50% to 65% PLT additive solution and up to 5 × 106 red blood cells (RBCs)/mL were prepared. Each component was contaminated with 105 to 106 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.

It has been shown that the COX2/PGE2 pathway is involved in the regulation of PD-L1 expression in tumor-infiltrating myeloid cells, and that angiogenesis inhibitors alter tumor stroma in a pro-immunogenic fashion. We recently developed Oomnicoxib, a compound that concurrently inhibits COX2 and soluble epoxide hydrolase (sEH), which work together to modulate expression a variety of prostaglandins of importance to inflammation and cancer. Oomnicoxib potently inhibits tumor angiogenesis in several tumor types via this mechanism of action that is unique compared to approved anti-angiogenesis drugs, which includes downregulation of COX2 and PGE2. Importantly, the compound is nontoxic to human PBMC. Accordingly, we hypothesize that Oomnicoxib modulates tumor stroma to favor immune cell infiltration and can potentiate immune activation of check point inhibitors. Progress towards comprehensive Oomnicoxib immune profiling and efficacy enhancement of a checkpoint inhibitor in mice will be presented. Note: This abstract was not presented at the conference. Citation Format: Maike Zimmermann, Sung Hee Hwang, Bruce D. Hammock, George Cimino, Paul T. Henderson. Enhancement of checkpoint inhibitor efficacy by combination treatment with a novel COX2/sEH dual inhibitor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B06.

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 111Indium-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

Cytokines generated in platelet concentrates (PCs) during storage have been implicated as possible mediators of febrile nonhemolytic transfusion reactions. Two potential methods of white cell inactivation were compared for their ability to reduce cytokine synthesis in pooled random-donor PC aliquots: treatment with gamma-radiation and photochemical treatment (PCT) using psoralens and ultraviolet A light.ABO-matched PC aliquots were pooled and divided into separate aliquots. Aliquots (20 mL) were taken from each pool to serve as an untreated control and to undergo gamma-radiation. Aliquots were treated by using either gamma-radiation (2500 or 5000 cGy) or virucidal PCT. PCT with the psoralens 8-methoxypsoralen (8-MOP), aminomethyltrimethyl psoralen (AMT), and S-59 was investigated. PC aliquots were stored for 7 days and analyzed for levels of interleukin 8 by use of an enzyme-linked immunosorbent assay. Levels of DNA adduct formation were determined by using 3H-labeled psoralens.Levels of interleukin 8 in the untreated random-donor PC aliquots increased with increasing white cell counts, but they were not affected by pooling. The untreated control aliquots and the aliquots treated with gamma-radiation had significant increases in levels of interleukin 8 after 5 to 7 days of storage (p0.05). PCT with S-59 resulted in a significant reduction in cytokine synthesis (p0.05). Day 5 to 7 levels of interleukin 8 did not differ significantly from Day 0 levels. Inhibition of interleukin 8 production by PCT increased with increasing levels of DNA modification (S-59AMT8-MOP).PCT that utilizes S-59 has been developed to inactivate potential viral and bacterial pathogens in PC aliquots while maintaining in vitro platelet function. These data demonstrate that PCT of aliquots of pooled PC aliquots before storage also prevents white cell cytokine synthesis during storage. PCT may therefore offer the potential for reducing cytokine-associated febrile nonhemolytic transfusion reactions.

A photochemical treatment (PCT) process using a novel psoralen and long wavelength ultraviolet light (UVA, 320-400 nm) has been developed to inactivate bacteria and viruses in platelet concentrates. This study evaluated the efficacy of PCT for inactivation of leukocytes that contaminate platelet preparations. Three psoralens, 8-methoxypsoralen (8-MOP), 4′-aminomethyl 4,5′,8-trimethylpsoralen (AMT), and the novel psoralen S-59, were compared using the following four independent but complementary biological and molecular assays. (1) T-cell viability: Treatment with 150 μmol/L S-59 and 1.0 to 3.0 Joules/cm2 UVA inactivated >5.4 ± 0.3 log10 of T cells in full-sized single-donor plateletpheresis units. Using 1.0 Joule/cm2 UVA, the lowest dose of S-59, AMT and 8-MOP required to reduce the number of T cells to the limit of detection was 0.05 μmol/L, 1.0 μmol/L, and 10.0 μmol/L, respectively. (2) Cytokine synthesis: Treatment with 1.9 Joules/cm2 UVA and 150 μmol/L S-59 or AMT completely inhibited synthesis of the cytokine IL-8 by contaminating leukocytes during 5 days of platelet storage. After treatment with 75 μmol/L 8-MOP and 1.9 Joules/cm2 UVA, only low levels of IL-8 were detected. (3) Psoralen-DNA adduct formation: The combination of 1.9 Joules/cm2 UVA and 150 μmol/L S-59, AMT, or 8-MOP induced 12.0 ± 3.0, 6.0 ± 0.9, and 0.7 psoralen adducts per 1,000 bp DNA, respectively. (4) Replication competence: Polymerase chain reaction (PCR) amplification of small genomic DNA sequences (242-439 bp) after PCT was inhibited. The degree of PCR amplification inhibition correlated with the level of adduct formation (S-59 > AMT > 8-MOP). In contrast, 2,500 cGy gamma radiation, a dose that inactivates >5 log10 of T cells in blood products, had minimal effect on cytokine synthesis and did not induce sufficient DNA strand breaks to inhibit PCR amplification of the same small DNA sequences. These results demonstrate that leukocytes are sensitive to PCT with psoralens and among the psoralens tested S-59 is the most effective. Therefore, PCT has the potential to reduce the incidence of leukocyte-mediated adverse immune reactions associated with platelet transfusion.

HIV-1 RNA extraction methodology, stability and cellular location in plasma were studied by quantitative analysis using reverse transcriptase (RT) and polymerase chain reaction (PCR). HIV-1 RNA as intact virus was stable in plasma at room temperature for at least for 24 h, or stable in RNAzol (Tel-Test, Inc. Texas) at −70 °C for at least 6 months. The HIV-1 RNA PCR signal did not decline significantly after freezing and thawing of the virus in plasma or in RNAzol. To assess the effect of plasma constituents from different individuals upon quantitative PCR, identical copy numbers of HIV LAI were spiked into plasma from 9 different, normal individuals. PCR detection of HIV-1 RNA did not show any significant variation in quantitative signals. Additionally, platelet-rich plasma from three seropositive subjects was fractionated into a platelet-free plasma fraction and a platelet pellet fraction. The quantitative analysis of HIV-1 RNA in these fractions, and in the corresponding peripheral blood lymphocytes (PBLs) from each patient, demonstrated that the majority of the HIV-1 RNA was distributed in the plasma, and the HIV-1 RNA in the plasma of these patients seemed not to be strongly platelet associated.

Photochemical decontamination (PCD) of platelet concentrates, with adequate preservation of platelet function, has been shown using 8- methoxypsoralen (8-MOP) and long wavelength UV light (UVA). To further evaluate this technique, models for the inactivation of pathogenic human cell-associated viruses and integrated proviral sequences are required. We have assessed the ability of the PCD technique to inactivate cell-associated human immunodeficiency virus 1 (HIV-1) in platelet concentrates. We correlated PCD inhibition of HIV-1 infectivity with 8-MOP-DNA adduct formation in contaminating nucleated cells, and measured the inhibition of polymerase chain reaction (PCR)- mediated amplification of cellular DNA sequences as a surrogate for inactivation of integrated proviral nucleic acid sequences. After PCD treatment (8-MOP 300 micrograms/mL, UVA 17 mW/cm2) for 60 minutes, 0.5 x 10(6) plaque-forming units (PFU)/mL of cell-associated HIV-1 were inactivated and no virus was detectable by infectivity assay. After 60 minutes of PCD, 15 8-MOP-DNA adducts per 1,000 bp were formed, while in the absence of UVA, no adducts were formed. PCR-mediated amplification of a 242-bp cellular DNA sequence (HLA-DQ-alpha) was inhibited when greater than eight psoralen-DNA adducts per 1,000 bp were present. These studies indicate that high titers of cell-associated HIV-1 in platelet concentrates were inactivated by PCD, and the numbers of 8-MOP- DNA adducts in nucleated cells were sufficient to inhibit amplification of DNA segments that encode for as few as 80 amino acids. Based on the frequency of 8-MOP-DNA adducts, for the 10-kb HIV-1 genome, the probability of an integrated genome without at least one 8-MOP adduct after 60 minutes of PCD was 10(-33).

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.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.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.This study demonstrated that the extent of amotosalen photodegradation can serve as an intrinsic actinometer which directly correlated with the level of pathogen inactivation.

We have developed an effective post-PCR sterilization process and have applied the procedure to a diagnostic assay for HIV-1. The method, which is based on isopsoralen photochemistry, satisfies both the inactivation and hybridization requirements of a practical sterilization process. The key feature of the technique is the use of isopsoralen compounds which form covalent photochemical adducts with DNA. These covalent adducts prevent subsequent extension of previously amplified sequences (amplicons) by Taq polymerase. Isopsoralens have minimal inhibitory effect on the PCR, are activated by long wavelength ultraviolet light, provide sufficient numbers of covalent adducts to impart effective sterilization, modify the amplified sequence such that it remains single-stranded, and have little effect on subsequent hybridization. The sterilization procedure can be applied to a closed system and is suitable for use with commonly used detection formats. The photochemical sterilization protocol we have devised is an effective and pragmatic method for eliminating the amplicon carryover problem associated with the PCR. While the work described here is limited to HIV-1, proper use of the technique will relieve the concern associated with carryover for a wide variety of amplicons, especially in the clinical setting.

356 Background: DNA adduct formation and incorporation of gemcitabine into genomic DNA are critical steps in cancer cell response to platinum (Pt) and gemcitabine chemotherapy, respectively. We hypothesize that levels of Pt-DNA adducts and gemcitabine in genomic DNA below a threshold are predictive of chemoresistance. Accelerator mass spectrometry (AMS) is an ultrasensitive method for measuring radiocarbon. By measuring 14C bound to DNA, AMS was used to quantify carboplatin-DNA damage and gemcitabine incorporation into DNA after mice or patients received nontoxic “microdoses” of 14C-labeled carboplatin or gemcitabine. Methods: Cancer cells and mice bearing tumor xenografts were treated with one microdose (1% of the therapeutic dose) or therapeutic dose of [14C]carboplatin or [14C]gemcitabine. Carboplatin-DNA adducts and gemcitabine incorporation in DNA were correlated with cell/tumor response to chemotherapy. In the Phase 0 trial, patients with advanced bladder or non-small cell lung cancer were treated with one microdose of [14C]carboplatin followed by tumor sampling 24 hours later. Carboplatin-DNA adducts and other relevant parameters, such as pharmacokinetics and repair of DNA damage, were measured and correlated with cancer response to chemotherapy. Results: The levels of microdose-induced carboplatin-DNA damage were linearly proportional to that caused by the therapeutic dose (R2=0.92, p

Background A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates. This process is based on the photochemical reaction of a novel psoralen, S-59, with nucleic acids upon illumination with long-wavelength ultraviolet light (UVA, 320-400 nm). Study design and methods High levels of pathogens were added to single-donor platelet concentrates containing 3 to 5 x 10(11) platelets in 300 mL of 35-percent autologous plasma and 65-percent platelet additive solution. After treatment with S-59 (150 microM) and UVA (0-3 J/cm2), the infectivity of each pathogen was measured with established biologic assays. In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays. The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model. Results The following levels of pathogen inactivation were achieved: >10(6.7) plaque-forming units (PFU) per mL of cell-free human immunodeficiency virus (HIV), >10(6.6) PFU per mL of cell-associated HIV, >10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus), >10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus), >10(6.6) colony-forming units of Staphylococcus epidermidis, and >10(5.6) colony-forming units of Klebsiella pneumoniae. Expression of integrated HIV was inhibited by 0.1 microM S-59 and 1 J per cm2 of UVA. In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment. Conclusion Photochemical treatment of platelet concentrates offers the potential for reducing transfusion-related viral and bacterial diseases.

Purpose: DNA adduct formation is a critical step in platinum(Pt)–induced cell death. The goal of this project is to measure carboplatin-DNA adducts after a single non-toxic microdose of [14C]carboplatin, use this information to predict efficacy before cancer patients receive toxic chemotherapy and assess the underlying chemoresistance mechanisms, thus enhancing personalized therapy. Methods: Six non-small cell lung cancer (NSCLC) and six bladder cancer cell lines were treated with 1 μM (microdose) or 100 μM (therapeutic dose) carboplatin, but with equal amounts of [14C]carboplatin. Carboplatin-DNA monoadduct levels were determined by measuring 14C content in genomic DNA with accelerator mass spectrometry (AMS). Cellular sensitivity to carboplatin (IC50 values) was correlated with drug-DNA adduct levels. To validate the in vitro findings, a pilot Phase 0 trial with microdosing [14C]carboplatin was initiated to determine the recommended Phase II dose (RP2D) of [14C]carboplatin for optimal measurement of DNA monoadducts and pharmacokinetics (PK) in lung and bladder cancer. PK and DNA monoadduct levels in peripheral blood mononuclear cells and tumor tissues were determined by liquid scintillation counting and AMS, respectively, validated with inductively coupled plasma mass spectrometry (ICP-MS), and correlated with tumor response to chemotherapy. Results: DNA monoadduct levels in cell lines induced by microdose and therapeutic concentrations of carboplatin were highly linearly proportional (r2 = 0.95, p < 0.001) as measured by AMS, and correlated to the total Pt-DNA adduct levels induced by therapeutic carboplatin (r2 = 0.80, p < 0.001) as measured by ICP-MS. DNA adduct levels were superior in predicting resistance to carboplatin when compared to the mRNA expression of ERCC1, a DNA repair gene commonly used as a resistant biomarker of Pt-based chemotherapy. Nine patients were recruited at the pilot Phase 0 trial. The Level I dose of [14C]carboplatin at 107 dpm/Kg with total carboplatin at 1% of therapeutic dose (area under curve of 6) was determined as the RP2D. No clinical toxicity was observed as a result of microdosing. The radiation exposure from 14C was less than a chest X-ray per patient. DNA monoadduct levels and PK parameters of carboplatin at the microdose and therapeutic doses were highly correlated (p Conclusions: This novel microdosing approach predicts DNA monoadduct levels and PK of therapeutic carboplatin dosing; preliminary data suggest the feasibility of predicting chemosensitivity and the possibility of guiding personalized dosing. Citation Format: Chong-xian Pan, Sisi Wang, Miaoling He, Tao Li, Tzu-yin Lin, Hongyong Zhang, Michael Malfatti, David Gandara, Tianhong Li, Ken Yoneda, Joyce Lee, Marc Dall'Era, George Cimino, Ralph de Vere White, Kenneth Turteltaub, Paul Henderson. Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2013-1183

Transmission of viral diseases through blood products remains a problem in transfusion medicine. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsorlan (8-MOP). This system is capable of inactivating 25-30 logs/hour of bacteria E. coli or S. aureus, 6 logs/hour of bacteriophage fd, 0.9 log/hour of bacteriophage R17, and 1.1 logs/hour of feline leukemia virus (FeLV) in PC. Immediately following 6 hours of PCD treatment, platelet integrity and function of PCD-treated and control PC were equivalent. After overnight storage, PCD-treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD-treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hours. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule (alpha g) content of PCD-treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 10(6)) were inactivated by PCD within 30 minutes. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ alpha locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.

We describe the advantages afforded by the use of short, crosslinkable oligonucleotide probes in hybridization assays. The ability to crosslink a probe to its target facilitates solution hybridization and permits the use of probes at sufficiently high concentrations so that equilibrium is established in short times. An elementary thermodynamic theory predicts that, at equilibrium, maximal discrimination occurs at the melting temperature (Tm) of the probe/target complex. Oligonucleotides with a site-specifically incorporated psoralen monoadduct are photocrosslinkable probes which also provide an additional tier of discrimination. Photochemical crosslinkage is very rapid compared to hybridization rates. We demonstrate kinetic covalent entrapment of a short oligonucleotide (25 bases) to its complementary sequence on a longer duplex DNA (2754 bases). Conditions which eliminate the problems of renaturation of the duplex genomic DNA are defined. Additionally, increased target coverage is obtained with photocrosslinkable probes since covalent entrapment provides a means of pumping the equilibrium between an oligonucleotide probe and its target sequence.

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5′ TpA 3′ sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.

The fluorescence (prompt emission) and phosphorescence (delayed emission) from a sample can be separated and recorded simultaneously by digital processing of the photon-induced electronic pulses they generate. A single rotating sector wheel (or “chopper”) periodically interrupts the beam of radiation incident upon the sample. Both prompt and delayed emissions from the sample pass through the same optical system to a photomultiplier. The electronic pulses they generate are processed by the same amplifiers and discriminator. Separation of the prompt and delayed components is a two-stage process. Pulses produced while the chopper is blocking the exciting beam are stored in one counter, the contents of which are a measure of the delayed emission. Pulses produced while the chopper is open are stored in another counter. The contents of this counter represent contributions of both prompt and delayed emissions. The contents of both counters are transferred to a computer for postexperimental deconvolution of the fluorescence and phosphorescence. Since the two components are measured simultaneously, the ratio of phosphorescence to fluorescence cannot be affected by changes in the sample as with sequential scans. The optical path, electronic amplification, and gating periods are identical so fluorescence and phosphorescence are measured with equal efficiency. This two-step method of signal processing recovers data which are lost in other arrangements.

PERSPECTIVES AND UMMARY 1152 INTRODUCTION 1155 ORGANIC AND STRUCTURAL CHEMISTRY OF PSORALEN AND ITS ADDUCTS 1158 Naturally Occurring Psoralens 1158 Synthesis and Radiolabeling of Psoralens 1158 Monofunctional Psoralens 1160 Relative Reactivity of Different Psoralens with Nucleic Acids 1161 Psoralen Monoadducts and Diadducts Formed With DNA and RNA 1162 PHOTOCHEMICAL PROPERTIES OFPSORALENS 1165 Spectroscopic Properties of Psoralen Molecules 1166 Reactive States for Monoadduct and Crosslink Formation in Polynucleotides ........ 1170 Controlled Production of Monoadducts and Diadducts in DoubleStranded Nucleic Acids 1172 THE USE OF PSORALENS AS PROBES FOR NUCLEIC ACID STRUCTURE AND FUNCTION 1172 Chromatin Structure 1172 Cruciforms 1175 Torsional Tension 1176 Secondary Structure in Single-Stranded Nucleic Acid 1177 Stabilization of Nucleic Acid Complexes 1184 FUTURE DIRECTIONS 1186

We report an action spectrum for the photoreversal of a psoralen cross-link joining two self- complementary DNA oligonucleotides. The cross-link was formed between two thymines (T) on opposite strands of the DNA oligomers and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). For comparison, we also present an action spectrum for the photoreversal of the isolated diadduct T-HMT-T. The wavelength dependence for the diadduct photoreversal parallels its absorption spectrum. Both the diadduct and the cross-linked DNA can be photoreversed by exposure to light with wavelengths between 240 and 3 13 nm. We did not observe photoreversal at 334 nm or above. At least two distinct absorption bands appear to contribute to photoreversal. We measured a quantum yield of 0.16 for photoreversal of the isolated diadduct at wavelengths between 240 and 266 nm. For wavelengths above 280 nm, the quantum yield is 0.30. We also observed a preferential photoreversal at the furan end of the psoralen in the T-HMT-T diadduct. In contrast, the cross-linked DNA oligonucleotides preferentially photoreversed at the pyrone end of the psoralen adduct. The rate constant for photoreversal of the cross-linked DNA is larger than that for the isolated diadduct at wavelengths below 300 nm.

Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60/sup 0/ C and subsequently cooling to 4/sup 0/ C released the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (..mu.. = 0.01), heating to 15/sup 0/ C or protease treatment at 4/sup 0/ C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4/sup 0/ C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairsmore » are present in equal concentrations and that about 20% are in a double-standard conformation in the native enzyme.« less

We have constructed a spectrometer which, using well established techniques, measures both fluorescence and circular dichroism (natural and magnetic) as well as several other parameters of interest in biophysical spectroscopy. This combination is feasible since the various measurements, aside from sharing many optical components, can all use a lock‐in amplifier to process electronic signals. Although fluorescence and circular dichroism provide greatly different information about the material under investigation, from the standpoint of instrumentation they are very similar. Combining several functions in a single spectrometer has the advantages of economy and flexibility.

A DNA oligomer 25 nucleotides long which contained an HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) furan side monoadduct to thymidine at a 5'-TpA-3' site was used as a probe for the polylinker sequence present in single-stranded M13 mp19 DNA and in double-stranded pUC 19 DNA. Hybridization and photofixation were carried out simultaneously in solution under conditions approximating the melting temperature of the probe-target hybrid. Use of probe concentrations greater than 10(-8) M permitted hybridization times of a few minutes. Irradiation with near ultraviolet light converted the HMT monoadduct present in hybrid complexes into an interstrand crosslink. Efficient photofixation removed hybrid from the equilibrium distribution and resulted in the formation of additional probe-target complex. After removal of excess probe by centrifugation through a semi-permeable membrane (Centricon-30), samples were electrophoresed through an alkaline agarose gel which was analyzed by autoradiography. When using an HMT-modified 25-mer probe end-labeled with 3,000 Ci/mmole 32P, 0.015 ng (3.8 X 10(6) copies) of M13 DNA could be detected. With this same probe 10 micrograms of denatured human DNA (corresponding to 3.0 X 10(6) copies) did not give a signal.

In photoreactivation, photochemical damage produced in DNA by far ultraviolet radiation (lambda < 320 nm) is repaired in an enzyme-mediated reaction using longer wavelength light (310 < lambda < 450 nm for E. coli). Photoreactivating enzyme (PRE) acts on a single class of photoproducts, cyclobutyl pyrimidine dimers, in an otherwise normal DNA strand at least nine bases long. PRE is one of the few DNA repair enzymes which has been purified to homogeneity in quantities sufficient for physico-chemical studies.