Comprehensive genomic profiling by liquid biopsy captures tumor heterogeneity and identifies cancer vulnerabilities in patients with RAS/BRAFV600E wild-type metastatic colorectal cancer in the CAPRI 2-GOIM trial.

BACKGROUND: Emerging evidence supports tumor tissue-based comprehensive genomic profiling (CGP) in metastatic colorectal cancer (mCRC). Data on liquid biopsy-based circulating tumor DNA (ctDNA) CGP are scarce and mainly retrospective. Prospective comparison between the two tests is not currently available. MATERIALS AND METHODS: The CAPRI 2-GOIM trial investigates efficacy and safety of ctDNA-driven, cetuximab-based sequence of three treatment lines in patients with RAS/BRAFV600E wild-type (WT) mCRC, as determined by the local laboratory. Before first-line therapy, CGP is carried out with FoundationOne (F1) CDx and F1 Liquid (F1L) CDx (324 genes) on tumor tissue DNA and plasma ctDNA, respectively. RESULTS: For 2/207 (0.96%) patients, no ctDNA was detected by F1L CDx. No patient displayed tumor fraction (TF) below 1%, whereas elevated ctDNA (TF ≥ 10%) was detected among 140/205 (68.3%) patients. One thousand and thirteen genomic variants were identified. F1L CDx found KRAS, NRAS, or BRAFV600E alterations in 19 patients, whose tumors were classified as RAS/BRAFV600E WT by the local laboratory. Both F1 CDx and F1L CDx were available for 164/205 (80%) patients. A concordance of 61.4% between the two tests was observed. The concordance increased to 72.7% for F1L CDx with TF ≥ 10%. Concordance for genes potentially involved in anti-epidermal growth factor receptor resistance was found in 137/164 (83%) patients, increasing to 91.5% for F1L CDx with TF ≥ 10%. A higher number of genomic alterations was detected by F1L CDx compared with F1 CDx, including six cases with KRAS and NRAS alterations. Overall, 109/205 (53.2%) patients displayed at least one actionable genomic alteration (I to IIIB), according to the European Society for Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT). CONCLUSION: Baseline liquid biopsy-based CGP is feasible, has high concordance with tumor tissue-based CGP, could better recapitulate tumor heterogeneity, and is clinically informative by identifying additional actionable genomic alterations in approximately half of RAS/BRAFV600E WT mCRC patients.

Antimetastatic effects of N-diazoacetyl-glycine derivatives in C57BL mice.

When three diazoacetyl-glycine derivatives, N-diazoacetyl-glycine amide (DGA), N-diazoacetyl-glycine hydrazide (DGI), and N-diazoacetyl-glycine ethyl ester (DGE), were tested against Lewis lung carcinoma in C57BL mice, DGA reduced sharply the number and weight of pulmonary metastases; the effects of DGI and DGE were less pronounced. The growth of the primary tumor was reduced slightly by DGA, but the greater effect was produced by DGI. The absence of correlation between the reduction of the growth of the primary implant and the number of lung secondary tumors for the tested compounds indicated that DGA possesses antimetastatic properties.

Antimetastatic action and hematological toxicity of p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide used as prophylactic adjuvants to surgical tumor removal in mice bearing B16 melanoma.

The treatment of mice bearing i.m. B16 melanoma with equitoxic dosages of the clinically used 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) and of its benzenoid water-soluble analogue p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt (DM-COOK) prior to surgical tumor removal results in a remarkable proportion of cures, even when the treatment is started on already palpable tumors for which surgery alone is ineffective. The survival time of mice which are not cured is also significantly increased with DM-COOK. At the same time, DM-COOK does not affect artificial metastases or spontaneous metastases in mice undergoing surgery and treated with DM-COOK postoperatively. Inhibition of i.m. tumor growth in surgical experiments, and of s.c. tumors in mice not treated with surgery, is significant, although not as pronounced as is necessary to obtain significant prolongation of the life span of the host; the survival time of mice with s.c. tumors treated with both drugs is indeed not significantly increased. DM-COOK thus appears to exert selective antimetastatic effects, unrelated to cytotoxicity for tumor cells, against B16 melanoma in addition to those reported for Lewis lung carcinoma and M5 ovarian reticular cell sarcoma; its therapeutic usefulness is evidenced in adjuvant surgical experiments. DM-COOK, unlike DTIC, is devoid of hematological toxicity for the host. Since, in leukemic mice, it is at least as active as DTIC in increasing the life span of the treated animals, it appears to be an advantageous substitute for DTIC that could undergo preliminary clinical trial.

Selectivity of the antimetastatic and cytotoxic effects of 1-p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt (+/-)-1,2-di(3,5-dioxopiperazin-1-yl)propane, and cyclophosphamide in mice bearing Lewis lung carcinoma.

The effects of two selective antimetastatic agents, 1-p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt (DM-COOK), and (+/-)-1,2-di(3,5-dioxopiperazin-1-yl)propane, have been examined in comparison with those of a cytotoxic agent, cyclophosphamide, in mice bearing Lewis carcinoma. Cyclophosphamide at the two highest dosages causes a strictly related and pronounced inhibition (to less than 10%) of the weight of the s.c. tumor, spontaneous metastases, and lung colonies formed after i.v. injection of tumor cells (artificial metastases); this behavior is consistent with a purely cytotoxic mechanism. At the three dosages used, (+/-)-1,2-di(3,5-dioxopiperazin-1-yl)propane reduces the weight of spontaneous metastases to less than 3%. A dose-dependent reduction of artificial metastasis weight is also observed. At the highest dose, artificial metastasis weight is reduced to about 5%, and s.c. tumor mass is significantly lowered to 40%. These effects are consistent with the combined occurrence of cytotoxic and selective antimetastatic action, although the latter appears to be predominant. At the three dosages used, DM-COOK markedly depresses the weight and number of spontaneous metastases to about 10%, leaving the formation of artificial metastases unaffected and causing no significant effect on primary tumor growth. The effects of these agents on the fractional incorporation of [3H]thymidine in tumor cells further indicate that only DM-COOK is devoid of cytotoxic effects for pulmonary and s.c. tumors. In hosts pretreated with DM-COOK, no reduction in the formation either of spontaneous or of artificial metastases is observed. These data indicate that DM-COOK acts directly on tumor cells and that it presumably inhibits their release from the primary tumor into the bloodstream.

Preparation and antitumor activity of 1-aryl-3,3-dimethyltriazene derivatives.

Several 1-aryl-3,3-dimethyltriazene derivatives have been synthesized and tested for their antitumor activity against the TLX5 lymphoma in mice. These compounds are characterized by the presence of a carbonyl group bound to the benzene nucleus in the para position to the triazene funciton. Three p-sulfamoyl derivatives have also been included and proved to be inactive. Among the carbonyl derivatives compounds 1 and 20, which can be used as reference, cause ILS of about 50%, respectively, at four and three dose levels. Compound 16, the o-nitro-phenylhydrazone of the hydrazide 1, is active at all six dose levels studied. The adduct 19, obtained from the same hydrazide and p-nitrobenzaldehyde, is active at four dose levels, and the ILS values at two optimum doses are significantly greater than those caused by compound 1.

Synthesis and anticancer activity of 5-diethylaminomethyl derivatives and nitrogen mustards of uracil and 2-thiouracils.

Several 5-diethylaminomethyl derivatives and nitrogen mustards of uracil and 2-thiouracil have been synthesized and tested for their potential anticancer activity in vitro on KB cells and in vivo on Ehrlich carcinoma. Among the alkylating derivatives tested several showed cytotoxic activity in vitro and compound V [5-[bis(2-chloroethyl) amino] methyl-6-propyluracil hydrochloride] showed both in vitro and in vivo anticancer activity.

Mechanism of the antileukemic effects of 1-p-carboxamidophenyl-3,3-dimethyltriazene and its in vitro metabolites.

DM-CONH2, a dimethyltriazene active in prolonging the survival time of mice bearing TLX5 lymphoma, requires metabolic activation by liver homogenate supernatant and cofactors in order to exert in vitro cytotoxic effects on the same tumor cells, as determined by in vivo bioassay of their viability. From the examination of the metabolites produced during these in vitro experiments, it is found that in vitro cytotoxicity is attributable to the generation of MM-CONH2 by oxidative N-demethylation of DM-CONH2. Also the generation of DM-COO is observed, although this compound is not cytotoxic in vitro. The in vivo effects of DM-CONH2 and CM-COOK on TLX5 lymphoma are not caused exclusively by cytotoxic effects of the drugs, since they are evident also when no reduction in the number or viability of peritoneal tumor cells is evident, whereas these parameters are significantly reduced by MM-CONH2. The increase in survival time of mice bearing TLX5 lymphoma caused by the dimethyltriazenes used appears to be caused by the drugs without being subjected to metabolic activation, with a mechanism different from cytotoxicity for tumor cells.

Antineoplastic action of p-(3-methyl-1-triazeno)benzoic acid potassium salt, a monomethyl derivative of the antimetastatic compound DM-COOK.

The antitumor and antimetastatic effects of p-(3-methyl-1-triazeno)benzoic acid potassium salt (MM-COOK) as compared with those of the parent 3,3-dimethyl derivative (DM-COOK) were examined using Lewis lung carcinoma, MCa mammary carcinoma of the CBA mouse and TLX5 lymphoma. Similarly to DM-COOK, MM-COOK reduces metastasis formation and significantly prolongs the survival of mice bearing the Lewis lung carcinoma when given at a daily dose corresponding to one-half that of DM-COOK. Unlike DM-COOK, MM-COOK exhibits significant cytotoxicity to metastatic foci and pronounced inhibition of primary tumor development. MM-COOK also causes cytotoxic effects on TLX5 lymphoma cell growing in the peritoneal cavity, even when used at low doses. The antimetastatic effects observed in mice bearing MCa mammary carcinoma are unrelated to the inhibition of primary tumor growth and are more likely due to the selection of clones endowed with lower metastatic ability. It appears that MM-COOK exhibits the same antineoplastic activity as DM-COOK, but the former does so at a lower daily dose and produces interesting cytotoxic effects other than those reflecting its antimetastatic properties. It thus seems to be a valid alternative to DM-COOK, in view of the possible introduction of newer aryltriazenes into clinical use.

Influence of serum albumins on decomposition rates of para-substituted 1-phenyl-3-methyltriazenes and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide in near physiological conditions.

The influence of human serum albumin on the decomposition rates of some arylmonomethyltriazenes in buffered aqueous solution was investigated. From the experimental data, a model for the triazene-albumin interaction was derived, and the thermodynamic parameters were systematically calculated by two independent methods. The results show marked dependence of the energetics of binding on the substituent in the triazene aromatic ring. For most of the triazenes studied, the binding with albumin was mainly enthalpy driven. Measurements also were performed using bovine and murine serum albumins.

Antitumor activity of hydrazones and adducts between aromatic aldehydes and p-(3,3-dimethyl-1-triazeno)benzoic acid hydrazide.

Hydrazones and adducts between aromatic aldehydes and p-(3,3-dimethyl-1-triazeno)benzoic acid hydrazide were synthesized and tested for antitumor activity. Two adducts derived from 2,6-dinitro- and 4-cyanobenzaldehyde were active against TLX5 lymphoma in mice. The hydrazone of the latter aldehyde was significantly more active than the adducts.

Synthesis of 1-aryl-3-formyl-3-methyltriazenes, potential metabolites of 1-aryl-3,3-dimethyltriazenes.

Some para-substituted 1-aryl-3,3-dimethyltriazenes were oxidized with tert-butyl hydroperoxide in the presence of vanadium pentoxide as a catalyst. Under these conditions, the corresponding 1-aryl-3-formyl-3-methyltriazenes, 1-aryl-3-tert-butylperoxymethyl-3-methyltriazenes, and p-nitrobenzenes were obtained. The 1-aryl-3-formyl-3-methyltriazenes might play a role in the metabolic oxidation of the 1-aryl-3,3-dimethyltriazenes, which are active as mutagenic, carcinogenic, and antitumor agents.

Kinetic study of the hydrolysis of 1-(4-nitrophenyl)-3-methyltriazene in aqueous solution and in the presence of surfactants.

The hydrolysis of 1-(4-nitrophenyl)-3-methyltriazene in aqueous solution has been studied over a pH range of 3-14. The effect of the anionic and cationic surfactants (sodium lauryl sulfate and hexadecyltrimethylammonium bromide) on the rate of hydrolysis was investigated. The quaternary ammonium bromide causes a rate decrease at all pH values studied, while sodium lauryl sulfate enhances the acid-catalyzed hydrolysis and decreases the observed rate constants in the pH-independent region. The results are discussed in terms of the current theory of micellar effects.

Kinetic study employing UV derivative spectroscopy of DM-COOK, antitumor and antimetastatic agent.

The hydrolysis of p-(3,3-dimethyl-l-triazeno)benzoic acid potassium salt (1;DM-COOK) a highly active antimetastatic and anti-disseminative agent, has been studied in buffered aqueous solution over a pH range of 2.8-8.8 degrees C. The pH dependence of the pseudo first-order rate constants showed two different routes. Under physiological conditions the hydrolysis reactions are carried out by acid catalysis. A procedure based on fourth-order derivative UV spectroscopy (D4) has been developed for the calculation of the kinetic constants at pH greater than or equal to 4.00 and no spectral interferences resulted from decomposition products. The application of derivative UV spectroscopy proved to be suitable fpr rapid, sensitive and reproducible studies of hydrolysis of this class of compounds.

Kinetic investigation of the aqueous stability and antitumor activity of a hydrosoluble diaryltriazene, AVIS, related to the antimetastatic agent DM-COOK.

The aim of this study is to investigate the hydrolysis of 1,3-di(p-carboxyphenyl)triazene dipotassium salt, AVIS (1), over a pH range of 2.60-8.50. This compound decomposes into p-aminobenzoic acid and the corresponding diazonium cation with no formation of alkylcarbo cations; the same compounds are formed from hydrolyses of DM-COOK (2), an antimetastatic agent, and of its possible demethylated metabolite, MM-COOK (3), a chemical xenogenization inducer. In these latter cases, however, a methylcarbo cation is formed. The pH dependence of the pseudo-first-order rate constants is intermediate between 2 and 3. Preliminary data on its toxicity and antitumor activity on both Lewis lung carcinoma and TLX5 lymphoma seem to indicate the essential role of alkylcarbo cation in mediating the antitumor action of aryldimethyltriazenes.

Prophylactic antimetastatic treatment with aryldimethyltriazenes as adjuvants to surgical tumor removal in mice bearing Lewis lung carcinoma.

The effects of two benzenoid dimethyltriazenes (1-p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt [DM-COOK] and 1-p-tolyl-3,3-dimethyltriazene), which have been previously shown to reduce the formation of spontaneous lung metastases in mice bearing subcutaneous Lewis lung carcinoma, have been investigated in mice implanted im with the same tumor in the calf of the hind leg. Primary tumor was removed surgically by amputation in mice treated with the tested compounds preoperatively, and the survival time of the animals was then determined. A high rate of cures, ranging from 23% to 43%, has been observed when the treatment consisted of eight or three daily doses prior to tumor removal; a single immediately preoperative dose was less effective and caused a 10% cure rate. Survival time of uncured mice was also significantly increased, particularly by DM-COOK. The division of the dose into two daily injections did not modify the activity of the triazenes, thus indicating that the antimetastatic coverage between two subsequent doses is not limited by fast pharmacokinetics or decomposition of the drugs. These results show that, at least in one animal system, aryldimethyltriazenes can be used also on palpable tumors as prophylactic adjuvants to surgery in order to reduce preoperative and intraoperative tumor spread.

Metabolism and mechanism of the antileukemic action of isomeric aryldimethyltriazenes.

Mice bearing TLX5 lymphoma or P388 leukemia were treated with ortho, meta, and para isomers of the salts of (3,3-dimethyl-1-triazeno)benzoic acid. Survival time was markedly increased in mice given the para isomer; effects were less pronounced for the meta isomer and absent for the ortho isomer. The in vivo effects of the tested compounds did not correlate either with the propensity of the drugs to undergo oxidative N-demethylation and hydrolysis to diazonium cations or with in vitro cytotoxicity for TLX5 lymphoma cells. The para isomer did not reduce the number and viability of peritoneal TLX5 lymphoma cells after in vivo and in vitro treatment, whereas a dose-dependent reduction that can even result in the absence of clonogenic tumor cells occurred in the brains of the treated animals. These data indicate that the increased survival time of the tumor-bearing mice treated with the para isomer should not be ascribed to cytotoxic effects of the drug and might be attributed to inhibition of tumor cell dissemination in various organs of the host, as already observed for solid metastasizing tumors in mice.

High-performance liquid chromatographic assay for the determination of p-(3,3-dimethyl-1-triazeno)benzoic acid in mouse plasma.

A reproducible and sensitive method is described for assaying p-(3,3-dimethyl-1-triazeno)-benzoic acid (pCOOH-DMT) and for identifying the N-desmethyl metabolite, p-(3-methyl-1-triazeno)benzoic acid (pCOOH-MMT) using high-performance liquid chromatography. The method measures concentrations as low as 1.25 nmol/ml of plasma. Extraction efficiency of internal standard or of added triazenes averages 88% and the coefficient of variation of the method is less than 10%. pCOOH-DMT is stable at room temperature at pH 7.4, whereas pCOOH-MMT undergoes rapid decomposition (half-life 6 min). pCOOH-MMT is more stable in an albumin-containing solution or in plasma, but not in boiled 9000 g mouse liver. After 80 min incubation with a 9000 g mouse liver fraction and reduced nicotinamideadenine dinucleotide phosphate, only 24% pCOOH-DMT was metabolized. Plasma pharmacokinetic studies in mice treated with 200 mg/kg intraperitoneally showed that the potassium salt of pCOOH-DMT has a half-life of 67 min.

Immunogenic changes of murine lymphoma cells following in vitro treatment with aryl-triazene derivatives.

A series of dimethyl aryl-triazene derivatives and related monomethyl compounds were studied for their efficacy in mediating a strong increase in immunogenicity (i.e., chemical xenogenization, CX) of murine leukemic cells following in vitro treatment. It was found that all compounds under investigation were able to induce CX. The dimethyl derivatives were able to induce CX only after metabolic activation, whereas related monomethyl compounds were active per se. The antigenicity acquired by triazene-treated leukemic cells was very marked; intact hosts histocompatible with the parental line were able to reject up to 10(7) cells. Antigenic tumor cells retained their immunogenic properties even after a large number of transplant generations in the absence of the drug. This means that marked immunogenicity of triazene-treated cells is a stable and heritable characteristic.